MiR-101 Inhibits Autophagy In Mouse Mesangial Cells Under High-glucose Conditions And Promotes Cell Proliferation By Regulating AMPK

(Background]Diabetic nephropathy (DN), a serious complication of diabetes, is one of the most common causes of end-stage renal disease. Diabetes mellitus and complications from the disease, including DN, has increasingly become major health problems worldwide. Therefore, there is an urgent need to identify new therapeutic target molecules or cellular processes that underlie the pathogenesis of DN.Mesangial cells (MCs) are important types of intrinsic renal cells that play an important part in the maintenance of extracellular matrix (ECM) homeostasis in the glomerular mesangial area. An increasing number of studies demonstrated that hyperglycemia plays a central role in the pathogenesis of DN, as shown by its promotion of MC proliferation in vitro. Early-stage DN is characterized by glomerular hypertrophy (caused by mesangial cell proliferation and extracellular matrix expression), which is eventually associated with glomerulosclerosis and the subsequent deterioration of kidney function. Therefore, we suspect that MCs are key players in these processes.Autophagy is an evolutionarily-conserved mechanism of cellular self-digestion in which proteins and organelles are degraded upon delivery to the lysosomes. Under physiological conditions, autophagy occurs at low levels in order to clear a small amount of damaged organelles and biological macromolecules to maintain normal cellular function. However, autophagy increases during stressful situations, such as nutritional deficiency and ischemia, leading to the degradation of cells and the release of free amino acids and fatty acids. In this way, autophagy provides supplemental energy for the synthesis of biological macromolecules under stressful conditions. Autophagy occurring at either insufficient or excessive levels is involved in numerous pathological processes of human disease. Several studies have found that autophagy is closely associated with cancer, senility, cardiovascular disease, lung disease, infectious disease, neurodegenerative disease, liver disease, renal disease and diabetes. In DN, damaging factors not only result in kidney injury, but also induce autophagy. The inhibition of autophagy aggravates the effect of the damaging factors on the intrinsic renal cells. The levels of the damaging factors increase when autophagy is inhibited. The contradictions between damage enhancement and weakened protection reveal that the dysfunction of autophagy in MCs may be involved in the occurrence and development of DN. Autophagy has a protective effect on the kidney, but the role of autophagy in DN and its underlying mechanisms remain largely undetermined.MicroRNA (miRNA) is a small, non-coding, single-stranded RNA with an average length of 22 nucleotides. In eukaryotic cells, most miRNAs reduce the expression of target genes by inhibiting translation in prokaryotic cells, miRNA works by degrading mRNA. miRNA is closely associated with cancer, endocrine disease and other diseases, and is now a commonly studied mechanism for the regulation of gene expression. Many studies found that miRNA is involved in the occurrence, progression and complications of diabetes. The aberrant expression of miRNA, such as the up-or down-regulation of miR-192, miR-200s, miR-29a, miR-29c, miR-21, miR-377, miR-323b-5p, miR-429 and miR-93, is closely related to the development of DN. miR-101 is a member of the miRNA family. It was recently reported that the expression of miR-101 is reduced in tumors and that miR-101 can inhibit tumor growth. These phenomena are associated with the inhibition of autophagy by miR-101. miR-101 can inhibit autophagy and enhance cisplatin-induced apoptosis in the hepatocellular carcinoma cell line HepG2 by regulating RAB5A, STMN1 and ATG4D. Chigusa Higuchi et al. found that the serum concentrations of miRNA, log10miR-101, were significantly increased in a T2D (type 2 diabetes) group compared with a NGT (normal glucose tolerance) group. It remains unknown whether the pancreas or orther tissues are the source of the increased circulating miR-101 levels in T2D. In tumor cells, miR-101 can inhibit autophagy and enhance apoptosis by negatively regulating target genes-such as E2H2, STMN1, RAB5A and ATG4D. In DN, autophagy was inhibited in MCs by high-glucose, and the expression of miR-101 was increased in the serum of diabetic rats. miR-101 can also inhibit autophagy in tumor cells. Based on the above information, we asked if the expression of miR-101 is increased in MCs and if autophagy in MCs is inhibited by miR-101.AMPK (AMP