Wei De

Lnc RNA HOTAIR functions as a competing endogenous RNA to regulate HER2 expression by sponging miR-331-3p in gastric cancer

BackgroundAccumulating evidence indicates that the long non-coding RNA HOTAIR plays a critical role in cancer progression and metastasis. However, the overall biological role and clinical significance of HOTAIR in gastric carcinogenesis remains largely unknown.MethodsHOTAIR expression was measured in 78 paired cancerous and noncancerous tissue samples by real-time PCR. The effects of HOTAIR on gastric cancer cells were studied by overexpression and RNA interference approaches in vitro and in vivo. Insights of the mechanism of competitive endogenous RNAs (ceRNAs) were gained from bioinformatic analysis, luciferase assays and RNA binding protein immunoprecipitation (RIP). The positive HOTAIR/HER2 interaction was identified and verified by immunohistochemistry assay and bivariate correlation analysis.ResultsHOTAIR upregulation was associated with larger tumor size, advanced pathological stage and extensive metastasis, and also correlated with shorter overall survival of gastric cancer patients. Furthermore, HOTAIR overexpression promoted the proliferation, migration and invasion of gastric carcinoma cells, while HOTAIR depletion inhibited both cell invasion and cell viability, and induced growth arrest in vitro and in vivo. In particular, HOTAIR may act as a ceRNA, effectively becoming a sink for miR-331-3p, thereby modulating the derepression of HER2 and imposing an additional level of post-transcriptional regulation. Finally, the positive HOTAIR/HER2 correlation was significantly associated with advanced gastric cancers.ConclusionsHOTAIR overexpression represents a biomarker of poor prognosis in gastric cancer, and may confer malignant phenotype to tumor cells. The ceRNA regulatory network involving HOTAIR and the positive interaction between HOTAIR and HER2 may contribute to a better understanding of gastric cancer pathogenesis and facilitate the development of lncRNA-directed diagnostics and therapeutics against this disease.

The long non-coding RNA HOTAIR indicates a poor prognosis and promotes metastasis in non-small cell lung cancer

BackgroundThe identification of cancer-associated long non-coding RNAs and the investigation of their molecular and biological functions are important for understanding the molecular biology and progression of cancer. HOTAIR (HOX transcript antisense intergenic RNA) has been implicated in several cancers; however, its role in non-small cell lung cancer (NSCLC) is unknown. 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.MethodsExpression 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).ResultsHOTAIR was highly expressed both in NSCLC samples and cell lines compared with corresponding normal counterparts. HOTAIR upregulation was correlated with NSCLC advanced pathological stage and 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.ConclusionsOur 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.

Downregulated long noncoding RNA MEG3 is associated with poor prognosis and promotes cell proliferation in gastric cancer

Long noncoding RNAs (lncRNAs) have emerged recently as major players in governing fundamental biological processes, and many of which are altered in expression and likely to have a functional role in tumorigenesis. Maternally expressed gene 3 (MEG3) is an imprinted gene located at 14q32 that encodes a lncRNA associated with various human cancers. However, its biological role and clinical significance in gastric cancer development and progression are unknown. In this study, to investigate the lncRNA MEG3 expression in gastric cancer, quantitative reverse-transcription polymerase chain reaction was conducted. We found that MEG3 levels were markedly decreased in gastric cancer tissues compared with adjacent normal tissues. Its expression level was significantly correlated with TNM stages, depth of invasion, and tumor size. Moreover, patients with low levels of MEG3 expression had a relatively poor prognosis. Furthermore, knockdown of MEG3 expression by siRNA could promote cell proliferation, while ectopic expression of MEG3 inhibited cell proliferation, promoted cell apoptosis, and modulated p53 expression in gastric cancer cell lines. By 5-aza-CdR treatment, we also observed that MEG3 expression can be modulated by DNA methylation. Our findings present that MEG3 downexpression can be identified as a poor prognostic biomarker in gastric cancer and regulate cell proliferation and apoptosis in vitro.

