Use of linc02806 in the preparation of a diagnostic or intervention product for liver cell steatosis with insulin resistance

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to application of LINC02806 in preparation of a diagnosis or intervention product for hepatocyte steatosis with insulin resistance. The present application discloses for the first time that the lncRNA marker LINC02806 is significantly up-regulated in the state of hepatocyte steatosis and insulin resistance, and verifies that the expression level of LINC02806 is closely related to insulin sensitivity. This discovery provides a new method for developing early diagnosis of non-alcoholic steatohepatitis and insulin sensitivity at the molecular level in clinic, and provides a new drug target for treatment of non-alcoholic steatohepatitis and insulin resistance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of LINC02806 in the preparation of diagnostic or interventional products for hepatic steatosis with insulin resistance. Background Technology

[0002] The liver, as a vital metabolic center and detoxification organ, directly impacts the body's homeostasis. Among numerous liver diseases, hepatocellular carcinoma and hepatocellular steatosis are two clinically common types. Hepatocellular carcinoma's core pathological feature is uncontrolled malignant proliferation of hepatocytes, a malignant disease driven by cellular carcinogenesis and abnormal tumor signaling networks. Its development is closely related to a series of complex biological processes, including gene mutations, abnormal epigenetic modifications, DNA damage repair defects, the acquisition of unlimited tumor cell proliferation, and immune escape. This disease progresses rapidly, has a poor prognosis, and severely impacts patient survival. Hepatocellular steatosis, on the other hand, is essentially a metabolic disorder caused by an imbalance in lipid metabolism within hepatocytes, leading to the accumulation of metabolic products. Its core is the excessive deposition of triglycerides in the hepatocyte cytoplasm, representing reversible cell damage caused by metabolic disturbances. Its pathogenesis involves multiple aspects, including an imbalance in lipid droplet formation and degradation, impaired fatty acid β-oxidation pathways, damaged mitochondrial energy metabolism, and decreased insulin signaling pathway activity. It does not involve malignant transformation of hepatocytes. It is worth noting that there is a close relationship between hepatocellular steatosis and insulin resistance. Most patients with hepatocellular steatosis also experience decreased insulin sensitivity. Decreased insulin sensitivity, as a major manifestation of insulin resistance, not only exacerbates the pathological process of hepatocellular steatosis but also plays a significant role in the etiological diagnosis and disease assessment of metabolic diseases such as diabetes. Although most patients with hepatocellular steatosis have a good prognosis, some patients, if their condition continues to progress, may further develop liver fibrosis, cirrhosis, and even increase the risk of cardiovascular disease, causing multifaceted harm to their health.

[0003] Long non-coding RNAs (lncRNAs) are a class of RNA molecules longer than 200 nucleotides that do not encode proteins. Although lncRNAs cannot directly encode proteins, recent studies have confirmed that these RNA molecules play crucial roles at multiple levels, including epigenetic regulation, gene expression, transcription, and post-transcriptional regulation, participating in the regulation of various physiological processes and the development of diseases in the human body. In the field of liver diseases, research has shown that lncRNAs have regulatory effects on biological processes such as hepatocyte proliferation, differentiation, apoptosis, and metabolism. Chinese invention patent application CN114525342A discovered abnormal expression of LINC02806 in hepatocellular carcinoma, and further found that knocking out LINC02806 can inhibit the growth of liver cancer cells. Using LINC02806 as a biomarker and therapeutic target for the diagnosis of hepatocellular carcinoma provides a new technical solution for the clinical diagnosis and treatment of hepatocellular carcinoma.

[0004] In current technologies, no lncRNA biomarkers with clear diagnostic value and application prospects have been found for hepatocellular steatosis and its associated insulin sensitivity abnormalities. Although some studies, such as CN114525342A, have explored the role of lncRNAs in other liver diseases, the pathogenesis and pathological characteristics of different liver diseases are quite different, and there are also significant differences in core regulatory pathways and molecular mechanisms. Furthermore, a specific association between lncRNA expression levels and hepatocellular steatosis and insulin sensitivity has not been established. As a result, there is currently a lack of effective molecular biomarkers in clinical practice that can accurately diagnose hepatocellular steatosis and assess insulin sensitivity, which cannot meet the clinical needs for early diagnosis, disease assessment, and monitoring of treatment efficacy for this type of disease. Summary of the Invention

[0005] To address the current lack of effective molecular markers for accurately diagnosing hepatocellular steatosis and assessing insulin sensitivity in clinical practice, which fails to meet the clinical needs for early diagnosis, disease assessment, and monitoring of treatment efficacy for this type of disease, this invention provides an application of LINC02806 in the preparation of diagnostic or interventional products for hepatocellular steatosis with insulin resistance.

