The Study Of RKIP About Its Role And Mechanism In Gastric Cancer Cells

Part One RKIP suppresses gastric cancer cell proliferation and invasion, improves apoptosis regulated by microRNA-224Backgroud:Gastric cancer is a malignant tumor with a relatively high incidence, and its tumorigenesis process has been closely correlated to precancerous lesions such as chronic atrophic gastritis and intestinal metaplasia. The development and progression of gastric cancer are related to multiple gene abnormalities. As a complicated biological process, gastric cancer metastasis is generally associated with certain changes in cytokines and signaling pathways. Investigations of tumor invasion and metastasis have demonstrated the important roles of miRNA during the course of these processes, as miRNAs regulate at the post-transcriptional level various critical genes related to invasion and metastasis. Therefore, the elucidation of the pathogenesis of gastric cancer and investigations of novel approaches and strategies for the diagnosis and treatment of gastric cancer are urgent tasks for improving the survival and prognosis of patients with gastric cancer. Raf kinase inhibitor protein (RKIP), one of the member of the phosphatidylethanolamine-binding protein (PEBP) family, was first isolated from bovine brain as a small cytoplasmic protein and was consequently identified as being extensively expressed in tissues from monkeys, chickens, rats and humans. As reported in other studies, RKIP was found to block the MAPK signal transduction pathway and suppress Raf-1-mediated MEK activation (also known as Raf kinase inhibitory protein). It was reported that RKIP could suppress the metastasis of tumor cells, RKIP has been highlighted in cancer studies. The purposes of this study were to determine the expression profile of RAF kinase inhibitor protein (RKIP) in human gastric cancer cells and its effect on the biological characteristics of SGC-7901 cell lines, to examine the modulatory effect of microRNA-224 (miR-224) on RKIP. The research will provide novel strategies for gastric cancer treatment in the future. Methods:Quantitative real-time PCR (qRT-PCR) was employed to determine the expression profile of RKIP in gastric cancer cell lines (SGC-7901. MGC80-3 and MKN45). A eukaryotic expression vector, pcDNA3.1-RKIP, was constructed and transfected into SGC-7901 cells. Changes in RKIP protein expression were examined by western blot assays, and the effect of RKIP overexpression on SCG-7901 cell viability was determined by MTT. The effect on SGC-7901 cell proliferation and apoptosis of RKIP overexpression was analyzed by flow cytometry and that on the migration of SGC-7901 cells was investigated by Transwell migration assays. RKIP was identified to be a regulatory target gene of miR-224 using a Luciferase Reporter Gene System, and the effect of miR-224 on intracellular RKIP protein expression was examined by western blot assays. The regulatory effect of miR-224 on the biological characteristics of RKIP was investigated by MTT, flow cytometry and Transwell invasion chamber assays. Results:The expression of RKIP in gastric cancer cells was decreased significantly in comparison to that of normal gastric mucosal cells (GES-1) (p<0.01), as demonstrated by qRT-PCR assays. After transfection of pcDNA3.1-RKIP, compared with the control group, the up-regulation of RKIP intracellular expression was observed in SGC-7901 cells (p<0.01). There were significant decreases in cell viability and the S-phase fraction (p<0.05), concomitant with a significant increase in apoptosis (p<0.01), as well as a significant reduction in cells migrating through Transwell chambers (p<0.05), as shown by MTT, flow cytometry and Transwell invasion chamber assays. A significant decrease in luciferase activities in cells transfected with a miR-224 mimic was observed compared with that of the control group (p<0.05), as suggested by the Luciferase Reporter Gene System. As shown by western blot assays, after transfected with the miR-224 mimic for 48 hours, there was a significant decrease in RKIP expression in SGC-7901 cells compared with the control group (p<0.05). As shown by MTT, flow cytometry and Transwell invasion chamber assays, the changes in biological characteristics induced by RKIP overexpression could be suppressed