SLP2 Promotes Proliferation And Invasion Of Gastric Cancer Through MAPK Signaling Pathway By Interacting With PHB

Background and objective:Gastric cancer(GC)is one of the most commonly malignant tumors and also is one of the main causes of cancer death.It has been estimated that the morbidity of GC was 169 per 100 thousand and more than 980 thousand GC patients have been diagnosed in 2013,moreover,more than 840 thousand person died in GC.Chen et al.estimated that the incidence of GC would be 490 per 100 thousand and more than 60 percent of global GC patients in our country.Nevertheless,the mortality would be 364 per 100 thousand.Resection the GC by surgery is the most efficient way for the cure of early-stage GC.However,there are more than 80%of GC patients are in advanced stage when they are diagnosed in our country.Unfortunately,chemotherapy and tumor-resection do not make great sense for the survival of those patients.The developments of GC are multi-step progressions that are regulated by various genes and protein.Therefore,it is of great important to strengthen the molecular mechanism of GC development and discover the efficient molecular targets that is critical for the survival promotion.The initiation of GC is progressive development as a result of multi-causes.Normally,there is a balance between the proliferation and apoptosis of gastric mucosal epithelial cells which is dependent on the regulation and harmony of oncogenes,anti-oncogenes and various grow factors.Moreover,several molecules,such as cyclooxygenase-2(COX2),are also make great importance in this regulation.The activation of oncogenes such as Ras and BCL2,suppression of anti-oncogene such as P53 and APC,the abnormal expressionof growth factors or DNA microsatellite instability will make epithelial cells into a stage of hyper-proliferation and the apoptosis signal will be inhibited.Consequently,the normal mucosa will develop into cancer.It has been well documented that Ras gene was closed to cancer and the mutation of Ras has been detected in multi-cancers.The mutation of Ras gene was merely detected in GC.However,the activation of Ras has demonstrated in the progression of GC.It has been reported that epidermal growth factor(EGF)interaction with epidermal growth factor receptor(EGFR)or human epidermal growth factor receptor-2(HER2)would promote the expression of Ras.As a consequence,the Rafl-MEKl/2-ERK1/2 signaling pathway was activated.Several inhibitors or monoclonal antibodies targeting HER2 or EGFR have been applied in the treatment of GC,while some were still need the demonstration of clinical trials.Therefore,enhancing the molecular mechanism study of GC is not only critical for the comprehension of GC,but also for the discovery of new targeting for treatment of GC.Previously,we were focus on identifying the differentially expressed protein in normal gastric mucosa and GC tissue by two dimensional gel electrophoresis(2-DIGE)and mass spectrometry.We has found 39 differentially expressed protein among which stomatin like protein 2(SLP2)has been identified.The expression level of SLP2 in Paraffin embedded GC tissue and normal mucosa has been detected by immunohistochemistry(IHC)that revealed that SLP2 was significantly increased in GC tissue compared to the normal mucosa and its overexpression was relevant to the poor prognosis of GC patients.Therefore,SLP2 was the object of this study.SLP2,named as STOML2,is a member of stomatin family and is also belong to the SPFH(stomatins/prohibitins/podocin/flotillins/HflK/C).Presently,there are five members of stomatin family has been identified that are stomatin,SLP1,SLP2,SLP3 and podocin.Stomatin,the first member of stomatin family,has been found its abnormal expression in tumors.There were special stomatin mRNA selected spliceosome were detected in Human Genome Project(HGP)and The Cancer Genome Atlas(TCGA).Meantime,results of several cDNA arrays have identified the overexpression of stomatin in GC and colorectal cancer(CRC).What's more stomatin was also involved in the mouse cross generation of cancer initiation.SLP2 has been detected in various tumors.Firstly,Zhang et al.found SLP2 was increased more than 6 folds in human esophageal squamous cell carcinoma by cDNA array and verified SLP2 was overexpressed in tumor tissue compared to normal tissue both in mRNA and protein level by RT-PCR and IHC.Moreover,they also testified the tumor-promoting function of SLP2 in cell and animal.Lately,researchers has identified that mRNA of SLP2 was also increased in CRC by gene chip.Furthermore,it has been reported that SLP2 showed higher expression in lung cancer and endometrial cancer tissues both in mRNA and protein.In 2012,it was reported that SLP2 was overexpressed in human glioma and indicated the poor survival of glioma patients.In addition,they revealed that SLP2 promote the proliferation and metastasis of glioma cell by activating the NF-kB/MMP9 signaling pathway.The International Gastric Cancer Congress(IGCC,Italy)has laid stress on our previous results that revealed relationship between the expression of SLP2 and the prognosis of GC patients in 2013.They proposed that SLP2 might be the potential molecular maker of GC.In 2014,Liu et al.testified