[0006] The technical solution of this invention is as follows: In a first aspect, the present invention provides an application of LINC02806 in the preparation of diagnostic products for hepatocellular steatosis with insulin resistance.

[0007] Furthermore, the diagnostic product diagnoses hepatocellular steatosis with insulin resistance by detecting the expression level of LINC02806 in biological samples.

[0008] Furthermore, the diagnostic product uses qPCR technology to detect the expression level of LINC02806 in biological samples.

[0009] Secondly, this invention provides an application of LINC02806 in the preparation of an intervention product for hepatocyte steatosis with insulin resistance.

[0010] Furthermore, the intervention products are drugs that have preventive, alleviating, or therapeutic effects on hepatocellular steatosis and insulin resistance.

[0011] Furthermore, LINC02806 affects insulin resistance by regulating the expression level of the EIF4EBP1 gene.

[0012] Furthermore, the intervention product exerts its effect by overexpressing the LINC02806 gene.

[0013] Furthermore, overexpression can be achieved through overexpression vectors of LINC02806.

[0014] The beneficial effects of this invention are as follows: 1. This invention reveals for the first time that the lncRNA marker LINC02806 is significantly upregulated in hepatocellular steatosis and insulin resistance, and its expression level is closely related to insulin sensitivity. This discovery provides a new molecular marker for the early diagnosis and targeted intervention of hepatocellular steatosis accompanied by insulin resistance. Experimental verification shows that LINC02806 does not affect lipid deposition in hepatocellular cells, but it can regulate the expression of EIF4EBP1 through a competitive endogenous RNA (ceRNA) mechanism, thereby improving glucose consumption and insulin signaling pathway activity. Compared with existing markers that only focus on phenotypic correlation, LINC02806 has a clear molecular mechanism of action.

[0015] 2. This invention can effectively distinguish between normal hepatocytes and hepatocytes with steatosis and insulin resistance by detecting the expression level of LINC02806 in biological samples. Overexpression of LINC02806 can significantly improve the glucose utilization capacity of hepatocytes, thereby improving insulin sensitivity. This invention provides a new intervention strategy and potential drug target for the treatment of non-alcoholic fatty liver disease and related metabolic disorders. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1The figures are the model verification results of each experimental group in Example 1. In the figures, (a) is the microscopic examination result of Oil Red O staining experiment, (b) is the semi-quantitative detection result of Oil Red O staining experiment, and (c) is the result of glucose consumption experiment.

[0018] Figure 2 The figure shows the screening results of lncRNA upregulated genes in Example 2. In the figure, (a) is the Venn intersection diagram of lncRNA upregulated genes; (b) is the expression cluster heatmap of lncRNA upregulated intersection genes.

[0019] Figure 3 The figures shown are qPCR verification results of lncRNA upregulation of intersecting genes in Example 2. In the figures, (a) is the verification result of the relative expression level of LINC02806 at different treatment times, (b) is the verification result of the relative expression level of LINC00582 at different treatment times, (c) is the verification result of the relative expression level of LINC-4061 at different treatment times, and (d) is the verification result of the relative expression level of LINC-8162 at different treatment times.

[0020] Figure 4 The figures show the results of LINC02806 expression intervention in Example 3 and the effect of LINC02806 expression intervention on the process of hepatocyte steatosis. In the figures, (a) is the verification result of the knockdown efficiency of LINC02806 by the LINC02806 interference sequence, (b) is the verification result of the overexpression efficiency of the LINC02806 overexpression vector, (c) is the microscopic examination result of Oil Red O staining of cells in each experimental group with LINC02806 expression intervention, (d) is the detection result of the transcriptional level of the adipogenesis marker SREBP1 in each experimental group with LINC02806 expression intervention, (e) is the detection result of the transcriptional level of the adipogenesis marker PPARγ in each experimental group with LINC02806 expression intervention, and (f) is the detection result of the transcriptional level of the adipogenesis marker ACACA in each experimental group with LINC02806 expression intervention.

[0021] Figure 5 This is a graph showing the regulatory effect of LINC02806 expression intervention on hepatocyte insulin sensitivity in Example 3. In the graph, (a) is the result of glucose consumption experiment of cells in each experimental group after LINC02806 overexpression, (b) is the result of glucose consumption experiment of cells in each experimental group after LINC02806 knockdown, (c) is the result of flow cytometry detection of glucose uptake experiment of cells in each experimental group after LINC02806 expression intervention, and (d) is the semi-quantitative result of glucose uptake experiment of cells in each experimental group after LINC02806 expression intervention.

[0022] Figure 6 The figure shows the verification results of the regulation of downstream target gene EIF4EBP1 by LINC02806; in the figure, (a) is the detection result of the relative expression level of EIF4EBP1 mRNA after overexpression of LINC02806, and (b) is the detection result of the relative expression level of EIF4EBP1 mRNA after knockdown of LINC02806.

