Knockdown Of XIST Inhibitors Cell Proliferation And Migration In Non-small Cell Lung Cancer

Background and objective: In the current environment, lung cancer has become one of the most common and high incidence of malignant tumor, and the mortality of lung cancer is the highest in all kinds of cancers according to past data. Among them, NSCLC in all lung cancer patients also occupy 80-85%. The overall 5 year survival rate for patients with NSCLC remains low which was only about 16%. In the course of the occurrence and development of lung cancer has been accompanied by non coding RNA(RNA non-coding) expression changes, some of NSCLC has become the target of nc RNA research. Nc RNAs can be subdivided into 2 kinds: small non encoding RNAs and long chain non encoding RNAs. Lnc RNA expression is specific to time, space, and organization, and its abnormal expression is related to many human diseases, including cancer. Lnc RNA can be changed by chromatin remodeling, DNA or histone methylation and the expression of genes, but also has the function of adsorption of mi RNA sponge. XIST is a major regulator of X chromosome inactivation in mammalian cells, and XIST plays an important role in human cell differentiation, proliferation and maintenance of genomic functions. However, the abnormal expression of XIST gene may be related to the occurrence of cancer. Although there are many reports about XIST lnc RNA, but there is a regulatory relationship between XIST and lung cancer, the development of NSCLC and the regulation of XIST gene expression has not been reported. The purpose of this study was to investigate the change of XIST gene expression in lung cancer, and to regulate the development and metastasis of NSCLC by the change of XIST gene expression.Materials and methods:(1) The XIST gene primers were designed and expression of XIST was detected in a variety of lung cancer cells by real-time PCR;(2) The application of si RNA interference experiments was used in A549 and H1299 cells for pupposes of XIST knockdown, scratch assay used for reseaching the change cell migration ability when XIST was at low level. Transwell experiments to investigate the lung cancer cell invasion ability changes when knockdown of XIST, usingfluorescence experiments again to verify cancer cell invasion ability when XIST was knockdown;(3) Use lentiviral included knockdown of XIST to infect cancer cells A549 and H1299, and screened stable knockdown of XIST lung cancer cells, flow cytometry and CCK-8 assay was used to detected the changes of proliferation and apoptosis of cells, cloning experiment can also be used to explore knockdown of XIST gene on cell proliferation;(4) The proliferation of tumor cells was detected in nude mice with subcutaneous tumor.(5) Western blot experiment inquiry Xist in modulating lung cancer growth and metastasis associated signaling pathway or protein, determine the target of Xist;(6) Bioinformatics analysis the target of XISTResults:(1) Real-time PCR experiments results showed that XIST was up-regulated in NSCL cells compared with the normal human bronchial epithelial cell.(2) Scratch test showed A549 and H1299 both were low wound healing ability, Transwell assay found that the metastasis and invision ability of A549 and H1299 was decreased with XIST knockdown.(3) The flow cytometry results show that when the Xist is knocked down, apoptosis of A549 and H1299 cells were at high level, CCK-8 assay results show that cell proliferation was significantly reduced with Xist knockdown.(4) Subcutaneous tumor formation in nude mice, the ability of A549 cells proliferation with stable low expression of XIST in mice was lower than that of the negative control group.(5) Western blot results showed that when XIST was knocked down, the proteins associated with apoptosis and metastasis were expressed in different degrees.(6) Luciferase assay showed that XIST can interact with mi R-449 a by complementary pairing of nucleotide sequences.Conclusion:(1) The up-regulated of XIST can promote the proliferation of lung cancer cells, and inhibit the apoptosis of lung cancer cells.(2) The metastasis and invasion of lung cancer cells were inhibited at low XIST, which indicated that XIST might have the ability to promote the metastasis and invasion of lung cancer.(3) the XIST gene may be down regulated by the mi R-449 a gene, thus increasing the expression of Bcl-2 protein to inhibit the apoptosis of lung cancer cells.