LincRNA HOTAIR Induces Breast Cancer Cells Metastases Through Epithelial To Mesenchymal Transition

IntroductionBreast cancer is one of the most malignant tumor in the world. About500,000women die from it each year around the world. The incidence of breastcancer rapidly increased in China over the past decade. Therefor, thepathogenesis and prevention of breast cancer are one of the most popular topicsin tumor research.LincRNA is pervasively transcribed in the genome, but the mechanism ofits potential involvement in human disease is not well understood. Recentstudies of dosage compensation, imprinting, and homeotic gene expressionsuggest that individual lincRNA can function as the interface between DNA andspecific chromatin remodelling enzyme. HOTAIR, one of lincRNA, is highly expressed in invasive breast cancer. And its expression level in primary breasttumour is a powerful predictor of prognosis. Enforced the expression ofHOTAIR in breast cancer cells can alter histone H3lysine27methylation, andincrease breast cancer invasiveness and metastasis. Conversely, loss of HOTAIRcan inhibit breast cancer metastasis.In our study, we identified HOTAIR as a key factor that can induce theepithelial to mesenchymal transition (EMT) progress. Our study also showedthat loss of HOTAIR can inhibit EMT process during breast cancer progressionand metastasis. And our results provided a new insight into molecularmechanism of HOTAIR-induced breast cancer metastasis.ObjectivesIn order to explore the relationship HOTAIR with EMT progress, weconstruct eukaryotic expression plasmid pcDNA3.0-HOTAIR with HOTAIRfull-length sequence and then detect the overexpression and loss of HOTAIRby qPCR and Western blot. Through detecting the human breast cancer andadjacent noncancerous tissue by Immunohistochemistry, we demonstratewhether HOTAIR has the positive correction with EMT progress.Methods1) We detected the relative levels of HOTAIR RNA in breast cancer cell lines.We cultured breast cancer cell lines SKBR3、 MDA-MB-231、MDA-MB-435、MDA-MB-468、MCF-7、T47D and BT549. Then in orderto construct HOTAIR full-length eukaryotic expression plasmid wescreened the breast cancer cell lines MDA-MB-231by qPCR and RT-PCR.2) After screening the breast cancer cell lines MDA-MB-231, we constructedHOTAIR full-length eukaryotic expression plasmid. HOTAIR full-length was cloned into pcDNA3.0vector to construct a plasmid. Then theexpressive vector was confirmed by restriction enzyme cut and sequencingso that we could confirm whether the HOTAIR full-length eukaryoticexpression plasmid was successfully constructed.3) We detected the overexpression or interference of HOTAIR in the breastcancer cell lines MDA-MB-231. After successfully constructing HOTAIRfull-length eukaryotic expression plasmid, HOTAIR was overexpressed inthe breast cancer cell lines MDA-MB-231. Then we detected overexpressionof HOTAIR by qPCR、 Western blot、 Transwell and cell scratchesexperiment. We interfered the expression of HOTAIR so that we couldreversely verify the raletive between HOTAIR and EMT.4) We detected the human breast cancer and adjacent noncancerous tissue byqPCR and Immunohistochemistry. We collected the human breast cancerand adjacent noncancerous tissue from the breast surgery of xijing hospital.Then we detected the expression of HOTAIR and EMT raletive molecule.Results1) The relative levels of HOTAIR RNA was different among the breast cancercell lines. The expression of HOTAIR was highest in the breast cancer celllines MDA-MB-231, and was lowest in the breast cancer cell lines SKBR3and BT549. In our study, we set up breast cancer cell lines MDA-MB-231as cell model.2) We successfully constructed the plasmid of HOTAIR full-length, namedpcDNA3.0-HOTAIR. First, HOTAIR was overexpressed in the breastcancer cell lines MDA-MB-231. The expression of HOTAIR was higher inthe transfected row than in the vetor row. Contrast with the vetor row, we found that the expression of E-cadherin was lower, but the expression ofSnail and β-catenin was higher in the transfected row. Transwell and cellscratches experiment also showed that the invasion and migration of cellsbecame more power.3) Second, through interference of HOTAIR, we found that the expression ofHOTAIR was remarkably depressed by qPCR. Contrast with NC row, wefound that the expression of E-cadherin was higher, but the expression ofSnail and β-catenin was lower in the siHOTAIR row. Transwell showed thatthe invasion of cells became weaker than NC. MTT showed the proliferationof cells was weakened, and FACS showed the apoptosis of cells wasenhanced in the siHOTAIR row.4) We demonstrated that the expression level of HOTAIR was much higher inthe breaset cancer than in the adjacent noncancerous tissue. And accordingto the pathologic grading of breast cancer rising gradually, the expression ofE-cadherin was lower, and the expression of Snail rose gradually, whichwas highly correspondent with the trend of HOTAIR.Conclusions1) The expression of HOTAIR was different in different breast cancer celllines. And it highly expressed in the breast cancer cell lines MDA-MB-231.2) HOTAIR was able to promote breast cancer cell lines metastasis throughinduction of EMT.3) HOTAIR highly expressed in the breast cancer, which was highlycorrespondent with the trend of EMT progress.