The Characteristics And Roles Of Non-coding RNAs MiR-141/miR-22 And PVT1 On Tumour Growth And Metastasis In Gastric Cancer

Gastric cancer(GC) is the fifth most prevalent type of malignancy and the third leading cause of cancer death worldwide. An estimated 951,600 new stomach cancer cases and 723,100 deaths occurred in 2012 based on Global Cancer Statistics. Although therapeutic methods are improving, the 5-years survival rates remain low and the prognosis for advanced stage patients is still very poor. Thus an improved and detailed understanding of the mechanisms underlying GC development and progression is urgently needed.Thanks to the general implementation of tiling microarrays and high-throughput sequencing technologies to whole genomes and transcriptomes, it is now evident that whereas less than 2% of the genome encodes proteins, at least 75% is actively transcribed into noncoding RNAs. It is increasingly evident that many of the genomic mutations in cancer reside inside regions that do not encode proteins. However, these regions are often transcribed into microRNAs(mi RNAs) and long noncoding RNAs(lncRNAs). The recent application of next-generation sequencing has indeed revealed thousands of miRNAs and lnc RNAs whose aberrant expression is associated with different cancer types. Notably, these mi RNAs and lnc RNAs have key roles in gene regulation and thus affect various aspects of cellular homeostasis, including proliferation, survival, migration or genomic stability.Mi RNAs are small noncoding RNAs, which regulate gene expression post-transcriptionally by base pairing to partially complementary sequences in the 3′-untranslated region of their target m RNA. Emerging evidence suggests that dysregulation of miRNAs is involved in the progression of many cancers through modulation of oncogene or tumor suppressor gene expression. In gastric cancer, many mi RNAs regulate the proliferation, apoptosis and invasion and correlate the carcinogenesis, processing and metastasis. The transcription of lncRNAs is subject to typical histone-modification- mediated regulation, and is processed by the canonical spliceosome machinery. In addition, the expression of lnc RNAs is strikingly cell type and tissue specific. The investigations on lncRNAs have demonstrated that these noncoding transcripts can serve as scaffolds or guides to regulate protein-protein or protein-DNA interactions, as enhancers to influence gene transcription, when transcribed from the enhancer regions, and as decoys to bind mi RNAs. The biological processes affected by lnc RNAs include cell proliferation, differentiation, migration, immune response, and apoptosis, all of which have been implicated in tumorigenesis.Despite an increasing number of studies on the biogenesis and mechanisms of non-coding RNAs(mi RNAs and lncRNAs) in the pathogenesis of GC, the accurate expression and mechanistic function of them in GC remain elusive. Illustrating the characteristics and roles of non-coding RNAs will enrich understanding of the development and progression of GC and provide new theoretical and experimental basis for future therapy of the GC.Objectives:1. Screen and identify the non-coding RNAs involved in gastric cancer and study the characteristics and roles.2. Investigate the roles of mi R-141 and mi R-22 in the regulation of gastric cancer growth and metastasis.3. Investigate the roles of PVT1 in the epigenetic regulation of gastric cancer invasion and metastasis.Methods:1. MiRNA-141 inhibits tumor growth and metastasis in gastric cancer by directly targeting TAZScreen and identify the miRNAs and signal pathways by high-throughput tiling microarrays. qRT-PCR was employed to detect the miR-141 expression in GC matched tissues. In vitro, CCK-8 proliferation, wound-healing and Transwell invasion and migration assay were employed to detect the GC cells proliferation rate, invasion and migration after HGC-27 transfected with mi R-141, TAZ siRNAs, pcDNA3.1-TAZ and AGS transfected with anti-mi R-141. The predicted target TAZ of mi R-141 was identified via bioinformatics prediction, microarrays screening and luciferase activation assays. q RT-PCR was also employed to detect the TAZ expression in GC matched tissues and the relationship between TAZ and mi R-141 were examined by correlation analysis. In vivo, for tumor xenograft studies, HGC-27 cells transfected with agomir-141 were injected subcutaneously into the axillary fossae of nude mice. Tumor diameters were measured every 7 days. At 35 days after injection, tumors were weighted after necropsy. For metastasis assays, agomir-141-HGC-27 cells were injected injected into the lateral tail veins of nude mice. Five weeks after injection, the metastases were counted in a double-blind manner by HE staining.2. MiRNA-22 inhibits tumor growth and metastasis in gastric cancer by directly targeting MMP14 and SnailqRT-PCR was employed to detect the mi R-22 expression in GC matched tissues. To investigate the clinicopathological and prognostic significance of mi R-22 levels in patients with GC, the levels of mi R-22 GC tissues were statistically analyzed. In vitro, CCK-8 proliferation, wound-healing and transwell invasion and migration assay were employed to detect the GC cells proliferation rate, invasion and migration after SGC-7901 transfected with miR-22, MMP14/Snail si RNAs, pc DNA3.1- MMP14/Snail and AGS transfected with anti-mi R-22. The predicted targets MMP14/Snail of mi R-22 was identified via bioinformatics prediction, microarrays screening and luciferase activation assays. q RT-PCR was also employed to detect the MMP14/Snail expression in GC matched tissues and the relationship between MMP14/Snail and miR-22 were examined by correlation analysis. In vivo, for tumor xenograft studies, SGC-7901 cells transfected with agomir-22 were injected subcutaneously into the axillary fossae of nude mice. Tumor diameters were