The Clinical Significance Of Bmi-1Expression In Uterine Cervical Cancer And Its Affection On Biological Behaviour Of Siha Cells

BackgroundUterine cervical cancer (UCC) is one of the most common malignancies, with an estimated493,000new cases and274,000UCC related deaths each year. Although incidence and mortality are decreasing owing to the implementation of routine screening, UCC still remains the second most common malignancy in women worldwide. Recently, the incidence of UCC has been rising in younger women and there has been a rise in the relative incidence of cervical adenocarcinoma, and these made the diagnosis and treatment of UCC face the new problems. Therefore, a scientific and feasible way to improve the early diagnosis and prognosis judgment of UCC, in order to achieve the early detection and treatment of UCC, has become a hot issue for the current study.High risk human papillomavirus (HPV) infection has been determined as the main risk factor for UCC, and the epidemiological data indicate high risk types of HPV DNA have been detected in95%～100%of UCC patients. However, only a few women with HPV infection have developed into UCC, indicating other unidentified genetic alterations are also involved.B cell-specific moloney murine leukemia virus integration site1(Bmi-1) gene was one member of polycomb group (PcG), originally identified as an oncogene that cooperated with c-myc in murine lymphomas in1991. Bmi-1acts as a transcriptional repressor by deregulating p16Ink4a and p14Arf tumor suppressor proteins, which can inhibit the apoptosis and senescence induced by p53and pRB growth regulatory pathways. Moreover, Bmi-1also plays an important role in the activation of human telomerase reverse transcriptase gene (hTERT), which can extend the replicative lifespan and immortalize cells. Recently, some researches have demonstrated that Bmi-1is overexpressed in hematological malignancies and a variety of solid cancers. All these suggested that the Bmi-1might play an important role in cell proliferation and tumor progression. However, the role of Bmi-1protein in human UCC has not been investigated so far.Base on these, our research aims to detect the expression of Bmi-1in UCC tissues and peripheral blood, and inhibit the expression of Bmi-1in UCC cell lines using RNAi method, and observe the expression of Bmi-1in UCC tissues and peripheral blood and its affection on cell proliferation, apoptosis and invasion, to discuss the value of Bmi-1in early diagnosis and prognosis judgment of UCC and the mechanism of UCC development, invasion and metastasis, in order to provide evidences for searching for new target of UCC diagnosis and treatment. Part1Overexpression of Bmi-1in uterine cervical cancer and its correlation with clinicopathology and prognosisObjective:To investigate the expression of Bmi-1protein in human uterine cervical cancer (UCC) and explore its associations with clinicopathological factors and prognosis.Methods:Western blot was used to detect the expression of Bmi-1in a normal cervical epithelial cell line (NCEC) and four human cervical cancer cell lines (Hela, SiHa, CasKi and C33A). In addition,152UCC and30adjacent normal cervical paraffin-embedded samples were collected to detect Bmi-1expression by immunohi stochemistry.Results:Western blot analysis showed Bmi-1was overexpressed in four human UCC cell lines (Hela, SiHa, CasKi and C33A) but not in the NCEC. Among of152paraffin-embedded archival UCC tissues,27cases showed negative expression (-),29cases showed weak expression (+),55cases showed moderate expression (++), and41cases showed strong expression (+++). Bmi-1was overexpressed in63.2%UCC tissues (Bmi-1++or+++). However, only negative or weak immunohistochemical staining was detected in the30adjacent normal cervical tissues. The overexpression of Bmi-1protein was significantly correlated with tumor size (P=0.046), clinical stage (P=0.021) and regional lymph nodes metastasis (P=0.010). However, no significant associations were found between Bmi-1protein overexpression and age (P=0.645), depth of stromal invasion (P=0.428), histological type (P=0.367), and cell differentiation (P=0.247). Survival analysis showed a significant difference between Bmi-1protein overexpression and poor overall survival (OS)(P=0.021). Cox proportional hazards risk analysis indicated that Bmi-1protein overexpression was an independent prognostic factor for OS.Conclusions:Bmi-1is overexpressed in UCC and correlated with adverse clinical characteristics and poor prognosis, which suggests that the Bmi-1might participate in the development and progression of UCC and have clinical potential not only as a useful predictor of aggressive phenotype but also a promising prognostic predictor. Part2Detection