Application of lncRNA-NONMMUG023935 and inhibitor thereof in regulation and control of diabetic nephropathy

Abstract

The invention discloses an application of lncRNA-NONMMUG023935 and an inhibitor of the lncRNA-NONMMUG023935 in regulation and control of diabetic nephropathy, and belongs to the technical field of biomedicine. It is found for the first time that MSC inhibits expression of lncRNA-NONMMUG023935 in a high glucose-induced renal tubular epithelial cell EMT model, so that epithelial-mesenchymal transformation and expression of TGF-beta and TNF-alpha are inhibited, and the effect of resisting renal tubular interstitial fibrosis is achieved. According to the discovery, the lncRNA-NONMMUG023935 can be used as a potential treatment target of the diabetic nephropathy, and can play an important role in diagnosis and treatment of the diabetic nephropathy, so that the lncRNA-NONMMUG023935 has a good practical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of lncRNA-NONMMUG023935 and its inhibitor in the regulation of diabetic nephropathy. Background Art

[0002] Diabetic nephropathy (DN) refers to the renal function impairment caused by microvascular lesions and glomerulosclerosis induced by diabetes. The clinical manifestations are mainly hypertension, proteinuria, edema, etc. It is one of the common and relatively serious complications of diabetic patients. According to statistics, by 2035, worldwide, diabetes will affect more than 550 million people, and diabetic nephropathy accounts for 20% to 40% of them. At the same time, DN is also one of the common causes of chronic kidney disease and accounts for a considerable proportion in end-stage kidney disease. Especially after renal failure, more than 70% of DN patients die rapidly within five years. Therefore, as a common disease threatening human health, DN has become a very serious and urgent social public health problem to be solved.

[0003] Mesenchymal stem cells (MSC) are derived from the mesoderm and are pluripotent stem cells with the ability of self-renewal and multi-directional differentiation potential. At the same time, the in vitro culture conditions of MSC are not high and it can be continuously passaged. It has pleiotropic effects such as anti-oxidative stress, anti-fibrosis and regulation of immune response, making stem cell transplantation become a research direction for the treatment of diabetes and its complications, especially a research hotspot for the treatment of DN caused by multiple incentives. Research shows that MSC can reshape the microenvironment in the renal tissue of DN and is a feasible means and research hotspot for the treatment of DN.

[0004] The extracellular matrix (ECM) increases and abnormally deposits, and continuously progresses to tubulointerstitial fibrosis and end-stage glomerulosclerosis. Among them, tubulointerstitial fibrosis plays a very important role in promoting the progression of diabetic nephropathy. Epithelial-mesenchymal transition (EMT) is an important mechanism for the occurrence and development of tubulointerstitial fibrosis. Research shows that EMT plays an important role in kidney diseases. Intervening in EMT can effectively improve renal fibrosis, and MSC can inhibit EMT by downregulating TGF-β and TNF-α, thereby inhibiting fibrosis.

[0005] Long non-coding RNAs (lncRNAs) are a class of gene transcripts located in the nucleus or cytoplasm with a length exceeding 200 nucleotides. They are involved in complex and precise gene regulatory networks and are also associated with the inflammation of various diseases. Research has confirmed that lncRNAs are abnormally expressed in various diseases such as cancer, acute leukemia, and myocardial infarction. The research group led by Zhang Zheng found that the upregulation of lncRNA-Gm4419 promotes the proliferation and fibrosis of mouse mesangial cells, and can promote the activation of NF-κB and the increase in the expression level of TNFα. However, in the intervention of MSCs to inhibit high-glucose-induced EMT of renal tubular epithelial cells and in the tubulointerstitial fibrosis of diabetic nephropathy, the expression status of long non-coding RNAs and their roles therein are still unclear. Summary of the Invention

[0006] In view of the above problems, the present invention aims to provide the use of lncRNA-NONMMUG023935 and its inhibitor in the regulation of diabetic nephropathy. In the treatment of diabetic nephropathy with MSCs, inhibiting the expression of lncRNA-NONMMUG023935 can inhibit epithelial-mesenchymal transition, as well as the expression of TGF-β and TNF-α, thereby playing an anti-fibrotic role.

