The Expression Of LncRNA DLX6-AS1 In Lung Adenocarcinoma And The Effect On Its Proliferation,Invasion And Apoptosis

Lung cancer is one of the most common malignant tumor in the world, and its morbidity and mortality in a variety of tumors, every year there are 1.6 million patients with newly diagnosed worldwide, which kills more than 1.4 million patients at the same time, 80% of lung cancer for non-small cell lung cancer(NSCLC). The main causes of high mortality for NSCLC patients were that patients with no obvious clinical symptoms in the early stage were diagnosised in the late stage with tumor cell invasion and metastasis. Current standard treatment of NSCLC include surgery,radiation therapy and chemotherapy based on platinum, but these treatments are hard to reach the purpose of cure NSCLC, therefore, the effect of these treatments in NSCLC patients is not beautiful, the prognosis is poor, the 5-year survival rate is still less than 15%. In order to raise survival rate of the NSCLC patients must further study of its pathogenesis, and inhibit the proliferation and invasion and metastasis of malignant tumor as soon as possible.Long non-coding(lncRNAs), is a diverse class of RNA transcripts>200nucleotides in length with limited protein coding potential, due to the lack of significant open reading frame. LncRNA involved in tumor biology function through regulating the expression and transcription of gene in epigenetics, and the transcription and posttranslation levels. So far, people know a bit about he regulatingmechanism of lncRNAs in the cancer.Several research datas show that expression levels of many lncRNAs changed in different tumor tissues.Therefore, its role in the tumorigenesis and development process. So people focused on its role in the tumorigenesis and development process. Although the specific mechanism of most lncRNAs are unclear, but the lncRNAs have become the new research hot spot and front after mi RNAs.LncRNA DLX6-AS1 is located on human chromosomal band 7q21.3. No report was found about lncRNA DLX6-AS1 in lung cancer at home and abroad. In order to discovery the function of lncRNA DLX6-AS1 in the proliferation and apoptosis of lung adenocarcinoma lines, and the contribution of lncRNA DLX6-AS1 to lung adenocarcinoma malignancy and the molecular mechanisms, the research includes three parts.The first part is expression and analysis of lncRNA DLX6-AS1 in lung adenocarcinoma tissues; the second part is biological effects of downregulating lncRNA DLX6-AS1 expression in lung adenocarcinoma cell; the third part is preliminary study of mechanism of lncRNA DLX6-AS1.Part One: Expression and analysis of lncRNA DLX6-AS1 in lung adenocarcinoma tissues.Methods:1. Seventy-two pairs of primary lung adenocarcinoma tissues and corresponding adjacent normal lung tissues were used.2. LncRNA chip was used to detect expression of lncRNA in 3 cases of lung adenocarcinoma tissue and corresponding normal adjacent tissue samples and analysis the differential expression of lncRNA.3. The q RT–PCR method was used to detect expressions of lncRNA DLX6-AS1 in seventy-two cases of lung adenocarcinoma tissue and Bivariate correlation analysis was used to verify the correlation between lncRNA DLX6-AS1 expression and gender, age, differentiation status, lymph node metastases and TNM stage.Results:1. Compared with adjacent normal lung tissues, tumor tissues exhibited 18 differentially expressed ln RNAs, including 9 lncRNAs upregulated and 9lncRNAs downregulated. LncRNA DLX6-AS1 is upregulated in lung adenocarcinoma tissue(P < 0.05).2. qRT-PCR detection showed that compared to normal tissues, expression level of lncRNA DLX6-AS1 has a significant upregulation in lung adenocarcinoma tissues(P < 0.05). The results of ln RNA chip and q RT-PCR are consistent.3. LncRNA DLX6-AS1 expression level in lung adenocarcinoma tissues was associated with tumor location and TNM stage(P>0.05). There was no statistically significant correlation between lncRNA DLX6-AS1 expression and either gender, age, lymph node metastases or differentiation status(P<0.05).Part Two Biological effects of downregulating lncRNA DLX6-AS1 expression in lung adenocarcinoma cell.Methods:1. Construct lentiviral vector with lncRNA DLX6-AS1 si RNA and non-sense si RNA, which were transfected into A549 and H1650 cells, screen a stable infected cell line that downregulation the expression level of lncRNA DLX6-AS1.2. The cells are divided into three groups: control group(recombined lncRNA DLX6-AS1 si RNA lentivirus infected cells), negative control(NC) group(lncRNA DLX6-AS1 non-sense si RNA lentivirus infected cells) and blank group(uninfected cells).3. Using CCK8 method and plate clone formation experiment to detect proliferation and growth ability of cells in each group.4. Using AnncxinV-FITC/PI double marker flow cytometry, Hoechst staining and Caspase-3/7 to test apoptosis ability of cells in each group.5. Using Western- blot to test the expression of apoptosis related proteins of cells in each group.6. Using transwell chambers experiment and scratch to test invasion and metastasis ability of cells in each group.7. Using Tumor xenograft model to detect the influence of downregulating lncRNA DLX6-AS1 expression in A549 cells