activated protein kinase) is a highly conserved sensor in eukaryotic cells and an important kinase in the regulation of energy homeostasis. AMPK is involved in cell growth and apoptosis, and the activation of AMPK has a positive effect on autophagy. AMPK is widely expressed in all types of renal cells, and the decreased phosphorylation of AMPK is associated with the occurrence and development of DN. In DN, AMPK activity is inhibited, leading to the autophagic dysfunction of MCs. However, the activation of AMPK can repair the autophagic dysfunction induced by high-glucose. Therefore, the inhibition of autophagy is related to the decrease in AMPK phosphorylation in MCs treated with high-glucose. The protective effect of AMPK in renal cells may be due to the activation of autophagy.According to the website TargetScan, the target gene of miR-101 is AMPK (Prkaal). Therefore, we report that miR-101 is highly expressed in MCs treated with high-glucose and that it can directly regulate the expression of AMPK. miR-101 inhibits autophagy and enhances MC proliferation under high-glucose conditions. This study reveals that miR-101, which inhibits autophagy, could be developed as a potential novel therapy for DN.This study has been divided into two parts:Part Ⅰ. Study the relationship between autophagy<. proliferation and theexpression of miR-101 induced by high-glucose and confirm the immediately regulatory relationship between miR-101 and AMPK in mouse mesangial cells[Objectives]1. To observe the effect of high-glucose on autophagy、proliferation and the expression of miR-101 in mouse mesangial cells (SV40 Mes) and the relationship between them.2. Verify the direct binding mechanism between miR-101 and AMPK 3’UTR in mesangial cells.[Methods]1. Cell culture:The mouse mesangial cells (SV40 Mes) were cultured in the low-glucose 1640 supplemented with 10% fetal calf serum (full-nutrient medium) at 37℃ in 95% air,5% CO2.2. SV40 Mes were cultured in high-glucose with 0、24、48、72h, meanwhile set the mannitol group(30mM). The group include in normal group (N), mannitol group, high-glucose group, HG Oh, HG 24h, HG 48h and HG 72h, a total of 7 groups. Measurements:a, using WB to test the expression level of LC3B Ⅰ, LC3B Ⅱ, AMPK, p-AMPK, PCNA in each group; b, using CCK8 assay to test cell proliferation rate in each group; c, using qRT-PCR to test cell expression levels of miR-101 in each group.3. SV40 Mes were transfected with mimic and inhibitor. The group include in N, miR-101-mimic, mimic-control, miR-101-inhibitor and inhibitor-control, a total of 5 groups. Measurements:using qRT-PCR to test the expression levels of miR-101 that confirm the transfection was successful.4. The bioinformatic softwares were used to predict the target gene:AMPK. Using the reoccupy dual luciferase report gene plasmid to test whether AMPK is the target gene of miR-101.5. MiR-101 regulated the expression level of AMPK mRNA and protein. The group include in N, miR-101-mimic, mimic-control, miR-101-inhibitor and inhibitor-control, a total of 5 groups. The intervention time of high-glucose is 72h, and the concentration is 30mM. Measurements:a, using the qPCR to text the expression level of AMPK; b, using the WB to text the protein expression level of AMPK.6. Statistical analysis:Qualitative data including qPCR and Western blot were repeated at least three times independently. The number of repetitions for experiments was given in figure legends (n) and data were presented as the mean±SEM. SPSS 19.0 for Windows was used for analysis. Comparisons between groups were made using one-way ANOVA. Comparisons in two groups were made using t-test. P<0.05 was considered significantly different.[Results]1. The protein expression of LC3B was reduced as the stimulus time extended gradually under high-glucose condition in SV40 Mes cells; the protein expression level of LC3B was the lowest in HG 72h. The expression of LC3B in HG group was lower than NG group significantly (P<0.05); but the protein expression between NG group and Man group had no difference in statistic (P>0.05).2. The protein expression of PCNA when high-glucose stimulated after 72h was higher than Oh (P<0.05); there has no obvious difference on the other time. The expression of PCNA in HG group were twice higher than NG group (P<0.05); but the protein expression between NG group and Man group had no difference in statistic (P>0.05).3. The cell proliferation of SV40 Mes was tested by using CCK8 assay infiltration method:the cell proliferation in HG group was higher significantly than NG group and Man group. the cell proliferation in SV40 Mes when high-glucose stimulated cell after 72h was higher than HG