Use of zhx2 / tipe1 in diabetes

Abstract

The application belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of Zhx2 / Tipe1 in diabetes. The application discloses that Zhx2 and Tipe1 are highly expressed in islet beta cells, and high glucose environment significantly reduces the expression of Zhx2 and Tipe1, which indicates that Zhx2 and Tipe1 have the potential to regulate beta cells. The application is studied through relevant cell and animal experiments. The application proves that Zhx2 targets the Pax6 promoter region, and enhances the activity of the Pax6 promoter; and on the protein level, Tipe1 interacts with Gsa, and Tipe1 inhibits the ubiquitination degradation of Gsa. The application provides a new therapeutic target for diabetes intervention, and therefore has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and particularly relates to application of Zhx2 / Tipe1 in diabetes. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Diabetes is primarily categorized as type 1 or type 2, with over 95% of cases occurring in type 2 diabetes. This type of diabetes is primarily caused by peripheral insulin resistance due to factors such as aging and / or obesity, leading to relatively insufficient insulin secretion. Insulin is secreted exclusively by pancreatic β-cells, and therefore plays a critical role in maintaining blood glucose homeostasis. Numerous studies, both domestic and international, have demonstrated that β-cells in patients with type 2 diabetes are damaged and have reduced function. Therefore, systematic and in-depth research on the development and functional maturation of β-cells to elucidate the pathogenesis of type 2 diabetes can provide new targets and approaches for intervention and treatment. Type 2 diabetes (T2D), the predominant type of diabetes, has become a global epidemic and a major risk factor associated with various metabolic syndromes, including heart disease, stroke, and cancer. Insulin secretion by β-cells is finely regulated through complex mechanisms to meet metabolic demands. Blood glucose is the primary stimulus for insulin secretion by β-cells. β-cell dysfunction, leading to deficient insulin secretion and elevated blood glucose levels, is a key hallmark of T2D. Furthermore, insufficient β-cell mass, resulting in insufficient insulin function, is a key cause of hyperglycemia. Therefore, maintaining the function of pancreatic β cells in the prediabetes stage and identifying potential therapeutic targets for preventing and treating T2D have become the focus of people's efforts.

[0004] Currently, patients with type 2 diabetes are primarily treated with oral hypoglycemic drugs and exogenous insulin injections, along with dietary control and moderate exercise to maintain stable blood sugar levels. However, long-term medication can easily burden the liver and kidneys, leading to drug resistance; long-term exogenous insulin injections can damage the patient's skin and cause dystrophic atrophy of the subcutaneous fat. Some patients undergo surgical resection of gastric tissue to achieve dietary control, but this can lead to death and complications. Transplantation of healthy allogeneic pancreatic beta cells can also be therapeutic, but its application is limited by a shortage of donors. There is currently no cure for diabetes, and much research is needed.

[0005] The Zinc Fingers and Homeoboxes (ZHX) family includes ZHX1, 2, and 3. Zhx2 is located on chromosome 8q24. The family members contain two zinc finger domains and five homeodomain (HD) domains. Initially, Zhx2 was a key transcriptional repressor of alpha-fetoprotein regulatory factor 1, which is an important oncogene in hepatocarcinoma. Previously, we first defined Zhx2 as a tumor suppressor in hepatocellular carcinoma (HCC) in our study. In addition, multiple studies have also shown that Zhx2 has an inhibitory effect on tumor development. However, in clear cell renal cell carcinoma and breast cancer, Zhx2 appears as a tumor oncogene. In addition, Zhx2 exerts various functions by inhibiting stem cell-like features, limiting NK cell maturation, inhibiting hepatitis B virus replication, promoting macrophage glycolysis and survival, or regulating neural precursor cell maintenance. These data show the diversity of Zhx2 functions. However, the role of Zhx2 in diabetes is still unclear.

[0006] The TIPE (Tumor necrosis factor alpha-induced protein 8) family is a new gene family discovered in 2008, which is involved in immune regulation and the occurrence and development of various tumors. The TIPE family includes four members, TIPE, TIPE1, TIPE2, and TIPE3. TIPE1 (also known as TNFAIP8L1) is the latest discovered member of the family. Homology analysis found that TIPE1 has about 55% homology with TIPE, TIPE2, and TIPE3, but the homology between human TIPE1 and mouse TIPE1 is as high as 82%.

[0007] Tipe1, located on chromosome 19 and composed of 188 amino acids, is a protein primarily expressed in the cytoplasm. In 2008, the journal Cell first reported the crucial relationship between TIPE1 and cell death, garnering significant attention for its role in immune regulation and tumor progression. Recent studies have shown that TIPE1 can regulate macrophage infiltration and inhibit the maturation and activation of dendritic cells, thereby exerting immunomodulatory effects. In cancer research, researchers have reported that TIPE1 functions as a tumor suppressor in gastric, breast, liver, lung, and colorectal cancers, while promoting tumor progression in cervical and nasopharyngeal cancers. Previous studies by our research group have revealed that TIPE1 is widely expressed in human tissues and organs, with particularly high expression in the liver, sparking our interest in further investigation. Our research has revealed that TIPE1 inhibits the development and progression of liver cancer by inducing apoptosis and inhibiting the growth and proliferation of liver cancer cells. Furthermore, studies have also implicated TIPE1 in the development and progression of non-alcoholic fatty liver disease. These reports demonstrate the diverse functions of TIPE1. However, the role of TIPE1 in the occurrence and development of diabetes and its specific expression and regulation mechanism are still lacking in-depth research. Summary of the Invention

[0008] To address the shortcomings of the prior art, the present invention aims to provide applications of Zhx2 / Tipe1 in diabetes. Through research, the present invention has discovered that downregulating the expression of Zhx2 and Tipe1, which are highly expressed in pancreatic β cells, can promote the development of diabetes. This discovery provides a new target for the intervention and treatment of diabetes, particularly type 2 diabetes. Based on these research findings, the present invention was completed.

[0009] In order to achieve the above technical objectives, the technical solutions provided by the present invention are as follows:

[0010] In a first aspect, the present invention provides a biomarker for (auxiliary) diagnosis, detection, monitoring or prediction of diabetes progression, wherein the biomarker includes but is not limited to the coding genes of Zhx2 and Tipel and their expression products.

[0011] The present invention discovered through research that both Zhx2 and Tipe1 are highly expressed in pancreatic beta cells, but their expression levels are significantly reduced in type 2 diabetes. Further studies have shown that both Zhx2 and Tipe1 positively regulate pancreatic beta cell function and beta cell mass. Therefore, during the development and progression of (type 2) diabetes, reduced expression of Zhx2 and Tipe1 also leads to further decline in pancreatic beta cell function, exacerbating the development and progression of diabetes. Therefore, by analyzing the expression of Zhx2 and Tipe1 in pancreatic beta cells, it can be used for (assisted) diagnosis, detection, monitoring, or prediction of the progression of diabetes, especially type 2 diabetes.

[0012] The second aspect of the present invention provides the use of substances for detecting the above-mentioned biomarkers in the preparation of products for (auxiliary) diagnosis, detection, monitoring or prediction of the progression of diabetes.

[0013] A third aspect of the present invention provides a system for (assisting) diagnosis, detection, monitoring or prediction of the progression of (type 2) diabetes, the system comprising:

[0014] i) an analysis unit, the analysis unit comprising: a detection substance for determining the expression level of the above-mentioned biomarkers in a test sample of a subject, and;

[0015] ii) an evaluation unit, comprising: performing (auxiliary) diagnosis, detection, monitoring or prognostic evaluation of diabetes mellitus on the subject according to the expression level of the biomarker determined in i).

[0016] Wherein, the sample to be tested includes the subject's pancreatic β cells and / or pancreatic islet tissue.

[0017] A fourth aspect of the present invention provides the use of the above-mentioned Zhx2 and / or Tipel as targets in the preparation and / or screening of (type 2) diabetes drugs.

[0018] The (type 2) diabetes drug may specifically be a drug for preventing and / or treating (type 2) diabetes.

[0019] In another embodiment of the present invention, the method for screening (type 2) diabetes drugs comprises:

[0020] 1) treating a system expressing and / or containing the Zhx2 and / or Tipe1 with a candidate substance; setting up a parallel control without treatment with the candidate substance;

[0021] 2) After completing step 1), detecting the expression level of Zhx2 and / or Tipe1 in the system; if the expression level of Zhx2 and / or Tipe1 in the system treated with the candidate substance is significantly reduced compared with the parallel control, the candidate substance can be used as a candidate (type 2) diabetes drug.

[0022] A fifth aspect of the present invention provides use of a substance for inhibiting the expression of Zhx2 and / or Tipel in any one or more of the following:

[0023] a) Disruption of glucose homeostasis;

[0024] b) inhibit pancreatic β-cell proliferation;

[0025] c) mediate a diabetes-like phenotype;

[0026] d) inhibiting the expression of key β-cell genes and / or insulin-related genes;

[0027] e) inhibit Pax6 transcription;

[0028] f) Promotes ubiquitination and degradation of Gsa protein;

[0029] e) Construct an animal model of (type 2) diabetes.

