Expression Of The MACC1in Cervical Cancer And Efffects Of MACC1SiRNA On Biological Behaviors Of Hela

Background and purposeMetastasis-associated in colon cancer1(MACC1) was recently identified a novel gene associated with colon cancer metastasis. It has been shown to play an important role in several types of cancer, and serve as biomarker for invasion metastasis. MACC1is the very important regulatory factor of the HGF/c-Met signaling pathway. However, information on the expression of MACC1in cervical carcinoma tissues is still very limited, the biological sigficance of MACC1and its molecular mechanism of MACC1overexpression induced cervical cancer cell occurrence and development remains unclear. The purpose of this study is to evaluate signficance of MACC1、hepatocyte growth factor (HGF) and c-Met expression in cervical neoplastic lesions tissues and investigate the relationships between MACC1、HGF and c-Met overexpression and the clinical pathological characters of cervical cancer. To evaluate the clinical significance of the expression of MACC1、HGF and c-Met in the development of the cervical cancer. To further explore relationship of MACC1, HGF and c-Met expression and human papillomavirus (HPV) infection in cervical cancer development. To reveal the mechanisms of the occurrence and development of cervical cancer, and provide new ideas to guide the clinical diagnosis and treatment.Methods1. Immunohistochemistry was performed to determine MACC1, HGF and c-Met expression in100cervical cancer tissues,20cervical intraepithelial neoplasia (CIN) and40normal cervical tissues. To compare the positive rate of MACC1, HGF and c-Met expression in cervical neoplastic lesions tissues.2. HPV DNA was detected by polymerase chain reaction (PCR)100cervical cancer tissues,20cervical intraepithelial neoplasia (CIN) and40normal cervical tissues. To compare the positive rate of HPV DNA expression in cervical neoplastic lesions tissues.3. The relationships between overexpressions of MACC1, HGF and c-Met and the clinical pathological characters of cervical cancer were analyzed.4. The correlation of overexpression in cervical cancer among MACC1, HGF and c-Met were analyzed.5. The association of MACC1, HGF and c-Met expression in cervical cancer with HPV infection was assessed.Results1. The expressions of MACC1, HGF and c-Met protein and HPV DNA were obviously higher in cervical carcinoma and CIN tissues compared to normal cervical tissues (P＜0.05).2. The overexpressions of MACC1, HGF and c-Met protein were related with clinical stage, pathologic stage and lymph node status, there were more advanced clinical stage, worse pathologic stage and accompanied lymph nodes metastasis, there were more high overexpressions of MACC1, HGF and c-Met in cervical cancer (P＜0.05). Furthermore, these was not a significant association between HPV DNA expression and clinicopathological variables(P＞0.05).3. There were positive correlations between overexpression of MACC1and overexpressions of HGF and c-Met(Spearman r=0.68, P=0.000; Spearman r=0.71, P＝0.008); there was also positive correlation between overexpression of HGF and overexpression of c-Met (Spearman r=0.75, P=0.000).4. MACC1, HGF and c-Met expression were sig&antly correlated with HPV DNA level (Spearman r=0.420, P=0.000).Conclusion1. MACC1, HGF and c-Met gene is overexpressed in cervical cancer tissues.2. MACC1-HGF/c-Met-HPV formed a positive feedback loop in cervical cancer tissues, which for the development of cervical cancer plays an important role.3. Overexpression of MACC1promotes proliferation and malignant transformation of HPV-infected cells.4. MACC1is expected to become a new target for cancer early diagnosis and gene therapy.5. Combined detection of MACC1and the expression of HPV infection, will contribute to the diagnosis of precancerous lesions, judgement of disease progression, and evaluation of the prognosis. Background and purposeMACC1was recently identified a key gene controlling tumor growth and metastasis, is over-expressed in a variety of tumors that have high metastasis. Preliminary studies have confirmed that MACC1was over-expressed in cervical cancer tissues, and its expression level was closely associated with the clinical stage, pathologic stage and lymph node status. We designed and synthesized the specific siRN A for MACC1, then transfect the HeLa cells to specifically inhibit the expression of MACC1gene, in order to better understand the role of MACC1in the pathogenesis of cervical cancer and the relationships between MACC1and the biological behaviors of cervical cancer cells. The final investigate the effects of MACC1knockdown on the growth, apoptosis, migration and invasion of ovarian cancer cells. To preliminary clarify MACC1molecular biological mechanisms of the occurrence and development of cervical cancer, and provides a new theoretical and experimental basis to define the screening, early diagnosis and RNAi treatment of cervical cancer cases.Methods1. We designed and synthesized MACC1specific siRNA strands, and then transfected the HeLa cells by the lipofectamine2000.2. The morphological changes of cytoskeleton in the MACC1siRNA-transfected cells were observed by immunofluorescence staining and laser confocal microscopy.3. Expressions of MACC1mRNA and protein were examined by semiquantitative reverse transcription polymerase chain(RT-PCR) and Western blot in HeLa-siRNA, HeLa-NC, and normal HeLa cells respectively.4. The speed of cell migration was measured by Transwell assay in HeLa-siRNA, HeLa-NC, and normal HeLa cells respectively.5. The MTT assay was used for observing cell proliferation and drawing a growth curve in HeLa-siRNA, HeLa-NC, and normal HeLa cells respectively.6. Apoptosis and cell cycle were assessed by flowcytometry(FCM) in HeLa-siRNA, HeLa-NC, and normal HeLa cells respectively.Results1. Analyses of immunofluorescence staining showed that reticular structure of microtubule fiber according to certain direction changed into clutter reticular structure, Without direction, part of it granular scattered in the cytoplasm. Integrated fluorescence intensity of cytoskleton in HeLa-siRNA, HeLa-NC, and normal HeLa cells were10.01±3.53,17.44±5.85and 18.53±3.61respectively. Integrated fluorescence intensity of cytoskleton in HeLa-siRNA is lower than HeLa-NC and normal HeLa cells (P＜0.05), but the difference between HeLa-NC and normal HeLa cells is not statistically significant(P＞0.05).2. The relative expression level of MACC1mRNA in HeLa-siRNA group was0.47±0.06, compared with the HeLa-NC group (0.88±0.09) and normal HeLa cells (1.00±0.00), the difference was significant (P＜0.05), there were no obvious differences between HeLa and HeLa-NC cells(P＞0.05).3. The positive rate of MACC1protein expression in HeLa-siRNA group is0.46±0.05, in HeLa-NC group is0.96±0.05, and in normal HeLa cells is1.01±0.27. MACC1protein expression in HeLa-siRNA group is lower than HeLa-NC group and normal HeLa cells (P＜0.05), but the difference between HeLa and HeLa-NC cells is not statistically significant(P＞0.05).4. Knockdown of MACC1gene in HeLa cells caused a significantly decrease in cell proliferation or migration, and increased cell apoptosis rate. These sequence specific siRNAs showed a blockbuster effect in arrestment of the cell cycle.Conclusion1. MACC1expression could be successfully inhibited by specific siRNA.2. Inhibition of MACC1gene could obviously suppress the biological behavior of HeLa cells, such as cell proliferation or migration, and cell apoptosis rate and cell cycle.3. To detect the expression of MACC1gene, has very important significance for the specific target of early diagnosis and gene therapy, prevention of cervical cancer.4. With the development of the RNA interference technology, siRNA MACC1may serve as a new strategy for gene therapy of cervical carcinoma.