Circ₀5381 Regulates The Proliferationof Rat Hepatocyte BRL-3A By Targeting MiR-138-5p/miR-370-3p

Circular RNA(circRNA)is a new type of long noncoding RNA with covalently closed continuous rings,which has been widely found in thousands of organisms including plants,animals and humans.It was found that many endogenous,stably and widely expressed circrna in mammalian cells usually showed cell type specificity,tissue specificity or development stage specific by using high-throughput RNA sequencing(RNA SEQ)technology.More and more evidences showed that some circRNAs may regulate microRNA(miRNA)as the function of microRNA sponge and play an important role in transcription control.The function of CircRNA is associated with miRNA,and the circRNA-miRNA is involved in serious disease pathways,such as apoptosis,angiogenesis,invasion and metastasis.In this study,the expression of circ₀5381 in rat liver cells BRL-3A/RH-35/CBRH-7919/HSC-T6 cells and its expression at seven time points of liver regeneration were verified by real-time fluorescence quantitative PCR(qRT-PCR).To study the mechanism of circ₀5381,which plays a key role in rat liver regeneration.circ₀5381 was infect into BRL-3A cells by lentiviral,and over-expressing circ₀5381 cell lines was obtained,and then the effects of circ₀5381 on the cell viability,cell cycle,cell proliferation and gene expression of BRL-3A cells were detected by use MTT,flow cytometry number,qRT-PCR and Werstern blot.The results showed that compared with the control,the activity of BRL-3A cells which over-expressed circ₀5381 was significantly increased,the proportion of cells in G1 phase decreased,the proportion of cells in S phase and G2/M phase increased significantly,and the expression of cyclin gene MYC and JUN also increased significantly at 48 and 72 h after inoculation,indicating that circ₀5381 promoted the cell cycle process.In summary,circ₀5381 could promote the proliferation of BRL-3A cells.Given that circRNA has been proven to be a miRNA sponge,potential miRNAs related to circ₀5381was predicted by miRanda,and found that 10 miRNAs contain at least one binding site(total 10)in the circ₀5381 region,including miR-1247-5p,miR-138-5p,miR-181b-1-3p,miR-210-5p,miR-326-3p,miR-330-5p,miR-3559-3p,miR-370-3p,miR-382-5p,and miR-505-5p.Due to the possible sponge function between circ₀5381 and miRNA,we initially identified 4 miRNAs(miR-138-5p,miR-370-3p,miR-382-5p,and miR-505-5p)based on their expression at the time points for further research.Then the expression of the above four miRNAs(miR-138-5p,miR-370-3p,miR-382-5p,and miR-505-5p)in BRL-3A cells was observed,as well as the effect of above four miRNAs on the cell viability and cycle of BRL-3A cells were detected by MTT and flow cytometry,it was found that miR-138-5p inhibited cell viability and cycle,that is,inhibited the cell proliferation,while miR-370-3p has no significant Inhibition effect.The targeting effects of circ₀5381 on miR-138-5p and miR-370-3p were detected by double fluorescein method,and it was found that circ₀5381-WT could bind with miR-138-5p and miR-370-3p respectively,suggesting that circ₀5381 is a sponge of miR-138-5p and miR-370-3p.Then,the functions of miR-138-5p and miR-370-3p were examined by MTT and flow cytometry,and found that the function of miR-138-5p on cell viability and cell cyclewqs opposite to circ₀5381,inhibiting BRL-3A cell viability and cell proliferation.while miR-370-3p showed the same function as circ₀5381.Therefore,miR-138-5p was selected for further research.The target mRNAs of miR-138-5p(Foxc1,Egfr,Atpllc1)were detected by through TargetScan prediction and querring literature,and it was found that Foxcl may be a potential target mRNA of miR-138-5p by qRT-PCR.