[0030] Specifically, the β-cell key genes and insulin-related genes include but are not limited to Pdx1, MafA, Ins1, Pax6, Glut2, Pcna and Ki67.

[0031] Among them, the substances that inhibit the expression of Zhx2 and / or Tipe1 include but are not limited to RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA against Zhx2 and / or Tipe1, substances for implementing lentiviral infection or gene knockout, and specific antibodies against Zhx2 and / or Tipe1 itself or its upstream and downstream molecules, such as anti-Zhx2 antibodies and anti-Tipe1 antibodies.

[0032] A sixth aspect of the present invention provides use of a substance for promoting the expression of Zhx2 and / or Tipel in any one or more of the following:

[0033] a) Promote glucose homeostasis;

[0034] b) Promote the proliferation of pancreatic β cells;

[0035] c) Promote insulin secretion;

[0036] d) promoting the expression of key β-cell genes and / or insulin-related genes;

[0037] e) Promotes Pax6 transcription;

[0038] f) inhibiting the ubiquitination and degradation of Gsa protein;

[0039] e) prevention and / or treatment of (type 2) diabetes.

[0040] The specific applications of the fifth and sixth aspects mentioned above may be in the form of preparing corresponding drugs or experimental reagents, which can be used for basic research.

[0041] According to the present invention, when the product is a medicine, the medicine further comprises at least one inactive pharmaceutical ingredient.

[0042] Beneficial technical effects of one or more of the above technical solutions:

[0043] The technical scheme provides application of Zhx2 / Tipe1 in diabetes, specifically, Zhx2 and Tipe1 are highly expressed in islet beta cells, and a high-sugar environment significantly reduces the expression of Zhx2 and Tipe1, indicating that Zhx2 and Tipe1 have the potential to regulate beta cells. In vivo, beta cell-specific knockout of Zhx2 (Zhx2BKO) or Tipe1 (Tipe1BKO) mice reduces beta cell proliferation and glucose homeostasis. Under diabetic conditions, we found that Zhx2BKO mice induced T2D glucose intolerance through HFD diet. The results show that the number of beta cells and insulin secretion of Zhx2BKO-db / db mice is reduced; compared with db / db mice, the number of beta cells and insulin secretion of Tipe1BKO-db / db mice is significantly reduced. Mechanistically, it is proved that Zhx2 targets the Pax6 promoter region, enhances the activity of the Pax6 promoter; and at the protein level, Tipe1 interacts with Gsa, and Tipe1 inhibits the ubiquitination degradation of Gsa. The technical scheme provides a new therapeutic target for diabetes intervention, and therefore has good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0045] Figure 1 Figure 1 is a diagram related to high expression of Zhx2 in islet beta cells in Embodiment 1 of the present application; wherein A is a diagram of detecting Zhx2 mRNA expression in different tissues and organs by qPCR; B is a diagram of detecting Zhx2 expression in pancreatic tissue by immunohistochemical staining; C is a diagram of detecting Zhx2 mRNA expression in beta cells and non-beta cells in islets by qPCR; D is a diagram of detecting insulin and Zhx2, glucagon and Zhx2, somatostatin and Zhx2 by immunofluorescence staining; E is a diagram of detecting Zhx2 mRNA expression in different cell lines by qPCR; F is a diagram of detecting Zhx2 protein expression in different cell lines by Western Blotting.

[0046] Figure 2Figure 1 is a graph showing that the diabetic environment reduces the expression of Zhx2 in β cells in Example 1 of the present invention; wherein, A is a graph showing the expression of Zhx2 mRNA in pancreatic tissue of db / m and db / db mice detected by qPCR; B is a graph showing the expression of Zhx2 protein in pancreatic tissue of db / m and db / db mice detected by Western Blotting; C is a graph showing the expression of Zhx2 protein in pancreatic tissue of db / m and db / db mice detected by Western Blotting; D is a graph showing the expression of Zhx2 mRNA in pancreatic islet tissue of db / m and db / db mice detected by qPCR; E is a graph showing the fasting blood glucose levels of wild-type (WT) and streptozotocin-injected combined with high-fat-fed (STZ+HFD) mice; F is a graph showing the expression of Zhx2 mRNA in pancreatic islet tissue of WT and STZ+HFD mice detected by qPCR; G is a graph showing the expression of Zhx2 mRNA in MIN6 cells after high-glucose stimulation detected by qPCR; H is a graph showing the expression of Zhx2 mRNA in pancreatic islet tissue of MIN6 cells detected by Western Blot analysis of Zhx2 protein expression in MIN6 cells after high glucose stimulation; I is the detection of insulin and Zhx2 in pancreatic tissues of db / m and db / db mice by immunofluorescence double staining; J is the detection of insulin and Zhx2 in pancreatic tissues of normal subjects and T2D patients by immunofluorescence double staining.

[0047] Figure 3 Figure 1 shows how the loss of Zhx2 in pancreatic β cells impairs glucose homeostasis in vivo; A shows a model constructed using β-cell-specific Zhx2 knockout mice; and B shows a model constructed using qPCR to detect Ins2. cre+ and Ins2 cre+ Zhx2 f / f Figure 3. Zhx2 mRNA expression in different tissues and organs of mice; C is the detection of Ins2 by Western Blotting cre + and Ins2 cre+ Zhx2 f / f Figure 3. Zhx2 protein expression in mouse pancreatic islet tissue; D is the detection of Ins2 by immunofluorescence staining cre + and Ins2 cre+ Zhx2 f / f Expression of Zhx2 and insulin in mouse islets; E is body weight; F is random blood glucose; G is fasting blood glucose; H is fasting insulin; I is glucose tolerance test; J is insulin secretion test; K is insulin sensitivity test.

[0048] Figure 4 This is a graph showing that in HFD-induced T2D mice, Zhx2 deficiency leads to a diabetic-like phenotype and reduced β-cell proliferation in Example 1 of the present invention; wherein A is the Ins2 of the 6-month HFD dietcre+ and Ins2 cre+ Zhx2 f / f Appearance of mice; B is the growth curve; C is fasting blood glucose; D is fasting insulin; E is glucose tolerance test; F is insulin secretion test; G is insulin sensitivity test; H is immunohistochemical staining of insulin in mouse pancreatic tissue; I is the statistics of pancreatic β cells occupying the area of ​​pancreatic tissue; J is the statistics of β cell mass; K is immunofluorescence staining of Ki67 and insulin in mouse pancreatic tissue.

[0049] Figure 5 Figure 1 is a graph showing the expression of related genes in MIN6 cells after Zhx2 overexpression for 48 hours, as shown in Example 1 of the present invention; Figure A shows the expression of related genes in MIN6 cells after Zhx2 overexpression for 48 hours, as shown in Figure B; Figure C shows the expression of related proteins in MIN6 cells after Zhx2 overexpression for 48 hours, as shown in Figure C; cre+ and Ins2 cre+ Zhx2 f / f The expression of related genes in mouse pancreatic islet tissue; D is the detection of Ins2 by Western Blotting cre+ and Ins2 cre+ Zhx2 f / f Diagram of the expression of related proteins in mouse pancreatic islet tissue.

[0050] Figure 6 This is a graph related to the transcriptional regulation of PAX6 by Zhx2 in pancreatic β cells in Example 1 of the present invention; wherein A is the detection of Ins2 by qPCR in high-fat-fed animals for 6 months cre+ and Ins2 cre+ Zhx2 f / f Figure 3. Pax6 gene expression in mouse islets; B is a graph showing the expression of Pax6 gene in Zhx2-overexpressing MIN6 cells detected by qPCR; C is the co-transfection of Zhx2 plasmid and Pax6 promoter plasmid in MIN6 cells, and the effect of Zhx2 overexpression on Pax6 promoter activity was detected by luciferase reporter gene; D is the analysis of the unique sequence bound by Zhx2 on the Pax6 promoter using the database (PMID: 30026228); E is the ChIP experiment detecting the binding of Zhx2 to the Pax6 promoter region in MIN6 cells.

[0051] Figure 7Figure 1 is a graph showing that the effect of Zhx2 in pancreatic β cells is partially dependent on Pax6 in Example 1 of the present invention; wherein, A is a graph showing the expression of Pax6 detected by qPCR 48-72 hours after co-transfection of MIN6 cells with Zhx2 plasmid and Pax6 small interfering protein; B is a graph showing the expression of pancreatic β cell functional genes (Pdx1, MafA, Ins2, Ins1, Glut2) detected by qPCR 48-72 hours after co-transfection of MIN6 cells with Zhx2 plasmid and Pax6 small interfering protein; C is a graph showing the expression of proliferation (Pcna, Ki67) related genes detected by qPCR 48-72 hours after co-transfection of MIN6 cells with Zhx2 plasmid and Pax6 small interfering protein; D is a graph showing the expression of pancreatic β cell proliferation (Pcna, Ki67) and functional genes (Pdx1, MafA, Ins2, Ins1, Glut2) detected by Western Blotting.

