The Roles And Mechanism Research Of RKIP Protein In Gastric Cancer

Backgroud:Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related mortality worldwide, both its incidence rate and mortality occupy the first place among the gastrointestinal malignant tumors, which threatens human’s health and lives severely. Stomach cancer is often asymptomatic or causes only nonspecific symptoms in its early stages. By the time symptoms occur, the cancer has often reached an advanced stage, of which the overall5-year survival rate is only about30%.And so far, the pathogenesis of gastric cancer has not completely clear, treatment of this cancer has not significantly break through, which can’t meet the clinical requirement in the prevention and treatment of this disease. In this regard, it is important to identify new tumor markers and therapy targets for diagnosis, metastasis and recurrence prediction, prognosis assessment and gene therapy of this cancer. In recent years, looking for molecular biology targets of tumor cell growth, apoptosis, cell cycle, invasion and infiltration has become key points and hot spots in basic research of this cancer.Raf kinase inhibitor protein (RKIP),belonging to the phosphatidylethanolamine-binding protein(PEBP) family, is a widely expressed and highly conserved small molecules cytoplasm protein with many physiological and pathological functions. In recent years, there has been an increased interest in RKIP due to the discovery of its ability to influence intracellular signaling cascades. RKIP binds to Raf-1, and blocks MAPK signaling pathway. Also it has been demonstrated to regulate G-protein-coupled receptor signaling pathway and NF-κB signaling. RKIP has been implicated in arange of normal and disease processes, such as cell growth, apoptosis, cell migration, angiogenesis, reproduction, and cancer metastasis. Evidence has emerged that RKIP can function as a suppressor of cancer metastasis, which suppresses metastasis in prostate cancer, breast cancer and melanoma cancer and so on. A variety of ablative interventions suggest that reduced RKIP function may influence metastasis, angiogenesis, resistance to apoptosis, and genome integrity. RKIP may constitute a useful prognostic marker for predicting the clinical outcome of certain cancers in human patients, and also may be a new therapeutic target in certain cancers.Our previous study found that the expression of Raf kinase inhibitor protein (RKIP) in gastric cancer was reduced or absent, such changes may be connected with the occurrence, differentiation, invasion and metastasis of the gastric cancer, but those specific effects and mechanism of RKIP in the occurrence and metastasis of the gastric cancer have not been reported at home and abroad.Based on the previous studies, we used immunohistochemistry to analyze expressions of RKIP protein in pre-cancerous conditions, precancerous lesions and gastric cancer tissues in this study, and to determine the the relationship between RKIP expression and carcinogenesis of gastric mucosa. Nextly, to explore the potential role of RKIP on the malignant biological characteristics of gastric carcinoma (GC) cell line SGC7901, cell proliferation, cell cycle, apoptosis rates and migration assays were analyzed after RKIP gene over expression GC cell lines in vitro or vivo. Finally, To investigate the molecular mechanisms by which RKIP inhibits GC cell proliferation or metastasis,we analysed Cyclin D1,STAT3and ADAM-12protein expression in normal gastric tissues, pre-cancerous conditions, precancerous lesions and gastric cancer tissues,and to detect the the relationship between RKIP and these proteins expression during the carcinogenesis of gastric mucosa. Our work would reveal the potential mechanisms that how RKIP inhibit the occurrence and development of GC, which provide the basis and the new clues for the diagnosis, prognosis monitoring and targeted gene therapy of GC.Chapter one Expression of RKIP at different stages in the course of gastric carcinoma and its significanceObject:To investigate the expression of