Decreased expression of long noncoding RNA GAS5 indicates a poor prognosis and promotes cell proliferation in gastric cancer

BackgroundGastric cancer is the second leading cause of cancer death and remains a major clinical challenge due to poor prognosis and limited treatment options. Long noncoding RNAs (lncRNAs) have emerged recently as major players in tumor biology and may be used for cancer diagnosis, prognosis, and potential therapeutic targets. Although downregulation of lncRNA GAS5 (Growth Arrest-Specific Transcript) in several cancers has been studied, its role in gastric cancer remains unknown. Our studies were designed to investigate the expression, biological role and clinical significance of GAS5 in gastric cancer.MethodsExpression of GAS5 was analyzed in 89 gastric cancer tissues and five gastric cancer 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 GAS5. The effect of GAS5 on proliferation was evaluated by MTT and colony formation assays, and cell apoptosis was evaluated by hochest stainning. Gastric cancer cells transfected with pCDNA3.1 -GAS5 were injected into nude mice to study the effect of GAS5 on tumorigenesis in vivo. Protein levels of GAS5 targets were determined by western blot analysis. Differences between groups were tested for significance using Student’s t-test (two-tailed).ResultsWe found that GAS5 expression was markedly downregulated in gastric cancer tissues, and associated with larger tumor size and advanced pathologic stage. Patients with low GAS5 expression level had poorer disease-free survival (DFS; P = 0.001) and overall survival (OS; P < 0.001) than those with high GAS5 expression. Further multivariable Cox regression analysis suggested that decreased GAS5 was an independent prognostic indicator for this disease (P = 0.006, HR = 0.412; 95%CI = 2.218–0.766). Moreover, ectopic expression of GAS5 was demonstrated to decrease gastric cancer cell proliferation and induce apoptosis in vitro and in vivo, while downregulation of endogenous GAS5 could promote cell proliferation. Finally, we found that GAS5 could influence gastric cancer cells proliferation, partly via regulating E2F1 and P21 expression.ConclusionOur study presents that GAS5 is significantly downregulated in gastric cancer tissues and may represent a new marker of poor prognosis and a potential therapeutic target for gastric cancer intervention.

Upregulation of the long non-coding RNA HOTAIR promotes esophageal squamous cell carcinoma metastasis and poor prognosis.

Recent studies of the individual functionalities of long non‐coding RNAs (lncRNAs) in the development and progression of cancer have suggested that HOX transcript antisense RNA (HOTAIR) is capable of reprogramming chromatin organization and promoting cancer cell metastasis. In order to ascertain the expression pattern of the lncRNA HOTAIR and assess its biological role in the development and progression of esophageal squamous cell carcinoma (ESCC), HOTAIR expression in ESCC tissues and adjacent noncancerous tissues were collected from 78 patients and measured by real‐time reverse transcription‐polymerase chain reaction (RT‐PCR). HOTAIR correlation with clinicopathological features and prognosis was also analyzed. Suppression of HOTAIR using siRNA treatment was performed in order to explore its role in tumor progression. Notably elevated HOTAIR expression levels were observed in cancerous tissues compared to adjacent noncancerous tissues (96%, P < 0.01), showing a high correlation with cancer metastasis (P < 0.01), elevated TNM (2009) stage classification (P < 0.01), and lowered overall survival rates (P = 0.003). Multivariate analysis revealed that HOTAIR expression (P = 0.003) is also an independent prognostic factor for comparison of TNM stage (P = 0.024) and lymph node metastasis (P = 0.010). Furthermore, in vitro assays of the ESCC cell line KYSE30 demonstrated that knockdown of HOTAIR reduced cell invasiveness and migration while increasing the response of cells to apoptosis. Thus, HOTAIR is a novel molecule involved in both ESCC progression and prognosis. Full elucidation of HOTAIR functionality relevant to ESCC may open avenues for the use of lncRNAs in identification of novel drug targets and therapies for ESCC and other prevalent cancers.

