Abnormal Expression And Mechanism Study Of SPIN1 In Gastric Cancer

Background:Gastric cancer(GC)is one of the most common cancers around the world,with the third highest lethality among all cancers worldwide.Due to the insidious symptoms and difficulty in early detection,GC patients are mostly in the middle and late stage when diagnosed,and the 5-year survival rate is less than 20%,causing serious harm to people's health.Due to the high heterogeneity of GC,the systemic treatment of GC is progressing slowly and the treatment means are limited.Although chemotherapy is the main treatment for middle and advanced GC patients and can prolong the overall survival period,it has prominent adverse reactions and is easily resistant to drugs.Except for trastuzumab,which can benefit a small number of patients with HER-2 positive advanced GC,the clinical efficacy of other targeted drugs is not satisfactory.Searching for sensitive and specific genetic changes in the development of GC is important for the diagnosis and therapeutic purposes,which is in line with the urgent demand of individualized treatment of GC in China.SPIN1,a member of the SPIN/SSTY family,is highly expressed in human malignant tumors such as ovarian cancer,lung cancer,breast cancer and liposarcoma.SPIN1 overexpression can promote the malignant transformation of mouse embryonic fibroblast NIH3T3 cells.Our previous studies in breast cancer revealed that SPIN1 is expressed in drug-resistant and metastatic tissues of breast cancer.SPIN1 promotes drug resistance and migration infiltration of breast cancer cells through positive regulation of PI3K/Akt pathway and drug metabolizing enzymes.All these results suggest that SPIN1 may be an important oncogene involved in tumorigenesis and development.However,the relationship between SPIN1 and GC has not been studied so far,and the mechanism of SPIN1's high expression in tumors is still unclear.In this study,the relationship between SPIN1 and the clinicopathological parameters and prognosis of GC was clarified at the clinical sample level,and the upstream regulatory mechanism of SPIN1 overexpression in GC was explored at the transcriptional level.We also explored the effect of SPIN1 on the proliferation,infiltration and metastasis of GC cells through in vitro and ex vivo functional experiments,and further elucidated the molecular mechanism by which SPIN1 promotes GC proliferation.Methods:(1)We investigated the expression of SPIN1 in GC tissues and cells by immunohistochemistry,real-time quantitative PCR and bioinformatics analysis,and further analyzed the relationship between SPIN1 expression and patients'clinicopathological parameters and prognosis.(2)In order to explore the upstream regulatory mechanism of SPIN1 expression in GC,we constructed a truncated plasmid of the upstream promoter region of SPIN1 and performed a dual luciferase assay to identify the core promoter region of SPIN1.We used a bioinformatics website to predict the transcription factors that might bind to the core promoter region of SPIN1,and further verified the regulation of SPIN1 by dual-luciferase assay,RT-qPCR and chromatin immunoprecipitation assay(ChIP).(3)The effects of overexpression and interference of SPIN1 on migration,infiltration and proliferation of GC cells were investigated by Transwell assay,MTS,EDU and plate cloning assay,and the effects of overexpression and interference of SPIN 1 on apoptosis and cell cycle of GC cells were investigated by flow cytometry.(4)Using gene expression microarrays,pathway enrichment analysis,real-time quantitative PCR,western blot and literature reports,we identified the target genes and signaling pathways involved in SPIN1 regulation,and further elucidated the direct regulatory effect of SPIN1 on target gene.The protein expression of target gene was detected by IHC in GC and non-tumor gastric mucosa,and analysis of its relationship with the prognosis of GC and its correlation with SPIN1.(5)In vivo verification of the effect of SPIN1 on the proliferation ability of GC cells and preliminary determination of its potential as a therapeutic target of GC.After nude mice were injected subcutaneously with GC cells,the experimental group and the control group were injected with lentivirus Lv-ShRNA-SPIN1 and Lv-NC intra-tumoraneously at multiple points,and the changes of tumor size in the two groups were observed.The expression of Ki67 in the tumor tissues of experimental and control groups were detected by immunohisto-chemistry,and the correlation between them was analyzed.RNA and protein were extracted from the tumors to verify the signaling pathways involved in the regulation of SPIN1 identified in vitro.Results:(1)SPIN1 is upregulated in human GC tissues and is associated with poor prognosisThe results of TCGA indicated that SPIN1 expression is markedly elevated in human