Functional Elucidation & Mechanistic Study Of MicroRNA-7 In Gastric Carcinogenesis

Background Gastric cancer remains the most malignant tumor throughout China and the world, with high morbidity and mortality. The development and progression of gastric cancer is a sequential process intertwined with multiple malignant phenotypes that mutually influence each other. Under the influence of all of these molecular events, epithelial cells are in a disturbed equilibrium state and undergo disorder and chaos. Thus, elucidation of the crucial molecular events during gastric carcinogenesis and clarification of the underlying molecular mechanisms will provide new strategies for gastric cancer treatment.Micro RNAs(mi RNA) are a group of recently identified endogenous non-coding RNAs that play an important regulatory role in eukaryotic cells. mi RNA functions in RNA silencing and the post-transcriptional regulation of gene expression by binding to the 3’-untranslated regions(UTRs) of target m RNAs, thus regulating the normal physiological functions and human disease. mi RNA functions in a “one to multiple” mode, which means that one mi RNA may regulate a set of genes as its targets. Such regulatory modes could cause alterations among multiple protein-protein interactions and among the entire pathways. Thus, mi RNAs are well known for their critical roles in regulating cell biological behaviors.micro RNA-7(mi R-7) was first discovered in 2001 in Drosophila. Since 2008, the function of mi R-7 in tumors has been reported. Some reports indicate that mi R-7 is downregulated in liver, lung and breast cancers and functions as a tumor suppressor, whereas others suggest that mi R-7 promotes carcinogenesis. However, the function of mi R-7 in tumors and its underlying mechanisms remain elusive. Our previous work identified mi R-7 as one of the most downregulated mi RNAs in highly metastatic gastric cancer cells based on mi RNA microarray analysis, suggesting that mi R-7 plays a tumor suppressive role in gastric cancer carcinogenesis and progression. Based on previous reports and our preliminary data, the current study aims to determine the role of mi R-7 in gastric cancer, elucidate its key functions and regulatory mechanisms, and thus provide novel understanding of the mechanism by which mi RNA functions in gastric cancer.Objectives1. To determine the correlation of mi R-7 levels with the clinic pathological parameters of gastric cancer;2. To determine the roles of mi R-7 in regulating the malignant biological characteristics of gastric cancers; 3. To elucidate the underlying mechanisms that mediate mi R-7 function; 4. To explore the causes of mi R-7 downregulation in gastric cancer.Methods 1. Real-time polymerase chain reaction(PCR) was used to determine mi R-7 expression in a normal gastric epithelial cell line and adenocarcinoma cell lines. Samples from fresh gastric cancer tissues and matched adjacent normal tissues were also collected and used for real-time PCR detection of mi R-7 expression. In situ hybridization(ISH) was utilized to detect mi R-7 expression levels in gastric cancer tissue arrays, and statistical analyses were performed to compare the correlation between mi R-7 expression and clinicopathological parameters.2. mi R-7 mimics and inhibitors were synthesized, and mi R-7 overexpression and sh RNA lentiviral vectors were also constructed. mi R-7 gain- and loss-of-function models were established via transient transfection and stable infection. The role of mi R-7 in gastric cancer cell proliferation, apoptosis and metastasis was studied using the MTT assay, colony formation assay, soft agar colony assay, flow cytometry, Transwell migration and invasion assay, and nude mice xenograft assay and tail vein metastatic assay.3. Via the combinational use of proteomic i TRAQ technology and c DNA microarrays, the direct targets of mi R-7 were screened using high throughput methods at the transcriptional and post-transcriptional levels. Bioinformatic analysis was performed to select the molecules that were differentially expressed. A dual-luciferase reporter gene system was used to verify the relationship between mi R-7 and its potential target genes. Western blot and real-time PCR were also performed to validate the expression changes of target genes after up- or down-regulation of mi R-7. Functional studies and rescue assays were also performed to evaluate the effect of mi R-7 on gastric malignant phenotypes mediated by those target molecules.4. Bioinformatic analyses were performed to predict the upstream transcription factor binding site in the mi R-7 promoter region. Real-time PCR was used to detect the change in expression of the mi R-7 initial transcript(primary mi R-7, pri-mi R-7) upon modulation of those transcription factors. Using chromatin immunoprecipitation(Ch IP) assays, the binding of candidate transcription factors to the mi R-7 promoter region was further confirmed. Immunofluorescence and Western blot were used to detect the