[0052] Figure 8 The figures are related to the fact that Tipe1 is highly expressed in pancreatic β cells and is reduced in type 2 diabetes in Example 2 of the present invention; wherein, A is the expression of Tipe1 in insulin (β cells), glucagon (α cells), and somatostatin (δ cells) detected by immunofluorescence staining; B is the expression of Tipe1 in pancreatic β cells and non-β cells detected by qPCR; C is the expression of Tipe1 mRNA in different cell lines detected by qPCR; D is the expression of Tipe1 in pancreatic tissue of db / m and db / db (type 2 diabetes model mice) detected by qPCR; E is the expression of Tipe1 in pancreatic tissue of db / m and db / db (type 2 diabetes model mice) detected by Western Figure 3 shows the expression of Tipe1 in pancreatic tissues of db / m and db / db (type 2 diabetes model mice) detected by blotting; F shows the expression of Tipe1 in pancreatic islets of type 2 diabetes patients detected by database analysis; G shows the expression of Tipe1 in pancreatic islet tissues of db / db (type 2 diabetes model mice) detected by qPCR; H shows the expression of Tipe1 in high glucose-stimulated Min6 cells detected by qPCR; I shows the expression of Tipe1 in high glucose-stimulated Min6 cells detected by Western blotting.

[0053] Figure 9 This is a diagram showing the disruption of blood glucose homeostasis by Tipe1 knockout in pancreatic β cells in Example 2 of the present invention; wherein, A is a diagram showing the Tipe1 knockout strategy in mouse pancreatic β cells; B is a diagram showing the knockout effect detected by qPCR; C is a diagram showing the effect of Tipe1 knockout detected by Western Blotting; D is body weight; E is random blood glucose; F is fasting blood glucose; G is fasting insulin; H is glucose tolerance; I is insulin secretion detected by GSIS experiment; and J is insulin sensitivity.

[0054] Figure 10 This is a graph related to the aggravated diabetic phenotype of pancreatic β-cell deficiency in type 2 diabetic mice in Example 2 of the present invention; wherein, A is a graph showing the Tipe1 knockout strategy in pancreatic β-cells of db / db mice; B is the appearance of mice at 16-20 weeks; C is a growth curve; D is fasting blood glucose; E is glucose tolerance; F is insulin secretion detected by the GSIS experiment; and G is insulin sensitivity.

[0055] Figure 11 These are graphs related to Tipe1 positively regulating pancreatic β-cell function, i.e., promoting proliferation and insulin secretion, in Example 2 of the present invention, wherein A is the expression of Pcna gene in MIN6 cells after interfering with Tipe1 expression by qPCR; B is the expression of Pcna protein in MIN6 cells after interfering with Tipe1 expression by Western Blotting; C and D are graphs of Pcna gene and protein expression in pancreatic islets after knocking out pancreatic β-cell Tipe1 by qPCR and Western Blotting, respectively; E is a graph of intracellular insulin content after knocking out pancreatic β-cell Tipe1 expression by insulin ELISA; F is a graph of high glucose-stimulated insulin secretion after overexpression of pancreatic β-cell Tipe1 by insulin ELISA; G is a graph of the expression of β-cell functional genes in pancreatic islets after knocking out pancreatic β-cell Tipe1 by qPCR; and H is a graph of the expression of β-cell functional genes in pancreatic islets after overexpressing Tipe1 by Western Blotting.

[0056] Figure 12: This is a diagram of the interaction between Tipe1 and related genes in Example 2 of the present invention, wherein A is HEK293T cells overexpressing Tipe1, and IP detection of the interaction between Tipe1 and Gsa; B is HEK293T cells overexpressing GNAS, and IP detection of its interaction with Tipe1; C is MIN6 cells detecting the interaction between endogenous Tipe1 and Gsa; D is a diagram of GNAS gene expression after Tipe1 knockout in pancreatic β cells detected by qPCR; E is a diagram of GSA protein expression levels in pancreatic islets of Tipe1BKO and control mice detected by Western Blotting; F is a diagram of GSA protein expression levels after Tipe1 interference in MIN6 cells detected by Western Blotting; G is a diagram of GSA protein expression levels after Tipe1 overexpression in MIN6 cells detected by Western Blotting; L is a diagram of Tipe1 affecting Gsa protein stability detected by Western Blotting; and M is a diagram of Tipe1 affecting Gsa protein degradation through the proteasome pathway detected by Western Blotting.

[0057] Figure 13 These are graphs related to Tipe1 inhibiting the ubiquitination and degradation of Gsa protein in Example 2 of the present invention, wherein A shows Tipe1 inhibiting endogenous ubiquitination and degradation of Gsa in HEK293T cells; B shows Tipe1 inhibiting exogenous ubiquitination and degradation of Gsa in HEK293T cells; and C shows Tipe1 inhibiting endogenous ubiquitination and degradation of Gsa in MIN6 cells.

[0058] Figure 14 This is a graph showing that the effect of Tipe1 in pancreatic β cells is partially dependent on the Gsa protein in Example 2 of the present invention, wherein A is a graph showing the protein expression levels of proliferation and insulin-related genes 72 hours after co-transfection of si-NC or si-Tipe1 with pcDNA3.0 or pcDNA-GNAS plasmid; B is a graph showing the interference efficiency of Gsa and the protein expression levels of proliferation and insulin-related genes detected by Western Blotting analysis. DETAILED DESCRIPTION

[0059] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0060] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0061] In a typical embodiment of the present invention, a biomarker for (assisting) diagnosis, detection, monitoring or prediction of diabetes progression is provided, wherein the biomarker includes but is not limited to the coding genes of Zhx2 and Tipel and their expression products.

[0062] The present invention discovered through research that both Zhx2 and Tipe1 are highly expressed in pancreatic beta cells, but their expression levels are significantly reduced in type 2 diabetes. Further studies have shown that both Zhx2 and Tipe1 positively regulate pancreatic beta cell function and beta cell mass. Therefore, during the development and progression of (type 2) diabetes, reduced expression of Zhx2 and Tipe1 also leads to further decline in pancreatic beta cell function, exacerbating the development and progression of diabetes. Therefore, by analyzing the expression of Zhx2 and Tipe1 in pancreatic beta cells, it can be used for (assisted) diagnosis, detection, monitoring, or prediction of the progression of diabetes, especially type 2 diabetes.

[0063] In another embodiment of the present invention, there is provided the use of substances for detecting the above-mentioned biomarkers in the preparation of products for (auxiliary) diagnosis, detection, monitoring or prediction of the progression of diabetes.

[0064] The substance specifically includes a substance for detecting the transcription of Zhx2 and / or Tipe1 in a sample based on RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing; or a substance for detecting the expression of Zhx2 and / or Tipe1 in a sample based on an immunoassay method.

[0065] The diabetes is specifically type 2 diabetes.

[0066] In another specific embodiment of the present invention, the transcription of Zhx2 and / or Tipe1 in the sample can be detected by methods including but not limited to liquid hybridization, Northern hybridization, miRNA expression profile chip, ribozyme protection analysis technology, RAKE method, and in situ hybridization; the expression of Zhx2 and / or Tipe1 in the sample can be detected by methods including but not limited to ELISA, colloidal gold test strips, and protein chips.

[0067] The sample is specifically pancreatic β cells and / or pancreatic tissue.

[0068] In another embodiment of the present invention, a system for (assisting) diagnosis, detection, monitoring or prediction of (type 2) diabetes progression is provided, the system comprising:

[0069] i) an analysis unit, comprising: a detection substance for determining the expression level of the above-mentioned biomarkers in a test sample of a subject, and;

[0070] ii) an evaluation unit, comprising: performing (auxiliary) diagnosis, detection, monitoring or prognostic evaluation of diabetes mellitus on the subject according to the expression level of the biomarker determined in i).

[0071] Wherein, the sample to be tested includes pancreatic β cells and / or pancreatic tissue of the subject.

[0072] In another specific embodiment of the present invention, the above detection substances include substances that detect the transcription of Zhx2 and / or Tipe1 in samples based on RT-PCR, real-time quantitative PCR, in situ hybridization, gene chips and gene sequencing; or substances that detect the expression of Zhx2 and / or Tipe1 in samples based on immunoassay methods.