GC samples compared with normal tissues.Moreover,survival analysis using the Kaplan-Meier Plotter showed that patients with high expression of SPIN1 had poorer overall survival and first progression survival than those with low expression of SPIN1.By IHC assay,SPIN1 expression exhibited a gradual increase from non-tumorous gastric mucosa via primary GC tissues to secondary LNM foci.More interestingly,we found that SPIN1 expression in GC with LNM was significantly higher than that without LNM,suggesting that SPIN1 may be associated with lymph node metastasis in GC.To further assess the clinical significance of SPIN1 in GC,we correlated SPIN1 expression with clinical variates of GC cohorts.The results showed that high SPIN1 expression was strongly correlated with positive LNM,positive distant metastasis,clinical stage,and differentiation.Kaplan-Meier analysis showed that high SPIN1 expression was correlated with poorer OS and DFS.Then,univariate and multivariate analyses by the Cox proportional hazards regression model were performed to explore factors associated with patient outcome.The results indicated that SPIN1 expression,tumor stage,LNM,and distant metastasis were significantly correlated with OS of GC patients,whereas only distant metastasis was an independent prognostic predictor for OS.To test the ability of SPIN1 as a biomarker to distinguish different clinical pathological parameters related to GC patients,ROC curves were established.ROC curves reflected clear separation between GC with and without LNM,early and late clinical stage,high and low differentiation,and with and without distant metastasis.(2)SPIN1 is directly regulated by transcription factor E2F1UCSC Genome Browser was used to find the promoter sequence of human SPIN1 and the 2000 bp sequence upstream of the transcription start site was downloaded.To identify the core promoter region of SPIN1,the promoter activities of the region?2,000 bp upstream of its TSS and seven deletion constructs were determined using dual-luciferase reporter assays.The results showed that the-374 to-75 bp region upstream of SPIN1 is its core promoter region.The transcription factor-binding site region from-374 to-75 bp was analyzed using the JASPER program and PROMO.There were potential binding sites for the transcription factor SP1,E2F1,YY1,and C/EBP? in the proximal promoter region of SPIN1.Dual luciferase,RT-qPCR and western blotting showed that E2F1 could activate the transcription of SPIN1.Finally,we verified that E2F1 can bind directly to the SPIN1 promoter region by ChIP experiments.(3)SPIN1 promotes GC cell migration,invasion and growth in vitroSPIN1 overexpression plasmid was constuced and SPIN1 small interfering sequences were synthesized by company.RT-qPCR analysis showed that the mRNA expression of SPIN1 was significantly up-or downregulated when transfected with SPIN1 plasmid or SiRNA-SPIN1 in GC cell lines.Migration and invasion assays showed that the cells transfected with the SPIN1 plasmid exhibited enhanced migration and invasion activities compared with the control,whereas the cells in which SPIN1 was knocked down had decreased migratory and invasive capacities.The proliferative capacity of SPIN1 was significantly increased when transfected with the SPIN1 plasmid,while its proliferative capacity was significantly reduced after interfering with SPIN1 by MTS,EdU,and colony formation assays.(4)SPIN1 promotes GC cell cycle progression and has no effect on apoptosis of GC cellsAs SPIN1 can promote GC cell proliferation,we next determined whether SPIN1 overexpression or knockdown could affect the cell cycle or apoptosis in GC cells.The results showed that SPIN1 overexpression promotes the transition from G1 to S phases of the cell cycle,whereas suppression of SPIN1 induces a decrease in the number of cells in the S phase and an unstable number of cells in the G2 phase.These results showed that SPESN1 plays a pivotal role in the G1/S phase transition.Next,we investigated the effects of SPIN1 on cell apoptosis.Annexin V-FITC assays indicated that there was no difference in the proportion of apoptotic cells between the experimental and control groups.These results revealed that SPIN1-mediated promotion of GC cell proliferation was due to cell cycle modulation rather than apoptosis(5)SPIN1 Promotes migration and infiltration of GC cells by regulating EMT Western blot assay was performed to detect the changes of EMT-related proteins Vimentin,Zeb1,Zo-1,?-catenin,Snail,Slug and Claudin-1 in GC cells after overexpression/silencing of SPIN1.The results showed that the expression of Slug was up-regulated and Claudin-1 was down-regulated after overexpressing SPIN1,and the expression of Slug was down-regulated and Claudin-1 was up-regulated after interfering with SPIN1,no significant changes were observed in the other proteins.The results suggest that SPIN1 may be involved in the EMT process.