The Role And Mechanism Of ITGA2 In Gastric Cancer Chemoresistance

Background:Gastric cancer(GC)is one of the most malignant tumors of the digestive system with high morbidity and mortality,which seriously endangers the health of humans.In the past few decades,chemotherapy has significantly improved the prognosis of patients with GC.Chemotherapy is the main postoperative adjuvant therapy for early gastric cancer and maintenance therapy for advanced GC.However,the development of chemotherapy resistance has become one of the main reasons for the failure of GC treatment.Many factors drive the development of chemoresistance.Different chemotherapeutic drugs present with different mechanisms of killing tumor cells,and tumor cells share multiple mechanisms of drug resistance.Therefore,it is of great significance for GC treatment to explore the mechanism of GC chemoresistance and find molecular targets for preventing and reversing from chemoresistance.The integrin family was firstly discovered in 1986,and it is the most crucial adhesion receptor family in organisms.Integrins are distributed in almost all types of cells,and the distribution of integrins is various in different kinds of cells,thereby exerting multiple physiological functions such as affecting cell proliferation and migration and regulating cell cycle.Abnormal expression of integrins is related to body dysfunction and various diseases.Integrin subunit alpha 2(ITGA2)is a member of the integrin family.Studies found that ITGA2 was involved in the initiation and progression of colorectal cancer,cervical cancer and breast cancer,and it is closely related to the migration and invasion of tumor cells.Recently,it has been reported that ITGA2 also plays an important role in tumor chemoresistance.However,the role and mechanism of ITGA2 in chemoresistance of GC deserve to be systematically and deeply studied.Objective:1.To clarify the relationship between ITGA2 and chemoresistance of GC;2.To explore the role of ITGA2 in regulating chemoresistance of GC;3.To elucidate the mechanism of ITGA2 regulating chemoresistance;4.To reveal the cause of aberrant ITGA2 expression in GC chemoresistance;5.To investigate the treatment strategy of ITGA2 mediated chemoresistance in gastric cancer.Methods:1.Western blot,quantitative real-time polymerase chain reaction(q RT-PCR),and immunohistochemistry(IHC)were used to detect the expression level of ITGA2 in different GC cell lines,GC tissues and adjacent tissues.The public databases were used to analyze ITGA2 expression and relationship between the prognosis of gastric cancer patients.2.Using ITGA2 overexpression and sh RNA lentiviral vector to construct a GC cell model with stable overexpression and down-regulation of ITGA2.Western blot and q RT-PCR assays were used to verify the transfection efficiency.Using the LIVE/DEAD viability/cytotoxicity kit to determine GC cells apoptosis and survival levels;Half maximal inhibitory concentration(IC50)experiments and tumorigenicity experiments in nude mice were used to study the effects of ITGA2 on chemotherapy resistance of GC.3.Nano String Pan Cancer Pathways analysis and bioinformatics were employed to identify the molecular pathways involved.Western blot was performed to detect the regulation of MAPK pathway and EMT activation by ITGA2.4.The potential mi RNA of targeting ITGA2 was analyzed by bioinformatics analysis method.The expression of candidate mi RNAs in GC cells was detected by q RT-PCR analysis.The GC cells were transfected with mi RNA mimics and the transfection efficiency was verified by q RT-PCR analysis,the regulation of ITGA2 by mi R-135b-5p was verified by Western blot.The binding of mi R-135b-5p and ITGA2 3’-UTR and the regulatory effect were confirmed by luciferase reporter analysis.The above functional experiments and functional rescue experiments were used to study the role of mi R-135b-5p in ITGA2 mediated chemoresistance of GC.Western blot was used to detect the inhibition of MAPK pathway and EMT by mi R-135b-5p.Results:1.Western blot and q RT-PCR results showed that ITGA2 was lowly expressed in GES.The expression was higher in 5 of the 7 GC cell lines.Further research found that ITGA2 expression was significantly increased in GC drug-resistant cell lines.IHC results found that ITGA2 is mainly located in the cytoplasm and membrane of the cells.Compared with normal gastric tissues,ITGA2 is more highly expressed in gastric cancer tissues.The GEPIA database results also in accordance with our conclusions.According to the KM-plotter database survival analysis,it was found that increased ITGA2 levels were associated with poor prognosis in GC patients treated with 5-fluorouracil(5-FU).The overall survival,first progression,and survival after progression of GC patients were negatively correlated with ITGA2 level.2.The results of functional experiments showed that down-regulated ITGA2 can inhibit the proliferation of GC cells,and this inhibition was enhanced when treated with5-FU.Up-regulation of ITGA2 promoted proliferation,and the effect was more significant after 5-FU treatment.Down-regulation of ITGA2 increased the sensitivity of GC drug-resistant cells to 5-FU and Adriamycin(ADR).Up-regulated ITGA2 could increase the resistance of parental GC cells to 5-FU and ADR.Down-regulated ITGA2 promoted5-FU induced apoptosis,and up-regulated ITGA2 inhibited apoptosis.In vivo drug resistance assay showed that down-regulated ITGA2 inhibited proliferation,promoted apoptosis,and enhanced the sensitivity to 5-FU and ADR.3.The results of Nano String analysis showed that the downstream signal pathway was changed after ITGA2 was down-regulated,and it was found that the MAPK pathway was one of the pathways with the most variation.Western blot experiments confirmed that up-regulated ITGA2 could activate the MAPK pathway,down-regulated ITGA2 inhibited the MAPK pathway.The MAPK / ERK pathway and epithelial-mesenchymal transition(EMT)were down-regulated when ITGA2 was down-regulated.4.Using bioinformatics analysis and mi RNA chips,we identified mi R-135b-5p as a possible upstream regulatory molecule for ITGA2.The luciferase reporter assays demonstrated that mi R-135b-5p can bind to the 3’-UTR region of ITGA2,and there are only two binding sites.mi R-135b-5p was identified as a direct upstream regulator of ITGA2.Functional experiments and Western blot results showed that overexpression of mi R-135b-5p reduced chemotherapy resistance and induced apoptosis in GC cells by inhibiting MAPK signaling pathway and EMT,and decreased ITGA2-induced chemotherapy resistance and anti-apoptosis effects.Conclusion:In this study,we clarified the relationship between ITGA2 and GC chemoresistance and revealed the function of ITGA2 to promote chemoresistance in GC.Our results suggested the novel mi R-135b-5p/ITGA2/MAPK axis as an epigenetic cause of chemoresistance.In summary,this study underscored the upstream and downstream molecular mechanisms of ITGA2 in promoting GC chemoresistance with diagnostic and therapeutic implications.