[0023] Figure 7 The figure shows the experimental results of EIF4EBP1 antagonizing the LINC02806 knockdown effect; in the figure, (a) is the verification result of the overexpression efficiency of the EIF4EBP1 overexpression vector, and (b) is the experimental result of glucose consumption of cells in each experimental group. Detailed Implementation

[0024] To address the shortcomings of existing technologies in diagnosing hepatocellular steatosis with insulin resistance—namely, the lack of specific molecular markers, the difficulty in achieving early and accurate diagnosis at the molecular level, and the limited effectiveness of interventions due to the lack of clear molecular mechanisms—this invention designs a series of cell experiments to verify the function and application value of LINC02806 (ENST00000427169): First, using the normal human hepatocyte line LO2 as the research subject, a hepatocyte steatosis model was constructed by oleic acid treatment, and a hepatocyte steatosis accompanied by insulin resistance model was constructed by oleic acid combined with palmitic acid treatment. Oil Red O staining was used to verify lipid droplet accumulation, and glucose consumption experiments were used to verify insulin sensitivity, successfully establishing the experimental models. Subsequently, lncRNA microarray analysis was used to detect the expression differences of lncRNAs in the two models. Intersection genes of 301 upregulated lncRNAs in the steatosis model and 298 upregulated lncRNAs in the insulin resistance model were identified. Cluster analysis and qPCR validation revealed that LINC02806 was significantly upregulated in both models with statistically significant differences. P <0.05. Further, LINC02806 interference and overexpression groups were constructed, and related indicators were detected in both models: Oil Red O staining and adipogenic gene (SREBP1, PPARγ, ACACA) detection results showed that LINC02806 intervention did not affect lipid deposition in hepatocytes; glucose consumption and glucose uptake experiments showed that overexpression of LINC02806 significantly improved the glucose consumption and uptake capacity of hepatocytes. P <0.05%, indicating improved insulin sensitivity. Simultaneously, downstream target genes of LINC02806 were screened using CNC and ceRNA analysis. This revealed and validated that LINC02806 positively regulates EIF4EBP1 expression through a competitive endogenous RNA (ceRNA) mechanism, and that overexpression of EIF4EBP1 antagonizes the effect of LINC02806 knockdown on insulin sensitivity.P <0.05).

[0025] Based on the above experiments and results, the present invention verifies the following conclusions: LINC02806 is significantly upregulated in hepatocytes with steatosis and insulin resistance. Its expression level can be used as a specific indicator to distinguish between normal hepatocytes and hepatocytes with steatosis and insulin resistance. Furthermore, regulation of the overexpression level of LINC02806 can improve the insulin sensitivity of hepatocytes.

[0026] Based on the above research, this invention provides a solution for applying LINC02806 to the preparation of diagnostic products for hepatocellular steatosis accompanied by insulin resistance.

[0027] In one preferred embodiment, the diagnostic product distinguishes between normal hepatocytes and hepatocytes exhibiting steatosis and insulin resistance by detecting the expression level of LINC02806 in biological samples. The biological samples are derived from the hepatocytes of the subject being tested, specifically obtained through routine clinical methods such as liver biopsy.

[0028] As a preferred embodiment, when the expression level of LINC02806 in a biological sample is significantly upregulated compared to the expression level of LINC02806 in normal hepatocytes, it is determined that the hepatocytes corresponding to the biological sample have undergone steatosis and are accompanied by insulin resistance. Specifically, qPCR detection technology can be used to obtain the relative expression levels of LINC02806 in the biological sample to be tested and the normal hepatocyte sample. t Tests or analysis of variance are used to determine whether a difference is statistically significant. P <0.05).

[0029] As a preferred embodiment, the diagnostic product includes a reagent for detecting the expression level of LINC02806. The reagent contains at least specific primers designed for LINC02806 and may further include qPCR reaction buffer, enzymes, fluorescent dyes, and other supporting reagents to form a complete detection system.

[0030] The present invention also provides a scheme for applying LINC02806 to the preparation of intervention products for hepatocyte steatosis accompanied by insulin resistance.

[0031] As a preferred specific implementation method, the intervention product is a drug that has preventive, alleviating or therapeutic effects on hepatocellular steatosis and insulin resistance. This drug can intervene in the pathological process of hepatocellular steatosis accompanied by insulin resistance at the molecular level by regulating the expression level of LINC02806 in hepatocytes. Compared with traditional intervention methods that only target phenotypic improvement, it is more targeted and can reduce the impact on the physiological function of normal hepatocytes.

[0032] As a preferred embodiment, the intervention product is a formulation capable of overexpressing LINC02806, wherein the formulation for overexpressing LINC02806 exerts its effect by increasing the expression level of LINC02806 in hepatocytes.