[0073] The system for (assisting) diagnosis, detection, monitoring, or prediction of diabetes progression of the present invention may be a virtual device, as long as it can realize the functions of the analysis unit and the evaluation unit. The analysis unit may include various detection reagent materials and / or detection instruments and equipment; the evaluation unit may be any computing instrument, module, or virtual device that can analyze and process the detection results of the analysis unit to obtain a type 2 diabetes risk assessment. For example, a corresponding data chart can be prepared in advance to match various possible detection results with corresponding disease risk conditions. By comparing the detection results of the detection unit with the data chart, a type 2 diabetes risk assessment and prognosis assessment result can be obtained.

[0074] In another embodiment of the present invention, there is provided the use of the above-mentioned Zhx2 and / or Tipel as targets in the preparation and / or screening of (type 2) diabetes drugs.

[0075] The (type 2) diabetes drug may specifically be a drug for preventing and / or treating (type 2) diabetes.

[0076] In another embodiment of the present invention, the method for screening (type 2) diabetes drugs comprises:

[0077] 1) treating a system expressing and / or containing the Zhx2 and / or Tipe1 with a candidate substance; setting up a parallel control without treatment with the candidate substance;

[0078] 2) After completing step 1), detecting the expression level of Zhx2 and / or Tipe1 in the system; if the expression level of Zhx2 and / or Tipe1 in the system treated with the candidate substance is significantly reduced compared with the parallel control, the candidate substance can be used as a candidate (type 2) diabetes drug.

[0079] In another embodiment of the present invention, the system can be a cell system, a subcellular system, a solution system, a tissue system, an organ system or an animal system.

[0080] In another embodiment of the present invention, the cells in the cell system may be pancreatic β cells;

[0081] In another embodiment of the present invention, the tissue in the tissue system may be pancreatic tissue;

[0082] In another embodiment of the present invention, the organ in the organ system may be the pancreas;

[0083] In another specific embodiment of the present invention, the animals in the animal system can be non-human animals, further non-human mammals, including but not limited to mice, rats, guinea pigs, rabbits, monkeys, gorillas, etc.

[0084] In another embodiment of the present invention, there is provided use of a substance for inhibiting the expression of Zhx2 and / or Tipel in any one or more of the following:

[0085] a) Disruption of glucose homeostasis;

[0086] b) inhibit pancreatic β-cell proliferation;

[0087] c) mediate a diabetes-like phenotype;

[0088] d) inhibiting the expression of key β-cell genes and / or insulin-related genes;

[0089] e) inhibit Pax6 transcription;

[0090] f) Promotes ubiquitination and degradation of Gsa protein;

[0091] e) Construct an animal model of (type 2) diabetes.

[0092] In another specific embodiment of the present invention, the β-cell key genes and insulin-related genes include but are not limited to Pdx1, MafA, Ins1, Pax6, Glut2, Pcna and Ki67.

[0093] In another specific embodiment of the present invention, the substance that inhibits the expression of Zhx2 and / or Tipe1 includes but is not limited to RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA against Zhx2 and / or Tipe1, substances for implementing lentiviral infection or gene knockout, and specific antibodies against Zhx2 and / or Tipe1 itself or its upstream and downstream molecules, such as anti-Zhx2 antibodies and anti-Tipe1 antibodies.

[0094] In another embodiment of the present invention, there is provided use of a substance for promoting the expression of Zhx2 and / or Tipel in any one or more of the following:

[0095] a) Promote glucose homeostasis;

[0096] b) Promote the proliferation of pancreatic β cells;

[0097] c) Promote insulin secretion;

[0098] d) promoting the expression of key β-cell genes and / or insulin-related genes;

[0099] e) Promotes Pax6 transcription;

[0100] f) inhibiting the ubiquitination and degradation of Gsa protein;

[0101] e) prevention and / or treatment of (type 2) diabetes.

[0102] In another specific embodiment of the present invention, the β-cell key genes and insulin-related genes include but are not limited to Pdx1, MafA, Ins1, Pax6, Glut2, Pcna and Ki67.

[0103] In another specific embodiment of the present invention, substances that promote the expression of Zhx2 and / or Tipe1 include but are not limited to substances that upregulate the expression of Zhx2 and / or Tipe1 based on gene-specific Mimics technology; such as artificially synthesized short hairpin RNA (shRNA) of Zhx2 and / or Tipe1 or promoters or lentiviruses that upregulate the expression of Zhx2 and / or Tipe1; and also include compound promoters.

[0104] The above-mentioned specific applications may be in the form of preparing corresponding drugs or experimental reagents, and the experimental reagents can be used for basic research.

[0105] According to the present invention, when the product is a medicine, the medicine further comprises at least one inactive pharmaceutical ingredient.

[0106] The inactive pharmaceutical ingredients may be carriers, excipients, and diluents commonly used in pharmacy. Furthermore, according to conventional methods, the pharmaceutical composition may be prepared into oral preparations, external preparations, suppositories, and sterile injectable solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays.

[0107] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0108] In another specific embodiment of the present invention, the carrier, excipient and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0109] In another embodiment of the present invention, the medicine of the present invention can be administered to the body in a known manner. For example, it can be delivered to the tissue of interest by systemic intravenous delivery or local injection. Alternatively, it can be administered intravenously, percutaneously, intranasally, through the mucosa, or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered in the present invention can vary depending on various factors to a great extent, such as the target cell, the type of organism or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0110] In another embodiment of the present invention, the subjects of drug administration can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, etc.

[0111] The present invention will be further described below in conjunction with the embodiments. The present invention will be further described below by way of the embodiments, but the present invention is not limited to the scope of the embodiments. Based on the embodiments of the present invention, any variation of the present invention by those skilled in the art without making any creative idea belongs to the protection scope of the present invention.

[0112] Example 1

[0113] Wild-type (WT) C57BL / 6 male mice were provided by the Experimental Animal Center of Shandong University (Jinan). Ins2-Cre (RIP-Cre) mice (stock number 003573) were purchased from Jackson Laboratory. Zhx2-floxed (Zhx2 f / f ) mice were donated by Professor BT Spear of the University of Kentucky. To eliminate the expression of Zhx2 in β cells, wef / f Mice with Ins2Cre + β-cell selective knockout mice (Ins2Cre + Zhx2 f / f or Zhx2BKO). In this study, Ins2 Cre+ After 1 week of adaptive diet, 4-week-old mice were fed a high-fat diet (HFD; 60% Kcal fat) for 16-24 weeks to induce obesity. db / m mice were provided by Professor Gong Yaoqin of Shandong University. + Zhx2 f / f Mice were mated to generate Zhx2 knockout and Lepr mutants (Zhx2BKO-db / db) or a control group (Con-db / db). All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Shandong University, and the experiments were approved by the Animal Ethics Committee of Shandong University (approval number: ECSBMSSDU2019-2-028). A high-fat diet (HFD) combined with streptozotocin (STZ) induced a T2D model. WT mice were randomly divided into two groups: a control group (4 mice) and an STZ+HFD-fed group (4 mice). The control group was fed a regular diet for 5 months. Mice in the STZ+HFD-fed group were fed an HFD diet for 1 month at 4 weeks of age. After fasting for 12 hours, 50 mg / kg STZ (Sigma) was intraperitoneally injected for 3 consecutive days, and then continued to be fed an HFD diet for 4 months.

[0114] cell

[0115] Human embryonic kidney 293T (HEK293T) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). MIN6 cell lines were obtained from the Provincial Hospital of Shandong University and cultured in Dulbecco's modified Eagle ...

[0116] Isolation and culture of pancreatic islets

[0117] Islets were isolated by digestion with 1 mg / ml type V collagenase (Solarbio-C8170, Beijing, China). After digestion at 37°C for 30 minutes, the islets were dispersed. Islets were manually extracted in d-Hanks' buffered saline solution. After at least two harvests, islets were collected and transferred to RPMI1640 medium supplemented with 10% FBS. Incubation was continued at 37°C for 12 hours in a humidified atmosphere containing 5% CO2 and 95% air.

[0118] DNA construction, siRNA, transfection and lentiviral infection

[0119] pcDNA3.0-zhx2-ha (pcZhx2) and pcDNA3.0 (pc3.0) plasmids were used for transfection. MIN6 cells were plated at 2×10 5 Cells were plated at a density of 10 cells / cm² on 35 mm culture dishes. After 24 hours, cells were transfected using Lipo2000 reagent according to the manufacturer's instructions. The corresponding cDNA sequence was cloned into the lentiviral expression vector pMX-Zhx2 to generate the lentiviral expression vector encoding mouse Zhx2. For lentiviral infection, MIN6 cells were cultured in basal medium to 70% confluence, then replaced with fresh medium containing lentivirus and incubated for 16-24 hours. After infection, cells were selected with 10 μg / ml puromycin.

[0120] To knockdown Zhx2 or Pax6, MIN6 cells were transfected with a 50 nM siRNA mixture for 48-72 hours using Lipo2000 reagent (Invitrogen, USA). The siRNA sequences used were 5'-CCGCUGAAUACUACCAAAUTT-3' (SEQ ID NO. 1) for Zhx2 and 5'-GGACCCAUUAUCCAGAUGTT-3' (SEQ ID NO. 2) for Pax6. NC-siRNA at the same concentration served as a negative control.

