Effect Of MicroRNA-455 On High Glucose-induced Proliferation And Extracellular Matrix Protein Expression In Human Mesangial Cells

BackgroundDiabetes mellitus is one of the most serious challenge to public health in the 21st century. According to the prediction of International Diabetes Federation, there are at least 387 million diabetic patients worldwide in 2014, and the number will rise to about 600 million in 2035, which means that one of ten adults will be diabetic patient in the world. Diabetic kidney disease (DKD) is one of the major complications of diabetes mellitus and the main reason for end-stage renal disease (ESRD). No matter in type 1 diabetes mellitus or type 2 diabetes mellitus the occurrence of DKD is related to poor blood glucose control. The accumulation of advanced glycation end products(AGE) produced by hyperglycemia through the non-enzyme pathway, the activation of protein kinase C (PKC) and polyol pathway, the increase of oxidative stress, the activation of vasoactive substance and cytokines, and the tissue damage caused by kallikrein---plasmakinin system and other factors influence each other. In the condition of hyperglycemia, mesangial cells (MCs) have a dysfunction, which causes the protein synthesis and degradation a relatively imbalanced of extracellular matrix (ECM), so they accumulate a lot in mesangial area and basilar membrane area, which makes the glomerular morphology, structure and function have pathological changes and eventually lead to a glomerular sclerosis.MicroRNA (miRNA) is a large family of small regulatory RNA, which is the single non-coding small RNA molecules of about 20 nucleotides, and it can be combined with target genes mRNA 3’-untranslated region(3’UTR), and regulate target gene expression by promoting mRNA degradation or blocking protein translation so as to regulate and control the body’s pathophysiological process. MicroRNA plays an important role in cell proliferation, growth, differentiation and apoptosis. MicroRNA has a different expression in different tissues, and its expressed changes involve in the happening and development of a variety of human diseases, such as tumor, cardiovascular disease, diabetes, kidney disease, etc. More and more evidence indicates that miRNA plays a regulatory role in the signaling pathway of pathological changes of DKD. For example, miR-192 can activate TGF β signaling pathway to lead to renal fibrosis and proteinuria. miR-21 can activate Akt signaling pathway to lead to kidney hypertrophy and fibrosis. miRNA which promotes DKD progress has been found that expression elevates in diabetic kidney. On the contrary, miRNA with decreased expression has protective effect on kidney.AMP activated protein kinase (AMPK) is the highly conservative serine /serine-threonine kinase in eukaryotic cells, it widely exists in eukaryotic organisms in heterotrimeric form, it is the energy receptor of cells and plays a very important role in the regulation of energy metabolism. Liver kinase B1 (LKB1) and CaMKKβ can activate AMPK by relying on ratio increase in AMP/ATP or ADP/ATP, and then regulate energy metabolism network of cells and improve its ability to respond to changes in the internal and external environment, so as to maintain the steady state of cell level and even the whole organism. Activated AMPK can enhance catabolism, inhibit anabolism, increase the level of ATP, and participate in glucose metabolism of cells, fat metabolism, and protein metabolism and other energy metabolism processes, and increase cellular energy reserve so as to cope with energy deficiency. At the same time, activated AMPK participates in cell growth, proliferation, apoptosis, autophagy and other basic biological processes. AMPK is the core of studying energy metabolic diseases such as obesity and diabetes mellitus, etc. Activity decrease of AMPK in the kidney may lead to kidney changes of obese patients and diabetic patients. In addition, the agonist AICAR of AMPK can significantly improve renal injury and reduce proteinuria in Akita mice.In the previous studies, we have found that miR-455 can promote the differentiation of brown fat, and ap2-miR-455 transgenic mice can withstand an obesity and abnormal glucose tolerance induced by a high-fat diet, it activates AMPK signaling pathway by its target gene HIFlan. We also found that the expression of miR-455 in kidney ranks only second to the fat. In a recent study, miR-455 was reduced to 0.428 in the podocyte of 5ng/ml TGF-β treatment, which suggests that miR-455 may take part in the progression of DKD. To sum up, we presume that