RKIP protein in pre-cancerous conditions, precancerous lesions and gastric cancer tissues, and to determine the relationship between RKIP expression and carcinogenesis of gastric mucosa. Methods:There were88cases of pre-cancerous conditions of gastric cancer including7cases of gastric adenomatous polyp (GAP),27cases of gastric ulcer (GU) and54cases of dysplasia (DYS),169cases of gastric carcinoma (GC) and36cases of the corresponding intra-abdominal metastatic lymph node tissues of GC involved in this research. Among the dysplasia cases,17cases with mild to moderate dysplasia,23cases with severe dysplasia, and26cases with intestinal metaplasia(IM). In169gastric cancer’s tissues,48were early gastric carcinoma (EGC),121were advanced gastric carcinoma(AGC). All of them were selected from Xiangya Hospital, Central South University and verified by pathology.All paraffin-embedded tissue samples were assessed by immunohistochemistry. We analyzed the diversification rule of RKIP expression from the precarcinomatous conditions to EGC and then to AGC in order to clarify the relationship between RKIP expression in the tumorigenesis of gastric mucosa and the clinical and pathological features of gastric cancer.Results:By using immunohistochemistry,the positive rates of RKIP protein expression were significantly lower in AGC (29.8%,36/121) and in metastatic lymph node tissues (2.8%,1/36) than in pre-cancerous conditions (70.5%,62/88).There is little difference between pre-cancerous conditions and EGC (66.7%,32/48),and P>0.05.Meanwhile,the experimental results were analyzed by using Image-Pro Plus image analysis software. The two methods gave the same determination.The positive expression rate of of RKIP in the GC invasion within mucosa and submucosa,that beyond muscularis and that beyond serosa was68.8%,35.3%,20.8%,respectively. The positive expression rate of RKIP in cases of I grade,cases of II grade,cases of III/IVgrade was68.3%,47.8%,and24.5%,respectively. The positive expression rate of RKIP in65cases without lymph-node metastasis and97cases with lymph-node metastasis was70.8%and20.7%. Positive expression of RKIP protein was negatively correlated with deeper invasion,TNM stage and lymphoid node metastasis(P<0.001). However RKIP protein expression has nothing to do with age, sex, the histological differentiation and Lauren type (P>0.05).A total of52GC cases with Hp test results, of which32cases positive.In those32positive cases, RKIP positive expression rate was43.8%; and in other20negative cases,RKIP positive expression rate was40.0%.RKIP protein expression has nothing to do with Hp infection in GC(P>0.05).Conclusion:RKIP protein was detected high expression in pre-cancerous conditions and EGC. RKIP protein expression has nothing to do with the histological differentiation,Lauren type and Hp infection in GC.The expression of RKIP protein in ACG and metastatic lymph node tissues was signifcantly lower than that in pre-cancerous conditions or EGC.Positive expression of RKIP protein is negatively correlated with deeper invasion,TNM stage and lymphoid node metastasis,which suggests that RKIP protein down-expression was associated with invasion and metastasis in GC. Abnormal alteration of RKIP is a relatively late event in stomach tumorigenesis and play an important role in the development of GC.Measurement of RKIP expression is of value in evaluating the prognosis of gastric carcinoma.Chapter two Effect of RKIP Gene Transfection on The Malignant Biological characteristics of Gastric Carcinoma CellsObject:To explore the potential role of RKIP on the malignant biological characteristics of gastric carcinoma (GC) cell line SGC7901.Methods:A recombinant plasmid carrying RKIP (pcDNA3.1(+)-RKIP) was transfected into SGC7901cells by Lipofectin-mediated method. Cells untransfected SGC7901cells and those transfected with empty pcDNA3.1(+) plasmid were used as controls. The expression of RKIP mRNA and protein were detected by q-RT-PCR and Western blotting analysis respectively. The cell proliferation of the transfected SGC7901cells were demonstrated