Expression Of RN181in GC And Mechanism Of RN181Regulating Cell Proliferation In Gastric Carcinoma Cell Lines

Introduction:Gastric Carcinoma (GC) is one of the most common human malignancies, and ranks first in the malignant tumors in China. In recent years, with improvement of diagnostic techniques and maturity of surgery and chemoradiation treatment strategies aiming at tumor, the overall5-year survival rate of GC has increased, but the postoperative recurrence rate is still high, and prognosis effect is not very satisfactory. Currently, a large number of research results have confirmed that the occurrence, development and prognosis of GC are the result of changes in multi-genes, multi-factors and multi-stages. Therefore, study of related gene and protein molecules in GC, is not only important to the understanding of the pathogenesis of GC, but also significant for clinically targeted therapy and improving the prognosis.Unlimited proliferation of tumor cells is closely related to cell cycle. Cell cycle refers to the whole process experienced by the continuously dividing cells, from the last end of mitosis to the next completion, and can generally be divided:G0phase (static phase), G1phase (pre-synthetic phase of DNA), S phase (DNA synthesis phase), G2phase (post-synthetic phase of DNA), M phase (mitosis phase). Meanwhile, cell cycle consists of two main checkpoints:G1/S checkpoint (DNA synthesis starting point) and G2/M checkpoint (mitosis induced conversion point). If through the inspection point, even though the stimulus signals are removed, cells will still begin DNA replication. The cell cycle is a precise and orderly regulated process in which multi-stages and multi-factors are involved. Regulation of cell cycle could be divided into exogenous and endogenous regulation. The exogenous regulation is mainly caused by externally physical and chemical stimulus, while endogenous regulation is realized by regulating the cell cycle proteins (Cyclins), cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CKIs). As we all know, the malignant tumor is characterized by apoptosis obstacle and uncontrolled proliferation, and a large number of studies have shown that the incontrolable cell cycle is closely related to the occurance and development of tumor. Hence, the study of changes about cycle-related molecules in tumors is helpful to reveal the mechanism of tumor development and has guiding significance on clinical treatment.Human RN181gene, also known as HSPC238, RNF181(ring finger protein181), has a total length of716bp, encoding153amino acids, and is named for its special RING finger domain formed by40th-60th amino acid residues. In2008, Brophy, et al first confirmed RN181possessing E3enzyme activity. Our previous study demonstrated that RN181through ERK/MAPK pathway inhibit growth of HepG2and SMMC7721hepatoma cell lines for the first time. However, the effect of RN181on other tumors and the regulation mechanisms of RN181in tumors are still poorly understood.Yeast two-hybrid system, as a common laboratory method that has been used to detect a direct intracellular protein-protein interaction, is to use a particular GAL4transcription factors in yeast cell. The two proteins (X, Y), which may have interaction, are respectively fused to the GAL4DNA binding domain (BD) and transcription activation domain (AD). Through the screening of the downstream reporter genes, the proteins possessing interaction with each other will be obtained.Therefore, this study took gastric carcinoma occurring high in clinic as object, though cytological experiments, molecular biology experiments, yeast two-hybrid and other commonly used experimental techniques, to research biological functions and mechanisms of RN181on GC cell cycle.Objectives:1To evaluate RN181expression in clinically gastric carcinoma tissues and analyze its relationship with development and progression of GC2To clarify definitely biological role of RN181in gastric cancer cells3To study the effect and mechanisms of RN181on cell-cycle regulation in gastric cancer cells in vitro and in vivo4To find the proteins that possess direct interaction with RN181and involve in cell-cycle regulation by the yeast two-hybrid systemMethods:1Immunohistochemical staining was used to evaluate RN181expression in182cases clinically gastric carcinoma tissues and adjacent normal tissues, and analyze its relationship with development and progression of GC.2Based on gene cloning techniques, the RN181fragment from pCDAN3.1(-)-flag/RN181was subcloned into the retro viral packaging plasmid plncx2-GFP-IRES. After identification by restriction enzymes digestion, DNA sequencing and Western blot, plncx2-GFP-IRES/RN181and pVSV-G were transfected into GP2-293cells for retrovirus packaging. The AGS cells infected by aforementioned retrovirus and AGS, MKN28and MKN45cells infected by RN181silencing lentivirus were sorted by FACS to obtain stable recombinant cell lines with RN181over-expression or interference. After identification by RT-PCR and western blot, the recombinant cells were cultured and cryopreserved at liquid nitrogen.3CCK-8assay and colony formation assay were performed to estimate the effect of RN181on cell proliferation in vitro in gastric carcinoma cell lines (AGS, MKN28and MKN45). Nude mice subcutaneously tumor forming assay was conducted to observe the tumor forming ability of RN181in GC cells.4Cell cycle blocking-release assay was carried out to investigate the effect of RN181on cell-cycle regulation in AGS cells, while cycle proteins Cyclin D1, Cyclin A, Cyclin B were detected by western blotting at the same time.5Western blotting analyses was used to test the changes of regulators at G1/S and G2/M cycle checkpoints in recombinant AGS and MKN28cells, and immunohistochemical staining was used to analyze the expression difference of the aforementioned molecules in the nude mouse tumors.6Yeast two-hybrid system and DNA sequencing technology were conducted to find proteins directly interacted with RN181. RPS27A, taken