[0033] As a preferred embodiment, LINC02806 affects insulin resistance by regulating the expression level of the EIF4EBP1 gene.

[0034] As a preferred embodiment, the intervention product is an overexpression vector of LINC02806.

[0035] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0036] In the following embodiments of the present invention, the materials, reagents, etc. used are commercially available unless otherwise specified; and the detection methods used in the following embodiments of the present invention are conventional methods unless otherwise specified.

[0037] Example 1 Establishment of hepatocyte steatosis cell models and hepatocyte steatosis with insulin resistance cell models Experimental methods: (1) The normal human hepatocyte line LO2 in the logarithmic growth phase was seeded in cell culture plates and cultured until the cell density reached 60%. The cells were then divided into the following 4 groups for treatment: Control group (Ctrl): Replace with RPMI-1640 complete medium containing 5% DMSO and culture continuously for 48 h; Fatty degeneration model group (OA): The culture medium was replaced with RPMI-1640 complete medium containing 1 mmol / L oleic acid, and the culture was continuously induced for 48 h. The RPMI-1640 complete medium with a final concentration of 1 mmol / L oleic acid was obtained by diluting a 20 mmol / L oleic acid stock solution with RPMI-1640 complete medium at a volume ratio of 1:20. The OA+PA model group was induced by first replacing the culture medium with RPMI-1640 complete medium containing 1 mmol / L oleic acid for 48 h, and then adding RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid for another 48 h. The RPMI-1640 complete medium with a final concentration of 0.5 mmol / L palmitic acid was prepared by diluting a 10 mmol / L palmitic acid stock solution with RPMI-1640 complete medium at a volume ratio of 1:20. Fatty transformation OA+PA control group (OA+PA Veh): The culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, it was changed to RPMI-1640 complete medium containing palmitic acid solvent control and cultured for another 48 h. The RPMI-1640 complete medium for palmitic acid solvent control was obtained by diluting the palmitic acid solvent control and RPMI-1640 complete medium at a volume ratio of 1:20.

[0038] (2) Cells from each group were stained with Oil Red O to verify lipid droplet accumulation. The distribution of lipid droplets in each group of cells was observed and photographed under an inverted microscope. Image J image analysis software was used to perform semi-quantitative analysis of the stained areas. The results are as follows: Figure 1 As shown in (a) and (b); (3) Four hours before the end of induction, the cells in each group were replaced with glucose at a final concentration of 1 g / L and glucose at a final concentration of 1 × 10⁻⁶ g / L. - 7 Low-glucose complete culture medium containing mmol / L insulin was cultured for another 4 hours. The supernatant was then collected, and glucose levels in each experimental group were measured to verify the insulin sensitivity of hepatocytes. The results are as follows: Figure 1 As shown in (c).

[0039] Depend on Figure 1 As shown in (a), compared with the control group (Ctrl), the number of lipid droplets in the fatty degeneration model group (OA), the fatty degeneration with insulin resistance model group (OA+PA), and the fatty degeneration OA+PA control solution group (OA+PA Veh) increased and showed a distinctly red color; Figure 1 As shown in (b), compared with the control group (Ctrl), the semi-quantitative absorbance of Oil Red O at 510 nm was significantly increased in the fatty degeneration model group (OA), the fatty degeneration with insulin resistance model group (OA+PA), and the fatty degeneration OA+PA control solution group (OA+PA Veh). P <0.05), where the absorbance difference between the fatty degeneration model group (OA) and the control group (Ctrl) was 2±0.125 fold, indicating a large accumulation of lipid droplets, suggesting the successful construction of the fatty degeneration model; Figure 1As shown in (c), compared with the control group (Ctrl), the cellular glucose consumption was significantly reduced in the fatty degeneration model group (OA), the fatty degeneration with insulin resistance model group (OA+PA), and the fatty degeneration OA+PA control group (OA+PA Veh). P <0.05); Compared with the control group (Ctrl), the glucose consumption of cells in the fatty degeneration model group (OA) was reduced by 0.35±0.064, indicating that the insulin sensitivity of hepatocytes decreased after fatty degeneration. In contrast, the glucose consumption of the fatty degeneration with insulin resistance model group (OA+PA) was reduced by 1.153±0.22. Palmitic acid further aggravated the insulin resistance of hepatocytes induced by oleic acid fatty degeneration, indicating that the hepatocyte fatty degeneration with insulin resistance cell model was successfully established.