in SGC-7901 cells after transfection of the miR-224 mimic. Conclusion: The down-regulation of RKIP expression was observed in human gastric cell lines. RKIP could be regulated negatively the expression and biological characteristics by miR-224. contributing to suppress the proliferation and invasion of gastric cells.Part Two RKIP promotes cisplatin-induced gastric cancer cell death through NF-κB/Snail pathwayBackgroud:Gastric cancer carries a poor prognosis and high mortality. The success rate of chemotherapy in treating gastric cancer is highly affected by the resistance to chemotherapy, it has been increasingly recognized that the apoptosis of tumor cells is a key indicator for measuring the effectiveness of chemotherapy, and the apoptosis is associated with the chemotherapy drugs in a time-and dose-dependent manner. In 2013, Martinho O et al reported that RKIP was correlated with chemo-sensitivity to cisplatin in cervical cancer. Cisplatin is one of the most commonly used drug for adjuvant chemotherapy and neoadjuvant chemotherapy, and also a common drug used in the testing for anti-cancer drug sensitivity preformed in in vitro cultured gastric cancer tissues. Research has shown that cisplatin can bind to DNA and form crosslinks, which can inhibit DNA synthesis and thus suppress cell division and induce apoptosis. However, the presence of drug resistance inside cancer cells will lower the efficacy of cisplatin. The research is designed to explore the expression profiles of RAF kinase inhibitor protein (RKIP) in SGC-7901/DDP (cisplatin-resistant cell line) and SGC-7901 (human gastric cancer cell line), and investigate the role of RKIP in the sensitivity of human gastric cancer cells to cisplatin and its signalling pathways, with an attempt to identify new approaches and strategies for the management of gastric cancer. Methods:The SGC-7901 and SGC-7901/DDP were cultured separately in vitro. The RKIP expression profiles in these two cell lines were detected by Western blotting. Forty-eight hours after the transfection of RKIP siRNA in SGC-7901 cells, the change of RKIP expression in the cells was detected using Western blotting, and the change of cell viability after the interference of RKIP expression was determined using MTT method. The effect of the ectopic expression of RKIP on the cisplatin-induced viability of gastric cancer cell was detected using MTT method. The effect of the ectopic expression of RKIP on the cisplatin-induced apoptosis of gastric cancer cell was detected using flow cytometry after having been double stained with Annexin V/PI. The effect of the ectopic expression of RKIP on the NF-κB and Snail expressions in cisplatin-induced gastric cancer cells were detected using Western blotting. Results:As shown by the Western blotting, the expression of RKIP in SGC-7901/DDP cells significantly decreased when compared with that in SGC-7901 cells (P<0.05). Compared with the control group, the expression of RKIP in SGC-7901 cells significantly decreased 48 hours after the transfection of RKIP siRNA (P<0.01). After the SGC-7901 cells were transfected with RKIP siRNA, the cell viability was significantly increased (P<0.05); after the SGC-7901/DDP cells were transfected with RKIP recombinant plasmid, the cell viability was significantly decreased (P<0.05). After the RKIP expression was suppressed in the cisplatin-treated SGC-7901 cells, the cell viability significantly increased (P<0.05) and the amount of apoptotic cells significantly decreased (P<0.05). In contrast, after the RKIP over-expression in the cisplatin-treated SGC-7901/DDP cells, the cell viability significantly decreased (P<0.05) and the amount of apoptotic cells significantly increased (P<0.05). The suppression of RKIP expression in SGC-7901 cells could significantly promote the increase of NF-κB expression (P<0.05); in contrast, the increased expression of RKIP in SGC-7901/DDP cells significantly inhibited the expression of Snail (P<0.05). Conclusions:The RKIP expression is down-regulated in cisplatin-resistant cell line (SGC-7901/DDP). The over-expression of RKIP can enhance the sensitivity of human gastric cancer cells to cisplatin, which may be achieved via the NF-KB/Snail signaling pathway.Part Three RKIP inhibits gastric cancer cell survival and invasion by regulating the exprssion