Effect Of Downregulated LncRNA HOTAIR Expression On The Invasion, Metastasis And Tumorigenicity Of Human Colorectal Cancer LoVo Cells And CSCs

Colorectal cancer (CRC) originated in the submucosal mucosal epithelial or mesenchymal tissue, which is made up of colorectal mucosa epithelium in a variety of carcinogenic factors such as environmental or genetic occur under the action of malignant transformation. It is one of the most common gastrointestinal malignancies. In all kinds of malignant tumors, the incidence of colorectal cancer ranks third, but it ranks second in the gastrointestinal tumors.The treatment of CRC is preferred to adopt the ways of surgery, radiation and chemotherapy. Because CRC is easy to infiltrate the blood vessels, and metastasize the lymph node and distant organs, it is a difficult to be radical cure. CRC is a malignant tumor origin of an epithelial cells, and the malignant lesions of epithelial cells may be involved in the epithelial to mesenchymoma transition (EMT).In recent years, non-coding RNA from small RNA to long chain non-coding RNA (LncRNA), as one of epigenetics research content, has been widely explored in the tumor fields. HOX transcript antisense RNA (HOTAIR) is one of LncRNA family members has published more research reports in the breast, lung, prostate and colorectal cancer and other tumors.In spite of this, HOTAIR in CRC and its molecular biological characteristics of cancer stem cells (CSCs) is still poorly understood. In this study, we chose human CRC cell line LoVo cells as the research object, and designed a small hairpin RNA that could knock down code gene HOTAIR by RNA interference. After the eukaryotic ShRNA expression vector was constructed, it was stably transfected into LoVo cells to reduce the HOTAIR expression. Then, the CRC CSCs were sorted from the LoVo cells stably transfected with ShRNA expression vector with magnetic activated cell sorting system. Finally, it will be explored that whether HOTAIR can be used as a new target for treatment of CRC CSCs by detecting the CSC biological characteristic changes.Objective:To investigate the effect of downregulated long non-coding RNA (lncRNA) HOTAIR expression on the invasion, metastasis and tumorigenicity of human CRC LoVo cells and CSCs, and to provide a fundament for CRC treatment based on HOTAIR target.Methods:The recombinant plasmids pSUPER-EGFP1-HOTAIR-shRNA and pSUPER-EGFP1-Scramble were transfected into LoVo cells respectively by liposome 2000 reagent, and stable transfected cells were selected with G418.The gene silencing efficiency was measured by real-time quantitative RT-PCR. And then, CD133+CSCs are isolated from the cells stably transfected with the HOTAIR-shRNA by magnetic activated cell sorting system. The biological characteristics of LoVo cells and CSCs with down regulated HOTAIR expression were evaluated by analyzing the ability of proliferation, colony forming and the migration in a common media and the tumorigenicity in Balb/c mice. In addition, an epithelial-mesenchymal transition (EMT) associated molecule expression was analyzed by immunohistochemical assay and Western blot.Results:1. The expression of HOTAIR was confirmed in CRC LoVo cells by RT-PCR detection, and HOTAIR expression in CD133+CSCs was higher than that in CRC LoVo cells, which was statistically significant (P＜0.05).2. A eukaryotic recombinant expression vector was transfected into LoVo cells by liposome, and the stable transfected cells were screened with G418. QRT-PCR detection result showed that the constructed HOTAIR-ShRNA had a certain inhibitory effect on expression of HOTAIR in LoVo cells.3. By scratch test, invasive experiment, plate clone formation, proliferation experiment, and 5-FU drug resistance in vitro as well as tumorigenic experiment in vivo, the HOTAIR-ShRNA transfected LoVo cells decreased the ability of migration and invasive, proliferation, clone formation, drug resistance in vitro, and tumorigenicity in nude mice compared with Scramble-ShRNA transfected LoVo cells and wild type LoVo cells, and the difference was statistically significant (P<0.05 or P＜0.01). Additionally, the downregulation of HOTAIR resulted in a significant down-regulation of the Vimentin expression and an upregulation of the E-cadherin expression.4. The CD133+CSCs transfected with HOTAIR-ShRNA were sorted by magnetic activated cell sorting system decreased its invasion, metastasis and tumorigenicity compared with wild type LoVo cells and CD133+CSCs transfected with pSUPER-EGFPl-scramble, and the difference was statistically significant (p<0.05 or p<0.01).The results of immuno-histochemistry and Western blot demonstrated at the same time that the CD133+CSCs transfected with HOTAIR-ShRNA decreased of Vimentin expression and increase of E-cadherin expression in tumor and lung tissues of nude mice, which was statistically significant (P<0.05).Conclusions:1. HOTAIR knockdown in LoVo cells exhibited the decreased the ability of proliferation, clonogenicity, invasion, metastasis, and tumorigenicity as well as inhibited the cell’s EMT.2. The invasion, metastasis and tumorigenicity of CRC CD133+CSCs can be reduced with down-regulated HOTAIR, which means that HOTAIR may serve as a new target for treatment of CRC.