| Objective: The present study is aimed to investigate the role of HOTTIP in the proliferation of lung cancer cells,and to reveal the underlying mechanisms.Methods:(1)A total of 80 patients with lung cancer who underwent lung cancer radical resection in Department of Thoracic Surgery of Tongling People’s Hospital were selected in this study.Once the tumor tissues and corresponding non-tumor tissues were cut,they were harvested and stored after resection.(2)Trizol is used to extract the total RNA from 80 of tumor tissues and non-tumor samples.The LncRNAs expression profiles were detected using LncRNA array between 3 cases of tumor samples and corresponding non-tumor samples.The significant differential expression of all LncRNAs were screened,and proliferation-related LncRNAs were analyzed by Pathway analysis.(3)These LncRNAs were identified using Real-time PCR in 77 cases of tumor tissues as well as lung cancer cell lines,and the most differentially expressed HOTTIP was subsequently selected for functional studies.(4)The location and expression level of HOTTIP was examined by in situ hybridization(ISH)and the correlation between HOTTIP expression and clinical features such as gender,age,tumor size and TNM stage were analyzed using Chi square test.(5)To up-regulate and down-regulate the expression of HOTTIP in lung cancer cells,the Lentivirus carrying full length of HOTTIP and the lentivirus carrying HOTTIP shRNA were constructed and transfected into lung cancer cell lines(95-D and A549).MTT assay is used to detect the viability of 95-D and A549 cells after transfection.(6)Annexin V/PI double staining assay is used to detect the apoptosis of 95-D and A549 cells after transfection.(7)Western blotting is used to examine the expression of proliferation-and apoptosis-related proteins such as p-AKT,p-mTOR,bcl-2,bax and caspase-3 in our study.(8)The expression of miR-137 in lung cancer tissues were tested using Real-time PCR and the association between HOTTIP and miR-137 expression was analyzed by Spearman.Results:(1)A total of 127 LncRNAs were screened out with differentially expression between lung tumor and non-tumor tissues(p<0.05).Among these differentially expressed LncRNAs,22 have foldchange more than 2(FoldChange>=2),and 15 were up-regulated in tumor tissues and 7 were down-regulated in tumor tissues.(2)As to these differentially expressed LncRNAs in tumor tissues,Real-time PCR were used to identify their expression in lung tumor tissues,and it is found that 7 LncRNAs were up-regulated and 6 LncRNAs were down-regulated.Among these,HOTTIP expression was most increased in lung tumor tissues with FoldChange=6.374.The further pathway analysis suggested that HOTTIP participates in several signaling pathways which may be involved in proliferation such as PI3K/AKT/mTOR signaling pathway,indicating that HOTTIP may participate in lung tumorigenesis via regulating cell growth.(3)Correlation analysis results showed that HOTTIP expression was significantly correlated with tumor size,TNM stage,local invasion,lymph node metastasis,PCNA,Ki67 and EGFR expression(P<0.05),while no significant correlation was observed between HOTTIP expression and CA-125,CA-199,CA50 and CEA expression(P>0.05).(4)The expression of HOTTIP in 95-D and A549 cells is much higher than that in human immortal lung epithelial cell line BEAS-2B.The Lentivirus carrying full length of HOTTIP(Lenti-HOTTIP)and Lentivirus carrying HOTTIP shRNA(HOTTIP-shRNA)were successfully constructed and transfection may up-regulate and down-regulate the expression HOTTIP in 95-D and A549 cells,respectively(P<0.05).(5)MTT result showed that HOTTIP overexpression by Lenti-HOTTIP could induce 95-D and A549 cell proliferation,while HOTTIP down-expression by HOTTIP-shRNA may suppress 95-D and A549 cell proliferation.(6)Annexin V/PI staining assay showed that HOTTIP overexpression by Lenti-HOTTIP did not affect 95-D and A549 cell apoptosis,while HOTTIP down-expression by HOTTIP-shRNA could apparently induce 95-D and A549 cell apoptosis.(7)Western blotting assay showed that HOTTIP overexpression by Lenti-HOTTIP could promote the expression of p-AKT,p-mTOR,bcl-2,while it did not affect expression of bax and caspase-3 in 95-D and A549 cells;In contrast,HOTTIP down-expression by HOTTIP-shRNA could down-regulate the expression of p-AKT,p-mTOR and bcl-2 protein,and it could up-regulate the expression of Bax and caspase-3 in 95-D and A549 cells.(8)The binding sites between miR-137 and HOTTIP was analyzed by miRDB online software,and there are several potential binding sites.Real-time PCR results showed that miR-137 expression is downregulated in lung cancers and its expression is negatively correlated with HOTTIP expression,with R=-0.412.Conclusion: HOTTIP expression is significantly up-regulated in lung cancer and it was significantly associated with tumor size,local invasion,TNM stage,lymph node metastasis and etc,which indicates that it may play a role in lung tumorigenesis.HOTTIP may promote lung cancer cell proliferation by competitively binding to miR-137,while may inhibit the tumor suppressive function of miR-137,leading to activation of AKT/mTOR pathway.Our data suggested that HOTTIP may serve as potential target for lung cancer diagnosis and treatment. |
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