Study On Molecular Mechanism Of Kidney Yin Deficiency In Postmenopausal Osteoporosis Based On MiRNA-mediated CLCF1 Gene Translation Regulation

ObjectiveUsing bioinformatics and dualluciferase assays to screen and verify microRNA(miRNAs)targeted to regulate postmenopausal osteoporosisassociated gene CLCF1,and screened miRNAs for in vivo validation,aiming at miRNAs regulation to explore the molecular substance of kidney yin deficiency syndrome in postmenopausal osteoporosis.Methods:1.MicroRNA screening:? Bioinformatics software was used to predict the complementation of miRNAs with the 3'Nunencoding region(3'UTR)of the target gene CLCF1 based on the principle of complementary base pairing;? The miRNAs screened by bioinformatics were tested with dual luciferase to verify theirrelationship with CLCF1.2.1n vivo validation of miR655:?Randomly selected postmenopausal females in Fuzhou Han nationality to conduct a questionnaire survey and screen the subjects according to the "Dialectical Criteria for TCM Deficiency Syndrome" and "Expert consensus on diagnostic criteria for osteoporosis in China" and dualenergy Xray absorptiometry.131 cases were divided into groups:50 cases with PMOP kidney yin deficiency syndrome,33 cases with PMOP kidney yang deficiency group,48 cases without kidney deficiency healthy control group;?Detecteing the expression of hsamiR655 and CLCF1 mRNA and protein in the three groups by qRTPCR and western blot was to study the correlation betwe en hsamiR655 and PMOP kidney yin deficiency syndrome.Results:1.MicroRNA screening and validation results:?Target miRNAs of CLCF1:44 TargetScans,21 miRandas,27 microRNAs,and 5 intersections of the three:hsamiR655,hsamiR300,hsamiR381,hsamiR374c,hsamiR515.?Two luciferases were used to verify the selected five target miRNAs,and the most significant ones were screened for further experimental analysis.The experimental results showed that compared with the control group,the fluorescence activity of hsamiR655 group was downregulated to 33.09%(P=0.001),and the inhibitory effect on the expression level of CLCF1 3'UTR was the most significant;hsamiR655 and CLCF1 After the 3'UTR binding site was mutated,the fluorescence activity was upregulated to 67.22%,suggesting that the gene expression level of CLCF1 3'UTR was specifically inhibited by hsamiR655.The experimental results show that the 3' UTR of CLCF1 gene can specifically bind to hsamiR655 and specifically inhibit the expression of CLCF1 gene.2.Relationship between hsamiR655 and PMOP Kidneyyin Deficiency Syndrome:? Three groups of experimental subjects:general data age,menopausal age,BMI were statistically compared,and there was no statistical difference in the three groups of general data;? The relative quantification of CLCF1mRNA and hasmiR655 expression levels in the three groups using 2??Ct method:The expression of CLCF1 mRNA in PMOP kidney yin deficiency group was significantly lower than that in healthy control group with statistically significantdiffer ence(P<0.01);The expression level of hasmiR655 was significantly higher in PMOP kidney yin deficiency group than in healthy control group(P<0.01);There was a statistically significant difference;There was no significant difference in the expression levels of CLCF1 mRNA and hasmiR655 between PMOP Kid-ney Yin Deficiency Syndrome and POP Kidney Yang Deficiency Syndrome(P>0.05);?Western blot was used to detectthe expression of three groups of CLCF 1 protein:control group:0.564±0.158,PMOP kidney yin deficiency group:0.325±0.125,PMOP kidney yang deficiency group:0.474±0.178;The expression of CLCF1 protein in PMOP kidneyyin deficiency syndrome group was significantly 1 ower than that in the control group,P<0.01,which was statistically significant;There was no significant difference between PMOM kidney yin deficiency syndro me group and PMOP kidney yang deficiency syndrome group(P>0.05).ConclusionHsamiR655 can negatively regulate postmenopausal osteoporosis kidney yin deficiency by targeting the 3' UTR region that binds to CLCF1 mRNA.