miR-455 may have important value in the prevention of DKD, but there is no report about miR-455 and DKD seen now.Therefore, this study takes in vitro human mesangial cells (HMCs) as model to observe the effects of miR-455 on mesangial cell proliferation level and ECM expression, and observes the effects of it on the above indexes of mesangial cells through adjusting the activity of AMPK, and then it explores the possible effects of AMPK signaling pathway in this process.ObjectivesTo explore the effect of microRNA-455 on high glucose-induced proliferation and extracellular matrix protein expression in human mesangial cells and its possible mechanism.Materials and MethodsCell cultureFrozen HMC recovered within 1 minute for water bath in 37℃ and was cultured in DMEM low gluose(5.6mM,LG) culture medium containing 10% fetal bovine serum, and incubated in the incubator with 37℃ and 5% CO2. New culture medium was replaced every 2-3 days.Cell proliferation (CCK-8) detectionCells were seeded into culture plate with 96 holes to make each hole contain about 4×103 cells. Firstly, cultured for 24h by using culture medium containing 1% fetal bovine serum, so that cells could synchronize in a quiescent stage, and then applied stimulation, 1h before ending, added 10μL CCK-8 solution to each hole and then continued to incubate for 1h in 37℃ after mixing, and OD value was detected in the 450nm wavelength of ELIASA.Cell transfectionCells were seeded in plate with 6 holes or 12 holes, the cell density of each hole was about 25% when transfection. Lipofectin reagent method was adopted to conduct transient transfection and operation was conducted in accordance with the instruction book for Lipofectamine(?) transfection reagent.Gene real-time fluorescent quantitation of PCR (qRT-PCR)The total RNA of cells was extracted, and nucleic acid was quantified on Nano Drop ND-1000 spectrophotometer. Reverse transcription of cDNA was conducted according to the instruction book for M-MLV reverse transcriptase. SYBR Select Master Mix was used for real-time fluorescent quantitation of PCR reaction. PCR reaction was conducted on the Roche 480 PCR instrument. Gene expression quantity was calculated by means of 2-△△Ct. β-actin served as references gene.miRNA real-time fluorescent quantitation of PCR (qRT-PCR)miRNA was extracted in accordance with the instruction book for miRcute miRNA extraction-separation kit of Tiangen Biotech (Beijing) Co., Ltd. The fist chain was synthesized in accordance with the instruction book for miRcute miRNA cDNA extraction-separation kit of the first chain synthesis kit of Tiangen Biotech (Beijing) Co. Ltd. PCR reaction liquid was prepared in accordance with the instruction book for miRcute miRNA fluorescent quantitative detection kit of Tiangen Biotech (Beijing) Co., Ltd. U6 was taken as reference and 2-△△Ct method was used for relative quantitation to calculate the relative expression quantity of miR-455 mRNA.Western blottingThe total cellular protein was extracted. BCA method was used for measuring protein concentration of samples.10% or 12% SDS-PAGE was casted to isolate the denatured sample proteins by electrophoresis, gel where the target protein was in was cut and target protein was transferred to PVDF membrane. TBST containing 5% skim milk powder was used to seal PVDF membrane, after dilution, primary antibodies (1:1000) were overnight incubated with PVDF membrane at 4℃ shaker. And then incubation away from light was conducted with fluorescent secondary antibodies (1:15000) for 1h. Luminescence development was carried out in the Odyssey near infrared imaging system, and gel image analysis system (Quantity One 4.4.0) was used for analyzing results.Statistical analysisThe data obtained were expressed by mean±standard deviation, and statistical software SPSS 20.0 was used for statistical and analysis processes. Two independent samples were compared using T test.One-way ANOVA was adopted for the comparison of multiple sample average, LSD method was adopted for multiple comparison of data with homogeneity of variance, Welch method was used for approximating variance analysis of data with heterogeneity of variance, and Dunnett’s T3 method was used for multiple comparison, and the opinion that P< 0.05 is variance has been considered to have statistical significance.ResultEffects of high glucose on HMCs function and AMPK signaling pathwayCell proliferation test demonstrated that high glucose(30mmol/l,HG) stimulation for 24h,48h and 72h could promote the proliferation of mesangial cells compared with LG(5.6mmol/l,LG) group. High glucose stimulation