[0121] Glucose tolerance test (GTT), glucose-stimulated insulin secretion test (GSIS), and insulin tolerance test (ITT)

[0122] Ins2Cre expression in normal chow (NCD) or high-fat diet (HFD), or in Con-db / db and Zhx2BKO-db / db mice after 16 h of fasting + 、Ins2Cre + Zhx2 f / f 、Zhx2 f / fMice were subjected to GTT testing. Blood glucose levels were recorded using a glucometer (Yicheng, China) 15, 30, 60, 90, and 120 minutes after fasting for 16 hours and intraperitoneal injection of 2g / kg body weight glucose. GSIS mice were fasted for 16 hours overnight and intraperitoneally injected with glucose (2g / kg body weight). Blood insulin levels at designated time points after glucose injection were measured using a mouse insulin ELISA kit (Elabscience, China). ITT tests were performed on mice fasting for 4-6 hours using the same glucometer. After recording fasting blood glucose values ​​for 4-6 hours, mice were intraperitoneally injected with human insulin (Sigma-Aldrich) at a dose of 0.7mU / g. Blood glucose levels were monitored at 15, 30, and 60 minutes after injection, respectively.

[0123] Immunohistochemistry (IHC) and immunofluorescence (IF) staining

[0124] Pancreatic tissue was fixed with 4% paraformaldehyde and embedded in paraffin. For immunohistochemistry, 5 μm sections were stained with antibodies or control IgG using the EnVision+ System-HRP (DAB) kit (Dako North America Inc, Carpinteria, CA, USA) according to the manufacturer's instructions. IF was performed with antibodies against insulin (ab7842, abcam), glucagon (sc-514592, Santa Cruz), somatostatin (sc-74556, Santa Cruz), Zhx2 (20136-1-AP, Proteintech), PAX6 (sc-81649, Santa Cruz), and Ki67 (ab16667, abcam) overnight at 4°C and incubated with AlexaFluor 594 goat anti-guinea pig IgG (ab150188, abcam), AlexaFluor 594 goat anti-mouse IgG (SA00006-3, Proteintech), and AlexaFluor 488 goat anti-rabbit IgG (SA00006-2, Proteintech) for 1 hour at 37°C. Microscopic analysis was performed with an Olympus IX51 microscope and Image J software.

[0125] Western blotting

[0126] Cell or tissue lysates were prepared using RIPA buffer (P0013B, Biotechnology). Equal amounts (20-30 g) were processed by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. After incubation with the corresponding antibodies ZHX2 (20136-1-AP, Proteintech), PCNA (ab92552, abcam), PAX6 (sc-81649, Santa cruz), MAFA (sc-390491, Santa cruz), PDX1 (sc-390792, Santa cruz), insulin (sc-377071, Santa cruz), GLUT2 (sc-518022, Santa cruz), HA (M180-3, MBL), β-actin (66009-1-Ig, Proteintech), and GAPDH (60004-1-Ig, Proteintech), the cells were incubated with secondary antibodies against rabbit IgG (SA00001-2) and mouse IgG (SA00001-1), respectively.

[0127] Quantitative reverse transcription PCR (RT-qPCR)-qPCR

[0128] Total RNA was isolated from cells or tissues using TRIzol reagent (Invitrogen). cDNA was synthesized using the RevertAid FirstStrand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific). Real-time quantitative PCR (qPCR) was performed using SYBR Green PCR reagent (FP205, TIANE).

[0129] Chromatin immunoprecipitation (ChIP) assay

[0130] Using EZMagnaChIP TMThe A / G Chromatin Immunoprecipitation Kit (17-10086; Germany) was used according to the manufacturer's instructions. Cells were fixed, and DNA was sheared to 200–1000 bp by sonication. The DNA was then incubated with anti-Zhx2 antibody (20136-1-AP, Proteintech) and rabbit IgG (sc-2027, Santa Cruz, CA, USA), respectively. A 1 / 50 ratio of the starting chromatin (input) was used as a control. The primers for PCR or qPCR were as follows: promoter segment 1 (-1740 to -1563) F: 5'-CCCAAGGCAGATGGTGGAAT-3' (SEQ ID NO.3), R: 5'-CCCGCGAATCCTGAGAGTTT-3' (SEQ ID NO.4), promoter region 2 (-1342 to -1148) F: 5'-CCAAAACTGAAGGAACCGGC-3' (SEQ ID NO.5), R: 5'-GGGAAGTTCGAGGGCTAGAG-3' (SEQ ID NO.6), promoter region 3 (-862 to -559) F: 5'-CTAAGCCGTCCTCTGTTCCAC-3' (SEQ ID NO.7), R: 5'-AACCCGGCCAAATCTAAGCC-3' (SEQ IDNO.8) and promoter region 4 (-251~+75) F: 5'-CTTAAGCCGAACCTCAGGGA-3' (SEQ ID NO.9), R: 5'-GTAGCAGATCTGACAACCGGG-3' (SEQ ID NO.10).

[0131] Results and Discussion

[0132] 1. Zhx2 is highly expressed in pancreatic β cells

[0133] RT-qPCR and IHC staining showed that Zhx2 was highly expressed in mouse pancreatic islet cells ( Figure 1 A and B). To further determine the expression of Zhx2 in pancreatic β cells, we sorted pancreatic β cells by flow cytometry. Consistently, the mRNA level of Zhx2 in pancreatic β cells was higher than that in non-β cells ( Figure 1 C). We then performed IF staining on mouse pancreatic sections using specific antibodies against Zhx2, insulin (β cells), glucagon (α cells), and somatostatin (δ cells). The results showed that Zhx2 was highly expressed in insulin-expressing β cells compared to α and δ cells ( Figure 1D). In addition, we also detected the mRNA and protein levels of Zhx2 in mouse insulinoma cell lines (MIN6, betaTC6), colon cancer cell line (CT26), liver cancer cell line (Hepa1-6), and lymphoma cell line (EL4). The results showed that the expression of Zhx2 in MIN6 cells was higher than that in other cells ( Figure 1 E and F), indicating that Zhx2 is highly expressed in pancreatic β cells, suggesting that Zhx2 has a potential regulatory role in pancreatic β cells.

[0134] 2. Diabetic environment reduces Zhx2 expression in β cells

[0135] Next, we attempted to determine the expression of Zhx2 under high glucose conditions. First, we examined the pancreatic mRNA and protein levels in 16-20-week-old db / m and db / db type 2 diabetes mice. The results showed that Zhx2 expression was significantly decreased in db / db mice ( Figure 2 A, B, C), we further detected the mRNA levels in the pancreatic islets of 16-20 week db / m and db / db mice, and found that Zhx2 was also significantly decreased in db / db mice ( Figure 2 D). It has been reported that HFD combined with streptozotocin (STZ) is a very effective method to induce T2D in rats. Then, we used HFD combined with streptozotocin (STZ) to induce T2D. After 5 months of induction, the fasting blood glucose of mice in the STZ and HFD feeding groups was higher than that in the WT group ( Figure 2 E). Zhx2 expression in pancreatic islets of mice fed with STZ and HFD was also decreased compared with that in the WT group ( Figure 2 F). Consistent with this finding, the expression of Zhx2 was significantly decreased in MIN6 cells exposed to high glucose (33.3 mM) for 48 h in vitro compared with the normal glucose (25 mM) and mannose (33.3 mM) groups ( Figure 2 G and h). We also observed that 20-week-old db / db mice ( Figure 2 I) and T2D patients ( Figure 2 J) The fluorescence intensity of Zhx2 is reduced in pancreatic β cells. In summary, Zhx2 is decreased under diabetic conditions, which may be related to β cell failure and diabetes.

[0136] 3. Loss of Zhx2 in pancreatic β cells impairs glucose homeostasis

[0137] The β-cell-specific expression of Zhx2 prompted us to investigate whether Zhx2 plays a role in β-cells. To clarify the role of Zhx2 in pancreatic β-cells, we transfected Zhx2-floxed (Zhx2 f / f) mice were crossed with RIP-Cre (Ins2-Cre) mice to obtain mice with conditional knockout of β cells, namely Zhx2 f / f ;Ins2 Cre+ mice (hereafter referred to as Zhx2BKO) Figure 3 A). Isolate pancreatic islets and different tissues to detect the knockout efficiency of Zhx2 in pancreatic β cells. qPCR ( Figure 3 B), Western blotting( Figure 3 C) and immunofluorescence staining ( Figure 3 D) shows that Zhx2 mRNA and protein levels were significantly reduced in Zhx2BKO islets relative to control islets. There was no significant difference in body weight between Zhx2BKO mice and control mice ( Figure 3 E), random blood glucose in Zhx2BKO mice tended to increase ( Figure 3 F), Fasting blood glucose in Zhx2BKO mice was significantly increased ( Figure 3 G), while fasting blood glucose and insulin tended to decrease in Zhx2BKO mice ( Figure 3 H). GTT results showed that glucose tolerance was significantly abnormal in 8-12 week old Zhx2BKO male mice compared with the sex- and age-matched control group ( Figure 3 I). Next, we performed GSIS experiments to investigate whether Zhx2 regulates insulin secretion in mice. Compared with the control group, insulin secretion in Zhx2BKO mice was significantly reduced ( Figure 3 J), although the ITT results showed no significant difference in insulin sensitivity between the two groups ( Figure 3 K). In conclusion, Zhx2 deficiency in pancreatic β-cells impairs glucose homeostasis.