(6)SPIN1 promotes tumorigenesis and proliferation via the activation of MDM2-p21-E2F1 pathwayBased on the Gene expression analysis and the literature review,four cancer-associated genes including MAP2,MDM2,MAPKBP1 and GPNMB were selected as candidates to be investigated.Then we validated the candidates by RT-qPCR and western blotting.The results showed that the mRNA levels and protein expression of MDM2 increased when SPIN1 was overexpressed,while no significant effects on mRNA and protein expression of MAP2,MAPKBP1 and GPNMB was observed.Hence,we chose MDM2 for further investigation.MDM2 is a crucial mediator of cell cycle regulation,we hypothesized that SPIN1 promotes cell-cycle progression in GC through MDM2-mediated signaling pathway.The expression of the MDM2,p21,p27,and cell-cycle regulators were examined by RT-qPCR and Western blot.The results revealed that the mRNA levels of MDM2,CDK4,CyclinDl and E2F1 were suppressed by SPIN1 siRNA,while little effect on the mRNA expression was found when SPIN1 was overexpressed.The protein expression of MDM2,CDK4,CyclinDl,P-RB,and E2F1 increased,whereas p21 expression decreased when SPIN1 was overexpressed in both GC cell lines.Conversely,knockdown of SPIN1 decreased the protein expression levels of MDM2 and its targets,CDK4,CyclinD1,P-RB,and E2F1,whereas p21 exhibited the opposite results.To investigate the relevance of SPIN1 and MDM2 in GC tissues,MDM2 expression level was quantified with immunohistochemistry in both GC and non-tumorous tissues.As expected,the results showed that MDM2 was negative in non-tumorous tissues,the expression of MDM2 in GC with LNM was significantly higher than that without LNM.Then Kaplan-Meier analysis showed that high MDM2 expression was related to poorer OS and DFS.Results of Spearman's correlation analysis showed a positive relationship between MDM2 and SPIN1.Consistently,the analysis of TCGA indicated that MDM2 expression is markedly elevated in human GC samples compared with normal tissues.Moreover,survival analysis using the Kaplan-Meier Plotter showed that patients with high expression of MDM2 had poorer overall survival.These data further demonstrated that SPIN 1-induced proliferation of GC was mediated via MDM2.(7)SPIN1 directly regulates MDM2 expression by binding to H3K4me3,independently of p53.To further investigate the mechanism by which SPIN1 regulates MDM2 expression,the UCSC genome browser was used to predict the presence of H3K4me1,H3K4me3 and H3K27AC enriched peaks upstream of MDM2.SPIN1 has been reported in the literature to be a histone code reader that recognizes H3K4me3.Therefore,we hypothesized that SPIN1 promotes transcriptional activation of MDM2 by recognizing H3K4me3.Subsequently,we performed CHIP-qPCR assay using anti-H3K4me3 antibody.The results showed the presence of H3K4me3 in the promoter region of MDM2.Finally,we verified the direct binding between endogenous SPIN1 and H3K4me3 in GC cells by Co-IP assay using the anti-SPIN1 antibody.The above results indicate that SPIN1 enhances the expression of MDM2 by binding to H3K4me3 of the MDM2 promoter.To further determine whether the regulation of MDM2 by SPIN1 is dependent on p53,we performed rescue experiments.The results indicated that the regulatory effect of SPIN1 on MDM2 was independent of p53.(8)SPIN1 could be used as a therapy target in suppressing GC growth in vivoTo explore whether SPIN1 can be used as a target for GC therapy,lentiviral packaged ShRNA-SPIN1 was injected into the tumor xenografts model every seven days thrice.After a 30-day follow-up period,we found a significant reduction in tumor volume in the LV-ShRNA-SPIN1 groupscompared with the LV-NC group.Besides,tumor nodules in the LV-ShRNA-SPIN1 group were non-invasive or well-encapsulated.However,those tumors in the NC group showed invasion of the margin and into the surrounding stroma or muscle tissues.Immuno-histochemistry for Ki-67 revealed that the GC cells in the LV-shRNA-SPIN1 group had a lower positivity rate than those in the LV-NC group.Taken together,these findings suggest that SPIN 1 could be used as a target for GC therapy.In order to further investigate the molecular mechanism of SPIN1 to promote the proliferation of GC cells in vivo,RNA and protein from tumor were extracted and verified by RT-qPCR and Western blotting,which was consistent with the results of in vitro cytology.Conclusions and significance:We have elucidated for the first time that SPIN1 is an oncogene that promotes GC development,and that high expression of SPIN1 is associated with poor prognosis of GC.E2F1 bind directly to the promoter region of SPIN1 and activate the transcription of SPIN1.SPIN1 promotes transcriptional activation of MDM2 by recognizing H3K4me3.SPIN1 can be a potential therapeutic target for GC.