| Cervical cancer (CC) is the second most common gynecologic cancer in women worldwide which threatened women's health seriously. Although cervical cancer screening is prevalent nowadays, but still some patients present with advanced disease without screening. The5-year survival rate is only30-60%in patients with advanced disease. Taxol and platinum-based chemotherapy is one of indispensable treatment for advanced cervical cancer. Drug resistance remains a major problem in the treatment of cervical cancer patients and an effective method for reversing drug resistance has not been found yet. Elucidate the mechanism of chemoresistence is very useful for improve the prognosis of advanced disease.A recent report demonstrated that epithelial mesenchymal transition (EMT) plays an important role in chemotherapy failure. In EMT, epithelial cells acquire fibroblast-like properties and exhibit a reduction in intracellular adhesion. This process is associated with the functional loss of the epithelial marker E-cadherin and the functional gain of the mesenchymal markers Vimentin, Fibronectin and N-cadherin, and an increase in the migratory and invasive behavior. EMT plays important roles in many normal biological processes such as development and differentiation. EMT-type cells that is unavailable to apoptosis and typically drug resistant. More importantly, recent studies have demonstrated that miRNAs regulate the acquisition of the EMT phenotype, which are critically associated with drug resistance. Since experimental evidence also revealed that miRNAs regulate anti-cancer drug resistance, suggesting that targeting specific miRNAs could be a novel therapeutic approach for the treatment of cancers by increasing the drug sensitivity of cancer cells or by eliminating EMT-type cells that are typically drug resistant.EMT is critical for tumor metastasis and drug resistance, however, the underlying mechanism is little known so far. It has been known that complex signaling pathways (such as Wnt pathway, TGF-(3and Notch pathways) are involved in this process. Recent studies have demonstrated that miRNAs can regulate the acquisition of the EMT phenotype though target the epithelial marker E-cadherin or EMT-related transcription factors and signaling pathway molecules. However, the underlying mechanisms of EMT remain unclear, and the involvement of microRNAs (miRNA) in this process is poorly understood. Only a few researches available described the relationship of miRNAs, EMT and drug resistance in cancer until now, the relationship of miRNAs, EMT and drug resistance in cervical cancer has not been reported yet.In this study, we aimed to find out miRNAs which related to taxol resistance in cervical cancer and clarify its underlying mechanisms. Therefore, targeting miRNA by natural agents could be a novel strategy for cancer therapy. First, we compare the miRNA expression not only between taxol sensitive and resistant squamous cell carcinomas (Sccs) tissues, but also between before and after chemotherapy in the same Sccs patient using a microarray platform. To confirm the microarray findings, the5miRNAs differentially expressed in microarray are selected and validated using real-time RT-PCR in cervical cancer cells and tissues. Secondly, to investigate the role of miR-375in taxol resistence in cervical cancer cells (Siha, Hela and Caski), we upregulate the expression of miR-375using lentiviral vector transfection and observe its impaction on taxol resistance using MTS. Lastly, to determine the relationship of miR375, EMT and taxol resistance, we performed realtime RT-PCR and westerm-blot analyses for E-cadherin, Vimentin, Fibronectin and N-cadherin expression and clarify their correlation with the miR-375level in cervical cancer cell during taxol chemotherapy. Then, we induced EMT in cervical cancer cell by TGF-β and EGF-β and observe its impaction on miR-375expression and taxol resistance. To confirm that E-cadherin was a direct target of miR-375, pmir-GLO-UTR containing the miR-375binding sites was constructed to perform the reporter assay. Taken together, our results would show the regulatory relations between miR375, EMT and taxol resistance in cervical cancer. Objective:To investigate the role of miR-375in taxol-resistance in cervical cancer.Methods:We construct cervical cancer cells (Siha, Caski and Hela) which stable over-expression miR-375using miRNA lentiviral transfection and flow cytometry screening assay. To determine the effect of miR-375on taxol resistance of cervical cell lines, the drug sensitivity testing was determined with MTS assay at72hours with different taxol doses (0,5,10,20,50.,100nM).Results:1. Cervical cancer cells (Siha, Caski and Hela) were stably transfected with miR-375lentiviral vector and screened by flow cytometry, which induced significant up-regulation (5000-6000fold increase) of miR-375compared with the negative control (P<0.000).2. The taxol sensitivity was significantly decreased after forced overexpression of miR-375in Siha, Caski and Hela cells compared with miRNA negative control (P <0.05).The IC50values were increase in Siha, Caski and Hela with overexpression miR-375(45.81±1.33nM,20.56±1.72nM and20.18±0.72nM), compared with miRNA negative control respectively (8.54±1.52nM,7.98±2.60,and13.28±0.52nM). Conclusions:1. Using lentiviral vector transfection, we construct cervical cancer cells (Siha, Caski and Hela) which stable over-expression miR-375.2. The taxol sensitivity is significantly decreased after forced overexpression of miR-375in Siha, Caski and Hela cells. Objective:To realized the potential mechanisms of miR-375regulate taxol resistance in cervical cancerMethods:To determine the relationship of miR375, EMT and taxol resistance, we performed realtime RT-PCR and westerm-blot analyses for E-cadherin, Vimentin, Fibronectin and N-cadherin expression and clarify their correlation with the miR-375level in cervical cancer cells (SiHa, Caski and HeLa) during taxol chemotherapy. Then, we induced EMT in cervical cancer cells (SiHa, Caski and HeLa) by TGF-(3and EGF-β and observe its impaction on miR-375expression and taxol resistance. To confirm that E-cadherin was a direct target of miR-375, pmir-GLO-UTR containing the miR-375binding sites was constructed to perform the reporter assay.Conclusions:1. The expression of E-cadherin and Vimentin at mRNA level in cervical cancer cells (SiHa,Caski and HeLa) after taxol chemotherapy72h were not significantly up-regulated compared with pre-chemotherapy (P>0.05).2. The expression of E-cadherin, Vimentin, Fibronectin and N-cadherin at protein level in cervical cancer cells (SiHa,Caski and HeLa) after taxol chemotherapy72h were significantly differented compared with pre-chemotherapy (P<0.05).3. There was an inverse correlation between miR-375and Ecadherin protein expression after chemotherapy with different taxol doses [rs=-0.345(Siha), rs=-0.476(Caski), P<0.05]4. Taxol could induce EMT in cervical cancer cells (SiHa,Caski and HeLa) EMT-type cervical cancer cells (SiHa,Caski and HeLa) are typically taxol resistant, but EMT can not regulate the expression of miR-375.5. MiR-375can regulate the acquisition of the EMT phenotype though targeted the epithelial marker E-cadherin, and mediated taxol resistance in cervical cancer cellsConclusions:1. Ecadherin is a key molecular in EMT in cervical cancer.2. EMT can regulate the taxol sensitivity in cervical cancer3. MiR-375downregulates Ecadherin and mediates EMT may be related to taxol resistance in cervical cancer... |
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