Application And Mechanism Of RGNEF In The Occurrence And Development Of Gastric Cancer And Gastric Cancer Targeted Chemotherapy Drug

Background and objective:Gastric cancer(GC)is one of the most common malignant tumors originated from digestive tract in China.Currently,the most diagnosed GC patients are already developed into the middle and late stages with infiltration and metastasis when it was diagnosed.Current chemotherapy usually results in a poor prognosis with low therapeutic efficacy and high side effects because of weak targeting specificity and fast elicitation of multidrug resistance(MDR).GC is caused by the accumulation of a series of abnormal gene expressions and signal pathways for a long time.It is still crucial to explore the functions and mechanisms of GC-related genes in oncogenesis and development of GC improving the GC diagnosis and targeted therapeutics,as well as developing novel chemotherapeutic drugs with high targeting efficiency with MDR inhibition.Rho guanine nucleotide exchange factor 28(ARHGEF28,also named as RGNEF)is over-expressed in multiple tumors and is a member of Rho guanine nucleotide exchange factors(RhoGEFs)that regulate the activation of Rho GTPases.Rho GTPases act as a molecular switch in signal regulation regulating cell adhesion,cell cycle,skeletal remodeling and other important processes,and its over-activation is closely related to oncogenesis.Integrins are α/β heterodimerize regulate a variety of biological activities.Integrin β1(ITGB1,also named as CD29)is a member of the integrin family and roles as a major adhesion receptor.ITGB1 is over-expressed in a variety of tumors and promotes malignant phenotypes of tumors by mediating cell migration,invasion,survival and apoptosis.Focal Adhesion Kinase(FAK)is up-regulated in GI tumors and involved in multiple processes of tumor development.Our previous results showed that the expression of RGNEF and ITGB1 would be down-regulated when FAK expression was suppressed,which suggested that there is a close interaction between these three genes in the oncogenesis,tumor development and the elicitation of MDR,which needs to be further explored.In this study,an RGNEF deleted cell line(RGNEF-/-SGC7901)was generated using the CRISPR/Cas9 technology,and the RGNEF was inhibited by shRNA in AGS cells.The roles of RGNEF in GC development were studied by combining the over-expression and rescue experiments of RGNEF in these two GC cell lines(SGC7901 and AGS).Simultaneously,the ITGB1-/-SGC7901 cell line was constructed,and the function of this RGNEF-related gene was studied by the same method.Finally,it was elucidated that the mechanism and signal pathway RGNEF promotes in GC oncogenesis and development.To explore the potential functions of RGNEF in GC targeted therapy,we developed a novel automatically GC targeted and MDR inhibited CRISPR delivery nano-drug:GP5-Lipo-RGNEF/Cas9-miR101-DOX(RGNEF/Cas9 was named as Cas9 in this study).The GP5 is a peptide that precisely and absolutely navigates to GC cells,and miR101-1-3p is a MDR inhibitor in this nano-drug.And this novel nano-drug was used in a series of experiments to investigate the cell behavior and therapeutic effect with or without the expression of RGNEF,and to test its efficacy of chemotherapy to GC and DOX resistant GC cells in vitro.It is of important potential to be further studied and developed a novel chemotherapeutic drug for GC targeted therapy with MDR inhibition,combined with gene deletion.Methods:1.The expression of RGNEF in GC cells/tissues and functions in the prognosis of clinical samples were assessed by the different databases(GEPIA,The Human Protein Atlas,UALCAN,Kaplan-Meier Plotter)and Western blot.2.The function of RGNEF in the EMT pathway and malignancy of GC cells was explored.(1)The RGNEF-/-SGC7901 cells was generated using CRISPR/Cas9 technology and the RGNEF expression in AGS cells was inhibited by the constructed shRNA vector,simultaneously,the functions RGNEF plays in these two GC cell lines was explored by the over-expression and rescue experiments.The key roles of RGNEF in regulating the malignant behavior and morphology of GC cells were investigated using MTT assay,transwell assay,flow cytometry(FCM),scanning electron microscope(SEM),confocal laser microscopy(LCSM),and so on.