The Long Noncoding RNA HOTAIR Contributes to Cisplatin Resistance of Human Lung Adenocarcinoma Cells via downregualtion of p21WAF1/CIP1 Expression

HOTAIR, a long intervening non-coding RNA (lincRNA), associates with the Polycomb Repressive Complex 2 (PRC2) and is reported to reprogram chromatin organization and promote tumor progression. However, little is known about the roles of this gene in the development of chemoresistance phenotype of lung adenocarcinoma (LAD). Thus, we investigated the involvement of HOTAIR in the resistance of LAD cells to cisplatin. In this study, we show that HOTAIR expression was significantly upregulated in cisplatin-resistant A549/DDP cells compared with in parental A549 cells. Knockdown of HOTAIR by RNA interference could resensitize the responses of A549/DDP cells to cisplatin both in vitro and in vivo. In contrast, overexpression of HOTAIR could decrease the sensitivity of A549 and SPC-A1 cells to cisplatin. We also found that the siRNA/HOTAIR1-mediated chemosensivity enhancement was associated with inhibition of cell proliferation, induction of G0/G1 cell-cycle arrest and apoptosis enhancement through regulation of p21WAF1/CIP1 (p21) expression. Also, pcDNA/p21or siRNA/p21 could mimic the effects of siRNA/HOTAIR1 or pcDNA/HOTAIR on the sensitivity of LAD cells to cisplatin. Importantly, siRNA/p21 or pcDNA/p21 could partially rescue the effects of siRNA/HOTAIR1 or pcDNA/HOTAIR on both p21 expression and cisplatin sensitivity in LAD cells. Further, HOTAIR was observed to be significantly downregulated in cisplatin-responding LAD tissues, and its expression was inversely correlated with p21 mRNA expression. Taken together, our findings suggest that upregulation of HOTAIR contributes to the cisplatin resistance of LAD cells, at least in part, through the regulation of p21 expression.

Long noncoding RNA PVT1 indicates a poor prognosis of gastric cancer and promotes cell proliferation through epigenetically regulating p15 and p16

BackgroundMounting evidence indicates that long noncoding RNAs (lncRNAs) could play a pivotal role in cancer biology. However, the overall biological role and clinical significance of PVT1 in gastric carcinogenesis remains largely unknown.MethodsExpression of PVT1 was analyzed in 80 GC tissues and cell lines by qRT-PCR. The effect of PVT1 on proliferation was evaluated by MTT and colony formation assays, and cell apoptosis was evaluated by Flow-cytometric analysis. GC cells transfected with shPVT1 were injected into nude mice to study the effect of PVT1 on tumorigenesis in vivo. RIP was performed to confirm the interaction between PVT1 and EZH2. ChIP was used to study the promoter region of related genes.ResultsThe higher expression of PVT1 was significantly correlated with deeper invasion depth and advanced TNM stage. Multivariate analyses revealed that PVT1 expression served as an independent predictor for overall survival (p = 0.031). Further experiments demonstrated that PVT1 knockdown significantly inhibited the proliferation both in vitro and in vivo. Importantly, we also showed that PVT1 played a key role in G1 arrest. Moreover, we further confirmed that PVT1 was associated with enhancer of zeste homolog 2 (EZH2) and that this association was required for the repression of p15 and p16. To our knowledge, this is the first report showed that the role and the mechanism of PVT1 in the progression of gastric cancer.ConclusionsTogether, these results suggest that lncRNA PVT1 may serve as a candidate prognostic biomarker and target for new therapies in human gastric cancer.

EZH2-mediated epigenetic suppression of long noncoding RNA SPRY4-IT1 promotes NSCLC cell proliferation and metastasis by affecting the epithelial-mesenchymal transition.

Recent evidence indicates that long noncoding RNAs (lncRNAs) have a critical role in the regulation of cellular processes such as differentiation, proliferation, and metastasis. These lncRNAs are dysregulated in a variety of cancers and many function as tumor suppressors; however, the regulatory factors involved in silencing lncRNA transcription are poorly understood. In this study, we showed that epigenetic silencing of lncRNA SPRY4 intronic transcript 1 (SPRY4-IT1) occurs in non-small-cell lung cancer (NSCLC) cells through direct transcriptional repression mediated by the Polycomb group protein enhancer of zeste homolog 2 (EZH2). SPRY4-IT1 is derived from an intron within SPRY4, and is upregulated in melanoma cells; knockdown of its expression leads to cell growth arrest, invasion inhibition, and elevated rates of apoptosis. Upon depletion of EZH2 by RNA interference, SPRY4-IT1 expression was restored, and transfection of SPRY4-IT1 into NSCLC cells resulted in a significant antitumoral effect, both in culture and in xenografted nude mice. Moreover, overexpression of SPRY4-IT1 was found to have a key role in the epithelial–mesenchymal transition through the regulation of E-cadherin and vimentin expression. In EZH2-knockdown cells, which characteristically showed impaired cell proliferation and metastasis, the induction of SPRY4-IT1 depletion partially rescued the oncogenic phenotype, suggesting that SPRY4-IT1 repression has an important role in EZH2 oncogenesis. Of most relevance, translation of these findings into human NSCLC tissue samples demonstrated that patients with low levels of SPRY4-IT1 expression had a shorter overall survival time, suggesting that SPRY4-IT1 could be a biomarker for poor prognosis of NSCLC.