[0040] Example 2 LINC02806 was upregulated in hepatocellular steatosis cell models and hepatocellular steatosis with insulin resistance cell models. Experimental methods: (1) The normal human hepatocyte line LO2 in the logarithmic growth phase was seeded in a cell culture plate and cultured for 18 hours. After the cell density reached 60%, the cells were divided into the following 4 groups for treatment: The control group (Ctrl) was replaced with RPMI-1640 complete medium containing 5% DMSO and cultured for 24, 36 and 48 hours. The fatty degeneration model group (OA) was induced and cultured for 24, 36 and 48 h by replacing the culture medium with RPMI-1640 complete medium containing 1 mmol / L oleic acid. The OA+PA model group was first induced by changing the culture medium to RPMI-1640 complete medium containing 1 mmol / L oleic acid for 48 h, and then adding RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid for 24, 36 and 48 h. The fatty OA+PA control group (OA+PA Veh) was cultured for 48 h in RPMI-1640 complete medium containing 1 mmol / L oleic acid, and then cultured for another 48 h in RPMI-1640 complete medium containing palmitic acid solvent control. (2) Total RNA was extracted from cells in each group using TRIzol RNA extraction reagent (purchased from Thermo Fisher Scientific, catalog number 9939001). lncRNA expression profiles were then detected using a lncRNA microarray. LncRNAs upregulated in the OA model group compared to the control group (Ctrl) were screened, yielding 301 upregulated lncRNAs, designated as group1 / 2. LncRNAs upregulated in the OA+PA model group with insulin resistance compared to the OA+PA control group (OA+PA Veh) were also screened, yielding 298 upregulated lncRNAs, designated as group3 / 4. The intersection of group1 / 2 and group3 / 4 was obtained, and the Venn diagram is shown below. Figure 2 As shown in (a), cluster analysis was performed on the intersecting lncRNAs, and a heatmap was plotted. The results are as follows. Figure 2 As shown in (b); (3) Total RNA was extracted from cells in the control group (Ctrl) and the fatty degeneration with insulin resistance model group (OA+PA) using TRIzol RNA extraction reagent. The RNA was then verified using qPCR. The specific steps were as follows: cDNA was reverse transcribed using the TAKARA reverse transcription kit (purchased from Baori Biotechnology Co., Ltd.), and then amplified using the Tiangen reagent kit (purchased from Tiangen Biotech Co., Ltd.) on a real-time PCR instrument (Thermo Fisher Scientific model: ABI 7500 Plus). The results are as follows: Figure 3 As shown in (a) to (d).

[0041] Depend on Figure 2 It can be seen that LINC02806 (corresponding to transcript number ENST00000427169 in the heatmap) showed a significant upregulation trend in both the hepatocellular steatosis model and the hepatocellular steatosis with insulin resistance model, and was shown as a significant high expression signal in the heatmap. Figure 3 Images (a) through (d) show the four genes LINC02806, LINC00582, LINC-4061, and LINC-8162, respectively. Figure 3 It can be seen that the expression change trends of LINC02806, LINC00582, LINC-4061, and LINC-8162 in the model group are consistent with the results of expression spectrum chip clustering analysis, which verifies the accuracy of the chip detection results.

[0042] Example 3 Regulatory Study of LINC02806 in Hepatocellular Steroid Cell Models and Hepatocellular Steroid with Insulin Resistance Cell Models Experimental methods (1) Constructing the LINC02806 interference sequence: 1.1 Three sets of siRNA interference sequences were designed targeting LINC02806, and a negative control siRNA (si-NC) was synthesized simultaneously. The specific sequences are as follows: siRNA-1: 5'-GGGAAGGUUAGGGUUUCAATT-3' (SEQ ID NO.1) of the positive strand, 5'-UUGAAACCCUAACCUUCCCTT-3' (SEQ ID NO.2); siRNA-2: 5'-GCCGAUCUGUUGAGAAGUATT-3' (SEQ ID NO.3), 5'-UACUUCUCAACAGAUCGGCTT-3' (SEQ ID NO.4); siRNA-3: 5'-CGGGAUGAAUAUGGGAAGUTT-3' (SEQ ID NO.5), 5'-ACUUCCCAUAUUCAUCCCGTT-3' (SEQ ID NO.6); si-NC: Justice chain 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO.7), Antisense chain 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO.8); 1.2 LO2 cells in logarithmic growth phase were seeded into 6-well plates and cultured for 16 hours until the cell density reached 60%, then transfected. The transfection steps are as follows: S1. Take four 1.5mL sterile centrifuge tubes, add 200μL of serum-free high-glucose DMEM medium and 4μL of Lipofectamine 2000 transfection reagent (purchased from Thermo Fisher Scientific) to each tube, mix well and let stand at room temperature for 5min. S2. Take another four 1.5 mL sterile centrifuge tubes, add 200 μL of serum-free high glucose DMEM medium to each, and then add 7.5 mmol of si-NC, siRNA-1, siRNA-2 or siRNA-3 respectively. Mix well and let stand at room temperature for 5 min. S3. Combine the sterile centrifuge tubes of S1 and S2, mix well, let stand for 20 minutes, and then drop the mixture into a 6-well plate. Incubate the cells at 37°C for 8 hours, then replace with RPMI-1640 complete medium and continue culturing for 24 hours to transfect the cells.

