Role And Molecular Mechanism Of MicroRNA 15a In Skeletal Muscle Dysfunction In Sepsis Based On Microarray Data Analysis

Objective: Sepsis is a life-threatening organ dysfunction caused by the imbalance of the host’s response to infection.It has a high morbidity and mortality rate worldwide.Sepsis,severe sepsis,and septic shock can lead to decreased muscle power generation capacity,unexcitable sarcolemma,loss of muscle mass,and subsequent muscle atrophy due to increased proteolysis.However,the mechanism has not yet been fully elucidated.Micro RNAs(miRNAs)are a new type of small regulatory molecules that participate in the post-transcriptional regulation of gene expression by binding specific sequences on the 3’untranslated region,and play an important role in a variety of biological processes.Studies have shown that mi RNAs have become key regulators of skeletal muscle development and function,and play a key role in maintaining physiological homeostasis and participating in skeletal muscle-related diseases.Some miRNAs have been found to be abnormally expressed in sepsis,which also indicates that they can be used as potential effective biomarkers for the early diagnosis and prognosis of sepsis.In recent years,gene chip technology has been widely used in the field of life sciences,which provides an ideal tool for analyzing large gene expression data sets in order to fully understand the underlying mechanisms of various diseases.This study is based on the gene chip GSE13205 containing septic skeletal muscle samples.Through further bioinformatics analysis,differentially expressed mi RNAs were screened to explore the molecular mechanisms and intervention strategies involved in septic skeletal muscle dysfunction.Methods: The gene chip GSE13205 containing septic skeletal muscle samples was screened in the gene expression comprehensive database GEO,and the online tool GEO2 R was used to analyze and screen differentially expressed miRNAs.Construct LPS models,including the murine sepsis model induced by intraperitoneal injection of LPS and treatment of C2C12 myoblasts with LPS in vitro,and use RT-PCR to detect hetero-expressed miRNAs to verify whether the trend is consistent with the data in the chip GSE13205.Screen out the miRNAs with the same trend,and further in-depth study.The literature shows that LPS can cause skeletal muscle atrophy by promoting autophagy.First observe the role of LPS and explore whether it is related to the autophagy pathway.Western Blot was used to detect the expression of ULK1,P62 and LC3;immunofluorescence method was used to observe the expression of endogenous LC3 in C2C12 myoblasts.Then verify whether the studied miRNA is involved in LPS-induced skeletal muscle autophagy,treat C2C12 myoblasts with LPS in vitro,and simultaneously transfect selected mi RNA mimics and inhibitors,and use Western Blot to detect the expression of ULK1,P62 and LC3;immunofluorescence The method to observe the expression of endogenous LC3 in C2C12 myoblasts.The Targetscan database was used to predict the potential target genes of the studied mi RNAs,the selected mi RNA mimics and inhibitors were transfected in C2C12 myoblasts in vitro,and the predicted target genes were detected by RT-PCR.In vitro experiments to verify whether the studied mi RNA participates in the LPS-induced skeletal muscle autophagy pathway by inhibiting its target genes.Western Blot was used to detect the expression of ULK1,P62 and LC3;immunofluorescence method was used to observe the expression of endogenous LC3 in C2C12 myoblasts.Results: 1)The expression of miR-15a-3p increases in LPS-induced in vivo and in vitro models,which is consistent with the trend of the data in the gene chip GSE13205;2)LPS can promote the expression of skeletal muscle autophagy-related proteins at overall and cellular levels;3)miR-15a-3p mimic can promote C2C12 myoblast autophagy;4)miR-15a-3p inhibitor can block LPS-mediated C2C12 myoblast autophagy;5)PGC1α is miR-15a-3p Target genes in C2C12 myoblasts;6)LPS can inhibit the expression of PGC1α protein at the whole and cellular level;7)miR-15a-3p inhibitor promotes the expression of PGC1α in C2C12 myoblasts.Conclusion: LPS promotes skeletal muscle autophagy,mi R-15a-3p can participate in mediating LPS skeletal muscle autophagy pathway by inhibiting PGC1α.