| BackgroundGastric cancer（GC）is one of the most common types of cancers worldwide.More than 70%of new cases and deaths occur in developing countries,particularly in Eastern Asia,Europe,and South America.In China,GC has the second highest incidence among common types of cancers.Although gastric carcinoma of certain patients can be resected,these patients still have a poor prognosis;their 5-year overall survival is only approximately 20-30%.Many patients with GC have no chance for surgery since they were already in the advanced or metastatic stage when their GC diagnosis was confirmed;the median survival time of these patients is often no more than one year.Recurrence and metastasis are the main reasons for the high mortality rate in patients with GC.Although several anticancer drugs and different surgical modalities have been presented,GC still cannot be cured,particularly in its late stages.Thus,understanding the molecular mechanisms underlying the pathogenesis of GC is important.RLIP76 is a 76-kDa splice variant,which is encoded by the human gene RalBP1（18p11.22）.It is a Ral-interacting protein of 76 kDa,also known as RalBPl.It was identified as a Ral GTPase effector protein that connects the Ral with Rho pathways originally.This protein participates in the ATP hydrolysis-dependent movement of substances,including glutathione conjugates（GS-E）and chemotherapy drugs,out of cells.It is a member of the Ras family.Ras is the upstream protein of many receptor proteins,including vascular endothelial growth factor（VEGF）and the activation of Ras is an important part in the pathogenesis of human malignant carcinomas.Meanwhile,Akt signaling pathway plays a fundmental role in the survival and apoptosis of cells.Futhermore,Akt is also the downstream molecule in Ras signaling pathway.Most early studies concentrated on the transport functions of RLIP76,whereas growing evidence has shown that RLIP76 is necessary in a variety of cellular functions,such as mitosis,proliferation,differentiation,apoptosis and endocytosis.It takes part in the formation of multi-functional protein complexes,like the mitotic spindle and the receptor signaling complexes of EGF,TGF-β,insulin and clathrin-dependent endocytosis and determines the rate of receptor-ligand signaling.RLIP76 are expressed in many human tissues,including liver,heart,ovary,lungs,muscles and kidneys.However,in multiple cancers,the expression of RLIP76 is increased and closely related with the sensitivity to radiotherapy and chemotherapy.In drug-resistant colon cancer cell line,HCT-8/VCR,the knockdown of RLIP76 incerased its sensitivity to VCR,and IC50 of VCR decreased.Blocking RLIP76 with targeting antibodies or antisense RNA is related to increasing sensitivity to chemotherapy and radiotherapy,and causes pronounced tumor regression in non-small cell lung cancers,colon cancers,prostate cancers and B16 melanomas in mice,and leads to apoptosis in wide varieties of histologic types of cancers in cell culture including melanoma,prostate cancer,non-small-cell lung cancer,small-cell lung cancer,ovarian cancer,colon cancer,lymphoma and myeloid leukemia.These data suggest that therapeutic strategies that target RLIP76 may provide a broad-spectrum anti-neoplastic approach.Based on previous studies,we hypothesized that RLIP76 may play an important role in gastric cancer.In this study,we first demonstrated that the expression level of RLIP76 in gastric cancer tissues and cells,and then explored the effect and mechanism of it in the gastric cancer cells by the knockdown of RLIP76.The results showed that the expression of RLIP76 in gastric carcinoma was significantly increased compared with the stomach mucosa.Compared with the corresponding adjacent tissues,the expression level of RLIP76 in gastric cancer tissues was also increased,and was associated with poor prognosis of patients.In gastric cancer cells,knockdown of RLIP76 can inhibit proliferation,induce apoptosis,reduce angiogenesis,and suppress the invasiveness of cells.Finally,we detected the changes of Akt/mTOR signaling pathway after RLIP76 knockdown,finding that the phosphorylation level of Akt and mTOR decreased significantly.The results provide a theoretical basis for the treatment of gastric cancer,providing new ideas for the treatment of gastric cancer.Part 1.The Expression of RLIP76 in gastric cancer and its clinicopathological significanceObjective1.To study the expression level of RLIP76 in normal gastric mucosa,GCand adjacent tissues.2.To analyze the correlation between the expression of RLIP76 and various clinical parameters in GC.Methods1.We selected 76 cases of patients with gastric cancer who were treated by surgery in Shandong Provincial Hospital Affiliated to Shandong University from January 2013 to January 2014,whose median age is 46 years