Study On The Mechanisms And Pharmacological Interventions Of SHCBP1 On Promoting Cell Proliferation And Migration In NSCLC

Objective:Lung cancer is the leading cause of cancer-related deaths worldwide.Non-small cell lung cancer(NSCLC)is the leading histological type of lung cancer,accounting for approximately 85% of these cases.Due to the highly aggressive and rapidly progressive nature of NSCLC,therapies such as surgical procedure and radiation treatment cannot eliminate tumors in an effective and timely manner.Therefore,targeted therapy,which kills tumor cells with high precision,is rapidly becoming a first-line treatment.However,the prevalence of acquired drug resistance in patients after targeted therapy poses a major challenge to the treatment of NSCLC.In order to achieve highly effective treatment of NSCLC,it is crucial to identify new targets for NSCLC progression and furt her elucidate their underlying molecular mechanisms.Src homolog and collagen homolog binding protein 1(SHCBP1)is an adaptor protein for SH2 domain in Src homolog and collagen homolog A(SHC A),and is involved in the formation of the central spindle in mitosis,as well as an essential regulator of the cytokinesis completion.Previous studies suggest that SHCBP1 can promote tumor cell proliferation and migration,and may be a prognostic biomarker for the development of various malignancies such as breast,gastric,and prostate cancers.Rucaparib is a PARP inhibitor that blocks the repair of damaged DNA in cells,thereby inhibiting the progression of multiple cancers.In cervical cancer,rucaparib inhibits cell proliferation by reducing the expression of cell cycle protein cyclin D1 and CDK4,which induces G2/M phase arrest.In addition,rucaparib has been shown to reduce the growth of lung adenocarcinoma and prostate cancer tumors in vivo.Moreover,rucaparib has been approved for clinical treatment of multiple cancer patients in the United States and Europe with specific conditions,indicating that rucaparib has become a well-established anti-cancer therapeutic agent.In this study,we aimed to clarify the role of SHCBP1 expression on cell proliferation and migration in NSCLC and explore its potential mechanism,as well as to preliminarily investigate whether rucaparib could regulate cell proliferation,cell migration and tumor growth through SHCBP1 in NSCLC.In addition,we proposed to identify SHCBP1 as a potential target for lung cancer patients who are more vulnerable to COVID-19 infection.In summary,this study intends to provide a theoretical basis for SHCBP1 to be a new target for treatment of NSCLC.Methods:1.The role of SHCBP1 in the cell proliferation and migration of NSCLCThe key genes promoting cell proliferation and migration of NSCLC were firstly screened by bioinformatics methods such as WGCNA,DEGs and correlation analysis.Meanwhile,the expression of SHCBP1 and its clinical stages were clarified in multiple cancer tissues.Then,the expression of SHCBP1 in NSCLC tissues and cells was detected by IHC and WB experiments.Finally,the ability of cell proliferation and migration in NSCLC after SHCBP1 knockdown or overexpression was investigated by CCK8,colony formation,cell immunofluorescence staining of Ki67,wound healing,Transwell,and cell adhesion assays.2.Mechanisms of SHCBP1 in promoting cell proliferation and migration of NSCLCThe biological functions of A549 cells affected by SHCBP1 knockdown were firstly analyzed by Cancer SEA and RNA-seq data.The ratio of cell cycle in A549 after SHCBP1 knockdown was detected by flow cytometry while the ability of cell extension was confirmed by cell immunofluorescence staining of F-actin.Then,the expression of G2/M phase related proteins such as CDK1 and Cyclin B1,and of EMT-related proteins such as E-cadherin,N-cadherin,MMP2 and MMP9 were detected in A549 after SHCBP1 knockdown by WB assay.Finally,CCK8,colony formation,cell immunofluorescence staining of Ki67,wound healing,Transwell and cell adhesion experiments were utilized to investigate cell proliferation and migration after overexpression of CDK1 in A549,as well as the expression of Cyclin B1,E-cadherin and N-cadherin was determined by WB assay after overexpression of CDK1 in A549.3.Study on the effect of rucaparib on inhibiting the cell proliferation,cell migration and tumor growth of NSCLC through SHCBP1Firstly,RNA-seq data were analyzed by CMap and compounds which could be potential small molecule inhibitors of SHCBP1 were screened.Then,the effects of rucaparib on cell proliferation and migration in A549 were detected by CCK8,RT-q PCR,colony formation,cell immunofluorescence staining of Ki67,wound healing and Transwell assays.Meanwhile,the expression of SHCBP1,CDK1,Cyclin B1,E-cadherin and N-cadherin was confirmed by WB assay.Finally,the effects of rucaparib on the tumor growth of A549 cells were investigated in vivo,and the expression of SHCBP1,CDK1,Cyclin B1,E-cadherin and N-cadherin in the isolated tumor tissues