The Long Noncoding RNA MEG3 Contributes To Cisplatin Resistance Of Human Lung Adenocarcinoma Cells

Background:Chemotherapy is the main clinical treatment of Ⅲb-Ⅳ stage non-small cell lung cancer (NSCLC). Currently, chemotherapy based on cisplatin (DDP) is the first-line chemotherapy regimen of NSCLC. Lung adenocarcinoma (LAD) is the most common histological subtype of NSCLC. Clinical practice confirms that acquired resistance often restricts the curative effect of DDP. Long non coding RNA (lncRNA) is one of the research hotspots at present, that palys an important role in epigenetics level, transcription level, and posttranscription level. Numerous studies have found that the abnormal expression of lncRNA in tumors has potential biological functions. Previous studies have confirmed that lncRNA MEG3 abnormally expressed in various human tumors, suggesting that MEG3 may be involved in tumorigenesis, development and metastas. However, the biological functions and specific molecular mechanisms of MEG3 in NSCLC chemotherapy sensitivity have not been reported.Objective:The aim of this study is to detect MEG3 expression in human LAD and to evaluate the role and mechanisms of MEG3 in LAD chemotherapy sensitivity of DDP.Methods and Methods:(1) Microarray expression profiling of lncRNA was undertaken in human LAD parental A549 cells and its DDP resistant A549/DDP cells to detect the significant deviation expressive lncRNA. (2) Real-time quantitative PCR (qRT-PCR) was used to detect the expression of MEG3 in parental A549 cells compared with A549/DDP cells. (3) qRT-PCR was preformed to detect the expression of MEG3 in transfected cells after transfection of A549/DDP cells with pCDNA-MEG3 and transfection of A549 cells with si-MEG3. The half maximal inhibitory concentration (IC50) values of DDP in A549/DDP/pCDNA-MEG3 and A549/si-MEG3 cells were determined by MTT assay. Hoechst staining assay and Flow cytometric analysis were preformed to analyze the apoptosis and cell cycle distribution of A549/DDP/pCDNA-MEG3 and A549/si-MEG3 cells when treated with various doses of DDP. MTT assay and cloning formation assay were applied to detect cell viability. (4) Western Blot was used to detect the expression of mitochondria apoptosis pathway associated protein in A549/DDP/pCDNA-MEG3 cells, including Bcl-2 protein family (Bcl-2, Bcl-xl) and p53 protein. (5) Nude mouse experiment was performed to observe the effect of MEG3 on A549/DDP cells sensitivity to DDP. The mRNA level of MEG3 and protein level of Bcl-xl、p53 in transplanted tumor were detected by qRT-PCR and Immunohistochemistry. (6) The expression of MEG3 in human LAD tissues was detected by qRT-PCR and the relationship of MEG3 expression level and chemosensitivity of patients to DDP was analyzed.Results:(1) The expression of MEG3 was significantly decreased in A549/DDP cells than that in A549 cells by microarray assay and qRT-PCR detection was consistent with results obtained. (2) The overexprssion of MEG3 in A549/DDP cells was shown to sensitize the cells to DDP. The MEG3 knockdown in A549 cells was shown to resistance the cells to DDP. (3) MEG3 was shown to induce cell apoptosis, decrease cell viability and increase cell cycle blocking in A549/DDP/pCDNA-MEG3 cells and decrease cell apoptosis, induce cell viability in A549/si-MEG3 cells compared with the control group when treated with various doses of DDP. (4) Western Blot verified that the overexpression of MEG3 increased p53 level and decreased Bcl-xl level. (5) Nude mouse experiment was shown that the overexpression of MEG3 reversed the DDP resistance of A549/DDP cells. (6) MEG3 expression level in human LAD tissues was proved to be related with chemosensitivity of patients to DDP.Conclusions:In our study, we confirme that overexpression of MEG3 can reverse the chemosensitivity of A549/DDP cells to cisplatin in vitro and in vivo by targeting mitochondria apoptosis pathway. Thus, MEG3 may represent as a new molecular target for for diagnosis and prognosis prediction of human LAD.