of circulating Bmi-1mRNA in plasma and its potential diagnostic and prognostic value for uterine cervical cancerObjective:To assess the diagnostic and prognostic potential of plasma circulating Bmi-1mRNA in uterine cervical cancer (UCC)Methods:The levels of circulating Bmi-1mRNA in plasma were detected by reverse transcription quantitative real-time PCR in109UCC patients,138cervical intraepithelial neoplasia (CIN) patients, and80healthy volunteers. Receiver operating characteristic (ROC) curve was established to discriminate the subjects with or without UCC. Cox model was employed to identify the independent prognostic factors.Results:GAPDH and EEF1A1mRNAs were detected in the plasma of247(91.8%) patients with cervical lesions and80(94.1%) healthy controls. No significant difference was observed between patients with cervical lesions and healthy controls (P>0.05). Median circulating Bmi-1mRNA levels were significantly higher in UCC compared with CINs and healthy controls (all at P<0.001). The levels of circulating Bmi-1mRNA were significantly higher in CIN2and CIN3patients than those in CIN1patients and healthy controls (all at P<0.05, respectively). The levels of SCC-Ag and CA125were significantly higher in UCC compared with CIN3, CIN2, CIN1and healthy controls (all at P<0.01, respectively). The high level of Bmi-1mRNA correlated significantly with advanced clinical stage (P<0.001) and lymph nodes metastasis (P=0.002). The area under the ROC curve (AUC) was0.881, and the optimal cut-off value was0.057, providing a sensitivity of69.7%and a specificity of95.9%. However, the levels of SCC-Ag and CA125were increased above the upper limits of normal in56.0%and33.9%of UCC patients, respectively. The AUC of Bmi-1mRNA was significantly larger than that for SCC-Ag (0.740,95%CI=0.689to0.787, P=0.035) or CA125(0.689,95%CI=0.636to0.739, P<0.001). Kaplan-Meier analysis showed a significant correlation between increased circulating Bmi-1mRNA level and reduced disease-free survival (DFS)(P=0.001) and overall survival (OS)(P=0.015). Cox analysis indicated that circulating Bmi-1mRNA was an independent prognostic factor for DFS and OS. Conclusion:Circulating Bmi-1mRNA is increased in plasma of UCC and correlated with adverse clinical characteristics and poor prognosis. Circulating Bmi-1mRNA can be served as a potential noninvasive molecular marker for diagnosis and prognosis of UCC. Part3Effects of Bmi-1expression on the biological behavior of uterine cervical cancer cell line SiHa cellsObjective:To analyze the affection on cell proliferation, apoptosis and invasion after silencing the Bmi-1expression in uterine cervical cancer (UCC) cell line SiHa cells with siRNA method, and further discuss the function of Bmi-1in genesis and development of UCC.Methods:The4sequences for siRNAs against Bmi-1were designed and synthesized according to Bmi-1gene sequence, then the siRNAs were transfected into UCC cell line SiHa cells as experiment groups, and transfected with negative control siRNA as negative group (NC group), transfected with transfection reagent only as transfection reagent control group (Mock group) and no treatment of the cells as blank group (Blank group). The expression of Bmi-1mRNA in SiHa cells after transfection was detected by RT-qPCR method. CCK8assay, Annexin V-FITC/PI double labeling experiments and transwell assay were used to examine the changes of cell proliferation, apoptosis and invasion, respectively.Results:Compared with NC group, Mock group and Blank group, the levels of Bmi-1mRNA in siRNA-1group, siRNA-2group, siRNA-3group and siRNA-4group after transfection24h and48h were significantly decreased (P<0.05), of which the infection efficiency of siRNA-4group was best. Compared with NC group, Mock group and Blank group, the proliferation ability in siRNA-4group was decreased after transfection24h,48h,72h and96h. After transfection48h, apoptosis rate in siRNA-4group was (62.43±9.86)%, which significantly higher than those in NC group with (10.17±5.15)%, Mock group with (8.56±4.27)%and Blank group with (8.12±3.89)%, respectively (P<0.05, respectively). In invasion experiment, the number of penetrating cells in siRNA-4group was (253.29±68.72), which significantly lower than those in NC group with (350.21±98.62), Mock group with (406.38±113.51) and Blank group with (418.46±119.78), respectively (P<0.05, respectively).Conclusion:siRNA can specifically inhibit the expression of Bmi-1gene in UCC cell line SiHa cells, thus inhibit the cell proliferation, induce cell apoptosis, and weaken cell invasion capabilities, which further demonstrate the function of Bmi-1in UCC and supply experimental evidences for gene therapeutic strategy in management of human cancers using RNAi method.