[0007] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0008] The use of lncRNA-NONMMUG023935 in the regulation of diabetic nephropathy, characterized in that: MSCs inhibit the expression of lncRNA-NONMMUG023935 in the high-glucose-induced EMT model of renal tubular epithelial cells, thereby inhibiting epithelial-mesenchymal transition.

[0009] Furthermore, MSCs inhibit the expression of TGF-β and TNF-α by downregulating the expression of lncRNA-NONMMUG023935, thereby playing an anti-tubulointerstitial fibrosis role.

[0010] Furthermore, the present invention also includes the application of lncRNA-NONMMUG023935 inhibitor in inhibiting epithelial-mesenchymal transition of renal tubular epithelial cells.

[0011] Furthermore, the present invention also includes the application of lncRNA-NONMMUG023935 inhibitor in improving renal fibrosis.

[0012] Furthermore, the present invention also includes the use of lncRNA-NONMMUG023935 inhibitor in the treatment of diabetic nephropathy.

[0013] Furthermore, the present invention also includes the use of an lncRNA-NONMMUG023935 inhibitor in the preparation of a medicament for treating diabetic nephropathy.

[0014] The beneficial effects of the present invention are as follows:

[0015] It is first discovered in the present invention that when MSC intervenes in the treatment of diabetic nephropathy, by inhibiting the expression of lncRNA-NONMMUG023935 in renal tubular epithelial cells, the epithelial-mesenchymal transition and the expression of TGF-β and TNF-α are inhibited, thus playing a role in anti-renal tubular interstitial fibrosis. This discovery can use lncRNA-NONMMUG023935 as a potential therapeutic target for diabetic nephropathy, and can play an important role in the diagnosis and treatment of diabetic nephropathy, so it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a technical roadmap for studying the effect of MSC on EMT of renal tubular epithelial cells by regulating lncRNA-NONMMUG023935 at the cellular level in the present invention.

[0017] Figure 2 It is a technical roadmap for studying the regulatory effect of MSC on renal interstitial fibrosis in a diabetic nephropathy model in the present invention.

[0018] Figure 3 It is a glomerular pathological section of a mouse with a diabetic nephropathy model in the present invention.

[0019] Figure 4 It is a section of the renal basement membrane of a normal mouse and a mouse with a diabetic nephropathy model in the present invention.

[0020] Figure 5 It is a microscopic photograph of in vitro cultured murine bone marrow-derived MSCs in the present invention.

[0021] Figure 6 It is immunohistochemical detection of the expression of E-cadherin in renal tissue in the present invention.

[0022] Figure 7 It is the result of differential expression analysis of lncRNA in diabetic nephropathy mice in the present invention.

[0023] Figure 8 It is the expression result of lncRNANONMMUG023935 in diabetic nephropathy in the present invention.

[0024] Figure 9 It is the expression result of fluorescence quantitative PCR for detecting NONMMUG023935 in high glucose-induced renal tubular epithelial cells in the present invention.

[0025] Figure 10 These are the experimental results of the animal model in the present invention. Detailed implementation manners

[0026] In order to enable those of ordinary skill in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described below in conjunction with the drawings and embodiments.

[0027] In order to study the use of lncRNA-NONMMUG023935 in the regulation of diabetic nephropathy, the main research contents and research methods in the present invention include the following two parts:

[0028] 1 Study the effect of mesenchymal stem cells (MSCs) on EMT of renal tubular epithelial cells by regulating lncRNA-NONMMUG023935 at the cellular level

[0029] 1.1 Experimental subjects

[0030] Isolation and culture of mouse renal tubular epithelial cells (mRTECs): The kidneys were removed from normal male C57BL / 6 mice under sterile conditions, chopped and digested with collagenase-I at 37 °C for 1 hour. The digestion was terminated with complete medium, and the suspension was filtered through two cell filters of 40 μm and 70 μm. Then the suspension was centrifuged at 1200 rpm for 10 minutes. After lysing and washing the red blood cells twice, the mRTECs were cultured in a flask of DMEM / F12 supplemented with 10% FBS, 1% penicillin and streptomycin and other growth factors. After one week, it was used for subsequent experiments.