for 48h could make mRNA and protein expression of cells FN and COLI significantly increase and the protein expression of cells phosphorylated AMPK and phosphorylated ACC significantly decrease.There was no significant difference in relative indexs between LG group and mannitol group(24.4mmol/l mannitol and 5.6mmol/l glucose).Effects of high glucose on the expression quantity of HMCs miR-455mRNAqRT-PCR detection found incubation of HMCs for 48h, the mRNA expression quantity of high glucose group miR-455 had significantly decreased compared with LG group.Effects of miR-455 mimics and inhibitor on expression quantity of HMCs miR-455 mRNAqRT-PCR detection found that the expression level of miR-455 transferring to miR-455-mimic group was significantly higher than scramble group, and the expression level of miR-455 transferring to miR-455-inhibitor group was significantly lower than scramble group, which indicated that transfection was successful.Effects of miR-455 mimics and inhibitor on HMCs functionCompared with HG+scramble group, the cell proliferation level as well as FN and COLI protein expression of HG+miR-455-mimic group were obviously decreased. Compared with HG+scramble group, the cell proliferation level as well as FN and COLI protein expression of HG+miR-455-inhibitor group were obviously increased.Effects of miR-455 mimics and inhibitor on HMCs AMPK signaling pathwayCompared with HG+scramble group, the protein expression quantity of cells PAMPK and PACC of HG+miR-455-mimic group was significantly increased. Compared with HG+scramble group, the protein expression quantity of cells PAMPK and PACC of HG+miR-455-inhibitor group was significantly decreased.Effects of AMPK inhibition on the HMCs function of overexpression of miR-455 under the stimulation of high glucoseOverexpression of miR-455 and 1mmol/L AICAR pretreatment could inhibit the proliferation of cells (compared with HG group), in addition,10μmol/L compound C could weaken the inhibiting effect of overexpression of miR-455 and enhance cell proliferation. Overexpression of miR-455 and 1 mmol/L AICAR pretreatment could inhibit the expression of FN and COLI (compared with HG group). In addition, 10μmol/L compound C pretreatment could weaken the inhibiting effect of overexpression of miR-455 and increase the expression of FN and COLI protein.Effects of AMPK inhibition on HMCs AMPK signaling pathway of overexpression of miR-455 under the stimulation of high glucoseOverexpression of miR-455 and 1mmol/L AICAR pretreatment could promote the expression of phosphorylated AMPK and phosphorylated ACC (compared with HG group). In addition, 10μmol/L compound C pretreatment could weaken the promoting effect of overexpression of miR-455, and reduce the expression of phosphorylated AMPK and phosphorylated ACC protein.Effects of AMPK activation on inhibiting the mesangial cell function of miR-455 under the stimulation of high glucoseInhibition of miR-455 expression could promote cell proliferation (compared with HG group), in addition, 1mmol/L AICAR pretreatment could weaken the promoting effect of inhibiting miR-455 expression and inhibit the proliferation of mesangial cells; inhibition of miR-455 expression could promote expression of cells FN and COLI protein (compared with HG group), in addition, 1mmol/L AICAR pretreatment could weaken the promoting effect of inhibiting miR-455 expression, and reduce the expression of FN and COLI protein.Effects of AMPK activation on inhibiting the mesangial cell AMPK signaling pathway of miR-455 under the stimulation of high glucoseInhibition of miR-455 expression could inhibit the expression of cell phosphorylated AMPK and phosphorylated ACC protein. In addition, lmmol/L AICAR pretreatment could weaken the inhibiting effect of inhibiting miR-455 expression, and increase the expression of phosphorylated AMPK and phosphorylated ACC protein.Conclusion1.In vitro high glucose culture can promote HMCs proliferation as well as expression of FN and COLI protein, inhibit AMPK and ACC phosphorylation and reduce the expression quantity of miR-455 mRNA;2.Overexpression of miR-455 can inhibit the high glucose induced cell proliferation as well as expression of FN and COLI protein, and promote AMPK and ACC phosphorylation. Compound C pretreatment can weaken the inhibiting effect of overexpression of miR-455.3.Inhibition of miR-455 expression can promote the high glucose induced cell proliferation as well as expression of FN and COLI protein, and inhibit AMPK and ACC phosphorylation. AICAR pretreatment can weaken the promoting effect of inhibiting mikR-455.