Profiling And Validation Of Metastasis Suppression Related MicroRNAs In Human Gastric Cancer

Profiling and validation of metastasis suppression related microRNAs in human gastric cancerAt present human gastric cancer ranks as the second most frequent cause of cancer-related death worldwide, distant metastasis of GC is the leading cause of death in patients, has become a major obstacle to clinical treatment of GC. In order to metastasize, tumor cells were regulated by multiple factors, associated with multiple genes and must undergo a series of sequential and selective cascading events, in theory the course of tumor cells metastasis include detachment, migration, local invasion, angiogenesis, intravasation, survival in the circulatory system, extravasation, and regrowth in distant metastatic site. At present, varieties of relevant factors involved in the gastric cancer cell adhesion, invasion,motion and migration of the metastatic cascade is gradually being recognized. However, distant metastasis of GC is an extremely complex process, and many studies have shown that a variety of genes, enzymes and cytokines involved in the regulation to promote or inhibit metastasis of GC. Till today the exact molecular mechanisms of GC metastasis have not yet been fully elucidated,so it will have a profound impact on the prevention and clinical prognosis of GC to explore and research actively until reveal the exact molecular mechanism of GC metastasis. MicroRNAs are endogenous, non-coding, single-stranded and high conserved small RNA molecules that negatively regulate target gene expression post-transcriptionally, and involve in almost all the biological events in the course of life activities. Growing research suggest that microRNAs have a close relationship with cancer, and not only regulate tumor cell proliferation and apoptosis, but also involve in tumor cell metastasis. A large number of microRNAs have been recently implicated in cancer metastasis, including miR-10b, miR-21, miR-126, miR-335, miR-373, miR-146, miR-520c, and miR-205 in breast cancer; miR-224 and miR-21 in prostate cancer; miR-29c in nasopharyngeal carcinomas; miR-182 in melanoma; miR-21 in colorectal cancer and miR-10a, miR-222, miR-125b, miR-7, and miR-452 in urothelial carcinomas; And miR-218 inhibits metastasis of GC through the regulation of Robo1 receptor. Since the damage caused by high metastatic ability of GC and the ambiguous situation of GC metastasis exact molecular mechanism,whether there are any other microRNAs involved in regulation of metastasis of GC deserves further study. In this study we detected and compared the microRNAs expression profile between the primary tumor tissue and the metastatic tumor tissue of GC patients by microRNAs array, and then verified the differentially expressed microRNAs by Realtime PCR and bioinformatics function prediction, the down regulated microRNAs probably involved in invasion and metastasis suppression of GC were validated further by Tumor cell proliferation experiments and Transwell invasion assay at cell level, expect to find out novel microRNAs related with metastasis suppression of GC, consequently it will contribute to reveal the molecular mechanisms of GC metastasis and explore the new therapeutic target of inhibiting metastasis of GC, and rich biological function of microRNAs further.PartⅠProfiling metastasis suppression related microRNAs in human gastric cancerObjective: To screen out the down regulated microRNAs in GC patients metastatic tumor tissue by detecting expression profile of microRNAs in human GC metastatic and primary tumor tissue,Methods: MicroRNAs array were used to detect expression profile of microRNAs in 3 cases of GC patients metastatic and primary tumor tissue, and screened out the down regulated microRNAs in human GC metastatic tumor tissue meeting the standard of (≤0.67 and >0)-fold down regulation.Results: The result of microRNAs array showed that hsa-miR-508-5p, hsa-miR-30c, hsa-miR-337-3p, hsa-miR-483-5p and hsa-miR-134 were down-regulated in 3 cases of GC patients metastatic tissue.Conclusions:1. Expression profile of microRNAs between GC patients metastatic and primary tumor tissue were differential.2. hsa-miR-508-5p,hsa-miR-337-3p,hsa-miR-30c,hsa-miR-483-5p and hsa-miR-134 down regulated in GC patients metastatic tissue, may be associated with metastasis suppression in human GC. PartⅡBioinformatics function prediction and quantitative analysis of candidate microRNAsObjective: To screen out low expressed microRNAs probably related with metastasis suppression in GC, we predicted potential target genes and their corresponding signal transduction pathway of candidate microRNAs by bioinformatics, and quantitativly analyzed their expression in human GC metastatic and primary tumor tissue at the basis of expanding sample size.Methods: TargetScan biological databases and Forecast engine self-developed by Shanghai QiMing bioinformation technology Co., Ltd were used to predict potential target genes of candidate microRNAs, and the fisher exact test were used to compute significant function and signaling pathways, then construct the candidate microRNAs and their Target genes regulatory network diagram; Realtime PCR method was used to detect candidate microRNAs expression in 16 cases of GC patients metastatic and primary tumor tissue to screen out low expressed microRNAs in human GC metastatic tissue.Results:1. Bioinformatics function prediction suggested that hsa-miR-508-5p, hsa-miR-337-3p, hsa-miR-30c and hsa-miR-134 have potential target genes and their corresponding Signaling pathways regulating tumor metastasis.2. Realtime PCR quantitative analysis showed that hsa-miR-337-3p and hsa-miR-134 significantly down regulated in GC patients metastatic tissue compared with primary tumor tissue(P<0.05).conclusions: hsa-miR-134 and hsa-miR-337-3p significantly down regulated in GC patients metastatic tissue, may be associated with metastasis suppression in GC.PartⅢInfluence of candidate microRNAs on proliferation and metastasis of human gastric cancer cell lineObjective: To study the influence of hsa-miR-337-3p and hsa-miR-134 on tumor cell proliferation and metastasis of human gastric cancer cell line in vitro. Methods: Realtime PCR method was used to detect expression of hsa-miR-337-3p and hsa-miR-134 in several human gastric cancer cell lines; CCK-8 cell proliferation experiments and Transwell cell invasion assay were used to observe the influence on tumor cell proliferation and invasion of human GC cell line MKN-45 by using the mimic and inhibitor of hsa-miR-337-3p and hsa-miR-134 to transfect human GC cell line MKN-45.Results:1. hsa-miR-134 up regulated in SNU-5, AGS, HGC-27, MGC-803, MKN-28 and MKN-45 compared with GES cells, while down regulated in SNU-1; hsa-miR-337-3p down regulated in GC, only trace expression of hsa-miR-337-3p could be detected in SNU-5, HGC27 and SGC-7901, and couldn't be detected in remaining gastric cancer cell lines.2. Cell proliferation experiments showed that the expression of hsa-miR-134 or hsa-miR-337-3p didn't influence proliferation status of GC cell line MKN-45.3. Cell invasion assay showed that the expression of hsa-miR-134 didn't influence the ability of metastasis of GC cell line MKN-45; The group transfected with mimic of hsa-miR-337-3p could inhibit metastasis of GC cell line MKN-45 significantly compared with control group.Conclusion: hsa-miR-337-3p could inhibit metastasis in GC in vitro.