[0043] 1.3 Total RNA was extracted from cells in each group using TRIzol RNA extraction reagent. The relative expression level of LINC02806 was detected by qPCR to verify the knockdown efficiency. The results are as follows: Figure 4 As shown in (a); (2) Construction of LINC02806 overexpression vector 2.1 Using pcDNA3.1 vector (purchased from Invitrogen) as the backbone, the full-length sequence of LINC02806 (as shown in SEQ ID NO.9) was inserted into the multiple cloning site of the vector to construct the LINC02806 overexpression vector; the pcDNA3.1 blank vector was used as a negative control. 2.2 LO2 cells in logarithmic growth phase were seeded into 6-well plates and cultured for 16 hours until the cell density reached 60%. Then, following the transfection steps in step 1.2 of this embodiment, the LINC02806 overexpression vector (denoted as LINC02806+) and the pcDNA3.1 blank vector (denoted as pcDNA3.1) were transfected into LO2 cells respectively. 2.3 Total RNA was extracted from cells in each group using TRIzol RNA extraction reagent. The relative expression level of LINC02806 was detected by qPCR to verify the overexpression efficiency. The results are as follows: Figure 4 As shown in (b); (3) Study the effect of LINC02806 on the process of hepatocyte steatosis. 3.1 The normal human hepatocyte line LO2 in logarithmic growth phase was seeded in cell culture plates and cultured for 18 hours. After the cell density reached 60%, the cells were divided into the following 7 groups for treatment: si-NC group: Following the procedure described in step 1.2 of this embodiment, si-NC was transfected into LO2 cells, and then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the cells were induced and cultured for another 48 h. siRNA-1 group: Following the procedure described in step 1.2 of this embodiment, siRNA-1 was transfected into LO2 cells, and then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the cells were induced and cultured for another 48 h. siRNA-2 group: Following the procedure described in step 1.2 of this embodiment, siRNA-2 was transfected into LO2 cells, and then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the cells were induced and cultured for another 48 h. siRNA-3 group: Following the procedure described in step 1.2 of this embodiment, siRNA-3 was transfected into LO2 cells, and then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the cells were induced and cultured for another 48 h. pcDNA3.1 group: Following the procedure described in step 1.2 of this embodiment, the pcDNA3.1 blank vector was transfected into LO2 cells, and then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the cells were induced and cultured for another 48 h. LINC02806+ group: Following the procedure described in step 1.2 of this embodiment, the LINC02806 overexpression vector was transfected into LO2 cells, and then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the cells were induced and cultured for another 48 h. Cells from each group were used to verify lipid droplet accumulation using Oil Red O staining. The distribution of lipid droplets within the cells of each group was observed and photographed under an inverted microscope. The results are as follows: Figure 4 As shown in (c); simultaneously, total RNA was extracted from cells in each group, and the transcriptional levels of adipogenesis markers SREBP1, PPARγ, and ACACA were detected by qPCR. The results are shown in the figure. Figure 4 As shown in (d), (f), and (e).

[0044] (4) Study the effect of LINC02806 on insulin sensitivity of hepatocytes 4.1 The normal human hepatocyte line LO2 in logarithmic growth phase was seeded in cell culture plates and cultured for 18 hours. After the cell density reached 60%, the cells were divided into the following 6 groups for treatment: Control group (Ctrl): Replace with complete culture medium containing 5% DMSO and culture continuously for 44 h; Fatty degeneration with insulin resistance model group (OA+PA): First, the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the induction culture was continued for 44 h. si-NC group: Following the procedure described in step 1.2 of this embodiment, si-NC was transfected into LO2 cells, and then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the cells were induced and cultured for another 44 h. siRNA group: Following the procedure described in step 1.2 of this embodiment, siRNA-1 was transfected into LO2 cells, and then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the cells were induced and cultured for another 44 h. pcDNA3.1 group: Following the procedure described in step 1.2 of this embodiment, the pcDNA3.1 blank vector was transfected into LO2 cells, and then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the cells were induced and cultured for another 44 h. LINC02806+ group: Following the procedure described in step 1.2 of this embodiment, the LINC02806 overexpression vector was transfected into LO2 cells, and then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the cells were induced and cultured for another 44 h. 4.2 Cells from each group were used to perform glucose consumption and glucose uptake experiments sequentially: Glucose consumption experiment: Replace with glucose containing a final concentration of 1 g / L and a final concentration of 10 g / L. -7 Low-glucose complete culture medium containing mmol / L insulin was cultured for another 4 hours, and the supernatant was collected. Glucose levels in each experimental group were then measured using an instrument. The results are as follows: Figure 5 As shown in (a) and (b); Glucose uptake experiment: Cells in each group were subjected to glucose uptake at a rate of 5 × 10⁻ 4 Cells were seeded at a density of 100 μg / well in 96-well plates, and glucose uptake fluorescent probe 2-NBDG (purchased from Abcam, USA, catalog number ab136955) was added to a final concentration of 100 μg / mL. Flow cytometry was used to detect the fluorescence intensity of cells in each experimental group, reflecting glucose uptake capacity. Results are as follows: Figure 5 As shown in (c) and (d).

