Expression, Effect Of NEAT1 In Lung Adenocarcinomaand The Interreaction Between NEAT1 And MiR-193a-3p

Background In the recent ten years,the incidence of lung cancer has increasing,take the first place of tumor incidence.Among lung cancer,the incidence of adenocarcinoma have been rising year by year,replace the row of lung squamous cell carcinoma,which was the most common subtype of lung cancer in past.For occurring insidiously,and often appear hematogenous metastasis in the early stage,patients can't get effective treatment after the diagnosis.Personalized and target treatment become the hotspots of tumor study recently.A lot of specific tumor targets remain to be discovered.Numerous of studies have found that long non-coding RNAs play an important role in the development of malignant tumors.NEAT1 is one kinds of long non-coding RNAs.It was found to be involved in the occurrence and development of tumors.This study aims to investigate the expression of NEAT1 in lung adenocarcinoma tissues,the effect of NEAT1 on the proliferation of lung adenocarcinoma and the interaction between NEAT1 and mi R-193a-3p.Materials and Methods1.Extract the expressive data of NEAT1 and miR-193a-3p in lungadenocarcinoma tissues from previous study,analyse of the expression of NEAT1 and miR-193a-3p in lung cancer tissues,and they relationship with clinical parameters.Collected the expressive data of NEAT1 and mi R-193a-3p in lung adenocarcinoma from TCGA database,and analysed the expression of NEAT1 and miR-193a-3p between lung adenocarcinoma and normal lung tissues.Survival analysis was performed by Kaplan-Meier survival curve.2.Searched and downloaded the arrays which had detected NEAT1 or mi R-193a-3p in lung adenocarcinoma tissues from GEO and oncomine databases.Extracted the expressive data of NEAT1 or miR-193a-3p in lung adenocarcinoma and normal lung tissues,combined with the data from previous study and TCGA database,the meta-analysis was done to explore the expression of NEAT1 and miR-193a-3p in lung adenocarcinoma.3.The expression of NEAT1 and miR-193a-3p in lung adenocarcinoma cells A549,H1299,HCC827 and human normal lung epithelial cells BEAS-2B by qRT-PCR.4.The effect of NEAT1 on the proliferation,viability and apoptosis of lung adenocarcinoma cell line A549 and Calu3 was detected by MTS,hoes+PI,viability kit and caspase3/7 activity after NEAT1 was silenced by siRNA.5.According to the expression data of previous studies and TCGA database,analysed the relativity between NEAT1 and mi R-193a-3p in lung adenocarcinoma.Explore the existence of gene complementary sequence between NEAT1 and miR-193a-3p by target prediction software.6.The directly targeted relationship between NEAT1 and mi R-193a-3p was detected by the dual luciferase gene report experiment.7.Based on the expressive data of TCGA database,seek the genes that was positively correlated with NEAT1,negatively correlated with miR-193a-3p,andwas the potential target genes of mi R-193a-3p that predicted by online predicted software.The genes meet the criterials mentioned above would be analysed by functional annotation.Results1.NEAT1 was up-regulated in lung adenocarcinoma tissues,and its up-regulated was correlated with vascular invasion and TNM advanced stage and lymphnode metastasis.The expressive data of NEAT1 in 514 lung adenocarcinoma and 59 normal lung tissues were collected on TCGA database,after analysis,it was found that the expression of NEAT1 was up-regulated in lung adenocarcinoma tissues,when compared to normal lung tissues.The overall survival time on patients with high level of NEAT1 was shorter that patients with low level of NEAT1.Eight arrays about the expression of NEAT1 in lung adenocarcinoma and normal lung tissues were found in oncomine database,combine with data from TCGA database and previous study,a meta-analysis about the expression of NEAT1 in lung adenocarcinoma was done,the result showed that NEAT1 was up-regulated in lung adenocarcinoma tissues significantly.The expression of NEAT1 was also up-regulated in lung adenocarcinoma cell lines.After knock down the NEAT1 by siRNA in lung adenocarcinoma cell lines,the proliferation and viability of cells were inhibited and the apoptosis of cells were promoted.2.Compared with adjacent normal tissues,the expression of mi R-193a-3p in 101 cases of lung adenocarcinoma was down-regulated,and its down-regulated was correlated with larger tumor nodules,lymph node metastasis and TNM advanced stage.The relative expressive data of mi R-193a-3p in 513 cases of lung adenocarcinoma and 46 cases of normal lungtissues were collected from TCGA database,the analytical result suggested that mi R-193a-3p was down-regulated in lung adenocarcinoma tissues but without statistical significant.11 arrays detected miR-193a-3p in lung adenocarcinoma and normal lung tissues were found in GEO database,combine with data from TCGA database and previous study,a meta-analysis about the expression of mi R-193a-3p in lung adenocarcinoma was done,the result showed that mi R-193a-3p was down-regulated in lung adenocarcinoma tissues significantly.The expression of mi R-193a-3p was also down-regulated in lung adenocarcinoma cell lines,compared with human normal lung epithelial cells.3.According to the expressive data of NEAT1 and mi R-193a-3p in 101 cases from previous study,514 case from TCGA database,correlated analysis found that the expression of NEAT1 was negatively correlated with mi R-193a-3p.Complementary sequences were found between the base sequence of NEAT1 and miR-193a-3p.Dual luciferase gene report experiment found the directly targeted relationship was existed between NEAT1 and miR-193a-3p,compared to NEAT1 3'UTR+miRNA-NC(miRNA plasmid)group,the fluorescence intensity in NEAT1 3' UTR+miRNA(miR-193a-3p plasmid)group was significantly decreased.Four potential down-stream target genes of NEAT1 and miR-193a-3p were found,they are PIGA ? TFCP2L1 ? C12orf76 and MRPS25.Conclusions1.NEAT1 is up-regulated in lung adenocarcinoma tissues and cells,and can promote malignant progression of lung adenocarcinoma;2.miR-193a-3p was down-regulated in lung adenocarcinoma tissues and cells,and negative correlation with NEAT1,complementary sequences werefound between NEAT1 and miR-193a-3p,directly binding relationship was existed between NEAT1 and mi R-193a-3p,the mechanism of the interaction between NEAT1 and miR-193a-3p may be related to PIGA ? TFCP2L1 ?C12orf76?MRPS25.