MicroRNA-203 Regulate The Cell Proliferation By Directly Targeting Bmi-1 In Hepatocellular Carcinoma

Cancer is one of the commonest causes of death worldwide,and has been a major public health challenge.Despite the increased survival rate of cancer patients in recent decades,there are still approximately 7.6 million cancer deaths reported in 2008.The successful treatment of cancer is dependent on not only early diagnosis,but also correct therapy strategies based on the prediction of patients’ outcome.Thus,in order to establish more accurate diagnosis and prognosis,newer tumor biomarkers with improved sensitivity and specificity are essential for this fatal disease.Recently,the B-cell-specific moloney leukemia virus insertion site 1(Bmi-1)seems to be a new putative marker for cancer.The human Bmi-1 gene,located on chromosome 10(10p11.23),comprises 10 exons and 9 introns.It encodes the Bim-1 protein that contains a conserved RING finger domain in the N-terminal end and a central helix-turn-helix-turn-helix-turn motif(H-T-H-T),which is required for inducing telomerase activity.Bmi-1 is one of the core members of the polycomb repressive complex1(PRC1)which can modify chromatin structure and thereby regulates the transcription of a number of important genes,such as the Ink4a/ARF locus,a gene code of two important tumor suppressor proteinsp16Ink4 aand p14ARF.It has been reported that Bmi-1 is associated with the initiation,self-renewal,proliferation,and chemoresistance of tumor.MicroRNAs(miRNAs)are small non-coding RNAs with a length of 19-25 nucleotides that post-transcriptionally regulate gene expression.They negatively regulate gene expression by base pairing to partially complementary sites on the target m RNAs,usually in the 3’ untranslated region.Binding of a miRNA to the target m RNA typically leads to translational regression and exonucleolytic m RNA decay.It has been found that numerous miRNAs exhibit dysregulated expression in multiple types of cancers and are often associated with diagnosis,staging,progression,prognosis and response to clinical therapies.Mi R-203 was known to be a putative tumor suppressor gene and its expression was downregulated in various tumor.Previous study demonstrated that miR-203 can directly target ZEB1/2 and SNAI1/2 m RNAs.These m RNAs play critical regulatory roles in two processes: epithelial-to-mesenchymal transition(EMT)and mesenchymal-to-epithelial transition(MET).The two processes are associated with metastasis and prognosis of malignant tumors.In addition,miR-203 plays a crucial role in regulating stem cell self-renewal and proliferation.In the present study,the expression level of Bmi-1 in 90 cases of HCC is detected to explore their prognostic role in patients with HCC.We also researched the relationship between Bmi-1 and miR-203 in HCC.Part I Prognostic role of high Bmi-1 expression in Asian and Caucasian patients with solid tumors: a meta-analysisObjective: Recently,many studies have shown that the B-cell-specific moloney leukemia virus insertion site 1(Bmi-1)exhibit altered expression in various cancers and may serve as prognostic biomarkers.We performed a meta-analysis to evaluate the prognostic role of Bmi-1 expression in solid cancers.Methods: Studies were recruited by searching Pub Med,Embase and the Cochrane Library(last search update was August 2014)and assessed by further quality evaluation.Results: 39 articles including 40 studies were involved in this meta-analysis.Our results indicated that the Bmi-1 showed the opposite prognostic effect in Asian and Caucasian populations.High Bmi-1 expression as a negative predictor for overall survival(OS)in Asian patients(HR=1.96,95% CI 1.62-2.36),but a positive predictor in Caucasian populations(HR=0.77,95% CI 0.63-0.93).Furthermore,we took a further subgroup analysis based on tumor type in these two populations respectively.In Asian cases,high expression of Bmi-1 was associated with poor OS in esophageal carcinoma(HR=1.93,95% CI 1.52-2.46),gastric cancer(HR=1.50,95% CI 1.22-1.85),lung cancer(HR=1.73,95% CI 1.05-2.85),cervical cancer(HR=2.80,95% CI 2.26-3.47)and colorectal cancer(HR=3.36,95% CI 2.19-5.15),rather than in breast cancer and HCC.In Caucasian populations,high expression of Bmi-1 was associated with better OS in breast cancer(HR=0.70,95% CI 0.51-0.97)but it showed no significance in esophageal carcinoma.Conclusion: High Bmi-1 