measured every 7 days. At 35 days after injection, tumors were weighted after necropsy. For peritoneal dissemination assays, agomir-22-SGC-7901 cells injected into the abdominal cavity of nude mice, the macroscopic nodules in abdominal cavity of the mice were counted at 28 days after injection. For metastasis assays, agomir-22- SGC-7901 cells were injected injected into the lateral tail veins of nude mice. Five weeks after injection, the metastases were counted in a double-blind manner by HE staining.3. lnc RNA PVT1 promotes tumour growth and metastasis in gastric cancerScreen and identify the lnc RNAs and signal pathways by high-throughput tiling microarrays. qRT-PCR was employed to detect the PVT1 expression in GC matched tissues. In vitro, wound-healing and transwell invasion and migration assay were employed to detect the GC cells invasion and migration after AGS transfected pc DNA3.1-PVT1. EMT related molecules were detected by qRT-PCR, immunofluorescence and western blotting. The binding target mi RNAs of PVT1 was identified via bioinformatics prediction, RIP and luciferase activation assays. In vivo, for tumor xenograft studies, AGS clone stable express PVT1 were injected subcutaneously into the axillary fossae of nude mice. Tumor diameters were measured every 7 days. At 35 days after injection, tumors were weighted after necropsy. For metastasis assays, AGS-PVT1 cells were injected injected into the lateral tail veins of nude mice. Five weeks after injection, the metastases were counted in a double-blind manner by HE staining.Results:1. MiRNA-141 inhibits tumor growth and metastasis in gastric cancer by directly targeting TAZGastric cancer involved mi RNAs has been screened and identified by tiling microarrays. The expression of mi R-141 was significantly reduced in GC compared with paired adjacent normal tissues and was significantly correlated with a more aggressive phenotype of GC in patients. Ectopic expression of mi R-141 mimics in GC cell lines resulted in reduced proliferation, invasion and migration, and inhibition of mi R-141 in GC cell lines promoted cell proliferation, invasion and migration in vitro. mi R-141 acted as tumor suppressors through targeting TAZ in GC. The inverse relationship between mi R-141 and its target was verified in patients and xenograft mice. Overexpression of mi R-141 suppressed tumor growth and pulmonary metastasis in nude mice.2. MiRNA-22 inhibits tumor growth and metastasis in gastric cancer by directly targeting MMP14 and SnailThe expression of miR-22 was significantly reduced in clinical GC tissues compared with paired adjacent normal tissues, and was significantly correlated with a more aggressive phenotype of GC in patients, and mi R-22 low expression correlated with poor overall survival. The introduction of mi R-22 markedly suppressed GC cell growth, migration and invasion, and inhibition of mi R-22 promoted GC cell proliferation, migration and invasion in vitro. MiR-22 acted as tumor suppressors through targeting ECM remodeling MMP14 and EMT inducer Snail in GC. Ectopic expression of MMP14 or Snail restored inhibitory effects of mi R-22 on cell migration and invasion in GC cells, and a negative relationship between the mi R-22 expression and MMP14 or Snail mRNA levels was observed in GC. Overexpression of mi R-22 suppressed tumor growth, peritoneal dissemination and pulmonary metastasis in vivo.3. lnc RNA PVT1 promotes tumour growth and metastasis in gastric cancerGastric cancer involved lnc RNAs has been screened and identified by tiling microarrays. The expression of PVT1 was significantly increased in GC compared with paired adjacent normal tissues and was significantly correlated with a more aggressive phenotype of GC in patients. Ectopic expression of PVT1 in GC cell lines enhanced cell invasion and migration in vitro. Stable expression of PVT1 promoted tumor growth and pulmonary metastasis in nude mice in vivo. qRT-PCR, immunofluorescence and western blotting analysis showed that Ectopic expression of PVT1 induces EMT in AGS cells in vitro. Bioinformatics prediction, RIP and luciferase activation assays demonstrated PVT1 could bind mi R-30 a and act as mi RNAs sponge.Conclusions:1. MiRNA-141 inhibits tumor growth and metastasis in gastric cancer by directly targeting TAZWe identified that miR-141 is a potent tumor suppressor in the stomach, and its growth inhibitory effects are, in part, mediated through its downstream target gene, TAZ. To the best of our knowledge, this is the first study to demonstrate that the mi R-141/TAZ axis regulates the proliferation, migration and invasion of GC cells. These findings provide a better understanding of the pathogenesis and development of GC and may be an important implication for future therapy of the GC.2. MiRNA-22 inhibits tumor growth and metastasis in gastric cancer by directly targeting MMP14 and SnailWe identified that miR-22 is a potent tumor suppressor in GC. MiR-22 downregulation promotes GC invasion and metastasis by upregulating MMP14 and Snail, and then inducing ECM remodeling and EMT. To the best of our knowledge, this is the first study to demonstrate that the mi R-22/MMP14/Snail axis regulates the proliferation, migration and invasion of GC cells. These findings provide a better understanding of the development and progression of GC and may be an important implication for future therapy of the GC.3. lnc RNA PVT1 promotes tumour growth and metastasis in gastric cancerWe identified that PVT1 is a potent oncogene lncRNA in GC. PVT1 promotes GC invasion and metastasis by binding mi R-30 a, and then inducing GC EMT. To the best of our knowledge, this is the first study to demonstrate that the PVT1/miR-30a/Snail axis regulates the migration and invasion of GC cells. Potentially, the findings of this study implicate the relevance of PVT1 as a possible therapeutic target for aggressive and metastatic breast cancers.