expression level and the sub-cellular localization of those transcription factors after up- or down-regulation of mi R-7 expression. The changes in downstream effector molecules were also detected.Results 1. Real-time PCR results demonstrated that mi R-7 expression was significantly decreased in gastric cancer cells and further reduced in gastric cancer cell lines with high metastatic potential compared with normal gastric epithelial cells. Compared with adjacent normal tissues, mi R-7 expression was greatly reduced in the gastric cancer tissues and further reduced in the metastatic sites compared with the primary tumor. ISH in tissue arrays revealed that mi R-7 is lowly expressed in most gastric cancer specimens and is negatively correlated with clinical stage. Furthermore, its expression is positively correlated with the patient survival time and could serve as an independent risk factor for the prognosis of gastric cancer.2. mi R-7 expression was successfully modulated either by transient transfection of mi R-7 mimics and inhibitors or stable mi R-7 lentiviral infection. MTT, colony formation and soft agar colony formation assays revealed that mi R-7 inhibits the in vitro growth and proliferation of gastric cancer cells. Cell cycle analysis by flow cytometry revealed that mi R-7 inhibits cell cycle progression in gastric cancer cells. Cell apoptosis detection by flow cytometry also revealed that mi R-7 promotes early apoptosis in gastric cancer cells. Transwell migration and invasion assays indicated that mi R-7 inhibits the migration and invasion of gastric cancer cells. Nude mice xenograft assays revealed that mi R-7 can inhibit the in vivo growth of gastric cancer cells. Tail vein metastatic assays demonstrated that mi R-7 has the capacity to inhibit gastric cancer cells from forming metastatic sites in the liver, lung and other organs.3. BGC823 cells were transiently transfected with mi R-7 mimics. Then, iTRAQ technology, c DNA microarray and bioinformatic analyses were performed in combination to identify 39 molecules that were significantly downregulated in the genome-wide high-throughput screen. Among them, 16 molecules contain potential mi R-7 binding sites. Our study mainly focused on mi R-7-mediated regulation of RELA, FOS and IGF1 R. Dual luciferase reporter gene assay results indicate that mi R-7 directly binds the 3’ untranslated region(UTR) of RELA, FOS and IGF1 R. q PCR and Western blot analysis revealed that mi R-7 expression is negatively correlated with both m RNA and protein expression of RELA and FOS. In contrast, mi R-7 expression is negatively correlated with IGF1 R expression at the protein level. By modulating RELA and FOS expression in the subsequent functional study, we demonstrated that FELA and FOS promote gastric cancer cell proliferation and contribute to resistance to apoptosis. Rescue experiments indicated that mi R-7 inhibits cancer cell proliferation through the suppression of RELA and FOS. Further studies also revealed that mi R-7 inhibited IKKε to reduce the activation of RELA. However, IGF1 R promotes gastric cancer cell invasion and metastasis according to gain and loss of function studies. Rescue studies indicated that mi R-7 inhibits gastric cancer cell invasion and metastasis by targeting IGF1 R expression. Further mechanical studies indicate that mi R-7 inhibits IGF1 R, reduces Snail expression and promote E-cadherin expression, which ultimately suppress the epithelial-mesenchymal transition(EMT) process in gastric cancer cells.4. Through bioinformatic analyses, 7 clusters of NF-κB binding site were identified in the upstream promoter region of human mi R-7. Real-time PCR results indicated that o IKKε and RELA overexpression in normal gastric epithelial cells inhibits pri-mi R-7 expression, whereas IKKε and RELA downregulation in gastric cancer cells enhances pri-mi R-7 expression. Ch IP assays also indicated that RELA could bind to the promoter region of both mi R-7-1 and mi R-7-2 genes. Immunofluorescence revealed that mi R-7 potentially suppresses the expression of IKKε and RELA and inhibits nuclear translocation. Real-time PCR results revealed that mi R-7 inhibits multiple effectors downstream of the NF-κB signaling pathway.Conclusions The current study defined mi R-7 expression levels in gastric cancer cells and tissues and revealed that decreased mi R-7 expression is closely related to the development of gastric cancer. The effects of mi R-7 in gastric malignant behaviors were also characterized. mi R-7 exerts inhibitory effects on cancer cell proliferation, apoptotic resistance and metastasis via regulation of the IKKε/RELA and IGF1R/Snail/E-cadherin axes. Furthermore, we also identified the important role of the NF-κB pathway, which is responsible for the aberrant expression of mi R-7 in gastric cancer. The current study elucidates a crucial role of mi R-7 in gastric carcinogenesis and progression and provides a potentially potent target for gastric cancer targeted therapy.