Screening Of MiR-34c-5p Target Gene And Its Effect On Apoptosis Of Mouse Brain Microvascular Endothelial Cells

Enterococcus faecalis(E.faecalis)is widely distributed in nature.When it is affected by the external environmental pressure,it will enter the dynamic but unculturable state.It will be revived when the conditions are favorable,which makes its survival ability enhanced in the harsh conditions of complex environment.However,the abuse of antibiotics and the spread of drug-resistant genes in the environment have led to an increase in drug resistance of Enterococcus faecalis,resulting in an increase in cases year by year,and the resulting infection is difficult to treat.Enterococcus meningococci faecalis was isolated in our laboratory in the previous stage,but the mechanism of its destruction of brain tissue structure leading to inflammation is still unclear.Our laboratory has conducted transcriptome sequencing on brain tissue samples of mice infected with Enterococcus meningitidis at different time periods,and screened out a large number of differentially expressed non-coding Rnas.In this study,differentially expressed miR-34c-5p was selected as the research object.Objective: The results of transcriptomic sequencing combined with test verification were used to screen out miR-34c-5p related to meningitis.Select the target gene of miR-34c-5p and determine the interaction between them;To explore the effect of miR-34c-5p and target gene on the proliferation and apoptosis of mouse brain microvascular endothelial cells.Methods:(1)After the mice were infected with Enterococcus faecalis causing meningitis,the brain tissues were collected for transcriptome sequencing,and the sequencing results were analyzed and verified by RT-qPCR.Determine the miRNA selected for the follow-up test,and carry out KEGG analysis on the target gene of differential miRNA;(2)Screening of miR-34c-5p target gene and verification of target relationship : Predict the binding site of miR-34c-5p and candidate target gene,transfect miR-34c-5p’s mimics,inhibitor and negative control into mouse brain microvascular endothelial cells,extract RNA and reverse transcript,and detect the expression of miR-34c-5p and candidate target gene by qPCR.Dual-luciferase wild-type and mutant vectors were constructed on psi-check 2 plasmids with fragments of the target gene containing miR-34c-5p binding sites and co-transfected with miR-34c-5p and miR-NC to 293 T cells,respectively,to detect luciferase activity;(3)Effects of raly targeted by mir-34c-5p on proliferation and apoptosis of mouse brain microvascular endothelial cells: The miR-34c-5p mimics,miR-34c-5p inhibitor,miR-NC and RALY-WT,psi-check 2empty vectors were transfected into the mouse brain microvascular endothelial cells,respectively or cotransfected.Cell proliferation was measured using CCK-8,apoptosis was measured using the TUNEL kit,and apoptotic protein caspase 3 and caspase 9 expression were measured by fluorescence quantitation and Western Blot assays.Results:(1)Clean readings accounted for more than 95% of high-throughput sequencing results,with low error rate and strong correlation between samples,indicating good sample selection and sequencing results.The proportion of miRNA with the known miRNA sequence length of 21-23 nt is the highest,there were 31 mirnas differentially expressed at 12 h,113 mirnas differentially expressed at 24 h,22 mirnas differentially expressed at 60 h,and 2 mirnas shared by the three groups.The results of sequencing and fluorescence quantitative detection of differentially expressed mirnas had the same trend;(2)RT-qPCR results showed that increased miR-34c-5p expression in MBMEC inhibited the expression of candidate target genes.The double Luciferase reporter vector was successfully constructed.Dual luciferin assay results showed that the relative luciferin activity of RALY-WT+miR-34c-5p mimic group was significantly decreased compared with RALY-WT+miR-NC group,while the mutant group and other candidate target gene groups had no significant differences;(3)CCK-8 results showed that the OD value of miR-34c-5p mimic group was significantly lower than that of negative control,and the OD value of miR-34c-5p inhibitor group was opposite.The OD value of miR-34c-5p mimic+RALY-WT group was significantly lower than that of miR-34c-5p mimic+psi-check 2group,and the OD value of miR-34c-5p inhibitor+RALY-WT group was significantly higher than that of miR-34c-5p inhibitor+psi-check 2 group.TUNEL assay showed that miR-34c-5p mimic group and miR-34c-5p mimic + psi-check 2 groups had higher numbers of TUNEL positive cells than miR-NC groups and miR-34c-5p mimic + RALY-WT groups.The results of fluorescence quantitative and Western Blot showed a significant increase in caspase 3 and caspase 9 expression when the expression of miR-34c-5p and RALY increased.Conclusion:(1)miR-34c-5p was differentially expressed in the brain tissue of mice infected with Enterococcus meningococci;(2)miR-34c-5p can bind to the 3’UTR region of RALY and inhibit its expression;(3)miR-34c-5p can target RALY to promote MBMEC apoptosis and inhibit its proliferation.