expression was significantly associated with poor survival in Asian patients with esophageal carcinoma,gastric cancer,lung cancer,colorectal cancer and cervical carcinoma,whereas high level of Bmi-1 can predict better prognosis in Caucasian patients with breast cancer.Part II Prognostic value of tissue and circulating microRNA-203 in solid tumors based on meta-analysis and The Cancer Genome Atlas(TCGA)datasetsObjective: Recently,many studies have shown that microRNAs(miRNA)exhibit altered expression in various cancers and may serve as prognostic biomarkers.We performed a meta-analysis and The Cancer Genome Atlas(TCGA)to evaluate the prognostic role of miR-203 expression in solid tumors.Methods: Studies were recruited by searching Pub Med,Embase and the Cochrane Library(last search update was March 2016)and assessed by further quality evaluation.The miR-203 expression and clinical data for TCGA solid tumors were extracted from the UCSC cancer genome browser project(https://genomecancer.ucsc.edu).Results: A total of 31 studies dealing with various carcinomas were included in the meta-analysis.Our results indicated that the expression of tissue-based miR-203 was not associated with OS and PFS in various carcinomas.In the subgroup analysis,upregulation of tissue miR-203 did predict poor OS colorectal cancer(HR=1.81,95% CI 1.31-2.49;P<0.001)and pancreatic cancer(HR=1.19,95% CI 1.09-1.31;P<0.001),while upregulation of tissue miR-203 showed the opposite results in liver cancer(HR=0.52,95% CI 0.28-0.97;P=0.040)and esophageal cancer(HR=0.41,95% CI 0.25-0.66;P<0.001).Based on TCGA datasets,the same results were found in pancreatic cancer and esophageal cancer,but not in colorectal cancer and liver cancer.Furthermore,overexpression of blood-based miR-203 was significantly related to poor OS and PFS in colorectal cancer and breast cancer.Conclusion: This research clarified that low expression of miR-203 in primary tissue was significantly associated with poor survival in esophageal cancer,whereas a high level of tissue-based miR-203 predicted poor prognosis in pancreatic cancer.High circulating miR-203 indicated poor prognosis in colorectal cancer and breast cancer.Part III Bmi-1 expression as an independent prognostic marker of hepatocellular carcinomaObjective: Many studies have shown that the B-cell-specific moloney leukemia virus insertion site 1(Bmi-1)exhibit altered expression in various cancers and serve as prognostic biomarkers.However,the expression level and prognostic value of Bmi-1 in hepatocellular carcinoma(HCC)is still controversial.Methods: We investigated Bmi-1 expression and its prognostic significance in HCC by performing immunohistochemical analysis,using a total of 90 HCC clinical tissue samples and The Cancer Genome Atlas(TCGA)datasets.Results: High Bmi-1 protein and m RNA expression were significantly higher in HCC tissues than that in the adjacent normal tissues(P<0.001).Cox regression multivariate analyses revealed that Bmi-1 expression was a significant and independent prognostic parameter(HR=2.378,95% CI=1.245-4.543,P=0.009)for HCC patients.We established a nomogram integrated Bmi-1 and TNM stage,which showed better performance in predicting clinical outcomes for patients with HCC.Conclusions: Bmi-1,an identified independent prognostic factor,could improve predictive accuracy for survival in patients with HCC.Part IV MicroRNA-203 regulates cell proliferation in hepatocellular carcinoma via directly targeting Bmi-1Objective: It has been reported that B-cell-specific moloney leukemia virus insertion site 1(Bmi-1)has important roles in various cancers,but its regulation through microRNAs(miRNAs)and its functions in hepatocellular carcinoma(HCC)remains unclear.Methods: We analyzed 367 HCC tissues from TCGA that have both m RNA and miRNA expression data.We performed luciferase assay in order to identify the relationship between Bmi-1 and miRNA.To investigate the effects of miR-203 and Bmi-1 on the proliferation and invasion of HCC cells,we transfected overexpression vector in HCC cells.Results: Our studies revealed that overexpression of miR-203 could decrease Bmi-1 levels in HCC cell lines and led to reduced proliferation.Dual-luciferase reporter assays confirmed that miR-203 could directly target the 3’ un-translated region(3’-UTR)of Bmi-1.Conclusions: Mi R-203 can directly target Bmi-1 and regulate HCC proliferation.