old.At the same time,40 patients with benign gastric tumor were selected as negative control.In addition,we collected cancer tissues and adjacent tissues from 114 patients who received surgery in Shandong Provincial Hospital Affiliated to Shandong University from February,2009 to July,2010 and their clinical data.Among them,69 were males and 45 were females.2.Immunohistochemistry was used to test the expression level of RLIP76.The scores of Immunohistochemistry were analyzed by SPSS 17.0.Survival analysis and responding curve were carried out with the Kaplan-Meier method.Chi square test was used to evaluate the relationship between RLIP76 and age,sex,lymph node metastasis,TNM stage and other clinical indexes.Results1.The expression level of RLIP76 in 76 cases of gastric carcinoma was significantly higher than that in the mucosa of 40 cases of benign gastric tumor.2.In 114 gastric cancer cases,there were 53 cases with positive expression of RLIP76 and 61 cases with middle-low（negative）expression in the immunohistochemical staining of RLIP76.Chi square test showed that the expression level of RLIP76 was positively correlated with lymph node metastasis and TNM stage,and there was no significant correlation with sex,age,tumor size and differentiation level of tumor.Survival curves showed that the expression level of RLIP76 was correlated with prognosis（p<0.05）.ConclusionThe expression level of RLIP76 was associated with lymph node metastasis,TNM stage and tumor recurrence,suggesting that it may play an important role in the occurrence and development of GC.Part 2.Downregulate RLIP76 and study its effects on biological behavior of gastric cancer cellsObjectiveTo establish RLIP76 low-expression cell model and study the effects of RLIP76 on the proliferation,invasion,migration and apoptosis of GC cells and research the mechanism.Methods1.We cultured DH5α cells,GC cells SGC7901and MGC803.2.Virus designWe searched RLIP76 gene sequence in Genebank of Pubmed,designed and synthesized the single stranded DNA and a scrambled sequence of DNA according to the gene sequence.The oligomeric chain will be doubled and the double strandeds ShRNA oligo were inserted into the lentiviral vector.Through the transformation,the recombinant plasmid got into the DH5α cells.After virus titer determination,we got PGMLV-SC5 mediated ShRNA RLIP76 with the green fluorescence and puromycin-resistant labeling.3.Transfeciton of virusAccording to multiplicity of infection（MOI）value,the pGMLV-SCS mediated RLIP76 ShRNA and scrambled ShRNA were transfected into the GC cells SGC7901and MGC803.In order to make sure the ratio of green fluorescent cells and fluorescence intensity,we observed cells by fluorescence microscope after transfection.We obtained successful transfection cells of SGC7901and MGC803 after continuous culture and screening by the puromycin.4.Verify efficiency of Lentivirus transfectionThe efficiency of knockdown of lentivirus mediated RLIP76 ShRNA was verified through qRT-PCR on mRNA level and through western blot on protein level.And then screen the ShRNA sequence with the best efficiency of knockdown.5.Cell functional experimentsIn order to research the clonality of GC cell lines SGC7901 and MGC803 after the transfection of RLIP76 ShRNA and scrambled ShRNA,we devised the experiment of clone formation.We designed CCK8 test to detect the proliferation of GC cell lines SGC7901 and MGC803 after transfection of RLIP76 ShRNA and scrambled ShRNA and render cell proliferation curve.We learned the alteration of migration and invasion ability of GC cell lines SGC7901 and MGC803 after transfection of RLIP76 ShRNA and scrambled ShRNA according to theTranswell and gelatin-zymography study.The changes of apoptosis of GC cell lines SGC7901 and MGC803 after transfeciton were studied by Hoechst 33342 staining;We studied the changes of related signaling pathways by western blot after transfection of lentivirus,finding the signaling pathway proteins that relative to RLIP76.6.Statistical analysisWe used Prism GraphPad 5 statistical software to analyze the experimental data.All data were evaluated with a two-tailed unpaired Student’s t-test or with a two-tailed paired Student’s t-test.A p-value<0.05 was considered to indicate a statistically significant result.Results1.We screened 3 SiRNA sequences with optimal kinetic parameters and built pGMLV-SC5 mediated RLIP76 ShRNA successfully:ShRNA1,ShRNA2 and ShRNA3.The lentivirus mediated scrambled sequence was taken as negative control.We found the efficiency of transfection of pGMLV-SC5 mediated ShRNA was as high as 80%and cells were well adherent-grew with the help of green fluorescence microscope.2.The results of qRT-PCR showed that the relative RLIP76 mRNA expression level in SGC7901 after transfecion of Sh-scrambled,ShRNA1,ShRNA2 