The Clinical Significance Of Expression In Gastric Cancer And The Regulation Mechanism Of Bmi-1

Worldwidely,gastric cancer(GC)is the most important cause for cancer-related deaths.Around 40% of GC cases are diagnosed in China,where GC is the second most common cancer and ranks the third place for the death of cancer patients.GC is highly malignant with unfavorable prognosis.Recently,the death rate of GC decreases remarkably but the 5-year survival rate remains low.Thus,early diagnosis and treatment are pivotal for increasing patients' quality of life and reducing the death rate.Nowadays,surgery,chemotherapy,radiotherapy and targeted therapy have encountered a bottle neck.It is very urgent to explore novel and more effective and sensitive biomarkers to diagnosing GC and evaluating the prognosis.B-cell specific moloney leukemia virus insertion site 1(Bmi-1)is a member of the polycomb group,which functions as a transcriptional repressor and presents high expression in many tumors,in most cases,indicating a poor prognosis.Several lines of evidence suggest that Bmi-1 blocks cell senescence and proliferation,and the Bmi-1 gene is also associated with tumor invasion and metastasis.Aberrant expression of Bmi-1 has been detected in several human cancers including lymphoma,acute myeloid leukemia,colorectal carcinoma,liver carcinoma,non-small cell lung cancer,breast carcinoma,prostate cancer,head and neck squamous cell carcinoma,medulloblastoma,and glioblastoma.Bmi-1 has been identified as a predictor of the response to therapy and survival in various tumors.MiRNAs are non–protein-coding small RNAs with length between 19 and 25 nucleotides cleaved from 70-to 100-nucleotide hairpin pre-miRNA precursors.MiRNAs post-transcriptionally regulate protein expression of many target genes by interacting with the 3'-UTR region of the m RNA transcripts.MiRNAs have pivotal functions in various biological processes including cellular differentiation,proliferation and apoptosis.MiRNAs have great potential as cancer biomarkers because of their superior stability,tissue-specificity and unique expression patterns in cancer cells.Expression profiling analysis has discovered a number of miRNAs deregulated in human malignancies.MiRNAs have been identified to be crucial in tumorigenesis by playing either oncogenic or tumor suppressive roles in gastric cancer.A number of studies have identified miRNAs that play significant roles in gastric cancer.The miRNA mediated Bmi-1 expression and functional significance in gastric cancer remained elusive.Bmi-1 is a key target for miR-15 a,miR-16,and miR-200 c based on several different tumor types.Mutiple miRNAs can target Bmi-1 m RNA in cancers.The development of GC is a multi-stage and complex process.In this study,we systemically investigated the expression and the function of Bmi-1 in GC.The study consists(1)the significance of Bmi-1 expression in various cancers;(2)the association of Bmi-1 expression and the clinicopathological characteristics of GC patients;(3)the correlation of Bmi-1 expression and miR-15 a expression in GC tissue;and(4)the mechanism of miR-15 a regulating Bmi-1 to impact on the biological behaviors of GC cells.Part ?.The significance of Bmi-1 expression for the prognosis in various cancers: a meta-analysisObjective: Bmi-1 is aberrantly expressed in various cancers and can be used as a predictor for the prognosis.A meta-analysis was performed to evaluate the role of Bmi-1 in the prediction for the prognosis.Methods: Studies were recruited by searching Pub Med,Embase and the Cochrane Library(last search update was December 2017)and assessed by further quality evaluation.Data searching and assessment of recruited literatures were done by 2 independent investigators.Positive or high Bmi-1 expression was defined according to the cut-off value from the literatures.Statistical heterogeneity was assessed by ?~2 test and calculating I~2 values.Results: A total of 60 articles including 61 studies including 8460 cases from multiple countries were involved in this meta-analysis.The average score of the study quality was 6.5.Overall,high Bmi-1 expression was associated with unfavorable prognosis of various cancers(HR = 1.80,95%CI: 1.55-2.09,P < 0.001).High Bmi-1 expression as a negative predictor for overall survival(OS)in Chinese,Japanese and Korean patients(HR = 2.02,95%CI 1.74-2.35,P < 0.001),but not significantely associated with Caucasion(HR = 1.15,95%CI 0.80-1.67,P = 0.448)or other populations(HR = 1.51,95%CI 0.69-3.32,P = 0.305).Moreover,we performed a further subgroup analysis based on cancer type.High Bmi-1 expression was associated with poor prognosis with gastric cancer(HR = 1.57,95%CI 1.31-1.89,P < 0.001),esophageal carcinoma(HR = 1.50,95%CI 1.07-2.08,P = 0.019),lung cancer(HR = 1.76,95%CI 1.29-2.41,P < 0.001),cervical cancer(HR = 2.81,95%CI 2.28-3.46,P < 0.001),colorectal cancer(HR = 2.42,95%CI 1.83-3.21,P < 0.001)and other cancers(HR = 2.09,95%CI 1.49-2.92,P < 0.001).However,it showed no significance in head and neck carcinoma(HR = 1.45,95%CI 0.90-2.34,P = 0.128),breast cancer(HR = 1.02,95%CI 0.64-1.61,P = 0.936)or hepatic cancer(HR = 1.64,95%CI 0.66-4.05,P = 0.238).In 2 studies on breast cancer,high Bmi-1 expression was found