MiR-145-3p Suppresses Stemness Of HCC Via Targeting DNMT3A

Objective:The purpose is to study the biological influence on stemness and malignancy of miR-145 and DNMT3A by revealing theinteraction and mechanism between miR-145 and DNMT3A and their biological function in HCC.This research might be able to provide a novel therapy of the treatment of HCC.Methods:1 The expression profile of miR-145 was analyzed using online tool ULCAN(http://ualcan.path.uab.edu/index.html),data were obtained from The Cancer Genome Atlas(TCGA).The Kaplan-Meier survival analysis was executed using Kaplan-Meier Plotter(http://kmplot.com/analysis/index.php?p=service).Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)were performws on 15 pairs of HCC and adjacent tissue specimens.2 The expressions of miR-145-3p in both HCC cell lines and normal hepatic cell line LO2 were detected using qRT-PCR.Lentivirus vectors were used to respectively up-and downregulate the expression of miR-145-3p in HCC cell lines,and stably transfected cell lines were established.The transfection efficiency was measured using qRT-PCR and flow cytometry.EdU proliferation,colony formation,transwell,wound-healing,and flow cytometry were performed to estimate the biological function of miR-145-3p in HCC.3 Sphere formation assay and flow cytometry,added to tumor formation assay were performed to assess the stem features of HCC cell lines.4 Target prediction of miR-145-3p was performed using miRabel,and DNMT3A was selected for further analysis.ULCAN and Kaplan-Meier Plotter were used to analyze the expression and prognostic association of DNMT3A in HCC.The expression of DNMT3A was validated using qRT-PCR and western blotting.In addition,a dual-luciferase reporter assay was used to confirm whether DNMT3A is a direct target of miR-145-3p.5 The expressions of DNMT3A were rescued in miR-145-3p stably transfected HCC cells to ascertain if DNMT3A can reverse miR-145-3p induced stem suppression.6 Identifying target genes of DNMT3A by assessing their expression of mRNA,protein,and methylation status of promoter.Results:1 MiR-145 is downregulated in HCC tissues,and lower expression of miR-145 is related to poorer prognosis.2 MiR-145-3p is down regulated in both HCC tissues and cell lines,forced expression of miR-145-3p can significantly suppress proliferation,colony formation,migration,invasion of HCC cells,and cause cell cycle arrest and apoptosis promotion.3 The ability of spere formation,and the proportion of CD133+CD44+ cells was significantly suppressed by miR-145-3p,indicating that the stemness of HCC cells was significantly suppressed by miR-145-3p.4 DNMT3A is a direct target of miR-145-3p,upregulating DNMT3A in HCC can reverse miR-145-3p induced stemness suppression.5 The promoters of DLC1,RUNX3,SOX1,and p16 are abnormally hypermethylated,causing silenced expression of those tumor suppressors.This methylation aberration might be prevented by miR-145-3p.Conclusion:MiR-145-3p was found to be a strong tumor suppressor,usually downregulated in HCC tissues and cell lines.It can significantly inhibit the stemness of HCC by targeting DNMT3A.MiR-145-3p might be able to provide new therapies for the treatment of HCC as a novel target of treating methylation aberrations.