Mechanism Of NcRNA Contributing To Non-small Cell Lung Cancer, Gastric Cancer Cells Proliferation And Invasion

High throughput transcriptiome analysis discovery that up to 98% of the human genome can be transcripted into noncoding RNA (ncRNA) that does not serve as templates for proteins. According to their size, ncRNAs are subdivided into two groups: small ncRNAs (microRNAs) and long ncRNAs (lncRNAs). ncRNAs constitute a very heterogeneous group of RNA molecules that allows them to cover a broad spectrum of molecular and cellular functions in the field of tumor biology.Recent studies show that ncRNA could play a similar role in oncogenes or tumor suppressor genes by regulating target genes to participate in related signaling pathways, and impact tumor invasion and metastasis process. Multiple lines of evidences link dysregulations of miR-196a and lncRNA HOT AIR to diverse human cancers. However, the contributions of these two ncRNAs to gastric cancer and NSCLC remain largely unknown.Therefore, the aim of present studies is to investigate the role of miR-196a and lncRNA HOTAIR in gastric and NSCLC carcinogenesis. In the first part of present study, we investigate the expression pattern of miR-196a in gastric cancer and its clinical significance as well as its biological role in tumor progression. In the second part, we demonstrate that increased HOTAIR expression is a characteristic molecular change in gastric cancer and investigate the effect of decreased HOTAIR levels on the phenotypes of gastric cancer cells in vitro and in vivo. We also show the ceRNA action of HOTAIR. In the third part, the effects and mechanism of endogenous HOTAIR on the migration of NSCLC cells were investigated.This studies will enhance our knowledge to better understand the pathogenesis and development of gastric cancer and NSCLC, and facilitate the development of ncRNA-directed diagnostics and therapeutics against these deadly diseases.Part I miR-196a is up-regulated in gastric cancer and promotes cell proliferation by down-regulating p27kip1AIM:The aim of the present study is to examine the expression pattern of miR-196a in gastric cancer and its clinical significance as well as its biological role in tumor progression.METHODS:Real-time quantitative PCR was performed to detect the relative expression of miR-196a in GC cell lines and tissues. Over-expression and RNA interference (RNAi) approaches were used to investigate the biological functions of miR-196a. The effect of miR-196a on proliferation was evaluated by MTT and colony formation assays, and subcutaneous injection of cells was used to study the development of tumors in nude mice. Luciferase reporter plasmids were constructed to confirm the action of miR-196a on downstream target genes including p27kip1. qRT-PCR and western blot assays verified that miR-196a reduced p27kip1 expression at both mRNA and protein levels. Results are reported as means±S.D. and differences were tested for significance using Student’s t-test (two-tailed).RESULTS:Here we report that miR-196a was highly expressed both in gatiric samples and cell lines compared with their corresponding counterparts. Higher expression of miR-196a in GC tissues was associated with tumor size, a higher clinical stage, and was also correlated with shorter overall survival of GC patients. Exogenous downregulation of miR-196a expression significantly suppressed the in vitro cell cycle progression, proliferation and colony formation of GC cells, and ectopic miR-196a expression significantly enhanced the development of tumors in nude mice. Luciferase assays revealed that miR-196a inhibited p27kipl expression by targeting one binding site in the 3’-untranslated region (3’UTR) of p27kipl mRNA. Finally, a reverse correlation between miR-196a and p27kip1 expression was noted in GC tissues.CONCLUSION:Our study demonstrates that aberrant over-expression of miR-196a and consequent down-regulation of p27kipl could contribute to gastric carcinogenesis, and would be targets for gastric cancer therapies and further developed as potential prognostic factors.Part Ⅱ Long noncoding RNA HOT AIR promotes gastric cancer cells invasion and proliferation by functioning as a competing endogenous RNAAIM:The aim of the present study is to examine the expression pattern of HOT AIR in gastric cancer and its clinical significance as well as its biological role in tumor progression.METHODS:Expression of HOTAIR was analyzed in 78 gastric cancer tissues by qRT-PCR. Over-expression and RNA interference (RNAi) approaches were used to investigate the biological functions of HOTAIR. The effect of HOTAIR on proliferation was evaluated by MTT and colony formation assays, and cell migration and invasion were evaluated by transwell assays. Subcutaneous injection of cells was used to study the development of tumors in nude mice. Luciferase reporter plasmids were constructed to confirm the ceRNA action of HOTAIR. Protein levels of HOTAIR targets were determined by western blot analysis and immunohistochemistry.RESULTS:In this study, we showed that HOTAIR expression was increased in 78 gastric tumors compared with non-tumor tissues and was associated with tumor size, pathological stage, distant metastasis, and shorter overall survival of gastric cancer patients. Knockdown of HOTAIR expression in gastric cancer cells was demonstrated to decrease the cell proliferation, migration, invasion, while lead to the promotion of cell apoptosis and induce tumor growth arrest in vitro and in vivo. Here we identified that HOTAIR may sponge miR-331-3p to regulate the expression of HER2 (Human epithelial growth factor receptor 2), which with a relevant function in development and progression of cancer and mediate its resistance to therapy. Moreover, we observed a positive correlation between HOTAIR and HER2 expression in advanced gastric cancer patients.CONCLUSION:We demonstrate that increased HOTAIR expression is a characteristic molecular change in gastric cancer and investigate the effect of decreased HOTAIR levels on the phenotypes of gastric cancer cells in vitro and in vivo. We also show the existence of a specific crosstalk between the lncRNA HOTAIR and HER2 mRNA through competition for miR-331-3p. This study suggest that ceRNA activity of HOTAIR and HOTAIR-HER2 interaction may contribute to a better knowledge of pathogenesis of gastric cancer and facilitate the development of IncRNA-directed diagnostics and therapeutics.Part III Long noncoding RNA HOT AIR contributes to non-small cell lung cancer cell invasion and metastasis through repressing HOXA5AIM:The aim of the present study was to examine the expression pattern of HOTAIR in NSCLC and to evaluate its biological role and clinical significance in tumor progression.METHODS:Expression of HOTAIR was analyzed in 42 NSCLC tissues and four NSCLC cell lines by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Over-expression and RNA interference (RNAi) approaches were used to investigate the biological functions of HOTAIR. The effect of HOTAIR on proliferation was evaluated by MTT and colony formation assays, and cell migration and invasion were evaluated by transwell assays. Tail vein injection of cells was used to study metastasis in nude mice. Protein levels of HOTAIR targets were determined by western blot analysis. Differences between groups were tested for significance using Student’s t-test (two-tailed).RESULTS:HOTAIR was highly expressed in NSCLC samples compared with corresponding normal counterparts. HOTAIR upregulation was correlated with advanced pathological stage, and also with NSCLC lymph-node metastasis. Moreover, patients with high levels of HOTAIR expression had a relatively poor prognosis. Inhibition of HOTAIR by RNAi decreased the migration and invasion of NSCLC cells in vitro and impeded cell metastasis in vivo. HOXA5 levels were affected by HOTAIR knockdown or over-expression in vitro.CONCLUSION:Our findings indicate that HOTAIR is significantly up-regulated in NSCLC tissues, and regulates NSCLC cell invasion and metastasis, partially via the down-regulation of HOXA5. Thus, HOTAIR may represent a new marker of poor prognosis and is a potential therapeutic target for NSCLC intervention.