on BALB/c nude mice.Results:1. Lentiviruses with lncRNA DLX6-AS1 si RNA and lncRNA DLX6-AS1 NC were successfully constructed. The titer were respectively 1.8×108TU/ml and2.3×108TU/ml. Compared with Blank and NC groups,the expression of lncRNA DLX6-AS1 was significantly decreased in si Lnc DLX6-AS1 group(P < 0.05).2. CCK8 results showed that compared to Blank and NC groups, OD450 value of si Lnc DLX6-AS1 group has a significant reduce in A549 and H1650 cells, which was more obvious with time passing. The difference has statistically significant(P < 0.05). Plate clone formation experiment results showed that compared to Blank and NC groups, the colony formation number of si Lnc DLX6-AS1 group has a significant reduce in A549 and H1650 cells. The difference has statistically significant(P < 0.05).3. AnncxinV-FITC/PI double marking flow cytometry assay and Hoechst staining method results showed that compared to Blank and NC groups, the apoptosis rate of si Lnc DLX6-AS1 group was significantly raised after infected in A549 and H1650 cells. Besides, the Caspase-3/7 activity also has a significantly raised when compared to Blank and NC groups in A549 and H1650 cells. The difference has statistically significant(P < 0.05).4. Western blot results showed that compared to Blank and NC groups,Pro-capase-9 and Bcl-2 in si Lnc DLX6-AS1 group were significantly reduce in A549 and H1650 cells, when Cleaved caspase3 and Cleaved caspase9 were significantly increased. The difference has statistically significant(P < 0.05).5. Transwell invasion experiment results showed that compared to Blank and NC groups, the cell number went through membrane of si Lnc DLX6-AS1 group has a significant reduce. The difference has statistically significant(P < 0.05). Scratch experiments results showed that compared to Blank and NC groups, after recombination of lentivirus infected cells, scratch healing speed after 48 h of si Lnc DLX6-AS1 group was significantly lower in A549 and H1650 cells. The difference was statistically significant(P < 0.05).6. To confirm the growth inhibitory effect of lncRNATP73-AS1 si RNA on lung adenocarcinoma in vivo, tumor xenograft transplant experiment was performed.Tumor size was significantly reduced in the si Lnc DLX6-AS1 mice group(A549cells transfected with lncRNA DLX6-AS1 si RNA) as compared to control mice(NC and Blank groups) at the fourth week(P<0.05).Part Three Preliminary study of mechanism of lncRNA DLX6-AS1Methods:1. Predict the potential interaction mi RNA of lncRNA DLX6-AS1 by bioinformatics analysis.2. Construct wildtype and seed region mutation expressional vector of lncRNA DLX6-AS1(pc DNA3.1-Wt DLX6 AS1、pc DNA3.1-Mt DLX6 AS1), which were transfected into A549 cells. Using q RT –PCR to test the expreesion of lncRNA DLX6-AS1 and mi R-497 in infected cells.3. Construct reporter gene recombinant vectors pmirGLO-Wt3’UTR-Bcl2 and pmir GLO-Mt3’UTR-Bcl2. Use dual-luciferase report experimental to validate that Bcl-2 is the target genes of mi R-497 and lncRNA DLX6-AS1 regulate Bcl-2expression by negative regulating mi R-497.4. Using CCK8, AnncxinV-FITC/PI double marker flow cytometry and Transwell invasion experiment to test the effect of upregulating mi R-497 and downregulating lncRNA DLX6-AS1 in lung adenocarcinoma cell.5. We constructed expression vectors containing Bcl-2 lacking the 3’UTR sequence.Transfected expression vectors alone or co-transfected with mi R-497 mimics into A549 and A549 infected si Lnc DLX6-AS1 cells. Restore assay was used to analyse mechanism that mi R-497 regulated the expression of Bcl-2.Results:1. We predicted the 3’ untranslated region(3’UTR) of mi R-497 contains one seed region for lncRNA DLX6-AS1 by the bioinformatics algorithms.2. Ln RNA DLX6-AS1 regulate negatively mi R-497 by binding the seed region in A549 cells.3. Dual luciferase report experiments show that Bcl-2 is the target gene of mi R-497.4. Overexpression of mi R-497 had similar function with lncRNA DLX6-AS1 si RNA in A549 cells.5. Serum starvation induced apoptosis experiments showed transfected the recombination vector pc DNA3.1-Bcl2 without 3’UTR regions of Bcl-2 into A549 cells led to the restoration of the negative function of mi R-497 and lncRNA DLX6-AS1. At the same time, the ability promote the apoptosis of cells had been restored.Conclusion:1. A total of 18 lncRNAs in lung adenocarcinoma tissues have abnormal expression,including 9 up-regulation and 9 down-regulation. LncRNA DLX6-AS1 expression level increased significantly when compared to corresponding adjacent normal lung tissues. LncRNA DLX6-AS1 expression level in lung adenocarcinoma tissues was associated with tumor location and TNM stage(P <0.05).2. Down-regulation of lncRNA DLX6-AS1 in lung adenocarcinoma cells in vitro can significantly inhibit cells growth, reduce the invasion ability of cells and promote cells apoptosis. The animal experiment indicated that the down-regulation of mi R-335 can significantly inhibit the transplant tumor growth in nude mouse models.3. LncRNA DLX6-AS1 regulate Bcl-2 expression by negative regulating mi R-497,thus exert its biological function.4. LncRNA DLX6-AS1 can play a role in cancer promotion and is expected to become new targets for lung adenocarcinoma gene therapy.