[0031] 1.2 Experimental protocol

[0032] The technical route of this experimental protocol is as shown in the appendix Figure 1As shown. C57BL / 6 mice were respectively used for mRTECs culture, and mRTECs were stimulated with high glucose. qPCR and western blot were used for detection and confirmation to establish an in vitro cell model induced by high glucose. Quantitative PCR was performed to compare the top 5 up-regulated and top 5 down-regulated 10 lncRNAs between mRTECs treated with high glucose and those cultured conventionally, to identify the target lncRNAs with statistically significant differences; then mRTECs stimulated with high glucose were co-cultured with BM-MSC, and the expressions of epithelial cell markers, mesenchymal cell markers, fibrosis indexes, inflammatory factors, and the target lncRNA-NONMMUG023935 in the co-cultured mRTECs cells were detected to study the effect of MSC on EMT of renal tubular epithelial cells induced by high glucose through regulating the expression of ncRNA-NONMMUG023935; among them, the co-culture of mRTECs stimulated with high glucose and BM-MSC included siRNA knockdown of the target lncRNA expression + MSC co-culture, pcDNA3.1(+)-amplification of the target lncRNA expression + MSC co-culture, and siRNA knockdown or pcDNA3.1(+)-amplification of the target lncRNA expression (control group).

[0033] High glucose stimulation: mRTECs were stimulated with high glucose for 24 hours. The specific high glucose stimulation is prior art and will not be elaborated in this invention.

[0034] qPCR and western blot detection: After treatment with high glucose, cell RNA and proteins were extracted, and the expressions of epithelial marker protein E-cadherin, mesenchymal cell marker proteins α-SMA, vimentin, and FSP-1, fibrosis indexes α-SMA, vimentin, collagen Ⅰ, and collagen Ⅲ, and lncRNA-NONMMUG023935 were respectively detected to confirm the establishment of an in vitro cell model induced by high glucose.

[0035] Quantitative PCR comparison of the top 5 up-regulated and top 5 down-regulated 10 lncRNAs between mRTECs treated with high glucose and those cultured conventionally was further performed to more clearly identify the target lncRNAs with statistically significant differences for subsequent research selection.

[0036] Co-culture: The above high-glucose-stimulated mRTECs were co-cultured with BM-MSCs, that is, BM-MSCs were cultured in transwell chambers and then cultured for another 24 hours. RNA and proteins of mRTECs cells were extracted, and the expressions of epithelial marker protein E-cadherin, mesenchymal cell marker proteins α-SMA, vimentin and FSP-1, fibrosis indicators TGF-β, α-SMA, vimentin, collagenⅠ, collagenⅢ, MCP-1 and TNF-α, and the expression of lncRNA-NONMMUG023935 were detected respectively.

[0037] siRNA interference of the expression of lncRNA-NONMMUG023935 in mRTECs: According to the NM sequence of lncRNA-NONMMUG023935, at least 3 siRNAs were designed using BLOCK-iT TM RNAi Designer, and the siRNAs were transfected by liposomes to verify the knockdown efficiency and transfection dose.

[0038] Overexpression of lncRNA-NONMMUG023935: The correctly constructed pcDNA3.1(+)-NONMMUG023935 plasmid was amplified and cultured, and the plasmid was extracted using an endotoxin-free plasmid extraction kit. At the same time, an empty vector was constructed for use as a control group.

