The Role And Molecular Mechanism Of Long Non-coding RNA HOTTIP In Non-small Cell Lung Cancer

Background and PurposeNon-small cell lung cancer(NSCLC)is the major cause of cancer death worldwide.Increasing evidences show that long non coding RNAs(lnc RNAs)are widely involved in the development and progression of NSCLC.The lnc RNA HOTTIP has been identified as an oncogene in several human cancers,but its role in NSCLC remains unknown.The present study was to determine the expression and function of HOTTIP in NSCLC;Effects of HOTTIP and HOXA13 on Cell Migration,Proliferation and Apoptosis in NSCLCadenocarcinoma Cell Line A549.MethodsQuantitative real-time PCR was used to detect the expressions of HOTTIP in 53 paired NSCLC tissues and cell lines.Furthermore,RNA interference(RNAi)and over-expression approaches were used to investigate the biological function of HOTTIP in lung cancer cell line.Wound scratch assay was used to assess the migratory ability of A549 cell in vitro.A549 cells were transfected with HOTTIP si RNA and HOTTIP-pc DNA3.1+ vector or HOXA13 si RNA and HOXA13-pc DNA3.1+ vector 24 h later after transfected Lipofectamine 2000.After 5h of transfection,a vertical horizontal wound was made with a sterile 10 ?l pipette tip and markers were made to allow observation of cells at the same point.The cell was cultured in the incubator at 37°C.Wound widthswere measured using a standard caliper 0 and 12 h after the wounds were made at the same points.The cell proliferation ability was determined using a MTT.A549 cell were transfected with HOTTIP si RNA and HOTTIP-pc DNA3.1+ vector or HOXA13 si RNA and HOXA13-pc DNA3.1+ vector.The fluorescence intensity was measured using a fluorescence microplate reader and absorbance was measured at 570 nm according to the manufacturer's protocol.Flow cytometry analysis was used to determine apoptosis.A549 cell was transfected with HOTTIP si RNA and HOTTIP-pc DNA3.1+vector or HOXA13 si RNA and HOXA13-pc DNA3.1+ vector at a confluence of approximately 70%.After 48 h,cells were re-suspended in 1×binding buffer.The fluorescence of stained cells was then analyzed by flow cytometry using 488 nm excitation within 30 min of staining,according to the manufacturer's protocol.ResultsThe results demonstrated HOTTIP expression in Lung cancer tissues was significantly higher compared with adjacent normal tissues.When normalized to normal bronchial epithelial cell line(16HBE),the expression level of HOTTIP was also up-regulated in Three NSCLC adenocarcinoma cell lines(A549,SPC-A1,NCI-H1975),a NSCLC squamous carcinomas cell line(SK-MES-1).Wound scratch assay showed that knock-down the expression of HOTTIP levels significantly impeded the migration of A549 cell compared with negative control while over-expression of HOTTIP increased the migration level in A549 cell.In contrast,knockdown of HOXA13 levels significantly increased the migration of A549 cells,whereas HOXA13 overexpression decreased the migration levels in A549 cells.MTT assays revealed that A549 cell growth was inhibited after transient transfected with HOTTIP si RNA compared with negative control.Meanwhile,over-expression of HOTTIP could increase the ability of cell growth.In contrast to HOTTIP,knocking low HOXA13 increased the ability to proliferate A549 cells.At the same time,overexpression of HOXA13 can reduce the ability of cell proliferation.Flow cytometry analysis showed that the apoptosis rate of A549 transfected with HOTTIP si RNA and negative control were 12.44% verse6.58%,while the apoptosis rates of A549 transfected with HOTTIP-pc DNA3.1+vector and pc DNA3.1+empty vector were 3.29% verse 6.23%.The apoptotic rates of HOXA13-pc DNA3.1 + vector and pc DNA3.1 + empty vector were 10.27% and4.51% respectively.The apoptotic rates of HOHA13 si RNA and negative control group were 10.25% and 17.56%,respectively.Indicating that upregulation of HOTTIP and downregulation of HOXA13 inhibited NSCLC cell apoptosis.In addition,we identified HOTTIP as a transcriptional regulator of HOXA13 in lung cancer cell.Ectopic expression of HOTTIP suppressed the endogenous level of HOXA13,while knock-down of HOTTIP increased HOXA13 expression.Knock-down of HOXA13 by RNA interference(si HOXA13)revealed that HOTTIP promoted lung cell proliferation,migration,and inhibited apoptosis,at least partly by regulating HOXA13.ConclusionsThe up-regulation of HOTTIP characterized as an oncogene in NSCLC progression.RNAi-mediated suppression of HOTTIP in A549 cell led to a significant inhibition of cell proliferation,migration and reduced cell apoptosis.Conversely,introducing HOTTIP into A549 cell induced malignant tumor cell behaviors.Si RNA-mediated HOXA13-knockdown induced the proliferation,migration and inhibited apoptosis of NSCLC cell,which was consistent with the functional changes that occurred after restoration the expression of HOTTIP in NSCLC cell.The present study is the first to identify that HOTTIP functions as an oncogene by regulating HOXA13 in NSCLC,which may represent a new biomarker and a potential therapeutic target for NSCLC intervention.