MiR-372Regulates Cell Cycle

Objects:To identify the cell cycle regulating microRNA and conform the target genes of this microRNA in cell cycle arrest HepG2with AFP silenced, and to give more understanding to the mechanism of hepatoma by partly recovering AFP signal pathway, hopefully this microRNA also would provide more mechanism and/or a new therapy choice to hepatoma the human fatal killer with very high incidence and mortality in the world especially in China.Contents:In former research, we have found that HepG2cells can be arrested when AFP the marker protein of hepatoma was silenced by RNA interfere. Michip showed that miR-372was upregulated and7other microRNAs were downregulated in HepG2-B5the mono clone with AFP persistently silenced compared with HepG2(-) the negative control mono clone.After screened miR-372was the one regulated cell cycle and miR-372ASO could rescue the growth speed of HepG2-B5as AFPsiRNA does, we got more evidences in HeLa229and OVCAR3that miR-372could regulated the growth speed and cell cycle in different cancer cells. The clone forming ability could be changed by miR-372in HeLa229and OVCAR3. CDK2CCNA1and other15genes are the targets of miR-372and they are the down stream regulated the cell cycles in the cells dyexpression miR-372. Methods1. Screen the regulating microRNA and overexpression of miR-372leads S phase to increase and G2/M decrease.1. Screen the key microRNA could change cancer cell growth speed.2. Investigate the growth speed of HepG2、HeLa229,OVCAR3after over expressed pSilencer2.1-U6neo-miR-372by growth curve assay.3. Perform FACS to investigate the cell cycle of HepG2-B5after blocking miR-372by miR-372ASO and HeLa229-933when miR-372overexpressed.4. Perform the clony forming ability of HeLa229、OVCAR3after transfected pSilencer2.1-U6neo/miR-372by clony forming assay.2. Block miR-372doesn’t change the distribution of S and G2/M phase 1) Investigate the growth speed of A549、LO2、 HepG2、HeLa229、HeLa229-933after transiently transfected miR-372ASO by growth curve assay.2) Perform FACS to investigate the cell cycle of HeLa229-933after blocking miR-372by miR-372ASO.3. Predict and verify the cell cycle target genes of miR-3721) Predict17target genes of miR-372by4bioinformatics softwares.2) Assay mRNA and protein level of target genes when miR-372dyexpressed.3) Get the direct evidence that miR-372effect on its targets by GFP fusion assay.Results and conclusionsWe screened that miR-372ASO could rescue the growth speed of AFP interfered HepG2-B5which was arrested. And overexpress miR-372could arrest the speed of HepG2(-)、HeLa22、OVCAR3and reduce the clony forming ability of HeLa229、 OVCAR3distinctively. FACS showed that overexpression of miR-372leads S phase to increase and G2/M decrease.The growth speed of A549、LO2、HepG2、HeLa229、HeLa229-933was slow too after transiently transfected miR-372ASO by growth curve assay and FACS showed that the distribution of S and G2/M phase was not change when miR-372blocked.We predicted17target genes of miR-372and chose6of them to identify. The mRNA levels of all target genes were negatively correlated with miR-372.Western blot showed that the protein level of PPP6C and CCNA1were negatively correlated with miR-372in several cells.GFP fusion assay showed that all6targets were negatively correlated with miR-372in different extent.The results suggest that miR-372could arrest cell cycle in AFP knockingdown HepG2-B5by targeting CDK2ect.miR-372regulate growth speed of cells negatively. It increases S decrease G2/M when miR-372overexpressed because the Synthesis speed slowed and M phase speedup. And it doesn’t change the distribution of S and G2/M phase when miR-372blocked because the target genes down regulated in all the phase simultaneously.