Downregulation of BRAF activated non-coding RNA is associated with poor prognosis for non-small cell lung cancer and promotes metastasis by affecting epithelial-mesenchymal transition

BackgroundRecent evidence indicates that long noncoding RNAs (lncRNAs) play a critical role in the regulation of cellular processes, such as differentiation, proliferation and metastasis. These lncRNAs are found to be dysregulated in a variety of cancers. BRAF activated non-coding RNA (BANCR) is a 693-bp transcript on chromosome 9 with a potential functional role in melanoma cell migration. The clinical significance of BANCR, and its’ molecular mechanisms controlling cancer cell migration and metastasis are unclear.MethodsExpression of BANCR was analyzed in 113 non-small cell lung cancer (NSCLC) tissues and seven NSCLC cell lines using quantitative polymerase chain reaction (qPCR) assays. Gain and loss of function approaches were used to investigate the biological role of BANCR in NSCLC cells. The effects of BANCR on cell viability were evaluated by MTT and colony formation assays. Apoptosis was evaluated by Hoechst staining and flow cytometry. Nude mice were used to examine the effects of BANCR on tumor cell metastasis in vivo. Protein levels of BANCR targets were determined by western blotting and fluorescent immunohistochemistry.ResultsBANCR expression was significantly decreased in 113 NSCLC tumor tissues compared with normal tissues. Additionally, reduced BANCR expression was associated with larger tumor size, advanced pathological stage, metastasis distance, and shorter overall survival of NSCLC patients. Reduced BANCR expression was found to be an independent prognostic factor for NSCLC. Histone deacetylation was involved in the downregulation of BANCR in NSCLC cells. Ectopic expression of BANCR impaired cell viability and invasion, leading to the inhibition of metastasis in vitro and in vivo. However, knockdown of BANCR expression promoted cell migration and invasion in vitro. Overexpression of BANCR was found to play a key role in epithelial-mesenchymal transition (EMT) through the regulation of E-cadherin, N-cadherin and Vimentin expression.ConclusionWe determined that BANCR actively functions as a regulator of EMT during NSCLC metastasis, suggesting that BANCR could be a biomarker for poor prognosis of NSCLC.

LncRNA HOXA11-AS promotes proliferation and invasion of gastric cancer by scaffolding the chromatin modification factors PRC2, LSD1 and DNMT1

Long noncoding RNAs (lncRNA) have been implicated in human cancer but their mechanisms of action are mainly undocumented. In this study, we investigated lncRNA alterations that contribute to gastric cancer through an analysis of The Cancer Genome Atlas RNA sequencing data and other publicly available microarray data. Here we report the gastric cancer-associated lncRNA HOXA11-AS as a key regulator of gastric cancer development and progression. Patients with high HOXA11-AS expression had a shorter survival and poorer prognosis. In vitro and in vivo assays of HOXA11-AS alterations revealed a complex integrated phenotype affecting cell growth, migration, invasion, and apoptosis. Strikingly, high-throughput sequencing analysis after HOXA11-AS silencing highlighted alterations in cell proliferation and cell-cell adhesion pathways. Mechanistically, EZH2 along with the histone demethylase LSD1 or DNMT1 were recruited by HOXA11-AS, which functioned as a scaffold. HOXA11-AS also functioned as a molecular sponge for miR-1297, antagonizing its ability to repress EZH2 protein translation. In addition, we found that E2F1 was involved in HOXA11-AS activation in gastric cancer cells. Taken together, our findings support a model in which the EZH2/HOXA11-AS/LSD1 complex and HOXA11-AS/miR-1297/EZH2 cross-talk serve as critical effectors in gastric cancer tumorigenesis and progression, suggesting new therapeutic directions in gastric cancer. Cancer Res; 76(21); 6299-310. ©2016 AACR.