(2)The expression of EMT-related genes regulated by RGNEF was detected by Western blot to explore the molecular mechanism RGNEF regulated in GC development.3.The interaction between RGNEF and ITGB1 was investigated.(1)The co-localization of RGNEF and ITGB1 was detected by LSCM.(2)The interaction between RGNEF and ITGB1 was analyzed by phosphorylated Western blot.4.It was studied that the mechanism of ITGB1 regulated in promoting GC malignancy(1)The expression of ITGB1 in GC cells/tissues and functions in the prognosis of clinical samples was analyzed by different databases and Western blot.(2)The ITGB1-/-SGC7901 cells was generated using CRISPR/Cas9 technology,and the ITGB1 expression was inhibited by the constructed miR153 vector in AGS cells,and the over-expression and rescue experiments of ITGB1 in these two GC cell lines was conducted.The functions of ITGB1 in regulating the malignant behavior and morphology of GC cells were explored using MTT assay,transwell assay,the wound healing assay,FCM,SEM,LCSM,and so on.(3)The mechanism of ITGB1 regulated in GC malignancy was investigated by Western blot.5.The regulatory relationship between RGNEF and ITGB1 was explored.(1)To explore the possible synergies between the two genes,it was studied that the effect on the behavior and morphology of SGC7901 cells in ITGB1 over-expressed RGNEF-/-SGC7901 cells,as well as in ITGB1 over-expressed cells with RGNEF inhibition.(2)To further explore the possible synergies between these two genes,it was explored that the effect on the behavior and morphology of SGC7901 cells in RGNEF over-expressed ITGB1-/-SGC7901 cells,as well as in RGNEF over-expressed cells with ITGB 1 inhibition.(3)The regulatory relationship between RGNEF and ITGB1 was finally confirmed by phosphorylated Western blot.6.The signaling pathway RGNEF regulated in GC development was investigated.(1)The relationship between RGNEF,ITGB1 and FAK was analyzed using different databases(STRING,TIMER),semi-quantitative RT-PCR and Western blot.(2)The signaling pathway RGNEF regulated to promote the development of GC was explored by phosphorylated Western blot.7.It was explored that the effect and mechanism of the GC targeted,MDR inhibited CRISPR nano-drug on GC therapy(1)The detection of the specificity and sensitivity of GP5 to GC cells/tissues by LSCM.(2)The MDR gene-related miRNAs’ vectors were constructed,and the targeting efficiency of miRNAs to their targets was confirmed by the dual-luciferase reporting assay.(3)The GP5-Lipo-Cas9-miR101-DOX was developed by membrane ultrasonic dispersion method,and the characteristics of nanoparticles was detect by laser particle analyzer,fluorescence microscope and TEM.(4)The targeting efficiency of the nano-drug to GC cells was detected by fluorescence microscopy and FCM in SGC7901/ADR,SGC7901,HepG2,Eca109,Caco2 and HEK293 cells transfected with GP5-Lipo-GFP(PCDNA6.2-GW/GFP).(5)It was confirmed that the cytotoxicity of GP5-Lipo-Cas9-miR101-DOX in SGC7901/ADR and SGC7901and control cells.(6)The mechanism of GP5-Lipo-Cas9-miR101-DOX in GC therapy was investigated by semi-quantitative RT-PCR and Western blot.Results:1.RGNEF is superabundant expressed in GC cells/tissues and induces the poor prognosis of GC patients.The methylation level of RGNEF is negatively correlated with GC stages/grades.2.The function of RGNEF in the EMT pathway and malignancy of GC cells was confirmed.(1)The RGNEF-/-SGC7901 cell line was generated using CRISPR/Cas9 and the RGNEF expression was inhibited by the constructed shRGNEF-2 vector in AGS cells,and the expression of RGNEF was rescued in these two GC cell lines by the constructed RGNEF over-expressed vector.(2)RGNEF deletion/inhibition significantly suppressed the GC cell proliferation and migration,and the development of motility related structures with the restoration of contact inhibition.The malignancy and morphology of GC cells were significantly re-enhanced by the RGNEF resumed expression.(3)The Western blot results showed that RGNEF regulates the EMT pathway of GC cells.3.The interaction between RGNEF and ITGB1 was confirmed.(1)The LSCM images showed that RGNEF and ITGB1 were co-localized in SGC7901 cells.(2)The Western blot results showed that RGNEF induced the expression and activation of ITGB1.4.The mechanism of ITGB 1 regulated in GC malignancy was investigated.(1)ITGB1 is over-expressed in GC cells/tissues and induces the poor prognosis of GC patients.