[0039] lncRNA-NONMMUG023935 interference experiment: The above siRNA-interfered mRTECs were stimulated with high glucose; or the mRTECs transfected with pcDNA3.1(+)-Gm4419 were stimulated with high glucose; RNA and proteins of mRTECs cells were extracted, and the expressions of epithelial marker protein E-cadherin, mesenchymal cell marker proteins α-SMA, vimentin and FSP-1, fibrosis indicators TGF-β, α-SMA, vimentin, collagenⅠ and collagenⅢ, as well as MCP-1 and TNF-α, and the expression of lncRNA-NONMMUG023935 were detected respectively.

[0040] 2 Studying the regulatory effect of MSCs on renal interstitial fibrosis in a diabetic nephropathy model

[0041] 2.1 Experimental subjects

[0042] Diabetes model: Male C57BL / 6 mice at 8 weeks of age were purchased. A type I diabetes (T1D) model was obtained by a single intraperitoneal administration of 150 mg / kg of STZ to 8-week-old C57BL / 6 mice to produce high-dose STZ-induced diabetic mice. A type II diabetes (T2D) model was obtained by intraperitoneal administration of 40 mg / kg of STZ for 5 consecutive days and feeding a high-fat diet to produce low-dose STZ-induced diabetic mice. Control group mice were injected intraperitoneally with buffer. Blood glucose levels were monitored using a blood glucose meter. When the blood glucose level of STZ diabetic mice was greater than or equal to 400 mg / dl, the type II diabetic nephropathy model was considered successful and used for experiments. All animal experimental methods were carried out in accordance with the relevant regulations of the hospital's animal experimental committee. Subsequently, blood glucose was detected weekly, urinary albumin (U-alb) and creatinine (Cr) were detected, and renal function was analyzed by U-alb / Cr.

[0043] 2.2 Experimental protocol

[0044] The technical route of this experimental protocol is shown in the appendix Figure 2 as follows. C57BL / 6 mice were induced to become type II diabetic mice using STZ, and BM-MSC transplantation was performed. Control group mice were treated with buffer. Blood glucose and urinary protein were detected at different time points to confirm successful model establishment. Blood, urine, and renal tissue of transplanted group and control group mice were detected respectively to study the effects of MSC on lncRNA expression and tubulointerstitial fibrosis in the diabetic nephropathy DN model. Among them, blood detection included detecting the levels of factors such as TGF-β and TNF-α, urine detection included detecting the urine protein / creatinine ratio, and renal tissue detection included immunohistochemical analysis such as HE staining and detecting EMT markers and lncRNA levels.

[0045] Among them,

[0046] Preparation of BM-MSC: Bone marrow was obtained from the tibia and femur of C57BL / 6J mice of the same age, and BM-MSC was obtained from these bone marrow samples. Then they were suspended in DMEM medium containing 10% fetal bovine serum and penicillin / streptomycin for amplification culture. BM-MSC was stained with PE-conjugated CD44 and CD49 antibodies and FITC-conjugated CD45 antibodies and analyzed by flow cytometry to identify the amplified and cultured BM-MSC.

[0047] Treatment of mouse MSC: Starting from the time of the first injection of STZ in the diabetes model, 4 weeks after induction, the diabetic mice were divided into two groups. One group of mice was given an intravenous injection into the tail vein every 4 weeks (1.0×10 4GFP-BM-MSC (2 × 10⁶ cells / g body weight, suspended in 200 μl PBS) was administered twice to the mice, and the control group received buffer. Renal function was then measured at the following time points: 0 weeks before STZ stimulation, before MSC intervention at 4 weeks after STZ stimulation, 4 weeks after MSC intervention at 8 weeks after STZ stimulation, and 8 weeks after MSC intervention at 12 weeks after STZ stimulation.

[0048] Biochemical examination of urinary albumin and glucose levels: Mice were housed in metabolic cages, and whole urine was collected for 3 hours. The levels of albumin and creatinine in the urine were analyzed. Albumin levels were measured by immunoturbidimetry, and creatinine levels were determined by an enzymatic method. Urinary albumin excretion was normalized to urinary creatinine excretion. Blood samples were collected from diabetic mice after a 12-hour fast.