Oh, there has an obvious difference in statistic significantly (P<0.05).4. The expression level of AMPK was increased as the stimulus time extended gradually under high-glucose condition (P<0.01). The expression of p-AMPK/AMPK in HG group was higher than NG group significantly (P<0.05); but the expression between NG group and Man group had no difference in statistic (P>0.05).5. The expression level of miR-101 was increased as the stimulus time extended gradually under high-glucose condition (P<0.01); the expression level of miR-101 was the highest in HG 72h. The expression of miR-101 in HG group was higher than NG group significantly (P<0.05); but the expression between NG group and Man group had no difference in statistic (P>0.05).6. The expression levels of miR-101 were confirmed by qRT-PCR. miR-mimic-tre ated cells showed a 37-fold higher miR-101 expression than mimic-control treated cells, whereas miR-inhibitor cells had an 80% lower expression when compared with inhibitor-control treated cells.7. Using bioinformatic softwares TargetScan to predicted target genes of miR-101, the research focus chosen AMPK. According to experimental group co-transfected into SV40 Mes cells, the transfected cells were measured of the firefly luciferase’s RLU and the Renilla luciferase’s RLU. According to data analysis, AMPK-3’UTR is capable of interacting with miR-101.8. SV40 Mes cells were transfected with miR-101-mimic and miR-101-inhibitor for detecting the changes in the expression level of AMPK mRNA and protein. The expression levels of AMPK mRNA and protein in the miR-101-mimic group were decreased significantly, but the miR-101-inhibitor group were increased, there has significant statistically (P<0.05).[Conclusions]1. High glucose inhibits autophagy in cultured mouse MCs (SV40 Mes).2. High glucose induces excess accumulation of PCNA in SV40 Mes cells.3. High glucose makes the expression of miR-101 increased in SV40 Mes cells.4. Activation of miR-101 reduced the autophagy and increased the level of PCNA accumulation in HG-treated SV40 Mes.5. The transcription and protein expression of AMPK is correlated negatively to miR-101 in HG-treated SV40 Mes.6. The seed sequence of miR-101 can directly combine with AMPK-3’UTR, resulting in a poor stability of AMPK mRNA and the degradation of it. Thereby, further reducing in the expression of AMPK protein is inevitable.7. May be miR-101 regulate autophagy and proliferation by targeting the AMPK.Part Ⅱ. Whether miR-101 can regulate autophagy and proliferation under high-glucose condition in SV40 Mes cells (Objectives]1. To confirm that miR-101 actually regulates the autophagy and proliferation of S V40 Mes under high-glucose condition.[Methods]1. Cell cultured:As mentioned before.2. MiR-101 inhibits autophagy of SV40 Mes cells under high-glucose condition. There are six groups:NG, HG, miR-101-mimic+HG, mimic-control+HG, miR-101-inhibitor+HG, inhibitor-control+HG. Measurement:a, using transmission electron microscopy to observe the autophagosomes of SV40 Mesa; b, the protein expression level of LC3B was tested by WB.3. MiR-101 induces proliferation of SV40 Mes cells under high-glucose condition. There are six groups:NG, HG, miR-101-mimic+HG, mimic-control+HG, miR-101-inhibitor+HG, inhibitor-control+HG. Measurement:a, the protein expression level of PCNA was tested by WB; b, the proliferation level of each group was tested by CCK8.4. Statistical analysis:As mentioned before.[Results]1. The protein expression of LC3B was lower significantly in miR-101-mimic group than the other groups treated by high-glucose (P<0.05). The protein expression of LC3B was higher significantly in miR-101-inhibitor group than the other groups (P<0.05).2. Quantification of autophagosomes per cellular cross-section revealed that autophagosomes were rarely detected in the non-transfected cell group. However, the number of autophagosomes was significant reduced in the miR-101-mimic group (P<0.05). Meanwhile, the number of autophagosomes in miR-101-inhibitor group was the most.3. The protein expression of PCNA was higher significantly in miR-101-mimic group than the other groups treated by high-glucose (P<0.05). The protein expression of PCNA was lower significantly in miR-101-inhibitor group than the other groups (P<0.05).4. The proliferation of SV40 Mes cells were detected by CCK8 under high-glucose condition. The proliferation in the miR-101-mimic group was higher t-han the other groups, but the lowest in the miR-101-inhibitor group.[Conclusions]1. miR-101 inhibits the autophagy of SV40 Mes cells treated with high-glucose.2. miR-101 induces the proliferation of SV40 Mes cells treated with high-glucose.