that SLP2 was overexpressed in thyroid papillary carcinoma and TGF-? could induce its expression.Recently,it was also demonstrated that SLP2 was also overexpressed in cervical cancer and epithelial ovarian cancer compared to the normal tissue and correlated with the prognosis of patients.Thus,it was clearly that SLP2 was closely with tumors.However,the study on SLP2 only preliminarily proved that SLP2 was overexpressed in GC and was correlated to the poor prognosis.It is still unclear the exact function of SLP2 and the regulation mechanism.This study revealed the influence of SLP2 on proliferation and metastasis of GC in vivo and in vitro and preliminarily elaborated the molecular mechanism:Firstly,we verified the results that we reported previously in multi-center paraffin embedded GC tissue.Secondly,we detected the ability of proliferation,colony forming and invasion of GC cells when the SLP2 were silenced or promoted and verified its function in vivo.Thirdly,we revealed that SLP2 activated the MAPK signaling pathway by interaction with PHB in GC.This study mainly elaborated the influence of SLP2 on cell proliferation and metastasis and express the involved mechanism and is critical for the recognition of GC which may provide a new target for the diagnosis and treatment of GC.Material and methods:1.To download the data from GEO public microarray data bank and analyze the expression of SLP2 in tumor and normal tissue.2.Analysis expression of SLP2 indifferent tissues based on BioGPS database.3.To detect the expression of SLP2 in paraffin embedded GC tissue and normal mucosa that come from our hospital and other hospital by IHC.To collect the clinical and follow-up data from GC data bank and analyze the clinical significance and prognosis value of SLP2 in GC.4.To silence or enhance the expression of SLP2 in GC cell by SiRNA or overexpressing vector,to detect the proliferation by CCK-8 assay,to detect the colony forming ability by plate colony formation assay and to detect the ability of invasion by Transwell chamber.5.To predict the interacting proteins of SLP2 by Genecards and its related website.6.To verify the interaction of SLP2 and PHB by co-immunoprecipitation(co-IP).7.Immunofluorescence(IF)staining of SLP2 and PHB,and analyze the location of them under laser scanning confocal microscope.8.To analyze the correlation of SLP2 and PHB based on cbioportal database.9.To download the array analysis results of GC in GEO public database and analyze the correlation of SLP2 and PHB in SPSS.10.To download data from the GEO public database and analysis the enriched genes in SLP2 overexpressed GC tissue by gene set enrichment analysis(GSEA).11.To silence or enhance the expression of SLP2 in GC cell by SiRNA or overexpressing vector and analyze variation of PHB and MAPK signaling pathway by Western-blot.12.To treat cell with Rafl inhibitor which results in MAPK signaling pathway inhabitation after enhance the expression of SLP2 and detect the variation of MAPK signaling pathway by Western-blot.At same time,to evaluate the proliferation,colony forming and invasion of GC cell.13.To Silence SLP2 in MGC803 cell and detect the mRNA level of PHB by Q-PCR.14.To analyze the post-translational modification of PHB in the on line datebase(post-translational modification function,PTMfunc).15.To Silence PHB in MGC803 cell and detect the mRNA and protein level of SLP2 in GC cell.16.To download data from GEO public database and analyze the enrichment genes in PHB overexpressed tissues by GSEA.17.To analyze the potential translational factors those regulates SLP2 and identify its binding sites.18.Binding sites of ELK1 in the promoter region of SLP2 were detected by Chromatin immunoprecipitation(ChIP).19.Total protein and phospholyration protein expression level of ELK1 were detected by Western-blot in SLP2 overexpression and silenced cells.20.Expression of endogenous SLP2 was detected when cell transfected with exogenous 3×Flag-SLP2 vector.Results:1.SLP2 is overexpressed in multiple tumors and correlated with the poor survival of GC patients.(1)Analysis the expression of SLP2 in GC and other tumors in public databaseSLP2 shows a higher expression in GC,CRC,prostate cancer and melanoma compared to the normal tissues in GEO public microarray database.SLP2 also shows an elevated expression in CRC tissue compared to normal mucosa in BioGPS database.(2)The expression level of SLP2 in GC tissues and matched normal tissuesMulti-center IHC results reveal that SLP2 was mainly located in cell membrane and cell cytoplasm.Internal validation shows that 75.2%(79/105)GC tissues are with higher level of SLP2 compared to the normal tissues.External validation shows that 73.3%(66/90)GC tissues are with higher level of SLP2 compared to the normal tissue.(3)Clinical significance and diagnosis value of SLP2 in GCInternal validation shows that expression of SLP2 is not correlated with the sex(P=0.276),age(P=0.158)and differentiation(P=0.875),While its expression was correlated with depth of invasion(P=0.004),lymph node metastasis(P=0.002),distance metastasis(P=0.037)and AJCC stage(P=0.001).Moreover,external validation shows that expression of SLP2 is not correlated with the sex(P=0.787),age(P=0.501)and distance metastasis(P=0.476),while its expression was correlated with depth of invasion(P=0.037),lymph node metastasis(P=0.036)and AJCC stage(P=0.037).