LncRNA HOTAIR Contributes To Cisplatin Resistance In Human Lung Adenocarcinoma Cells By Regulating P21^WAF1/CIP1

Background: Lung cancer, the leading cause of cancer-related deaths, has the most rapidly increasing incidence rate in the world. Non-small cell lung cancer(NSCLC) accounts for more than 80% of primary lung cancer, more than 60% of patients with advanced, often unresectable. Chemotherapy is the main treatment for stage IIIb-IV NSCLC. Cisplatin(DDP), an important component of chemotherapy in NSCLC, is still first-line drug. However, its acquired resistance restricts the play of its efficacy. Long non-coding RNAs(lnc RNAs) regulate gene expression at the level of epigenetic, transcriptional and post-transcriptional processing, playing an important role in tumorigenesis. Recently, lnc RNAs HOTAIR overexpression has been observed in various human cancers. Additionally, accumulating evidence indicates a significant relationship between HOTAIR and tumorigenesis, development, metastasis, and prognosis in cancer patients. However, the mechanism of its function in chemoresistance remains unclear.Objective: The aim of this study is to evaluate the efficacy and mechanism of HOTAIR in the chemoresistance of DDP in NSCLC cisplatin-resistant A549/DDP cells.Materials and Methods:(1) Real-time quantitative PCR was performed to detect the relative expression of HOTAIR in cisplatin-resistant A549/DDP cells compared with parental A549 cells.(2) A549/DDP cells were treated with HOTAIR si RNA or si NC and the expression of HOTAIR in transfected cells was detected by q RT-PCR. The half maximal inhibitory concentration(IC50) values of DDP in resistant A549/DDP, parental A549 and transfection A549/DDP cells were determined by MTT assay. Flow cytometric analysis and Hoechst staining assays was used to analyze the apoptosis and cell cycle distribution of transfection A549/DDP cells treated with DDP.(3) To evaluate the mechanism of HOTAIR. Western blot were used to detecte the expression of Bcl-2 protein family(Bcl-2, Bax, Bcl-xl and Bim) and cell cycle regulating protein p21WAF1/CIP1 in A549/DDP/si NC +/-DDP 、A549/DDP/si HOTAIR +/-DDP at the level of protein.(4) Pc DNA3.1/ p21WAF1/CIP1 vector was constructed and transfected into A549/DDP cells. The expression of p21WAF1/CIP1 was detected by q RT-PCR and western blot in transfection A549/DDP cells. The half IC50 value of DDP in transfection A549/DDP cells was determined by MTT assay. Flow cytometric analysis was used to analyze the apoptosis and cell cycle distribution of transfection A549/DDP cells treated with DDP.(5) In vivo experiments were used to observe the effect of HOTAIR on cisplatin resistance by regulating p21WAF1/CIP1. To validate the effect of HOTAIR on tumor size of A549/DDP cells to cisplatin in vivo.(6) q RT-PCR and immunohistochemistry was performed to detect the relative expression of HOTAIR and p21WAF1/CIP1 in lung adenocarcinoma ‘‘sensitive’’ and ‘‘insensitive’’ tissues and analyze the relationship between HOTAIR and p21WAF1/CIP1.Results:The current study examines the increase of lnc RNAs HOTAIR expression in cisplatin-resistant A549/DDP cells compared with parental A549 cells. Moreover, HOTAIR knockdown in A549/DDP cells was shown to resensitized A549/DDP cells to cisplatin, indicating that HOTAIR plays an important role in DDP resistance in NSCLC cells. Moreover, HOTAIR knockdown in A549/DDP cells was shown to induce cell apoptosis, decrease cell proliferation and increase proportion of G0/G1 cell cycle. Western Blot assays verified that HOTAIR knockdown increased p21WAF1/CIP1 expression at protein levels. Simultaneously, p21WAF1/CIP1 overexpression was shown to recapitulate the functions of HOTAIR knockdown in vitro. The inverse correlation between HOTAIR and p21WAF1/CIP1 expression was also detected in clinical lung adenocarcinoma ‘‘sensitive’’ and ‘‘insensitive’’ tissues.Conclusions: In the present study we provide the ?rst evidence that HOTAIR reverses resistance to DDP in A549/DDP cells, at least in part, by regulating p21WAF1/CIP1 in vitro and vivo.