[0045] Depend on Figure 4As shown in (a), compared with the si-NC group, the relative expression level of LINC02806 was significantly reduced after transfection with siRNA-1, siRNA-2, and siRNA-3 (P<0.01). The knockdown efficiency of the three siRNA groups was all above 90%, with a knockdown efficiency of -0.9433±0.01764, indicating good interference effect. Figure 4 As shown in Figure (b), compared with the blank vector group (pcDNA3.1), the relative expression level of LINC02806 after transfection with the LINC02806 overexpression vector (LINC02806+) was 1122±127.8 times that of the blank vector, and the difference was highly statistically significant (P<0.01), indicating a significant overexpression effect.

[0046] Depend on Figure 4 As shown in (c), compared with the OA+PA treatment group, there was no significant difference in the number of hepatocyte lipid droplets and the degree of staining in the siRNA-1 group; similar results were obtained in the siRNA-2, siRNA-3, pcDNA3.1, and LINC02806+ groups (not shown in the figure). Figure 4 As shown in (d), (f), and (e), there were no significant differences in the transcriptional levels of SREBP1, PPARγ, and ACACA between the si-NC group and the siRNA-1 group, and between the pcDNA3.1 group and the LINC02806+ group (P>0.05); similar results were obtained for the siRNA-2 and siRNA-3 groups (not shown in the figure). These results indicate that intervening in the expression level of LINC02806 cannot affect hepatocyte lipid deposition by regulating adipogenesis factors, meaning that LINC02806 does not participate in the regulation of hepatocyte steatosis.

[0047] Depend on Figure 5 As shown in (a) and (b), compared with the pcDNA3.1 group, the glucose consumption of hepatocytes in the LINC02806+ group was significantly increased (P<0.05); compared with the si-NC group, there was no significant difference in glucose consumption in the siRNA group (P>0.05), which is presumably related to the low basal expression level of LINC02806 in hepatocytes. Figure 5 As shown in (c) and (d), the trend of change is consistent with the glucose consumption experiment, and the LINC02806+ group can significantly improve the glucose uptake capacity of hepatocytes. P <0.05). The above results collectively indicate that overexpression of LINC02806 can significantly improve insulin sensitivity in a hepatocellular steatosis model accompanied by insulin resistance.

[0048] Example 4 LINC02806 affects insulin resistance by regulating the expression level of the EIF4EBP1 gene through a ceRNA mechanism. Experimental methods: (1) Screening of candidate target genes 1.1 The normal human hepatocyte line LO2 in logarithmic growth phase was seeded in cell culture plates and cultured for 18 hours. After the cell density reached 60%, the cells were divided into the following two groups for treatment: pcDNA3.1 group: Following the procedure described in step 1.2 of Example 3, the pcDNA3.1 blank vector was transfected into LO2 cells, and then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the cells were induced and cultured for another 48 h. LINC02806+ group: Following the procedure described in step 1.2 of Example 3, the LINC02806 overexpression vector was transfected into LO2 cells, and then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the cells were induced and cultured for another 48 h. 2.1 Cells from each group were collected, and total RNA was extracted using TRIzol RNA extraction reagent for transcriptome sequencing. Combining competitive endogenous RNA co-expression network (CNC) analysis and ceRNA analysis, the intersection of the two analysis results was used to screen candidate target genes that have potential regulatory relationships with LINC02806. A total of 7 candidate target genes were screened: HAPLN4, PCDH1, CLEC16A, THSD4, EIF4EBP1, POLR3D, and RNPEPL1. (2) Validation of target gene expression 2.1 The normal human hepatocyte line LO2 in the logarithmic growth phase was seeded in cell culture plates and cultured for 18 hours. After the cell density reached 60%, the cells were divided into the following 4 groups for treatment: si-NC group: si-NC was transfected into LO2 cells according to step 1.2 in Example 3; siRNA group: siRNA-1 was transfected into LO2 cells according to step 1.2 in Example 3; pcDNA3.1 group: Following the procedure described in step 1.2 of Example 3, the pcDNA3.1 blank vector was transfected into LO2 cells; LINC02806+ group: Following the procedure described in step 1.2 of Example 3, the LINC02806 overexpression vector was transfected into LO2 cells; 2.2 Cells from each group were collected, and total RNA was extracted using TRIzol RNA extraction reagent. After reverse transcription to obtain cDNA, the relative mRNA expression levels of LINC02806 and EIF4EBP1 were detected using qPCR. The results are as follows: Figure 6 As shown in (a) and (b); Depend on Figure 6 It was found that, compared with the pcDNA3.1 group, the LINC02806+ group showed a significant upregulation of EIF4EBP1 mRNA expression after overexpression of LINC02806; compared with the si-NC group, the relative expression level of EIF4EBP1 mRNA was significantly downregulated after knockdown of LINC02806 expression. These results confirm that LINC02806 can positively regulate EIF4EBP1 expression, consistent with the regulatory pattern of competitive endogenous RNA.