(2)The ITGB1-/-SGC7901 cell line was generated using CRISPR/Cas9 and the ITGB1 expression was inhibited by the constructed miR153 vector in AGS cells,and the expression of ITGB1 was rescued in these two GC cell lines by the constructed ITGB1 over-expressed vector.(3)ITGB1 deletion/inhibition significantly suppressed the GC cell proliferation and migration,and the development of motility related structures with the restoration of contact inhibition.The malignancy and morphology of GC cells were significantly re-enhanced by the ITGB1 resumed expression.(4)The Western blot results showed that ITGB1 regulates the EMT and Src-mediated FAK/PT3K/AKT pathway to promote the development of GC.5.The regulatory relationship between RGNEF and ITGB1 was explored.(1)The over-expressed ITGB1 induced the cell growth and motility in RGNEF-/-SGC7901 cells to some extent,and the RGNEF inhibition significantly suppressed the cell growth and motility in ITGB1 over-expressed cells,indicating that RGNEF was stronger than ITGB1 in maintaining GC malignancy.(2)The Western blot results showed that RGNEF induced the expression and activation of ITGB 1.6.The results are consistent with database analysis that RGNEF regulates the expression of ITGB 1,FAK and Src.The Western blot results indicated that RGNEF regulates GC oncogenesis,development and malignancy via ITGB 1-mediated FAK/Src/PI3K signaling pathways.7.The effect and mechanism of the GC targeted,MDR inhibited CRISPR nano-drug on GC therapy was investigated.(1)GP5 targets to the GC cells/tissues with specificity and sensitivity and locates in the cell membrane of SGC7901/ADR and SGC7901 cells.(2)The miR-101-3p vector was successfully constructed,and the dual-luciferase reporter assay confirmed that miR101 significantly targets to P-gp,ABCC5 and EZH2.(3)The GP5-Lipo-DOX-Cas9-miR101 was synthesized.The particle size ranges of GP5-Lipo and GP5-Lipo-DOX were119.8 nm and 143.4 nm.The PDI was approximately 0.2 represents good homogeneity of these constructed liposomes.And the entrapment efficiency of DOX was 89.25%.The drug-loaded nanoparticles were constructed homogeneously with moderate dispersion under an inverted fluorescence microscope and TEM(4)The nano-drug targeted to SGC7901/ADR and SGC7901 cells with high targeting efficiency(5)GP5-Lipo-DOX-Cas9-miR101 targeted GC cells with RGNEF deletion and MDR inhibition promoted GC cell apoptosis with DOX,and significantly inhibited cell proliferation,migration,and the development of motility relevant micro structures.(6)The novel GC targeted chemotherapeutic formulations induce GC and DOX-resistant GC cells death and growth inhibition via regulation to the expression of MDR and EMT relevant genes.Conclusions:(1)The over-expression of RGNEF regulates the EMT pathway to promote the proliferation and migration,and the development of motility relevant micro structures of GC cells,as well as the poor prognosis of GC patients.(2)ITGB1 regulates the EMT and Src-mediated FAK/PI3K/AKT pathway to promote the proliferation,migration and the development of motility relevant micro-structures of GC cells.(3)RGNEF expression induces the expression and activation of ITGB1,and regulates GC oncogenesis and malignancy via ITGB1-mediated FAK/Src/PI3K signaling pathways.(4)GP5-Lipo-DOX-Cas9-miR101 targets to GC cells with RGNEF deletion and MDR inhibition,and promotes to GC cells apoptosis with DOX,and significantly inhibits cell proliferation,migration,and the development of motility relevant micro structures.(5)The novel GC targeted chemotherapeutic formulations induce GC and DOX-resistant GC cells death and growth inhibition via regulation to the expression of MDR and EMT relevant genes.The important roles of RGNEF and ITGB1 in GC oncogenesis and development were confirmed in this study,and the molecular mechanism and signal pathway in which RGNEF and ITGB1 participate were explored.It was first time confirmed that RGNEF regulates the GC malignancy via ITGB1-mediated FAK/Src/PI3K signaling pathways.Then a novel GC targeting with MDR inhibited CRISPR nano-drug with a good therapeutic effect on DOX resistant GC cells was developed.The role and mechanism of RGNEF gene in GC targeted chemotherapy and MDR inhibition were verified,which is of great significance for GC diagnosis and targeted therapy as well as the development of GC targeted drugs supported by some molecular mechanisms in the future.