associated with favorable prognosis.The heterogeneity of recruited literatures was not correlated with publication time(P = 0.560)or cancer type(P = 0.334),and there was no significant publication bias.Conclusions: High Bmi-1 expression is associated with poor prognosis of GC,esophageal carcinoma,lung cancer,cervical cancer,colorectal cancer and other cancers,while not with head and neck carcinoma,breast cancer or hepatic cancer.Generally,the significance of Bmi-1 in prediction of cancers is still controversial.Part ?.Bmi-1 expression as an independent prognostic marker of GCObjective: The expression level and prognostic value of Bmi-1 in cancers remains controversial.Here we aimed to analyze the association of Bmi-1 expression with patients' clinicopathological characteristics and prognosis.Methods: Bmi-1 expression in 352 cases of gastric cancer was detected in high-density tumor tissue chips using immunohistochemistry method.Together with the results from data analysis of TCGA database,the association between Bmi-1 expression and patient clinopathological characteristics and survival were analyzed.Meta-analysis was performed to observe the value of high Bmi-1 expression for various malignant cancers.Results: In 352 cases of gastric cancer,Bmi-1 protein was found in cell nuclear and plasma of tumor cells.Bmi-1 expression in tissue was significantly higher than that in corresponding normal tissue(P < 0.001).Results from data analysis of TCGA database revealed that Bmi-1 m RNA level was significantly higher than that in corresponding normal tissue(P < 0.001).Bmi-1 level was not significantly with any patient clinopathological characteristics(P > 0.05).Higher Bmi-1 level,larger tumor size(< 5cm),higher pathological degree(III-IV),higher positive rate of lymph node(> 70%),deeper tumor invasion(later T stage),lymph node metastasis or more metastatic lymph nodes(> 70% or later N stage)?distant metastasis(M1),later AJCC stage were the risk factors for the prognosis of patients.Bmi-1 expression of patients of stage I-II significantly increased than that of stage III-IV(P = 0.021).Bmi-1 expression of patients of stage II significantly increased than that of stage III(P = 0.035).The median overall survival time of patients with higher Bmi-1 expression was significantly than that of patients with lower Bmi-1 expression(43.5 months vs.24.5 months,P < 0.05).In patients with positive lymph nodes,the survival time of those with higher Bmi-1 expression was significantly longer than that with lower Bmi-1 expression(P = 0.007).Conclusions: High expression of Bmi-1 in tumor tissue of gastric cancer is associated with favorable prognosis of GC patients.Part ?.The correlation of miR-15 a and Bmi-1 expression in GCObjective: This part aimed to search for the upstream miRNA(target miRNA)regulating Bmi-1 expression in GC,and detect the target miRNA expression and Bmi-1 and investigate the association between them.Methods: By searching for literatures,3 candidate miRNAs,miR-15 a,miR-16 and miR-200 c were detected in 21 formalin-fixed paraffin-embedded(FFPE)GC tissues and the paired normal tissues by real-time q PCR.The difference of the candidate miRNA expression was analyzed statistically.The expression of target miRNA and Bmi-1 was detected by in situ hybridization and immunohistochemistry(IHC)methods,and the association between them was analyzed.Results: MiR-15 a level in tumor tissue was significantly lower than that in corresponding normal tissue(P = 0.0239),while miR-16 or miR-200 c not.The expression of Bmi-1 was quantified by IHC from 21 gastric tumor tissues.The expression of miR-15 a was quantified by q RT-PCR analysis from total RNA isolated from the same set of GC tissues.The protein expression of Bmi-1 and miR-15 a RNA levels was inversely correlated(P = 0.034,R2 = 0.22,r =-0.48)based on two-tailed Spearman correlation in the GC patient samples.In addition,the results are consistent with q RT-PCR analysis that elevated miR-15 a staining is associated with reduced expression level of Bmi-1 by IHC.Conclusions: In gastric cancer tissue,miR-15 a expression is negatively associated with Bmi-1 expression.Part ?.MiR-15 a participates in the regulation of Bmi-1 on proliferation and invasion of GC cellsObjective: This part aimed to explore the mechanism of regulation of miR-15 a on Bmi-1 and observe the impact of Bmi-1 on biological behaviors of gastric cancer cells.Methods: MiR-15 a was transfected into two gastric cancer cell lines.Bmi-1 3'UTR vector was established.The impact of Bmi-1 on the cell proliferation and migration/invasion of tumor cells were verified by cell proliferation assay and transwell migration/invasion assay.Results: In gastric cancer AGS and SNU-5 cell lines,miR-15 a binded to Bmi-1 m RNA to negatively regulate the expression of Bmi-1 protein.After miR-15 a was transfected into AGS and SNU-5 cell lines,the velocity of tumor cell proliferation significantly decreased(P < 0.001),and the tumor cells invading extracellular matrix reduced by nearly 50%.Conclusions:MiR-15 a negatively regulates Bmi-1 in GC cells.Bmi-1 promotes the proliferation and migration/invasion ability of gastric cancer cells in vito.This effect may be involved in multiple mechanisms.