| Gastric cacinoma is one of the most commen malignant tumors in the world. It’s the third common incidence of cancer in male, and the fourth common incidence of cancer in female. East Asia including China is the area with highest prevalence and mortality of the gastric carcinoma. The carcinogenesis is a complicated multistage process include poly-molecular change, polygene. And the activation of oncogene and inactivation of tumor suppressor gene are the most important events in this process.SLP-2(stomatin-like protein2) is a new gene which first discovered by the pathology department of Yale university in2000. It belongs to the stomatin super family.Human SLP-2gene presents on chromosome9p13.1. It encodes a1.5kilobase mRNA and derived a38537Da protein including357amino acid sequences. SLP-2protein is a membranin. It may link to the cytoskeleton and thereby play a role in regulating ion channels. SLP-2is also a mitochondria associate protein and functions in ATP production. Recently research showed that the expression of SLP-2increased in esophageal squamous cell cancinoma、lung cancinoma、 endometrial carcinoma、breast carcinoma、colonic carcinoma、liver carcinoma and prostate carcinoma. Knockdown of SLP-2expression can inhibit cell growth、 proliferation、motility and increase cell apoptosis in certain kinds of cancer cell lines. So we hypothesized that SLP-2is an new alternative oncogene. But now there were no reports on study of SLP-2gene in gastric carcinoma. For this reason, we investigated the SLP-2expression in gastric carcinoma and the effect of SLP-2on gastric cancer cell line, trying to finding the mechanisms of SLP-2gene in gastric carcinoma.Methods1. Expression of SLP-2mRNA and protein in gastric carcinoma and adjacent tumor-free tissueThe expression of SLP-2mRNA and protein in40cases and60cases gastric carcinoma and the normal counterparts were detected by RT-PCR and IHC technique. The association between SLP-2protein expression and clinicopathologic parameters were analysed.2. Transfection of siRNA into gastric cancer cell line and transfection effect detectionDetect the expression of SLP-2mRNA and protein in gastric cancer cell line SGC-7901by RT-PCR and westernblot. Separated the cell line into three groups: SLP-2siRNA transfected group、negative control group and blank control group. Chemical modificated SLP-2siRNA and negative control siRNA were designed and synthetized. Transfected the chemical modificated SLP-2siRNA and negative control siRNA into SLP-2siRNA transfected group and negative control group, respectively.The blank control group only added transfection reagent. Then, use RT-PCR and westernblot to detect the expression of SLP-2mRNA and protein in the three groups of SGC-7901cells.3. Effect of SLP-2siRNA on proliferation of gastric cancer cell lineUse MTT colorimetric method to detected growth of the three groups of SGC-7901cells. Calculated the cell growth inhibition ratio, and ploted the growth curves.4. Effects of SLP-2siRNA on cell cycle and apoptosis of gastric cancer cell line Cell cycles and apoptosis of the three groups of SGC-7901cells were analyzed by flow cytometry system.5. Effects of SLP-2siRNA on migration and invasion of gastric cancer cell lineThe three groups of SGC-7901cells were incubated in the transwell caves for24h, to investigate the invasion, after the polycarbonate membrane of transwell cave was covered by Matrigel. Or, cells were incubated for24h, to investigate the migration. The number of cells which gone through the membrane was counted. Calculate the invasion inhibition rate and migration inhibition rate.6. Establishment of nude mice model of tumor xenografts and tumor xenografts treatmentTumor xenografts were established by inoculation of SGC-7901cells in BALB/c nude mice subcutaneously. All mice were randomized separated into three groups: SLP-2siRNA transfected group、negative control group and blank control group. Chemical modificated SLP-2siRNA and negative control siRNA were injected into tumor xenografts of SLP-2siRNA transfected group and negative control group, respectively. Every three days,5times in total.The blank control group only receied an injection of same amount of saline.7. Effects of SLP-2siRNA on proliferation and apoptotic of tumor xenograftsTumor volume and weight of nude mice were recorded every three days. After gotten the samples, the changes of histopathology and apoptotic index were observed by microscope after HE stain. The metastasis of liver and lung of nude mice were also observed.8. Effects of SLP-2siRNA on SLP-2expression of tumor xenograftsUse IHC and RT-PCR to detect SLP-2protein and mRNA expression in tumor xenografts tissue. Statistical AnalysisData were expressed as mean±SD. The results were evaluated by χ2test, t test and analysis of variance with SPSS16.0. Comparison among groups was evaluated by LSD-t test. The statistical level of significance was set at P<0.05。Results1. RT-PCR and IHC showed that the expression of SLP-2mRNA and protein in gastric carcinoma were both higher than that of the adjacent tumor-free tissues (both.P<0.05). And the increase of SLP-2protein was more notable in terminal gastric carcinoma and cases with lymph nodes metastasis (both P<0.05)2. RT-PCR and westernblot showed that the expression of SLP-2mRNA and protein in gastric cell line were lower in SLP-2siRNA transfected group than the negative control group and blank control group (both P<0.05)3. MTT assay showed that, compared with the negative control group and blank control group, proliferation of SLP-2siRNA transfected group was restrained (P<0.05)4. Flow cytometry system showed that, compared with the negative control group and blank control group, the G1phase percentage of SLP-2siRNA transfected group was increased(P<0.05),whereas the cell proliferation index(PI) was decreased(P<0.05). But the rate of apoptosis did not show any difference (P>0.05).5. Transwell detection showed that, compared with the negative control group and blank control group, the number of invasion and migration cells of SLP-2siRNA transfected group was decreased (both P<0.05). The migration inhibition ratio compared with the negative control group and blank control group was30.08%and32.40%respectively, and invasion inhibition ratio was32.65%and27.11%respectively.6. When nude mice were putting to death, the body weights of the three groups were no notable difference (P>0.05). Compared with the negative control group and blank control group, the tumor volume of SLP-2siRNA transfected group was diminished (P<0.05), and the inhibitory rate of tumor growth was26.74%and 30.15%.7. The xenografts pathology conformed with the characteristics of moderately differentiated human gastric carcinoma. Compared with the negative control group and blank control group, the apoptosis cell number and apoptosis index of the SLP-2siRNA transfected group were no obvious difference (both P>0.05). The metastases of liver and lung of the three groups were no found.8. RT-PCR and IHC showed that the expression of SLP-2mRNA and protein were both lower in SLP-2siRNA transfected group tumor xenografts than in the negative control group and blank control group (both P<0.05).Conclusions1. The expression of SLP-2mRNA and protein both increase in gastric carcinoma, and the expression of SLP-2protein in gastric carcinoma tissues was associated with lymph nodes metastasis and TNM staging.2. SLP-2siRNA can suppress the proliferation, invasion and migration, block at G1phase of the gastric cancer cell line, but has no significant influence on cell apoptosis. SLP-2may involve in the proliferation and metastasis of gastric carcinoma.3. SLP-2siRNA can inhibit the growth of the gastric cancer xenografts, but has no significant influence on cell apoptosis of the gastric cancer xenografts.4. Our data demonstrate the increase of SLP-2gene expression in gastric carcinoma and the inhibition of SLP-2siRNA on human gastric carcinoma in vivo and in vitro. The mechanisms of the effects need further research. |
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