[0138] 4. In HFD-induced T2D mice, Zhx2 deletion leads to a diabetic-like phenotype and reduced β-cell proliferation

[0139] Since prolonged exposure to high lipid concentrations (3-6 months) can increase β-cell number by inducing β-cell proliferation, we used a HFD-induced T2D model in mice to determine whether HFD stress affects β-cell function and proliferation in Zhx2BKO mice during the pre-diabetic stage. We found that Zhx2BKO mice had no significant difference in body weight after HFD feeding compared with the control group ( Figure 4 A and B). We then examined the effects of Zhx2 deficiency on glucose homeostasis in mice. First, we measured fasting blood glucose in Zhx2BKO and control mice. The results showed that fasting blood glucose in Zhx2BKO mice was significantly increased ( Figure 4 C). Consistent with this, fasting insulin levels were also significantly reduced in Zhx2BKO mice ( Figure 4D). Next, we tested the GTT of the control and Zhx2BKO mice exposed to HFD for 3-6 months. The results showed that Zhx2BKO mice showed obvious glucose intolerance compared with the control mice ( Figure 4 E). HFD-fed Zhx2BKO mice consistently showed reduced insulin secretion in the GSIS test ( Figure 4 F), but there was no significant difference in insulin resistance between Zhx2BKO mice and control mice ( Figure 4 G). Insulin was detected by immunohistochemical staining of pancreatic tissues of Zhx2BKO mice ( Figure 4 H) and statistical analysis, we observed that the islet β-cell area ratio (per mouse pancreas) and β-cell number were significantly decreased in HFD-fed Zhx2BKO mice ( Figure 4 IJ). In addition, the proportion of Ki67-positive β cells in pancreatic islet tissue of Zhx2BKO mice was reduced under HFD stress compared with control mice ( Figure 4 K). These results indicate that Zhx2BKO mice develop a diabetic phenotype and reduced β-cell proliferation after high-fat diet exposure.

[0140] 5. Zhx2 promotes β-cell proliferation and insulin-related gene expression

[0141] Next, we aimed to investigate whether Zhx2 affects the expression of key genes in β cells, so we interfered with or increased the level of Zhx2 in β cells. We used RT-qPCR ( Figure 5 A) or Western blotting ( Figure 5 B) Detection of key β-cell genes in MIN6 cells transfected with pc3.0 or pcZhx2 plasmids for 48 hours. As expected, proliferation and insulin-related genes were increased in MIN6 cells overexpressing Zhx2 ( Figure 5 A and B). We further examined the expression of key β-cell genes in the pancreatic islets of Zhx2BKO and control mice at 8-12 weeks. Compared with control mice, these key genes were significantly reduced in the pancreatic islets of Zhx2BKO mice ( Figure 5 C and D). These data indicate that Zhx2 regulates β-cell function by promoting β-cell proliferation and insulin-related gene expression.

[0142] 6. Zhx2 Transcriptionally Regulates PAX6 in Pancreatic β Cells

[0143] Next, we attempted to determine the molecular mechanism by which Zhx2 regulates pancreatic beta cells. To identify the direct target genes of Zhx2, we performed cluster analysis on Zhx2 target genes in ccRCC chip sequencing data, as well as genes reported to be decreased in human T2D and genes highly expressed in beta cells. Through database analysis, we found Pax6, a key gene that has been reported to regulate beta cell function. Multiple studies have confirmed that Pax6 binds to the promoter regions of several key genes to regulate beta cells, such as Glut2 (encoding SLC2A2), Pdx1, MafA, and Ins. Therefore, we believe that Zhx2 may regulate beta cell function through Pax6. To verify the effect of Zhx2 on Pax6, we detected the expression of Pax6 in the islets of Zhx2BKO and control mice after HFD feeding. The results of qPCR detection showed that the expression of Pax6 in the islets of Zhx2BKO mice was significantly reduced ( Figure 6 A). We further found that overexpression of Zhx2 significantly increased the expression of Pax6 ( Figure 6 B). Further dual luciferase assay showed that Zhx2 overexpression significantly promoted the activity of Pax6 promoter in MIN6 cells ( Figure 6 C). We searched for potential unique binding sites of Zhx2 in the Pax6 promoter region and found that Zhx2 has three unique motifs in the Pax6 promoter region ( Figure 6 D). ChIP assay using anti-Zhx2 showed that Zhx2 clearly bound to the Pax6 promoter in MIN6 cells ( Figure 6 E) These results indicate that Zhx2 can bind to the Pax6 site and positively regulate Pax6 transcription.

[0144] 7. The role of Zhx2 in pancreatic β cells is partially dependent on Pax6

[0145] To verify the involvement of Pax6 in Zhx2-mediated regulation of pancreatic β-cell proliferation and function, we added siRNA targeting Pax6 to MIN6 cells. Si-NC or si-Pax6 were co-transfected with pc3.0 or pcZhx2 plasmids for 72 h. The results showed that Pax6 interference partially reversed the upregulation of proliferation and insulin-related genes in the Zhx2 overexpression group ( Figure 7 BD). The interference efficiency of Pax6 was detected by RT-qPCR and Western blotting analysis ( Figure 7 A and D). Taken together, these data suggest that Zhx2 promotes β-cell proliferation and function by promoting Pax6 transcription.

[0146] Example 2

[0147] Materials and Methods

[0148] Wild-type (WT) C57BL / 6 male mice were provided by the Experimental Animal Center of Shandong University (Jinan). Ins2-Cre (RIP-Cre) mice (stock number 003573) were purchased from Jackson Laboratory. To eliminate Tipe1 expression in β cells, we induced TIPE1 f / f Mice and Ins2 Cre+ β-cell selective knockout mice (Ins2Cre + Tipe1 f / f or Tipe1BKO). In this study, Ins2-Cre + or Tipe1 f / f As a control, db / m mice were provided by Professor Gong Yaoqin of Shandong University. Cre+ Tipe1 f / f Mice were cross-linked to generate Tipe1 knockout and Lepr mutants (Tipe1BKO-db / db) or control groups (Con-db / db). All animal operations were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Shandong University and were approved by the Animal Ethics Committee of Shandong University (approval number: ECSBMSSDU2019-2-028).

[0149] cell

[0150] Human embryonic kidney 293T (HEK293T) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). MIN6 cell lines were obtained from the Provincial Hospital of Shandong University and cultured in Dulbecco's modified Eagle ...

[0151] Isolation and culture of pancreatic islets

[0152] Islets were isolated by digestion with 1 mg / ml type V collagenase (Solarbio-C8170, Beijing, China). After digestion at 37°C for approximately 30 minutes, the islets were dispersed. Islets were manually extracted in d-Hanks' buffered saline solution. After at least two harvests, islets were collected and transferred to RPMI 1640 medium supplemented with 10% FBS. Incubation was continued at 37°C for 12 hours in a humidified atmosphere containing 5% CO2 and 95% air.

[0153] DNA construction, siRNA, transfection and lentiviral infection

[0154] Construct PRK5-Tipe1 overexpression plasmid. During transfection, MIN6 cells were plated at 2×10 5 cells / cm 2 Cells were plated at a density of 100 μg / mL on 35 mm culture dishes and transfected with Lipo2000 reagent 24 hours later according to the manufacturer's instructions. To knock down Tipe1 or Gnas, MIN6 cells were transfected with a 50 nM siRNA mixture using Lipo2000 reagent (Invitrogen, USA) for 48-72 hours. The siRNA sequences used were 5'CUCUUGUUGUACCAGACUAdTdT-3' (SEQ ID NO. 11) for Tipe1 and 5'-GCAGCUACAACAUGGUCAUTT-3' (SEQ ID NO. 12) or 5'-CUGCAUGUUAAUGGGUUUATT-3' (SEQ ID NO. 13) for Gnas. NC-siRNA at the same concentration was used as a negative control.