as a further study object, was cloned into eukaryotic expression vector pCMV-myc. After identification by restriction enzymes digestion, sequencing and western blot, pCMV-myc/RPS27was co-transfected with pCDAN3.1(-)-flag/RN181into AGS cells to investigate the protien expression changes, meanwhile, to observe the co-localization of RPS27A and RN181by confocal microscopy after immunofluorescence technology. Finally, co-immunoprecipitation was used to confirm the direct interaction between RPS27A and RN181.Results:1In165cases of clinical specimens, the result of immunohistochemical staining showed that RN181was low-expressed in gastric cancer tissues and high expression in the adjacent tissues. Moreover, the lower the degree of tumor differentiation, RN181protein expression levels were lower in cancer tissues. With gastric cancer into the middle and late ages, the expression of RN181declined gradually. In addition, tissues derived form bigger tumors had lower expression of RN181. And the survival time of patients who had less RN181expression was significantly lower than those who had more RN181expression. As to RN181expression in para-neoplastic tissues, there were no significantly differences in gender, age, pathological grades, clinical stages and tumor size.2Retroviral packaging vector plncx2-GFP-IRES/RN181was successfully constructed, confirmed by restriction enzymes digestion, sequencing, and Western blot, and could correctly package RN181high-expressed retro virus with pVSV-G in GP2-293cells. The recombinant GC cells obtained from AGS cells infected by aforementioned retrovirus and AGS, MKN28and MKN45cells infected by RN181silencing lentivirus via FACS, were verified successful after identification by RT-PCR and Western blot.3CCK-8assays showed the growth speed of AGS recombinant cells with RN181high-expressed (AGS-RN181) is significantly slower than its control cells (AGS-RV), and the growth speeds of AGS, MKN28and MKN45recombinant cells with RN181silenced are faster than their own control cells. It was found by colony formation assay that RN181high expression could inhibit clone forming in AGS recombinant cells, versus RN181interference promoted it in AGS, MKN28and MKN45cells. Nude mice subcutaneously tumor forming assay verified that tumor growth of AGS-RN181cells was much lower than its control cells, and RN181interference accelerated AGS cells subcutaneously tumor forming.4Cycle blocking-release assay showed RN181high expression inhibited AGS cell cycle process from G1to S phase, but RN181interference relieved this phenomenon. Besides, RN181interference may speed up the process from G2phase into M phase in AGS cells. Western blot results showed that the changes of Cyclin D1and Cyclin B were consistent with the phenomenon.5Western blot analysis of expression difference of regulators at G1/S and G2/M checkpoints demonstrated the inhibition of RN181on AGS cell cycle occurred mainly at the G1/S checkpoint. With RN181high-expressed, the expression of Cyclin D and CDK4decreased significantly, and the tumor suppressor P21rised obviously. And, these changes in the cells with RN181silenced were opposite. The changes of Cyclin D, CDK4and P21in nude mice xenografts kept consistent with the change in the AGS recombinant cells in vitro, confirmed by immunohistochemical staining.6Through yeast two-hybrid assay, we obtained initially41proteins interacted potentially with RN181, most of which were involved in apoptosis, cycle, metabolism, adhesion, et al. pCMV-myc/RPS27A eukaryotic expression vector was successfully constructed after identification by restriction enzymes digestion, sequencing and western blot. In co-transfection analysis, RPS27A expression reduced remarkably, when RPS27A and RN181co-expressed in AGS cells, and this down-regulation was not affected by MG132. There was obviously spatial overlap of RN181and RPS27A in AGS cells in co-localization assay by confocal microscopy. Co-IP experiments confirmed the direct interaction between RN181and RPS27A in AGS cells.Conclusion:The expression of RN181is low in cancer tissues and high in neighboring noncancerous tissues, and is positively related with degrees of differentiation and survival time, but is negatively related to clinical stages and tumor size. There are no remarkable differences of RN181expression at adjacent tissues in gender, age, clinical stages, pathological grades and tumor size. The distinctive differences prompt that RN181might a new inhibitory molecule of GC. Through gene cloning techniques, retroviral packaging technology and flow sorting technology, we successfully constructed stable AGS recombinant cell lines with RN181high-expressed and stable AGS, MKN28and MKN45recombinant cell lines with RN181silenced. CCK-8and colony formation experiments confirm that high expression of RN181inhibits the growth of AGS cells in vitro, and RN181interference promotes the growth of AGS, MKN28and MKN45cells in vitro. Nude mice subcutaneously tumor-forming assay verifies high expression of RN181represses tumorigenic effect in AGS cells, while the phenomenon could be reversed by RN181interference. Up-regulation of RN181can inhibit AGS cell cycle process from Gl to S phase, the phenomenon could be relieved by RN181interference, which may speed up the process of AGS cell cycle from G2phase into M phase. RN181suppress AGS cell cycle at the G1/S checkpoint, through down-regulating Cyclin D and CDK4expression and up-regulating the tumor suppressor P21. Likewise, changes of Cyclin D, CDK4and P21in nude mice transplanted tumor derived from AGS cells remains consistent with the changes in the AGS recombinant cells. By yeast two-hybrid experiments,41RN181interacting molecules were obtained. One of which is RPS27A, which may involve in cell cycle regulation. Via expression analysis after co-transfection, confocal co-localization technique and Co-IP experiments, the interaction of RN181and RPS27A was confirmed. And the mechanism of RPS27A involved in cell cycle regulation is still under study.