| Objective: Gastric cancer is one of the most common diseases threatening people's health.It is the fifth most common cancer in the world and ranks third among cancer-related deaths,seriously jeopardizing human health and life.The incidence of gastric cancer in China has been as the second only inferior to Japan and South Korea,with more than 42% of gastric cancer patients in the world.Although both diagnostic and treatment strategies of gastric cancer have been continuously improving,the overall prognosis of gastric cancer is still dismal.Many studies have demonstrated that the occurrence and outcome of tumors are still closely related to the interaction of multiple genetic abnormalities.The occurrence and progression of gastric cancer are the multi-factor,multi-stage and multi-step process,involving a large number of molecular participations and complex network regulations.To clarify the key biological mechanisms and molecular indicators in the occurrence and progression of gastric cancer to be contribute to the diagnosis,treatment and recurrence prevention of clinically advanced gastric cancer is the key scientific issue to improve the prognosis of gastric cancer patients.Protocadherin-10(PCDH10)gene,as a tumor suppressor gene in the protocadherin family,is capable of participating in many molecular events in eukaryotic cells,including cell sorting and recognition,boundary formation,induction and cell-cell adhesion.Early researches on PCDH10 mainly focused on its neurophysiological aspects,indicating that PCDH10 could act an key role in the development of brain.PCDH10 has also been identified as a new kind of indicator used to predict the occurrence and progression of human malignant tumors in recent years.The low expression or sliencing of PCDH10 has been founded in a variety of malignant tumor tissues(colorectal cancer,gastric cancer,bladder cancer,lung cancer,prostate cancer,and endometrial cancer),which is closely related to the malignant biological behaviors such as proliferation and invasion of tumor cells.Therefore,it is certainly worth exploring whether PCDH10 can affect the cell function of gastric cancer,and investigating the mechanism of cellular biological function regulated by PCDH10.This study was to investigate the expression of PCDH10 in gastric cancer and matched adjacent normal tissues and then to to explore the relationship between clinicopathological features of gastric cancer patients and the expression of PCDH10 in gastric cancer tissues.Furthermore,The protein and m RNA expression difference of PCDH10 in different human gastric cancer cell lines(HGC-27,MGC-803,SGC-7901,BGC-823,SUN-1,KATO-III and AGS)and normal gastric mucosa cells(GES-1)was also detected in our study.In addition,the effects of PCDH10 gene on the biological behavior of gastric cancer cells were further clarified through the experiments of proliferation,migration and invasion after overexpression of PCDH10 in gastric cancer cells.Method: 1.The surgical paraffin sections of 69 gastric adenocarcinoma,including cancer tissues and matched adjacent normal tissues,and the corresponding complete clinical follow-up information were collected from the Department of Gastroenterology of the Tianjin Medical University Cancer Hospital from November 2003 and September 2007.Immunohistochemistry(IHC)was used to evaluate the protein expression level of PCDH10 in 69 cases of gastric cancer tissues and its corresponding adjacent normal tissues.The difference of PCDH10 expression in gastric cancer and paracancerous normal tissues was analyzed,and then the relationship between the expression level of PCDH10 and the clinicopathological characteristics and prognosis of gastric cancer and its clinical significance was also explored in this study.2.The effect of PCDH10 gene on proliferation,migration,apoptosis and cell cycle of gastric cancer cells.The gastric cancer cell lines MGC803 and HGC27 were selected as experimental subjects.CCK8(Cell Counting Kit-8)and clone formation experiments were performed to explore the proliferation ability of gastric cancer cells.scratch experiments were conducted to investigate The effect of gene PCDH10 on the migration ability of gastric cancer cells.In addition,Transwell experiments were carried out to identify the effect of gene PCDH10 on the invasion ability of gastric cancer cells.Also,flow cytometry(apoptosis and cell cycle experiments)is conducted to investigate the effect of PCDH10 gene on cell cycle and apoptosis of gastric cancer cells.3.The effect of PCDH10 on Bcl-2,MMP-2,HGF,VEGF-A and VEGF-C was verified by RT-PCR: gastric cancer cells MGC803 and HGC27 were selected as the research object and transfected with empty vector plasmid and overexpressed PCDH10 plasmid.semi-quantitative reverse transcription polymerase chain reaction.was administrated to detect the expression of Bcl-2,MMP-2,HGF,VEGF-A and VEGF-C.4.The establishment of an animal model of subcutaneous injection into transplanted tumor in vivo was conducted to explore the effect of PCDH10 on the