and ShRNA3 was:0.947±0.038,0.246±0.021,0.706 ± 0.109,0.361 ± 0.093,respectively;In MGC803 the relative RLIP76 mRNA expression level was:0.973±0.061,0.225±0.009,0.469±0050,0.377±0.061.The results of qRT-PCR showed that ShRNA 1 had the best efficiency of knockdown and there was a statistically significant difference compared with scrambled ShRNA（p<0.05）.3.The results of western blot showed that the relative expression level of RLIP76 in SGC7901 after transfecion of Sh-scrambled,ShRNA1,ShRNA2 and ShRNA3 was:1.016 ± 0.181,0.245 ± 0.103,0.709 ± 0.191,0.416 ± 0.107,respectively.While in MGC803,the relative expression level of RLIP76 was:0.953±0.143,0.199±0.113,0.565±0.198,0.384±0.205.The expression level of RLIP76 in GC cell lines SGC7901 and MGC803 was lowest after transfected with ShRNA1,and there was statistical difference compared with transfection of scrambled sequence,so we chose ShRNA1 to help us to study alterations of cell functions after RLIP76 knockdown.4.The results of clone formation experiment revealed that there were less and smaller clones formation in the GC cell lines transfected with ShRNAl（KD）compared with cell lines transfected with scrambled ShRNA（NC）.CCK8 results showed that the proliferation rate of GC cell lines SGC7901 and MGC803 after transfection of ShRNAl（KD）was slower than that of GC cell lines transfected with scrambled ShRNA（NC）（p<0.05）,suggesting that knockdown of RLIP76 could decrease the proliferation of GC cells.5.In the migration study of Transwell,the migration cells were 486.7±128.8 in the SGC7901 NC cells,noticeably more than that in the KD cells（219.7 ±43.6）（p<0.05）.At the same time,the migration cells were 630 ±95 in MGC803 NC cells,and 333.7±46.5（p<0.05）in MGC803 KD cells.In the invasion study of Transwell,the invasion cells were 306 ± 33.5 in SGC7901 NC cells,while they were 97.7±24.3 in SGC7901 KD cells（p<0.05）.On the other hand,the invasion cells were 350±50.9 in MGC803 NC cells,and 163.3 ±87.5（p<0.05）in MGC803 KD cells.In the gelatin-zymography,the MMP2 in cell supernatant decreased in both KD cells.6.In the Hoechst 33342 staining analysis,there were more apoptotic cells in SGC7901 and MGC803 KD cells than that in the NC cells（p<0.05）.In western blot,the expression levels of apoptotic protein:Bax.cleaved-PARP,cleaved-caspase-8 and cleaved-caspase-9 were higher in SGC7901 and MGC803 KD cells,compared with that in the NC cells.Furthermore,transfection of lentivirus changed the Akt/mTOR signaling pathway obviously.The expression level of p-Akt and p-mTOR decreased in the KD cells of SGC7901 and MGC803（p<0.05）,while the t-Akt and t-mTOR kept stable.Conclusion1.The suppression of the RLIP76 gene by ShRNA can inhibit proliferation and clone formation,suppress the migration and invasion of GC cells.2.RLIP76 knockdown can induce apoptosis which is regulated through Akt/mTOR signaling pathway.Part 3.Effects of RLIP76 on VEGF secretion and tubular formation in vitroObjectiveTo study the effect of RLIP76 expression on VEGF secretion,explicating the influence of RLIP76 on tubular formation.Methods1.We cultured HUVEC in vitro.2.We cultured KD and NC GC cells in 12-well plates,and collected the cell supernatants after 24h.3.The supernatants were then tested by ELISA to measure the VEGF protein level,which was performed according to the manufacturer’s protocol.4.Tubular formation assay in vitro:A 96-well plate was coated with cold Matrigel and incubated for 1 h at 37℃ and we mixed the collected cell supernatants with HUVEC respectively.The mixture was added to the wells and was incubated at 37℃ with 5%C02.The cells were monitored every half an hour under a microscope after 2 hours,and tube formation was imaged at 8 h.5.Western blot:To verify VEGF alteration on protein level according to western blot.Results1.In the ELISA assay,the concentration of VEGF（pg/ml）in supernatants of SGC7901 and MGC803 KD cell was 158.582±6.931,264.024±11.177 respectively,while in supernatants of SGC7901 and MGC803 NC cells,the concentration of VEGF was 465.048±2.121,463.453±13.350 respectively.We found that there were more secreted VEGF in NC GC cells（p<0.05）.2.In tube formation assay,the tube formation in KD SGC7901 and MGC803 cells were 44.5±3.69,71.8±8.83（%control）（p<0.05）respectively.While in NC SGC7901 and MGC803 cells,they were 202.8±83.3,193±3.5（%control）（p<0.05）respectively.3.In western blot,VEGF decreased in both KD cells.In NC SGC7901 cells,the relative protein level of VEGF was:1.351 ±0.107,while in KD cells it was:0.553 ±0.071（p<0.05）.In NC MGC803 cells,the relative protein level of VEGF was:1.230±0.230,while in KD cells it was:0.823±0.191（p<0.05）.ConclusionRLIP76 knockdown could reduce the protein level of VEGF and secretion of VEGF,and inhibit tube formation. |
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