were detected by IHC assays.4.Study of SHCBP1 interaction with GINS1 as a potential factor of vulnerability to COVID-19 infection in lung cancer patientsFirstly,differential gene expression,GO,KEGG and Venn methods were used to analyze the key targets vulnerable to COVID-19 infection in cancer patients.Then,the biological function of GINS1 was noted by Cancer SEA and the correlation between SHCBP1 and GINS1 was determined by correlation analysis.Finally,RNA-seq analysis and RT-q PCR assay were performed to detect the transcriptional level of GINS1 after SHCBP1 knockdown in A549.Results:1.SHCBP1 promotes cell proliferation and migration in NSCLCEight key genes(SHCBP1,WDHD1,DLGAP5,CDCA4,CENPN,POLE2,CDKN3 and GNPNAT1)that drive the progression of NSCLC were screened by WGCNA and DEGs,and correlation analysis was used to detect that SHCBP1 had the highest positive correlation with cell proliferation and migration genes among these eight genes.Meanwhile,the results of IHC and WB experiments showed that SHCBP1 was highly expressed in NSCLC tissues and cells,and was positively correlated with poor prognosis.Moreover,knocking down SHCBP1 induced cell viability(A549),colony numbers and size(A549 and PC9),the ratio of Ki67 positive cells(A549 and PC9),wound closure rate(A549 and PC9),the cell number across Transwell chamber(A549),and cell adhesion ability(A549 and PC9)were decreased.Finally,overexpression of SHCBP1 increased cell viability,colony numbers and size,the ratio of Ki67 positive cells,wound closure rate,the cell number across Transwell chamber,and cell adhesion ability in A549.2.SHCBP1 promotes cell proliferation and migration via CDK1 upregulation in NSCLCFirstly,the analysis of Cancer SEA and RNA-seq data showed that downregulation of SHCBP1 could affect G2/M phase and EMT process.Meanwhile,the result of flow cytometry showed that inhibition of SHCBP1 could block A549 cells in G2/M phase.Moreover,the ability of cell extension after SHCBP1 knockdown in A549 was inhibited.Secondly,SHCBP1 knockdown can lower the expression of CDK1,Cyclin B1,N-cadherin,MMP2,MMP9 and up-regulate the expression of E-cadherin.Finally,overexpression of CDK1 increased cell viability,colony numbers and size,the ratio of Ki67 positive cells,wound closure rate,the cell number across Transwell chamber and cell adhesion in A549 knockdown SHCBP1 cells.Meanwhile,overexpression of CDK1 increased Cyclin B1,N-cadherin expression and decreased E-cadherin expression.3.Rucaparib inhibits cell proliferation,cell migration and tumor growth of NSCLC through SHCBP1Firstly,the top 10 compounds that could be potential small molecule inhibitors of SHCBP1 were screened out by CMap.Then,the result of CCK8 showed that rucaparib could inhibit cell viability of A549,and the results of RT-q PCR and WB determined that rucaparib could reduce the transcription and expression level of SHCBP1.Meanwhile,colony numbers and size,the ratio of Ki67 positive cells,wound healing rate and the cell number across Transwell chamber were all decreased,and the expression of CDK1,Cyclin B1 and N-cadherin was decreased while E-cadherin was increased after rucaparib treatment.Finally,the results in vivo showed that rucaparib significantly inhibited the tumor growth of A549 cells.The result of IHC assays confirmed that the expression of SHCBP1,CDK1,Cyclin B1 and N-cadherin was decreased and E-cadherin was increased in rucaparib-treated A549 tumor tissues.4.The interaction between SHCBP1 and GINS1 might be an influential factor in the vulnerability to COVID-19 infection in lung cancer patientsThe results of bioinformatic methods found that GINS1 was a key gene in cancer patients who are susceptible to COVID-19 infection,and its high expression promoted poor prognosis of cancer.Cancer SEA analysis showed that both GINS1 and SHCBP1 are involved in the cell cycle,and there was a significant positive correlation between GINS1 and SHCBP1.Finally,it was found in RNA-seq data that the transcription level of GINS1 decreased after SHCBP1 knockdown,and the result of RT-q PCR also confirmed that the transcription level of GINS1 decreased in A549 cells with SHCBP1 knockdown.Conclusion:This study demonstrated that SHCBP1 is an oncogene driving the progression of NSCLC,and SHCBP1 promotes the cell proliferation and migration by CDK1 upregulation in NSCLC.Initially,rucaparib was identified as a potential small molecule inhibitor of SHCBP1 to inhibit the cell proliferation,cell migration and tumor growth of A549 cells in vitro and in vivo.In addition,the interaction between SHCBP1 and GINS1 may also be an impact on the vulnerability of lung cancer patients to COVID-19 infection.In summary,SHCBP1 could promote the cell proliferation,migration and susceptibility to COVID-19 infection in NSCLC.This study provides the theoretical and experimental basis for SHCBP1 to become a new target for NSCLC treatment and for rucaparib to become a potential target drug for SHCBP1.