tumorigenic ability of gastric cancer cells in nude mice and the biological characteristics and gene expression differences of related tumor cells.5.Western Blot and RT-PCR experiments were used to verify the effect of overexpressed RNF180 on PCDH10.We selected gastric cancer cells MGC803 and HGC27 as the research objects and transfected the expression plasmid RNF180 and the corresponding empty vector plasmid respectively.And then the semi-quantitative reverse transcription polymerase chain reaction and Western blot were applied to detect the expression of PCDH10.Results: 1.Immunohistochemical results showed that the expression of PCDH10 in paracancerous normal tissues was significantly higher than that in gastric cancer tissues.The positive expression rate of PCDH10 protein in gastric cancer tissues was 20.3%(14/69)and the positive expression rate of PCDH10 protein in paracancerous normal tissues was 53.6%(37/69).The positive rate of PCDH10 protein expression in gastric cancer tissues was significantly higher than that in paracancerous normal tissues.The results were statistically different(c2=20.803,P < 0.001).Further statistical analysis of the relationship between the expression level of PCDH10 and clinicopathological characteristics of gastric cancer and prognosis showed that patients with positive PCDH10 expression had longer survival time than patients with negative PCDH10 expression(49 vs 29 months,p=0.006).PCDH10 protein expression in GC tissues was identified as an independent predictor of outcome(HR =2.784,p =0.009),along with Lauren classification.Therefore,positive PCDH10 expression in tumour samples indicated a tendency of comparatively longer OS in GC patients.The number of lymph node metastasis(p N stage)was validated as more relevant to PCDH10 expression(p =0.033).As the number of lymph node metastasis increases,the positive rate of PCDH10 expression in tumor tissues of GC patients is reduced obviously(50% for p N1 patients,30% for p N2 patients,20% for N3 a patients and 0% for N3 b patients,p =0.033).2.The m RNA and protein expression levels of PCDH10 in gastric cancer cell lines was significantly lower than these in GES-1 cell line(P<0.05).3.The CCK8 and colony formation experiments showed that PCDH10 can inhibit the proliferation of gastric cancer cells.The scratch test and transwell experiments confirmed that PCDH10 can inhibit the migration and invasion of gastric cancer cells.The results of flow cytometry(apoptosis and cell cycle experiments)indicated that PCDH10 can promote the apoptosis of gastric cancer cells and induce G1 arrest of gastric cancer cells.4.RT-PCR experiments confirmed that compared with the control group(empty vector transfection),the Bcl-2,MMP-2,HGF,VEGF-A and VEGF-C expression were down-regulated in the gastric cancer cell group transfected with the plasmid of overexpressed PCDH10(MGC-803-GFP-PCDH10 and HGC27-GFP-PCDH10).Therefore,overexpressing PCDH10 can inhibited the expression of Bcl-2,MMP-2,HGF,VEGF-A and VEGF-C.5.The in vivo experiments(establishment of an animal model of subcutaneous injection into transplanted tumor)indicated that The mean tumor size was significantly smaller in MGC-803-GFP-PCDH10 injected subcutaneously in nude mice as compared with MGC-803-vehicle cell injected subcutaneously in nude mice at 3-weeks time-point after cell implantation,indicating that PCDH10 does function as a tumor suppressor gene in GC.6.Western Blot and RT-PCR experiments confirmed that compared with the control group,both protein and m RNA levels of PCDH10 was up-regulated in gastric cancer cell lines transfected with the plasmid of overexpressed RNF180.It is indicated that overexpressing RNF180 can enhance the expression of PCDH10.Conclusions: 1.The expression of PCDH10 related to the occurrence and development of gastric cancer suggested that PCDH10 plays an important role in gastric cancer development.2.The expression of PCDH10 in gastric cancer tissues was significantly lower than that in adjacent normal tissues.Survival analysis showed that the expression of PCDH10 in gastric cancer tissues was closely related to the survival of patients.The number of lymph node metastasis(p N phase)is significantly correlated with the expression of PCDH10.Therefore,the expression of PCDH10 can be used as a predictor of prognosis in patients with gastric cancer.3.PCDH10 exerts its effects to inhibit lymph node metastasis of GC cells by suppressing the several biological events of GC cell including the proliferation,viability,invasion,migration,anti-apoptosis and tumor lymphangiogenesis.4.RNF180 can be used as a new biomarker to improve the accuracy of lymph node metastasis prediction in gastric cancer.This study speculates that PCDH10 is regulated by RNF180,which may be an important participating molecule in the mechanism of RNF180 inhibiting lymph node metastasis of gastric cancer cells.The specific regulation mechanism is still unclear,and further research is needed to be further explored. |
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