by drawing growth curve and soft agar assay. The invasion ability of the cells was detected by scratch assays and transwell assay. Cell cycle distribution and the percentage of apoptosis were determined by flow cytometric analysis. Tumor formation in nude mice was used to assess the tumorigenic characteristics in vivo.Results:QRT-PCR and Western blot analysis showed that the mRNA and protein levels of RKIP in the transfected cell lines were significantly higher than those in controls,which suggested that a stable cell line pcDNA3.1(+)-RKIP/SGC7901containing overexpressed RKIP gene was successfully established. Compare with vector transfected and untansfected SGC7901cells, the cell proliferation of pcDNA3.1(+)-RKIP/SGC7901cells was significantly inhibited from the4th day (P<0.001), colony formation numbers of pcDNA3.1(+)-RKIP/SGC7901cells in soft agar showed significant decreases (57±7,59±9vs20±4, both P<0.05). Scratch woundhealing assay show that the scratch width after18h was2.91±0.21mm in pcDNA3.1(+)-RKIP/SGC7901cells, significantly higher than that in pcDNA3.1(+)/SGC7901cells (1.31±0.34mm) and SGC7901cells (1.26±0.41mm), P<0.05. The results of Transwell invasion assay showed that the number of cells invaded the membrane was107.0±20in pcDNA3.1(+)-RKIP/SGC7901cells, significantly less than that of pcDNA3.1(+)/SGC7901cells (256.5±37) and SGC7901cells (276.5±40)(P<0.05). Flow cytometry analysis demonstrated that the percentage of cells in GO and G1phase was69.34±1.37%in pcDNA3.1(+)-RKIP/SGC7901cells, higher than that in pcDNA3.1(+)/SGC7901cells (63.42±0.49%) and SGC7901cells (62.55±0.91%). In contrast, the percentage of cells in G2and M phase was8.21±1.64%in pcDNA3.1(+)-RKIP/SGC7901cells, significantly lower than that in pcDNA3.1(+)/SGC7901cells (1.07±0.29%) and SGC7901cells (13.71±2.36%)(P<0.05). In addition, the apoptosis index was3.59±0.51%in pcDNA3.1(+)-RKIP/SGC7901cells, significantly higher than that in pcDNA3.1(+)/SGC7901cells (1.17±0.87%) and SGC7901cells (1.07±0.29%)(P<0.05). We employed xenograft nude mice model in which SGC7901and derived cells were injected subcutaneously. We observed that visual tumor developed7days in average after the injection in pcDNA3.1(+)/SGC7901and SGC7901groups, but developed21days in average after the injection in pcDNA3.1(+)-RKIP/SGC7901group. The tumors were dissected6weeks after the injection and their weight was significant less in pcDNA3.1(+)-RKIP/SGC7901group (7.36±2.10g) than in pcDNA3.1(+)/SGC7901group (13.63±1.61g) and SGC7901group (14.03±3.51g)(P<0.05,). In addition, tumor growth curve assay showed that transfection of RKIP expression plasmid inhibited the growth of xenografted SGC7901cells,the average volume of xenograft tumors on the7th,14th,21th,28th,35th,42th days was significantly lower than those in the controls at the same time points(P<0.05).Conclusions:1) SGC7901cell line display decreased expression of RKIP mRNA and protein,which indicated that down-regulation of RKIP expression is associated with gastric cancer.2) Restoration of RKIP can reduce cell proliferation, migration and invasiveness in SGC7901cells in vitro.3) Human gastric carcinoma implant tumor model in nude mice was successfully established. RKIP can suppress incidence of gastric cancer xenograft and decrease tumorigenicity in nude mice, inhibit growth of transplanted tumor in vivo.4) RKIP inhibits SGC7901cell proliferation,migration and invasiveness, which is closely related with altering the cell cycle and promoting apoptosis.5) RKIP gene may play an important role in gastric cancer cell proliferation, invasion and apoptosis; the expression of RKIP gene could favor the malignant phenotype revision of GC cells,which indicated that RKIP may serve as an effective target for cancer gene therapy.Chapter three Mechanisms of RKIP inhibiting invasion and metastasis in GCObject:Our investigation is aim to analyze the correlation among RKIP,Cyclin D1,STAT3and ADAM-12expression,especially to explore the correlation and significance with clinicopathological features of gastric carcinoma. These results provide a