[0049] Optical microscopy of renal tissue: Paraffin-embedded kidney tissues were sectioned, stained with H&E, PAS, and Azan, and photographed with an optical microscope to evaluate the degree of glomerular disorder, changes in renal tubular epithelial cells, and interstitial changes in the renal tissue. Glomerular damage was expressed as the percentage of glomeruli showing mesangial expansion or glomerulosclerosis. Renal tubular epithelial injury was evaluated based on tubular dilation, protein cylinders, and atrophic changes (0, no change; 1, change of 25% or less; 2, change from 25% to 50%; 3, change from 50% to 75%; 4, change exceeding 75%).

[0050] Detection of TGF-β and TNF-α in serum: Detection was performed by ELISA according to a commercially available kit.

[0051] Detection of EMT markers: Renal tissue proteins were extracted using RIPA reagent, and the expression of epithelial marker protein E-cadherin, mesenchymal cell marker proteins α-SMA, vimentin, and FSP-1, and renal fibrosis indicators α-SMA, vimentin, collagen I, and collagen III was detected by western blot; the expression of lncRNA-NONMMUG023935 was detected by fluorescence quantitative PCR in renal tissue sections.

[0052] Research results:

[0053] The pathological analysis results of the kidney tissue sections of the diabetic nephropathy model mice established in the present invention are shown in Appendix Figure 3 and Appendix Figure 4 (the left figure is the kidney basement membrane of normal mice, and the right figure is the basement membrane of diabetic nephropathy model mice). As can be seen from Appendix Figure 3 the glomerular GBM of diabetic nephropathy model mice was thickened, the mesangial area of the glomeruli was widened, and the matrix increased; some renal tubular epithelial cells showed vacuolar degeneration, and focal fibrosis of the renal interstitium (Masson × 400). From Appendix Figure 4It can be seen that; Right: The basement membrane of the diabetic nephropathy model mice is homogeneously thickened compared with that of the normal mice (electron microscopy, 60 kV, ×6000). From the appendix Figure 3 and the appendix Figure 4 It can be obtained that the diabetic nephropathy model in the present invention is successfully established.

[0054] In the present invention, the in vitro cultured mouse MSCs are shown in the figure below in the appendix Figure 5 As shown, where A is ×10 under an inverted microscope, and B is ×20 under an inverted microscope. The in vitro culture of mouse bone marrow-derived MSCs provides a basis for stem cell supply.

[0055] Furthermore, the expression of E-cadherin in the renal tissue was detected by immunohistochemistry, and the results are shown in the appendix Figure 6 As shown, in the appendix Figure 6 N - normal animals; D - diabetic nephropathy animals; D+M - diabetic nephropathy animals received MSCs transplantation; N+M - normal animals received MSCs transplantation. The results showed that in the diabetic nephropathy animal model, the expression of E-cadherin was inhibited, while after peripheral intravenous injection of MSCs, the expression of E-cadherin increased, suggesting that MSCs can inhibit the occurrence of EMT in renal tubular epithelial cells in the diabetic nephropathy animal model.

[0056] Furthermore, the differential lncRNAs between mRTECs treated with high glucose and mRTECs cultured conventionally were screened, and the data IDs and distribution were sorted out. According to the FPKM values of the data, genes with FPKM of 0 in all samples were removed. For the 2 controls and 6 samples in the data, differential lncRNAs were screened in 3 groups, and the differential genes of each group were calculated using the moderate t test and Bayesian model. Based on the screening criterion of p < 0.01, a list of differential genes was obtained. From the list of differential genes obtained in the above steps, the screening criterion was further set as adjusted p value < 0.05 and expression difference |log2(FC)| > 1.5, and 104 differentially expressed genes related to diabetic nephropathy were obtained, as shown in the appendix Figure 7 As shown, the figure shows the FPKM values of each sample for a single lncRNA. Red represents high FPKM levels of lncRNAs, and blue represents low-expressing lncRNAs. Tables 1 and 2 are the top 20 up-regulated and top 20 down-regulated lncRNAs.

