Differential Expression Of MiR-7-5p Of Hepatocytes In Insulin Resistance And Its Targeting Effect With ITCH

Insulin res istance is a typical manifestation of metabolic syndrome induced by obesity.Because of its extensive organ function and complex mechanisms,it becomes a major problem in the treatment of metabolic diseases such as diabetes.The biological function of insulin depends on the sequential activation of many proteins in its signaling pathway.The inhibition or degradation of any protein molecule will lead to insulin resistance phenomena such as disorder of glucose and lipid metabolism.The pathogenesis of insulin resistance presents an extremely complex network of effects,including the interaction between multiple signaling pathways and insulin signaling pathways,and the extens ive participation of multiple regulatory factors.Therefore,it is very important to identifying the mechanism of insulin resistance by clarifying the specific action modes of the relevant signal pathways and the regulatory factors and insulin signaling pathways.Ubiquitin proteasome system(UPS)is the main pathway of protein degradation and can selectively participate in the degradation of more than 80% proteins in cells.It is closely related to the normal transduction of insulin signaling pathway.Micro RNAs(mi RNAs)are a new class of highly effective regulatory molecules that have attracted much attention in recent years.They can directly target protein molecules that regulate insulin and its related signaling pathways,and induce insulin resistance directly or indirectly.At present,there is no report on the interaction among insulin signal pathway,UPS and mi RNAs.In our previous reaearch,we found that the mmu-mi R-7a-5p(MIMAT0000677)levels were significantly decreased in liver tissue of insulin resistant mice induced by high fat diet us ing gene chip screening method.Target gene prediction software Target Scan and mi RDB analysis confirm that mmu-mi R-7a-5p shares 100% homology with human hsa-mir-7-5p.in this study,we established a human hepatoma Hep G2 cell insulin res istance model,and detected the differential e xpression of mi R-7-5p under insulin resistance,and studyed its targeting relationship with the predicted target gene E3 ubiquitin ligase Itch gene.Our aim is to explore the relationship among mi R-7-5p and Itch genes and insulin resistance for enriching the mechanism of insulin resistance,and to provide an experimental evidence for further study the biological role of mi R-7-5p and Itch genes and finding molecular targets of the diagnosis and treatment of insulin resistance.Firstly,Hep G2 cells were induced to establish an insulin resistance model by suitable concentration of palmitic acid(PA)in vitro.The expression of mi R-7-5p in insulin resistance status was detected by real-time fluorescence quantitative PCR(RT-q PCR).The results showed that when Hep G2 cells were treated with 0.25 mmol/L PA for 24 h,the cell activity was better,glucose utilization ratio was decreased,and lipid accumulation in the cytoplasm was increased,which was the most suitable concentration for inducing insulin res istance in Hep G2 cells.The expression of mi R-7-5p in Hep G2 cells was down-regulated nearly 2 times under insulin resistance(P<0.01),suggesting that mi R-7-5p may be involved in the process of insulin resistance in Hep G2 cells.Secondly,the bioinformatics analyses were performed.The Targetscan database and mi RDB database were used to predict the mi R-7-5p target genes,and KEGG database was used to analyze the biological signaling pathways that the differentially expressed target genes of mi R-7-5p might be enriched.The protein-protein interaction net was further analyzed between the predicted target genes and insulin signaling pathway proteins by STRING 10.0 software to identify the most related target gene to insulin resistance.The results showed that a considerab le number of target genes were enriched in UPS,and E3 ubiquitin ligase ITCH could interact directly with Phosphatidylinositol-4,5-bisphosphonate 3-kinase,catalytic subunit,alpha polypeptide gene(PIK3CA),to reduce the production of p110 catalytic subunit of phosphatidylinositol 3-kinase(PI3K),which directly affects the normal transduction of PI3K-AKT pathway,an important branch of insulin signaling pathway.Thirdly,in order to confirm the correlation between the expression of mi R-7-5p and Itch gene,RT-q PCR and Western blot techniques were used to detect the changes of Itch gene at transcription and trans lation level in insulin resistance.The results showed that there was no significant difference at transcription level between the insulin res istance group and the control group(P>0.05),and the level of translation was higher than that in the normal control group.The difference was statistically significant(P<0.01),indicating that there was a negative correlation between the expression of mi R-7-5p and Itch gene in insulin resistance.Finally,in order to further verify the targeting relationship between the two,firstly,the appropriate concentration of mi R-7-5p inhibitor was transfected to inhibit the expression of mi R-7-5p in Hep G2 cells in vitro.The expression differences of Itch gene at transcription and translation level were detected again by RT-q PCR and Western blot to explore the targeting relationship preliminarily.Next,pmir GLO-Itch-3?UTR wild-type vector and pmir GLO-mut-Itch-3?UTR mutant vector were constructed,respectively.The two constructs were cotransfected with mi R-7-5p mimics into Hela cells,respectively.According to the ratio of firefly luciferase activity to sea kidney luciferase activity,the direct targeting relations hip was determined between them.The results showed that the expression of mi R-7-5p was more than 2 times down-regulated after transfection of 200 n M mi R-7-5p inhibitor into Hep G2 cells,and the expression of Itch gene was up-regulated at translation level(P<0.05).It is suggested that mi R-7-5p may be bound to the 3?UTR seed sequence of Itch m RNA,which may affect the expression of Itch gene at translation level,thus participating in the occurrence and development of insulin resistance.The results of double luciferase reporter gene analys is showed that the ratio of firefly luciferase activity to sea kidney luciferase activity was significantly down-regulated in pmir GLO-Itch-3?UTR wild-type vector transfected cells in mi R-7-5p mimic group compared with negative control group.The difference was statistically significant(P<0.05),while there was no significant difference in the ratio of cells transfected with pmir GLO-mut-Itch-3?UTR mutant vector(P>0.05).The results indicate that Itch gene is the target gene of mi R-7-5p,which could bind to 3?UTR of Itch gene and inhibit gene expression at translation level.In conclusion,on the basis of Hep G2 cells insulin res istance model and bioinformatics analys is,the targeting effects between mi R-7-5p and Itch genes,and their relationship with insulin resistance were preliminarily verified by RT-q PCR,Western blot,cell transfection and double-luciferase reporter gene experiments.It is suggested that mi R-7-5p may be involved in the pathophysiological process of insulin res istance,and may directly affect the normal transduction of PI3K-AKT signal pathway by targeting the expression of Itch gene to induce insulin resistance.