(4)Relationship between expression level of SLP2 and the prognosis of GC patients.Internal validation reveals that SLP2 is correlated to the prognosis of GC patients(P<0.001).Internal validation also reveals that SLP2 is correlated to the prognosis of GC patients(P=0.006).2.Level of SLP2is associated with the proliferation,colony forming and invasion of GC cell.(1)Analyze the biological function of GC cell after silencing the expression of SLP2.The proliferation,colony forming and invasion of GC cell are suppressed in cell that SLP2 is silenced by SiRNA.(2)Analyze the biological function of GC cell after enhancing the expression of SLP2.The proliferation,colony forming and invasion of GC cell are promoted in cell that SLP2 is upregulated by overexpressing vector.3.Interaction between SLP2 and PHB in GC.(1)Database analysis of Interaction of SLP2There are 10 interaction proteins of SLP2 are predicted by searching STRING database including PHB.In addition,50 interaction proteins of SLP2 are predicted in BioGrid database.(2)Interaction of SLP2 and PHB is verified by co-IPWe verified the interaction of SLP2 and PHB by co-IP in MGC803 cell.(3)Location of SLP2 and PHB detected by IFWe found the co-localization of SLP2 and PHB in MGC803 cell using IF staining and laser scanning confocal microscope.4.mRNA levels of SLP2 and PHB are positive correlated.(1)Analyze the correlation between SLP2 and PHB based on public databaseWe analyzed the correlation between SLP2 and PHB based on cbioportaldatabase and found that mRNA levels of SLP2 and PHB were moderate positive correlation.We analyzed the correlation between SLP2 and PHB by downloading GSE1313911(n=38)and GSE15460(n=200)from GEO public microarray database.It shows that SLP2 is moderate positive correlated with PHB both in GSE13911(Pearson r=0.685,Spearman r=0.691)and GSE15460(Pearson r=0.690,Spearman r=0.725).5.Analysis of the gene enrichment in tissues with high SLP2 expressionWe tried to analysis the differentiated genes between SLP2 high expressed tissues and low expressed tissues by GSEA.It revealed that cell cycle related genes were significantly enriched both in GSE13911(Enrichment Score,ES=0.567,P<0.001)and GSE15460(ES=0.578,P<0.001).6.The mechanism of SLP2 involved in the progression of GC(1)The down-stream signaling pathway of SLP2We silenced the SLP2 in MGC803 cells and detected the variation of MAPK signaling pathway.It revealed that PHB,p-RAf1,p-MEK1/2,p-ERK1/2 were decreased when SLP2 was suppressed.Besides,expression of PHB,p-RAf1,p-MEK1/2,p-ERK1/2 were increased when SLP2 was promoted in AGS cell line.(2)The influence of Rafl inhibitor on SLP2 induced variation of MAPK signaling pathwayWe increased the expression of SLP2 in AGS cell and then treated cell with Raf1 inhibitor and detected variation of MAPK signaling pathway.We found that Raf1 inhibitor significantly suppressed the activation of p-Raf1,p-MEK1/2 and ERK1/2 in AGS cell.In addition,the proliferation,colony forming and invasion was also inhibited.7.Detection of the up-stream regulation mechanism of SLP2(1)Influence of SLP2 on the level PHB mRNAmRNA level of PHB do not changed when SLP2 was inhibited in MGC803 cell.(2)Influence of PHB on the mRNA and protein level of SLP2We found that mRNA and protein of SLP2 were significantly decreased inPHB silenced MGC803 cell.(3)Enrichment of genes regulated by transcript factors in PHB highly expressed tissuessSet transcript factor as an enrichment parameter,we analyzed the enriched genes in PHB highly expressed tissues by GSEA.We noticed that transcript factor ELK1 potentially regulated genes were significantly enriched both in GSE13911(ES=0.444,P<0.001)and GSE15460(ES=0.480,P<0.001).(4)Analysis of transcript factors of SLP2We predicted the transcript factors of SLP2 in Consite online analyzing system and found that there were 4 potential binding sites of ELK1 in the promoter region of SLP2.Furthermore,we predicted the transcript factors of SLP2 in JASPARonline analyzing system and found 2 potential binding sites of ELK1 in the promoter region of SLP2.8.Influence of SLP2 on the phosphorylation of ELK1Up-regualtion of SLP2 increased phosphorylation of ELK1,while silence of SLP2 inhibited the phosphoration of ELK1.9.Influence of transfenction of exogenous 3×Flag-SLP2 on the expression of endogenous SLP2Expression of endogenous SLP2 was promoted when AGS cell were transfected with exogenous 3×Flag-SLP2 vector.Conclusion:1.SLP2 is significantly increased in GC tissue compared to the normal mucosa.Up-regulation of SLP2 in GC tissues is correlated with the depth of invasion,lymph node metastasis and AJCC stage of GC,and is a predict maker for prognosis.2.The proliferation,colony forming and invasion ability of GC cell are inhibited when SLP2 were silenced,while overexpression of SLP2 promotes the proliferation,colony forming and invasion of GC cell.3.SLP2 promotes the MAPK signaling pathway and activation of ELK1 by interacting with PHB.4.ELK1 binds to promoter region of SLP2 that contributes to the SLP2-PHB-MAPK-ELK1-SLP2 positive feedback loop.