new experimental evidence for clarifying the mechanisms of RKIP inhibiting invasion and metastasis in GC.Methods:Firstly,We analyzed Cyclin D1,STAT3and ADAM-12mRNA expression in the RKIP transfected SGC7901cell lines by q-RT-PCR in order to demonstrate whether RKIP inhibit invasion and metastasis in GC by regulating Cyclin D1,STAT3and ADAM-12expression. Secondly,we analyzed the rule of Cyclin D1,STAT3and ADAM-12protein expression from the precarcinomatous conditions to GC and then to metastatic lymph node tissues by using immunohistochemistry,in order to clarify the relationship between Cyclin D1,STAT3and ADAM-12protein expression in the tumorigenesis of gastric mucosa and the clinical and pathological features of gastric cancer.Furthermore the correlation among RKIP,Cyclin D1,STAT3and ADAM-12expression was analyzed.Results:The mRNA expression of CyclinD1、STAT3、ADAM12in pcDNA3.1(+)-RKIP/SGC7901group was0.405±0.073times,0.578±0.274times,0.673±0.447times than those in SGC7901group, and the mRNA expression of CyclinD1、STAT3、ADAM12in pcDNA3.1(+)/SGC7901group was1.038±0.543times,1.772±1.078times,1.003±0.434times than those in SGC7901group. The mRNA expression of CyclinD1,STAT3in pcDNA3.1(+)-RKIP/SGC7901group was significantly lower than that of the control group or the vector group (P<0.05),however there were no significant differences in the expression levels of ADAM12mRNA between pcDNA3.1(+)-RKIP/SGC7901group and the control group or the vector group (P>0.05).By using immunohistochemistry,STAT3protein expression was significantly higher in EGC (66.7%,20/30),AGC (71.6%,43/60)and in metastatic lymph node tissue(64.0%,16/25)than that in precarcinomatous conditions (26.1%,23/88),P<0.001.Expression of STAT3protein was positive correlated with deeper invasion,TNM stage,the histological differentiation and lymphoid node metastasis (P<0.05),however,there was little difference between intestinal type and diffuse type gastric carcinoma (P>0.05). The positive rates of CyclinD1protein expression were significantly higher in EGC (63.3%,19/30) AGC (73.3%,44/66) and in metastatic lymph node tissue (52.0%,13/25) than those in precarcinomatous conditions (23.9%,21/88),P<0.05.Expression of CyclinD1protein was positive correlated with the histological differentiation, Lauren type and lymphoid node metastasis(P<0.05),however CyclinDl protein expression has nothing to do with deeper invasion,TNM stage(P>0.05). The positive rates of ADAM12protein expression in precarcinomatous conditions,EGC,AGC and metastatic lymph node tissues were44.3%(39/88),53.3%(16/30),55.0%(33/60),36(9/25),respectively.There was little difference among these groups (P>0.05).Meanwhile,the immunohistochemistry results were analyzed by using Image-Pro Plus image analysis software. The two methods gave the same determination.T h e expression of RKIP was negatively correlated with that of CyclinDl (r=-0.411,P<0.001) and STAT3(r=-0.640,P<0.001). Expression of CyclinD1and STAT3was positively correlated in GC (r=0.305, P=0.004).Conclusion:1.Overexpression of both STAT3and CyclinD1protein was observed in EGC,AGC and metastatic lymph node tissues. Expression of these protein was positive correlated with the histological differentiation and lymphoid node metastasis of GC.These observations suggest that the up-regulation of STAT3and CyclinDl may play important roles in the progression,invasion and matastasis of gastric cancer.2.Inverse association between Raf Kinase Inhibitory Protein and signal transducers and activators of transcription3or cyclinD1expression in gastric cancer patients and inhibiting expression of cyclinD1and STAT3gene in gastric cell line SGC7901by overexpression RKIP gene suggest that RKIP inhibit the progression, invasion and matastasis of gastric cancer may through regulation of its downstream target genes STAT3and CyclinD1’s expression and function. RKIP gene may play an important role in gastric cancer cell proliferation, invasion, apoptosis by inhibiting expression of STAT3and its downstream target gene CyclinD1’s transcription.52Figures,22tables and148references.