The Molecular Mechanism Of Circ₁366 Regulating The Proliferation Of Rat Normal Liver Cells BRL-3A

Recent studies have shown that circRNA is a class of endogenous non-coding RNA molecules,which are widely present in a variety of biological cells to regulate gene expression.circRNA can act as a miRNA sponge,regulate gene transcription,interact with RNA-binding proteins,and translate protein.Studies have shown that circ RNA is involved in the expression and regulation of endogenous competitive RNAs,and is associated with the growth and development of tissues and certain diseases,such as atherosclerosis,neurological diseases,and prion diseases,especially in the development of tumors.Development,diagnostics and other research have far-reaching potential value.However,a large number of circRNAs involved in the regulation of liver regeneration have not yet been discovered.Their function and mechanism of action in liver regeneration remains to be studied.In this experiment,we completed the high throughput sequencing and bioinformatics analysis of 0h,2h and 6h regenerated liver circ RNA after 2/3hepatectomy in rats,and found 2412 kinds of circRNAs.Based on GO analysis and KEGG functional enrichment analysis,it was speculated that circRNA may participate in physiological activities.Among them,various circRNAs are closely related to the physiological activities of cell proliferation,growth,differentiation,and apoptosis.According to the results of high throughput sequencing and transcriptome and proteomics microarray,there were 159 kinds of circRNA in the experimental group,which were significantly changed in the regenerative liver induced by 2/3 hepatectomy,96 up-regulated,61down-regulated,two kinds of reduction in 6h and 2 h up-regulated..By comparing the significance of the differential expression of target genes corresponding to the circRNAs in the regenerating liver at 10 time points and consulting related literatures,we finally screened circ₁366,which was significantly related to liver regeneration,and detected its effect on proliferation of BRL-3A cells in rats.Firstly,the expression level of circ₁366 was detected by the method of gene intervention in rat BRL-3A.Then the cell viability was detected by MTT assay,cell cycle was detected by flow cytometry,qRT-PCR and Western blot were used to study the expression of proliferation-associated mRNA/protein,and the analysis of circ₁366 regulation on rat BRL-3A cell proliferation.The results showed that aftertransfection of circ₁366 overexpression plasmid for 48 h,the expression level of circ₁366 in the overexpression group was about 9 times that of the no-load control group;MTT assay showed that the activity of the overexpression group was significantly higher than that of the no-load control group;flow cytometry Tests showed that the proportion of S+G2/M phase cells in the overexpression group was not significantly higher than that in the no-load control group;qRT-PCR and Western blot showed that the mRNA and protein expressions of hepatocyte proliferation-related genes CCND1,CCNA2,JUN,MYC,and PCNA were up-regulated,and the mRNA and corresponding protein expression levels of the apoptosis-related genes CASP3 and CASP9 were down-regulated after circ₁366 was over-expressed in the BRL-3A cells.The ratio of CASP9 protein level decreased significantly.After transfection of circ₁366interference fragments,the interference efficiency was 73% compared with the negative control group.MTT assay showed that the activity of the cells in the interference group was significantly lower than that in the negative control group;flow cytometry showed that the intervention group was significantly less than the negative control group cell proportion of S+G2/M phase;qRT-PCR and Western blot showed that the mRNA and protein expressions of hepatocyte proliferation-related genes CCND1,CCNA2,JUN,MYC,and PCNA were down-regulated in BRL-3A cells after circ₁366 interference,and the mRNA and corresponding protein expression levels of apoptosis-related genes CASP3 and CASP9 were up-regulated,indicating that overexpression of circ₁366 promoted cell proliferation in rat BRL-3A,and circ₁366interference could inhibit BRL-3A.Cell Proliferation.Therefore,circ₁366 can affect the cell proliferation of rat normal hepatocyte BRL-3A by regulating the expression level of genes/proteins associated with cell proliferation.