[0155] Glucose tolerance test (GTT), glucose-stimulated insulin secretion test (GSIS), and insulin tolerance test (ITT)

[0156] Ins2re in normal diet (NCD) or Con-db / db and Tipe1BKO-db / db mice after 16 h fasting + 、ins2re + Tipe1 f / f Tipe1 f / f Mice were subjected to GTT testing. Blood glucose levels were recorded using a glucometer (Yicheng, China) at 15, 30, 60, 90, and 120 minutes after fasting for 16 hours or intraperitoneal injection of 2g / kg body weight glucose. GSIS mice were fasted for 16 hours overnight and intraperitoneally injected with glucose (2g / kg body weight). Blood insulin levels at designated time points after glucose injection were measured using a mouse insulin ELISA kit (Elabscience, China). ITT tests were performed on mice fasted for 4-6 hours using the same glucometer. After recording blood glucose values ​​after fasting for 4-6 hours, mice were intraperitoneally injected with human insulin (Sigma-Aldrich) at a dose of 0.7mU / g. Blood glucose levels were monitored at 15, 30, and 60 minutes after injection.

[0157] Immunohistochemistry (IHC) and immunofluorescence (IF) staining

[0158] Pancreatic tissue was fixed with 4% paraformaldehyde and embedded in paraffin. For immunohistochemistry, 5 μm sections were stained with antibodies or control IgG using the EnVision+ System-HRP (DAB) kit (Dako North America Inc, Carpinteria, CA, USA) according to the manufacturer's instructions. IF was performed using antibodies against insulin (ab7842, abcam), glucagon (sc-514592, Santa Cruz), somatostatin (sc-74556, Santa Cruz), and Tipe1 (BS60529, Proteintech). The sections were incubated overnight at 4°C and incubated with AlexaFluor 594 goat anti-guinea pig IgG (ab150188, abcam), AlexaFluor 594 goat anti-mouse IgG (SA00006-3, Proteintech), and AlexaFluor 488 goat anti-rabbit IgG (SA00006-2, Proteintech) at 37°C for 1 hour. Microscopic analysis was performed using an Olympus IX51 microscope and imageJ software.

[0159] Western blotting

[0160] Cell or tissue lysates were prepared using RIPA buffer (P0013B, Biotechnology). Equal amounts (20-30 g) were processed by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. After incubation with the corresponding antibodies Tipe1 (BS60529, Bioworld), PCNA (ab92552, abcam), MAFA (sc-390491, Santa cruz), PDX1 (sc-390792, Santa cruz), insulin (sc-377071, Santa cruz), GLUT2 (sc-518022, Santa cruz), HA (M180-3, MBL), DDDK (M185-3, MBL), β-actin (66009-1-Ig, Proteintech), and GAPDH (60004-1-Ig, Proteintech), the cells were incubated with secondary antibodies against rabbit IgG (SA00001-2) and mouse IgG (SA00001-1), respectively.

[0161] Quantitative reverse transcription PCR (RT-qPCR)-qPCR

[0162] Total RNA was isolated from cells or tissues using TRIzol reagent (Invitrogen). cDNA was synthesized using the RevertAid FirstStrand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific). Real-time quantitative PCR (qPCR) was performed using SYBR Green PCR reagent (FP205, TIANE).

[0163] Co-immunoprecipitation (Co-IP)

[0164] First, cells were lysed using WB-IP lysis buffer + protease inhibitor (PMSF) (100:1); 1 / 10 of the lysed solution was reserved as input, and 2-4 μg of the corresponding primary antibody was added to the remaining 9 / 10 and shaken at 4°C overnight; the next day, 15-30 μl of Protein A / G beads were added and incubated at 4°C for 2-4 hours; the samples were washed five times with PBS and collected; 1×SDS-PAGE was added to both the input and IP groups, and the proteins were denatured by boiling at 100°C for 5 minutes; and protein electrophoresis was performed.

[0165] Results and Discussion

[0166] 1. Tipe1 is highly expressed in pancreatic β cells and is downregulated in type 2 diabetes

[0167] To determine the expression of Tipe1 in pancreatic β cells, we first performed IF staining on mouse pancreatic sections using specific antibodies against Tipe1, insulin (β cells), glucagon (α cells), and somatostatin (δ cells). The results showed that Tipe1 was highly expressed in insulin-expressing β cells compared to α and δ cells ( Figure 8 A). Next, we sorted pancreatic β cells by flow cytometry. Consistently, the mRNA level of Tipe1 in pancreatic β cells was higher than that in non-β cells ( Figure 8 B). In addition, we also detected the mRNA level of Tipe1 in mouse insulinoma cell lines (MIN6, betaTC6), colon cancer cell line (CT26), liver cancer cell line (Hepa1-6) and lymphoma cell line (EL4). The results showed that the expression of Tipe1 in MIN6 cells was higher than that in other cells ( Figure 8 C), indicating that Tipe1 is highly expressed in pancreatic β cells, suggesting that Tipe1 has a potential regulatory role in pancreatic β cells.

[0168] Next, we attempted to determine Tipe1 expression under high glucose conditions. First, we examined pancreatic mRNA and protein levels in 16-20-week-old db / m and db / db type 2 diabetes mice. The results showed that Tipe1 expression was significantly reduced in the pancreas of db / db mice ( Figure 8 D and E). We analyzed the expression of Tipe1 in pancreatic islets of normal subjects and T2D patients in the database (GSA50397), and the results showed that Tipe1 expression was significantly downregulated in pancreatic islet tissues of T2D patients ( Figure 8 F). Subsequently, we further examined the mRNA levels in pancreatic islets of db / m and db / db mice at 16-20 weeks and found that Tipe1 expression was significantly decreased in db / db mice ( Figure 8 G). We detected a significant decrease in the expression of Tipe1 at both RNA and protein levels in MIN6 cells exposed to high glucose (40 mM) for 48 h in vitro ( Figure 8 H and I). In summary, Tipe1 expression is reduced under diabetic conditions, which may be associated with β-cell failure.

[0169] 2. Knockout of Tipe1 in pancreatic β cells disrupts blood glucose homeostasis

[0170] The specific expression of Tipe1 in β cells prompted us to investigate whether Tipe1 plays a role in β cells. f / f ) mice were crossed with RIP-Cre (Ins2-Cre) mice to obtain mice with conditional knockout of β cells, namely Tipe1 f / f ;Ins2 Cre+ Mice (hereafter referred to as Tipe1BKO) Figure 9 A). Isolate pancreatic islets and other tissues, and detect the knockout efficiency of Tipe1 in pancreatic islets and other tissues and organs by qPCR ( Figure 9 B), Western blotting to detect the knockout efficiency of Tipe1 in pancreatic islets ( Figure 9 C) The results showed that Tipe1 mRNA and protein levels were significantly reduced in the islets of Tipe1BKO mice compared with the control islets. There was no significant change in the body weight of Tipe1BKO mice compared with the control mice ( Figure 9 D), there is no significant change in random blood glucose ( Figure 9 E), Fasting blood glucose tends to increase in Tipe1BKO mice ( Figure 9 F), there was no significant difference in fasting insulin ( Figure 9 GTT results showed that Tipe1BKO male mice had significantly abnormal glucose tolerance compared with the sex- and age-matched control group ( Figure 9H). Next, we performed GSIS experiment to investigate whether Tipe1 regulates insulin secretion in mice. Compared with the control group, the insulin secretion level of Tipe1BKO mice was significantly reduced Figure 9 I). ITT results showed that the insulin sensitivity of Tipe1BKO mice was increased Figure 9 J), indicating that the abnormal blood glucose of Tipe1BKO mice was caused by insufficient insulin secretion rather than insulin resistance. In summary, Tipe1 deficiency in pancreatic beta cells impairs glucose homeostasis.

[0171] 3. Tipe1 deficiency in pancreatic beta cells of type 2 diabetic mice exacerbates diabetic phenotype

[0172] Next, we performed specific knockout of Tipe1 molecules in beta cells in T2D model mice db / db mice. The knockout strategy is as follows Figure 10 A. We found that the body size of Tipe1BKO-db / db mice at 16 weeks was significantly smaller than that of control db / db mice. During the growth of mice, the increase in body weight of Tipe1BKO-db / db mice was significantly less than that of control mice Figure 10 B and C). We detected the fasting blood glucose level of mice, and the results showed that the fasting blood glucose of Tipe1BKO-db / db mice was significantly increased Figure 10 D). Then we detected the GTT of db / db and Tipe1BKO-db / db mice, and the results showed that compared with the control mice, Tipe1BKO-db / db mice showed more severe and obvious glucose intolerance Figure 10 E). Consistent with this, Tipe1BKO-db / db mice always showed a significant decrease in insulin secretion in GSIS test Figure 10 F). But in terms of insulin resistance, Tipe1BKO-db / db mice were less insulin resistant than control mice Figure 10 G), indicating that the abnormal blood glucose homeostasis of Tipe1Bko mice was caused by the decrease of insulin secretion rather than the insulin resistance of the body. These results show that Tipe1 deficiency in pancreatic beta cells of type 2 diabetic mice exacerbates diabetic phenotype.

