| Objective:Breast cancer is a common malignancy in women. Breast cancer cellinvasion and metastasis is an important factor threatening breast cancerpatients’ lives. Epithelial-mesenchymal transition (EMT) makes breast cancercells lose epithelial markers and obtains mesenchymal phenotype, showingstrong invasion, migration, result of tumor metastasis. And stem cells promotemetastasis of tumor cells, resulting in resistance to chemotherapeutic drugs.E-cadherin and vimentin were respectively expressed in epithelial cells andmesenchymal cells, which were signs of the occurrence of EMT. It has beenreported that OCT4was an important transcription factor induced stem celland it related to maintain the self-renewal and differentiation potential ofcancer stem cell. And OCT4was involved in many types of tumor cellproliferation and apoptosis regulation. However, it was still little studied thatOCT4on the impact of tumor invasion and metastasis. This experimentmainly study was the expression of EMT related factor E-cadherin, vimentintwo and stem cells factor OCT4in breast cancer in order to investigate thecorrelation between EMT and cancer stem cells. We knockdown OCT4expression in breast cancer cells by RNA interference technology, toinvestigate the silence effect of OCT4expression on breast cancer cellinvasion, metastasis，proliferation and apoptosis.Method:1Quantitative real-time PCR was used to detect the mRNA expression ofOCT4, EMT associated protein E-cadherin and vimentin in invasive breastcancer tissue and the corresponding adjacent tissues.2Immunohistochemical staining was used to detect the protein expression of cancer stem cell marker OCT4and EMT associated proteinE-cadherin and vimentin in invasive breast cancer tissue the clinical/biologicalfactors of breast cancer were analyzed.3The three plasmid system was used to package293T cells and generatelentivirus. Lentivirus packaged was used to infect MDA-MB-231, usingcontrol-shRNA-pLKO.3as negative contrast. The cells were detected afterinfection for72h.4Fluorescence microscopy and flow cytometry were used to detected thetransfection efficiency of control-shRNA-pLKO.3and OCT4-shRNA-pLKO.3in the breast cancer.5Quantitative real-time PCR and Western blot were used to identify theinhibitory effect of OCT4expression.6Quantitative real-time PCR and Western Blot method was used todetect the expression of OCT4inhibition of EMT marker molecules ofE-cadherin and vimentin expression levels.7Transwell experiments was used to detect the effects on cell invasionand migration capabilities of MDA-MB-231cells.8The proliferation activity of MDA-MB-231cells was used detected bycolony formation assay.9The rate of apoptosis and cell cycle distribution of MDA-MB-231cellswere used to detect by Flow cytometry.Result:1The results of quantitative real-time PCR showed that, in40cases ofinvasive breast cancer tissues, the mRNA expression of OCT4negativelycorrelated with the mRNA expression of E-cadherin (R=0.119，P＜0.05),positively correlated with the mRNA expression of vimentin (R=0.245，P＜0.05). And the mRNA expression of E-cadherin correlated negtively with themRNA expression of vimentin (R=0.227，P＜0.05).2Immunohistochemical staining discovered that, in40cases of invasivebreast cancer tissues, the expression rate of OCT4was30%(12/40),E-cadherin55%(22/40), vimentin65%(26/40). OCT4gene expression was correlated with the age of breast cancer patients, histological grade, lymphnode metastasis and the expression of Her-2(P＜0.05). E-cadherin geneexpression was correlated with the lymph node metastasis (P＜0.05).Vimentin gene expression was correlated with the histological grade andlymph node metastasis (P＜0.05). The expression of OCT4negativelycorrelated with the expression of E-cadherin (R=0.395，P＜0.05), positivelycorrelated with the expression of vimentin (R=0.366，P＜0.05). And theexpression of E-cadherin correlated negatively with the expression of vimentin(R=0.453，P＜0.05).The protein expression levels of OCT4were different significantlybetween metastasis and non-metastasis breast cancer (P＜0.05). The proteinexpression levels of E-cadherin, vimentin were no significantly differentbetween metastasis and non-metastasis breast cancer (P＞0.05).Survival analysis showed that, the survival rate with OCT4positiveexpression breast cancer patients was significantly lower than patients withOCT4negative expression (P＜0.05). There was no significant correlationbetween the expression of E-cadherin and vimentin in patient survival (P＞0.05).3Fluorescence microscopy was used to detected the infection efficiency(Fig.1). The result of flow cytometry assay showed that, the transfectionefficiency of control-shRNA-pLKO.3and OCT4-shRNA-pLKO.3were22.6%and20.8%respectively when100l virus solution infected293T cell,54.9%and41.6%respectively when200l virus solution infected293T cell,98.4%and86.1%respectively when500l virus solution infected MDA-MB-231cell.4The results of quantitative real-time PCR and Western blot showed that,the mRNA and protein expression levels of OCT4were remarkably lowerafter silenced the expression of OCT4(P＜0.05). And after transfected withOCT4-shRNA, the expression levels of E-cadherin protein or mRNA weresignificantly higher (P＜0.05), and the expression level of vimentin protein ormRNA were significantly lower (P＜0.05). 5The results of transwell assay showed that, migrating invasion abilitywas significantly lower that of parental and OCT4-shRNA group cells (P＜0.05).6The results of colony formation assay showed that, the numbers of cellcolony in parental group (MDA-MB-231), control-shRNA group andOCT4-shRNA group were344.55±29.67,318.20±23.22,102.65±9.87,respectively. The third one was remarkably lower than the parental group,control-shRNA group (P＜0.05). There was no significant difference betweenthe control-shRNA group and OCT4-shRNA group (P＞0.05).7The results of flow cytometry assay showed that, the rate of earlyapoptosis of MDA-MB-231cells were remarkably increase after silenced theexpression of OCT4(P＜0.05). And the silence of OCT4in MDA-MB-231cells induced G1arrest in cell cycling.Conclusion:In breast cancer tissues, the expression levels of OCT4negativelycorrelated with the expression of E-cadherin, positively correlated with theexpression of vimentin. These indicated that OCT4may promote themetastasis of breast cancer cells via EMT. OCT4gene expression is anindependent prognostic factor for breast cancer. OCT4-shRNA plasmid cansilenced the expression of OCT4effectively, which inhibited tumor cellproliferation, and induced apoptosis and cell cycle arrest in tumor cells. Whilethe expression of EMT associated markers E-cadherin upregulated, vimentindownregulated. These results explained that OCT4-mediated tumorigenesiswas correlated with the regulation of EMT. And OCT4could become a newtherapeutic target in clinical. |
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