[0057] Table 1 Top 20 up-regulated lncRNAs

[0058]

[0059] Table 2 Top 20 down-regulated lncRNAs

[0060]

[0061] The expression of lncRNA NONMMUG023935 in diabetic nephropathy is shown in the appendix Figure 8 as follows. As can be seen from Figure 8 A in the appendix, the expression of lncRNA NONMMUG023935 was significantly increased in the diabetic nephropathy model, while the expression of lncRNA NONMMUG023935 was inhibited after MSC intervention. After treatment with si-lncRNA, the expression of lncRNA NONMMUG023935 could be significantly reduced, as shown in Figure 8 B in the appendix, which can be used for subsequent experiments.

[0062] The fluorescence quantitative method was used to detect the expression of lncRNA NONMMUG023935 in renal tubular epithelial cells, and the results are shown in the appendix Figure 9 as follows. In Figure 9 the appendix, ctrl: normal control group; NC: si-negative control group; si-lncRNA: group interfering with the expression of lncRNA NONMMUG023935; HG: high glucose-induced group; HG+si-lncRNA: high glucose-induced and group interfering with the expression of lncRNA NONMMUG023935; HG+BMSC: co-culture group of renal tubular epithelial cells and MSC (quantity 1:1); HG+BMSC+lncRNA: co-culture group of renal tubular epithelial cells and MSC (quantity 1:1) and overexpression group of lncRNA NONMMUG023935. As can be seen from Figure 9 the appendix, the expression of E-cadherin in renal tubular epithelial cells decreased under high glucose induction, while the expressions of α-SMA, vimentin, and collagen I increased. However, under high glucose induction, if renal tubular epithelial cells were co-cultured with MSC, the expression of E-cadherin in renal tubular epithelial cells could be increased, and the expressions of α-SMA, vimentin, and collagen I could be inhibited. The expression levels of the above factors changed due to MSC intervention, showing the same change trend in the si-lncRNA group, and this change due to MSC intervention was significantly weakened in the HG+BMSC+lncRNA group.

[0063] The results of the animal model experiment are shown in the appendix Figure 10 as follows. Among them, ctrl: normal control group; T2D: type 2 diabetes artery model group; T2D+MSC: animal model and MSC transplantation group; From Figure 10It can be seen that in the type 2 diabetes arterial model, the level of E-cadherin decreases, while the expressions of α-SMA, vimentin, and collagen I increase. After transplantation of MSCs, the decrease in E-cadherin can be improved, and the expressions of α-SMA, vimentin, and collagen I can be inhibited. The results are consistent with those of the cell experiment.

[0064] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification are only used to illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. Use of lncRNA-NONMMUG023935 in the regulation of diabetic nephropathy, characterized in that: MSC inhibits the expression of lncRNA-NONMMUG023935 in the high glucose-induced EMT model of renal tubular epithelial cells, thereby inhibiting epithelial-mesenchymal transition.

2. The use according to claim 1, characterized in that: MSC exerts an anti-renal tubulointerstitial fibrosis effect by downregulating the expression of lncRNA-NONMMUG023935 and inhibiting the expression of TGF-β and TNF-α.

3. Application of lncRNA-NONMMUG023935 inhibitor in inhibiting epithelial-mesenchymal transition of renal tubular epithelial cells.

4. Application of lncRNA-NONMMUG023935 inhibitor in improving renal fibrosis.

5. Use of lncRNA-NONMMUG023935 inhibitor in the treatment of diabetic nephropathy.

6. Use of lncRNA-NONMMUG023935 inhibitor in the preparation of drugs for the treatment of diabetic nephropathy.

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