[0173] 4. Tipe1 positively regulates pancreatic beta cell function-promotes proliferation and promotes insulin secretion

[0174] To further investigate the role of Tipe1 in pancreatic β-cells, we knocked down Tipe1 expression in MIN6 cells and detected the expression of the proliferation gene Pcna in MIN6 β-cells transfected with NC or Tipe1 siRNA for 48 h by qPCR and Western blotting. As expected, knockdown of Tipe1 reduced the expression of Pcna ( Figure 11 A and B). Next, we isolated pancreatic islet tissues from Tipe1BKO and control mice and detected the expression of Pcna by qPCR and Western blotting. The results showed that the expression of Pcna in pancreatic islet tissues of Tipe1BKO mice was significantly reduced ( Figure 11 C and D). We further examined the insulin levels in the pancreatic islets of Tipe1BKO and control mice, and the results showed that the insulin levels in the pancreatic islets of Tipe1BKO mice were lower than those in the control mice ( Figure 11 E). In vitro stimulation of pancreatic islet tissue using high glucose showed that insulin secretion in Tipe1BKO mouse islets was significantly reduced ( Figure 11 F). We detected the expression of genes related to β-cell function in the pancreatic islets of Tipe1BKO mice by qPCR. The results showed that the expression of genes related to β-cell function was significantly downregulated in the pancreatic islets of Tipe1BKO mice ( Figure 11 G). Subsequently, we overexpressed Tipe1 in MIN6 cells and used Western blotting to detect the expression of β-cell function-related gene protein levels in MIN6 cells transfected with control or Tipe1 overexpression plasmids for 48 hours. The results showed that Tipe1 overexpression increased the expression of β-cell function-related gene protein levels ( Figure 11 H). These data indicate that Tipe1 regulates β-cell function by promoting β-cell proliferation and insulin-related gene expression.

[0175] 5. Tipe1 interacts with Gsa and inhibits Gsa protein degradation through the proteasome pathway

[0176] Next, we attempted to determine the molecular mechanism by which Tipe1 regulates pancreatic beta cells. By analyzing the mass spectrometry results of the Tipe1 molecule and the database, we found the coding gene for Gnas (Gsa), a key gene that regulates beta cells. We then verified the interaction between Tipe1 and Gsa using Co-IP technology for exogenous verification in HEK293T cells and endogenous verification in MIN6 cells. The results showed that Tipe1 had a strong interaction with Gsa at both exogenous and endogenous levels ( Figure 12A, B, and C). Next, we first determined the effect of Tipe1 on the expression of Gnas gene levels. We used qPCR to detect the expression of Gnas gene in the pancreatic islets of Tipe1BKO and control mice. The results showed that Tipe1 did not affect the expression of Gnas gene levels ( Figure 12 D). We then examined the effect of Tipe1 on Gsa protein levels. Western blotting was used to detect changes in Gsa protein levels in the pancreatic islets of Tipe1BKO and control mice. The results showed that Gsa expression was significantly reduced in Tipe1BKO mice ( Figure 12 E). To further clarify the effect of Tipe1 on the expression of Gsa protein in β cells, we intervened the expression of Tipe1 in MIN6 cells and detected it by Western blotting. We found that interfering with Tipe1 expression reduced the expression of Gsa protein ( Figure 12 F), consistent with this, overexpression of Tipe1 upregulated the expression of Gsa protein level ( Figure 12 G). Next, we treated HEK293T cells transfected with control and Tipe1 overexpression plasmids with cycloheximide and used Western blotting to detect the effect of Tipe1 on exogenous Gsa protein degradation. The results showed that Tipe1 overexpression reduced the protein degradation rate of Gsa ( Figure 12 H). To determine the pathway through which Tipe1 affects the degradation of Gsa protein levels, we treated HEK293T cells transfected with control and Tipe1 overexpression plasmids with chloroquine (CQ) and MG132, and detected the expression of Gsa protein levels by Western blotting. The results showed that Tipe1 inhibited the degradation of Gsa protein through the proteasome pathway ( Figure 12 I). The above experiments demonstrated that Tipe1 interacts with Gsa and inhibits Gsa protein degradation through the proteasome pathway.

[0177] 6. Tipe1 inhibits Gsa ubiquitination and degradation

[0178] Previous results showed that Tipe1 inhibited the degradation of Gsa protein. By predicting the protein modification sites on Gsa through the database, we found that Gsa was enriched with multiple ubiquitination sites and only a small number of other types of protein modification sites. We first verified the ubiquitination modification of Gsa. We transfected control and human Tipe1 overexpression plasmids into HEK293T cells and detected the ubiquitination level of endogenous Gsa and the effect of Tipe1 on Gsa ubiquitination by Western blotting. The results showed that Gsa protein was significantly ubiquitinated and Tipe1 inhibited the ubiquitination degradation of Gsa protein ( Figure 13A). Next, we verified whether Tipe1 affects the exogenous ubiquitination of Gsa. The results also showed that Tipe1 inhibited the ubiquitination and degradation of Gsa ( Figure 13 B). To clarify the effect of Tipe1 on Gsa ubiquitination in β cells, we overexpressed Tipe1 in MIN6 cells and performed Western blotting. The results showed that Tipe1 inhibited the endogenous ubiquitination and degradation of Gsa in MIN6 cells ( Figure 13 C) The above experiments demonstrated that Gsa is ubiquitinated and Tipe1 inhibits Gsa ubiquitination and degradation.

[0179] 7. The role of Tipe1 in pancreatic β cells is partially dependent on Gsa

[0180] To verify that Gsa is involved in Tipe1-mediated regulation of pancreatic β-cell proliferation and function, we added siRNA-1 and siRNA-2 targeting GNAS to MIN6 cells. Figure 14 As shown in A, si-NC or si-Tipe1 was co-transfected with pc3.0 or pcGnas plasmid for 72 h. The results showed that GNAS overexpression partially reversed the downregulation of proliferation and insulin-related genes induced by Tipe1 interference ( Figure 14 A); In B, si-NC or si-Gnas1, si-Gnas2 were co-transfected with Ctrl or Tipe1 overexpression plasmid for 72 h. The results showed that Gsa interference partially reversed the upregulation of proliferation and insulin-related genes in the Tipe1 overexpression group ( Figure 14 B). Taken together, these data suggest that Tipe1 promotes β-cell proliferation and function by promoting Gsa.

[0181] It should be noted that the above examples are only intended to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the examples given, those skilled in the art may modify or replace the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. The use of substances for detecting biomarkers used to diagnose, detect, monitor or predict the progression of diabetes in the preparation of products for diagnosing, detecting, monitoring or predicting the progression of diabetes; The diabetes is type 2 diabetes; The biomarkers include genes encoding Zhx2 and Tipe1 and their expression products.

2. The use of substances for detecting biomarkers used to assist in the diagnosis, detection, monitoring or prediction of diabetes progression in the preparation of products used to assist in the diagnosis, detection, monitoring or prediction of diabetes progression; The diabetes is type 2 diabetes; The biomarkers include the coding genes of Zhx2 and Tipe1 and their expression products.

3. The use according to claim 1 or 2, characterized in that The substance comprises a substance for detecting the transcription of Zhx2 and Tipe1 in a sample based on real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing; or a substance for detecting the expression of Zhx2 and Tipe1 in a sample based on an immunoassay method.

4. The use according to claim 3, characterized in that The transcription of Zhx2 and Tipe1 in samples was detected by liquid phase hybridization, Northern hybridization, miRNA expression microarray, ribozyme protection analysis, RAKE method, and in situ hybridization; the expression of Zhx2 and Tipe1 in samples was detected by ELISA, colloidal gold test strips, and protein microarray. The sample is pancreatic β cells and / or pancreatic tissue.

5. Use of a substance that inhibits Zhx2 expression in constructing an animal model of type 2 diabetes, characterized in that: The substance that inhibits Zhx2 expression is 5'-CCGCUGAAUACUACCAAAUTT-3'.

6. Use of a substance that inhibits the expression of Zhx2 and Tipe1 in constructing an animal model of type 2 diabetes, characterized in that: The substance that inhibits the expression of Zhx2 is 5'-CCGCUGAAUACUACCAAAUTT-3'; the substance that inhibits the expression of Tipe1 is 5'-CUCUUGUUGUACCAGACUAdTdT-3'.

7. Use of a substance that promotes Zhx2 expression in the preparation of a medicament for preventing and / or treating type 2 diabetes, characterized in that: The substance that promotes Zhx2 expression is a lentivirus that overexpresses Zhx2.

8. Use of a substance that promotes the expression of Zhx2 and Tipe1 in the preparation of a drug for preventing and / or treating type 2 diabetes, characterized in that: The substance that promotes the expression of Zhx2 and Tipe1 is a lentivirus that overexpresses Zhx2 and Tipe1.

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