[0049] Example 5 Overexpression of EIF4EBP1 can antagonize the regulatory effect of LINC02806 on insulin sensitivity. Experimental methods: (1) Construction of EIF4EBP1 overexpression vector 2.1 Using the pcDNA3.1 vector as a backbone, the full-length EIF4EBP1 sequence (as shown in SEQ ID NO.10) was inserted into the multiple cloning site of the vector to construct an EIF4EBP1 overexpression vector; the pcDNA3.1 blank vector was used as a negative control. 2.2 The normal human hepatocyte line LO2 in logarithmic growth phase was seeded into 6-well plates and cultured for 16 hours until the cell density reached 60%. Then, following the procedure described in step 1.2 of Example 3, the EIF4EBP1 overexpression vector (EIF4EBP1+) and the pcDNA3.1 blank vector (pcDNA3.1) were transfected into LO2 cells respectively. 2.3 Total RNA was extracted from cells using TRIzol RNA extraction reagent. The relative expression level of EIF4EBP1 was detected by qPCR to verify the overexpression efficiency. The results are as follows: Figure 7 As shown in (a); (2) The normal human hepatocyte line LO2 in the logarithmic growth phase was seeded in a cell culture plate and cultured for 18 hours. After the cell density reached 60%, the cells were divided into the following 4 groups for treatment: Control group (Ctrl): Replace with RPMI-1640 complete medium containing 5% DMSO and culture continuously for 44 h; Fatty degeneration with insulin resistance model group (OA+PA): First, the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the induction culture was continued for 44 h. siRNA group: siRNA-1 was transfected using step 1.2 in Example 3, and then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then, RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the culture was induced for another 44 h. EIF4EBP1+ group: The co-transfected siRNA-1 and EIF4EBP1 overexpression vector in step 1.2 of Example 3 were used. Then the culture medium was changed to RPMI-1640 complete medium containing 1 mmol / L oleic acid and cultured for 48 h. Then RPMI-1640 complete medium containing 0.5 mmol / L palmitic acid was added and the induction culture was continued for 44 h. (2) Replace each group of cells with glucose at a final concentration of 1 g / L and glucose at a final concentration of 10 g / L. -7 Low-glucose complete culture medium containing mmol / L insulin was cultured for another 4 hours, and the supernatant was collected for glucose level detection. The results are as follows: Figure 7 As shown in (b).

[0050] Depend on Figure 7 As shown in (a), transfection with the EIF4EBP1 overexpression vector significantly increased the intracellular expression level of EIF4EBP1. P <0.05), verifying the overexpression effect; by Figure 7 As shown in (b), compared with the siRNA group, the cellular glucose consumption in the EIF4EBP1+ group was significantly increased. P The value <0.05 indicates that overexpression of EIF4EBP1 can antagonize the inhibitory effect of knockdown of LINC02806 on insulin sensitivity in hepatocytes, further confirming that LINC02806 improves insulin sensitivity by positively regulating the expression of EIF4EBP1.

[0051] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. The application of LINC02806 in the preparation of diagnostic products for hepatocellular steatosis with insulin resistance.

2. The application as described in claim 1, characterized in that, The diagnostic product diagnoses hepatocellular steatosis with insulin resistance by detecting the expression level of LINC02806 in biological samples.

3. The application as described in claim 2, characterized in that, The diagnostic product uses qPCR technology to detect the expression level of LINC02806 in biological samples.

4. The application as described in claim 1, characterized in that, Diagnostic products include chips, formulations, or kits.

5. Application of LINC02806 in the preparation of an intervention product for hepatic steatosis with insulin resistance.

6. The application as described in claim 5, characterized in that, LINC02806 affects insulin resistance by regulating the expression level of the EIF4EBP1 gene.

7. The application as described in claim 5, characterized in that, The intervention product works by overexpressing the LINC02806 gene.

8. The application as described in claim 5, characterized in that, The intervention products include an overexpression vector of LINC02806.

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