MiR-486-5p Inhibits Liver Cancer Stem Cells By Targeting SIRT1

In addition to a mass of cancer cells,there are also a small number of cells in solid tumor which have the ability of self-renewal and generate heterogeneity of tumors and show the stem cell properties.The population is called cancer stem cells(CSCs)or tumor initiating cells(TICs).Investigators have proved that the cancer stem cells may be the source of tumor growth,recurrence,metastasis and drug resistance.Currently,cancer stem cells have become a new and important research direction to tumor treatment.MicroRNAs(miRNAs)are a class of small non-coding RNAs between 19 and 24 nucleotides.MiRNAs are usually transcribed from a hairpin intermediate of about 70-90 nucleotides which is called pre-miRNA processed by the ribonucleases Dicer.Then the mature miRNAs complement with 3'untranslate region(3'UTR)of target gene,thereater,the expression of target genes is suppressed,either by blocking the translation process or by initiating the cleavage.Thus,these small mi RNAs have vital roles in numerous biological processes,such as cell differentiation and apoptosis or pathological processes including cancer.More and more studies have shown involvement that mi RNAs are abnormally expressed in various human cancers involving tumor formation,metastasis,recurrence and drug resistance.In addition,some of the mi RNAs are closely related with liver cancer stem cells.Thus,to screen the miRNAs that differentially expressed between CSCs and non-CSCs may help to diagnose and treat liver cancer.This topic research idea: First,we screened significantly down-regulated miRNA for liver cancer stem cells.Further,we verify the candidate mi RNAs affect the cell biology of cancer stem and its target genes.Content of study includes four parts:The first part,down-regulated mi RNA screened for liver Cancer tissues by The Cancer Genome Atlas(TCGA)database.The results showed that 59 miRNAs are significantly down-regulated in primary HCC tissuescompared with adjacent non-tumor tissues;Tumor sphere-forming assays have been widely used to retrospectively identify stem cells based on their capacity to evaluate self-renewal and differentiation in vitro.The sphere-forming assay has also been applied to cultivate various CSCs.We examined the expression of miRNAs in the adherent cells and tumor sphere(Huh7 and PLC)by Real-time quantitative PCR(q RT-PCR),screening for special micoRNA in liver cancer stem cells.Our results showed that the expression of miR-99 a,mi R-10 a,miR-486,miR-195,miR-154 and miR-138 was significantly down-regulated;Using flow cytometry to sort CD13– and CD13+cells,EpCAM-and EpCAM+cells,we tested the expression of mico RNA in CD13+ EpCAM+ cells.Results show that mi R-486-5p in EpCAM+ CD13+cells were significantly downregulated.In the first part of study,we found that miR-486-5p was significantly downregulated in liver cancer stem cells.Many studies have shown that miR-486-5p was an antioncogene in some tumor,such as lung cancer,gastric cancer.To definitude functions of specific miR-486-5p on liver CSCs,such as cell proliferation,cell differentiation and metastasis,we explore the molecular mechanism of miR-486-5p in maintenance of liver CSCs phenotype.The second part,to investigate the tumor suppressive role of miR-486-5p,lentiviral constructs expressing miR-486-5p were packaged using the lentivectorpackaging kit.Pseudovirus particles were subsequently used to infect HCC cells.HCC cell lines constitutively expressing miR-486-5p.To further confirm the tumor suppressive function of miR-486-5p,in vitro assays were performed to assess the tumorigenicity of miR-486-5p-expressing Huh7 cells in which miR-486-5p expression was over-expression.Both in vitro and in vivo functional assays were applied to investigate the tumor suppressive role of miR-486-5p.Functional assays indicated that miR-486-5p significantly inhibit the tumor cell growth rate by tumor sphere assays.We also studied the effect of miR-486-5p up-regulation on HCC tumor invasion and metastasis.Matrigel invasion and wound healing assays found miR-486-5p to efficiently suppress the invasive and migratory abilities,respectively.We next investigated whether miR-486-5p could suppress the chemo-sensitivity of HCC cells by treating Huh7-miR-486 and Huh7-Vec cells with doxorubicin.CCK8 assay showed that the cell viability was significantly decreased in Huh7-miR-486 cells.FACS was applied to investigate the potential regulatory effect of miR-486-5p on stemness-associated marker CD13 and qRT-PCR was used to confirm whether miR-486-5p can regulate stemness-associated genes at the transcriptional level,and results showed that these genes were significantly downregulated in miR-486-transfected cells compared with empty vector-transfected cells.The third part,in order to further explore the mechanism of mi R-486-5p in liver cancer stem cells,potential molecular target of miR-486-5p were analysed by bioinformatic algorithms in the three commonly used databases including TargetScan and Pictar and microRNA.org.We predicted candidate target genes of miR-486-5p from three aspects,such as tumor,proliferation and metastasis.qRT-PCR was used to investigate the expression of candidate target genes in miR-486-transfected HCC cells compared with emptyvector-transfected HCC cells and tumor sphere compared with adherent cells.Immunohistochemical technology and qRT-PCR were used to further verify the relationship between miR-486-5p and target genes in HCC tissues;Dual-luciferase reporter gene assay confirmed mi R-486-5p binding site in3'-untranslated region(3'UTR)of human SIRT1.Results show that in the transcription and translation level,the expression of SIRT1 is negatively related with miR-486-5p.Luciferase activity assays demonstrated a direct binding of miR-486-5p to the 3'UTR of SIRT1.The fourth part,to investigate the role of SIRT1,we built short hairpin RNA interference lentiviral vector of SIRT1 gene(shSIRT1);lentiviral constructs expressing shSIRT1 were packaged using the lentivector packaging kit.Pseudovirus particles were subsequently used to infect HCC cells.HCC cell lines constitutively down-regulated expressing SIRT1.To further confirm the tumor suppressive function of SIRT1,in vitro assays were performed to assess the tumorigenicity and chemo-sensitivity of shSIRT1-expressing HCC cells in which SIRT1 expression was knocked down.Functional assays indicated that SIRT1 significantly promote the tumor cell growth rate by CCK8 proliferation assay found.Tumor sphere and foci formation assays found shSIRT1 to significantly reduce the frequency of colony formation.For in vivo tumor formation assay,tumor sizes and tumor volumes of tumors induced by empty vector-transfected cells were significantly larger than SIRT1 down-regulated cells.CCK8 assay showed that the cell viability was significantly decreased in SIRT1 down-regulated cells.These findings demonstrate that shSIRT1 could suppress both the in vitro and in vivo tumorigenicity of HCC cells and increase the chemo-sensitivity of HCC cells by treating SIRT1 down-regulated HCC cells with doxorubicin and sorafenib.In conclusion,we firstly confirmed the miR-486-5p inhibits liver cancer stem cells,such as self-renewal,invasion,migration,drug-resistant and stemness.Second,we clear reveal the miR-486-5p suppress liver cancer stem cells by regulating SIRT1 expression.The meanings of this research are cluding the following two aspects.First,the function of artificial mircoRNA is similar to a small molecule compounds which can be used to treat diseases.Our results remind that miR-486-5p can be as a new targets and mocular markers of HCC in clinical diagnosis and treatments.