of HMGA2 and OPNBackgroud:The high mobility group AT-hook 2 (HMGA2) plays a key role in the early embryonic development and during the occurence and development of many malignant tumors. As an oncogenic protein, HMGA2 is highly expressed in many malignant tumors including pancreatic cancer, non-small-cell lung cancer, colorectal cancer, breast cancer, and ovarian cancer, indicating that HMGA2 plays an important role in the tumorigenesis. Sugiko et al found that HMGA2 is the indirect target gene of MEK in human pancreatic carcinoma. HMGA2 can directly bind Snaill gene promoter regions and thus promotes epithelial-mesenchymal transition in pancreatic cancer cells by inhibiting the transcription of E-cadherin. Osteopontin (OPN) is a secreted phosphorylated protein. Our preliminary results have shown that the RKIP expression is down-regulated in human gastric cancer cell lines, which can inhibit the invasion and proliferation of gastric cancer cells. Then, what are the mechanisms via which RKIP suppresses the proliferation and invasion of gastric cancer cells and promote their apoptosis? Recent studies have also shown that RKIP can also block the activity of MAPK signaling pathway and inhibit the Raf-1-mediated MEK activation. HMGA2 is an indirect target gene of MEK. It has been proposed that RKIP suppresses the biological activities of HMGA2 by suppressing the activation of MEK, thus exerting its roles in inhibiting the proliferation and invasion of gastric cancer cells and promote their apoptosis. The objective of this study was to explore the mechanism via which RKIP suppress the invasion of gastric cancer cells and promote apoptosis, with an attempt to provide evidences for the application of RKIP for treating gastric cancer. Methods:The RKIP and HMGA2 recombinant expression plasmids pcDNA3.1-RKIP and pcDNA3.1-HMGA2 were constructed. The recombinant plasmid pcDNA3.1-RKIP or RKIP-shRNA were transfected into the gastric cancer cell line SGC-7901 using liposome. After the ectopic expression of RKIP in SGC-7901 cells was detected using real-time quantitative PCR and Western blotting, the mRNA and protein expressions of RKIP, HMGA2, and OPN in cells were determined. After the recombinant plasmids pcDNA3.1-HMGA2 or HMGA2-shRNA were transfected into the gastric cancer cell line SGC-7901, the expression of HMGA2 in SGC-7901 cells were detected using Western blotting. The effects of HMGA2 on the proliferation, apoptosis, and invasion of SGC-7901 cells after the ectopic expression of HMGA2 in SGC-7901 cells were detected using flow cytometry and Transwell assay. To further explore the regulatory effect of PKIP on the biological activities of HMGA2, we over-expressed RKIP and HMGA2 simultaneously in SGC-7901 cells or interfered the RKIP and HMGA2 at the same time; meanwhile, we applied flow cytometry and Transwell assay to detect its effects on the proliferation, apoptosis, and invasion of SGC-7901 cells. Results:As shown by real-time quantitative PCR and Western blotting, After the transfection of pcDNA3.1-RKIP in SGC-7901 cells, the mRNA and protein expressions of RKIP significantly increased (p<0.01), whereas the mRNA and protein expressions of HMGA2 and OPN significantly decreased (p<0.01). In contrast, the transfection of RKIP-shRNA in the SGC-7901 cells resulted in opposite results. After the transfection of pcDNA3.1-HMGA2 in SGC-7901 cells, the protein expression of HMGA2 significantly increased (p<0.01); however, it significantly decreased after the transfection of HMGA2-shRNA (p<0.01). As shown by Transwell assay and flow cytometry, After the over-expression of HMGA2 in SGC-7901 cells, the (G2+S) phase fraction significantly increased (p<0.01); the apoptotic cells declined (p<0.05) and the number of invasive cells significantly increased (p<0.05). However, the interference of the expression of HMGA2 resulted in opposite results. The simultaneous over-expression of RKIP and HMGA2 in SGC-7901 cells or the simultaneous interference of RKIP and HMGA2 showed no significant difference with the control group in terms of (G2+S) phase fraction, percentage of apoptotic cells, and number of invasive cells (p>0.05). Conclusion:RKIP can inhibit the survival and invasion of gastric cancer cells and promote apoptosis, possibly by regulating the expression of HMGA2 or OPN.