The Mechanism Of APEX1/miR-27a-5p Axis In The Occurrence And Development Of Gastric Cancer And Targeted Therapy

Background and ObjectiveGastric cancer(GC)is a prevalent yet heterogeneous malignancy in the digestive system.The oncogenesis and development of GC is a dynamic and complex pathological process involving multiple genes and factors,and clinical surgery is the main strategy for GC treatment,while chemotherapy is one of the most efficient methods for most of the patients that missed the best opportunity for surgical treatment.However,it presents an ongoing challenge for conventional therapeutics primarily for low targeting specificity and subsequent elicitation of multiple drug resistance(MDR).Therefore,improving the targeting specificity and anti-MDR properties of chemotherapeutics is the current focus of cancer targeted therapy research and is urgently warranted.Here,we intended to explore the roles of apurinic/apyrimidinic endodeoxyribonuclease 1(APEX1)and vascular endothelial growth factor A(VEGFA),two effectors of focal adhesion kinase(FAK)that contribute to the heterogeneity ofcancer and have been investigated in our previous study,in the tumorigenesis,progression and targeted chemotherapy of GC.APEX1 is a multifunctional protein that takes part as a DNA repair enzyme in the repair of genomic DNA damage through the base excision repair(BER)manner and modulates the transcriptional factors in redox signaling.Its aberrant expression has been reported in a variety of cancers for its contribution to cancer metastasis,poor prognosis and DNA repair-induced chemo-resistance.VEGF is well known as a key regulator of blood vessel development and it induces several pathological responses,including tumor angiogenesis,tumor invasion and tumor growth.Both APEX1 and VEGF are potential targets for cancer diagnosis,prognosis and therapeutic intervention.Besides,as one of the main regulators of gene expression,microRNAs(miRNAs)induced RNA interference(RNAi)is a novel and promising therapeutics.However,the specific roles miRNAs and their target genes play in modulating GC tumorigenesis,progression and MDR development remain unclear.Thus,the prediction of miRNAs specifically targeting APEX1 was adjusted for online databases,and we found that miR-27a-5p is down-regulated in GC and it is predicted to modulate the expression of APEX1 in the tumorigenesis and progression of GC,and it potentially inhibits the formation of MDR induced by chemotherapeutics and can be utilized as a method of cancer treatment.Based on the above considerations,APEX1 and VEGF were deleted in SGC-7901 cells to established a biallelic knockout cell model by CRISPR/Cas9(n)[Cluster regularly interspaced short palindromic repeats/Cas9(nick),CRISPR/Cas9(n)],and the roles APEX1 and VEGF play in the GC tumorigenesis,progression and development of MDR were explored.To further investigate the potential of APEX 1 in clinical chemotherapy,a GC-specific peptide(GP5)functionalized liposomal drug delivery formulation(GP5/Lipo/DOX/miR-27a-5p)was constructed and optimized by a previously screened gastric cancer targeted peptide(GP5)with good specificity and sensitivity with GC cells,and miR-27a-5p as a MDR inhibitor that negatively regulates APEX1 and is closely related to the formation of MDR in gastric cancer.Then the efficacy of drug system was evaluated in vitro and the roles APEX1/miR-27a-5p axis played in the oncogenesis,development and targeted therapy of GC were investigated,especially in APEX1 deleted cell model.This provides a theoretical and experimental basis for the investigation of the oncogenesis and development of GC and new ideas and strategies for the development of targeted chemotherapeutics for GC in the future.Methods1.Online database analysis(GEPIA,GTEx,cBio Cancer Genomics Portal):the APEX1 expression profiles and Kaplan-Meier survival curves of GC patients were analyzed.2.Real-time PCR:the relative expression of APEX 1 was detected.3.CRISPR/Cas9(n)was utilized to generate an APEX 1-deleted SGC-7901 cell line and VEGF-deleted SGC-7901 cell line,and the deletion was validated by DNA sequencing and western blot assay.4.After deletion of APEX1 and VEGF:The cell proliferation was determined by CCK-8 assay,colony formation assay and cell culture observation.The cell mobility was detected by wound healing assay and transwell chamber assay.The cell cycle was detected by flow cytometry.The cell apoptosis was detected by DAPI staining assay.The cell mobility-related cytoskeletal microstructures were detected by coomassie brilliant blue staining,crystal violet staining,SEM observation,TEM observation,immunofluorescence(IF)assay and immunocytochemistry(ICC)assay.Semi-quantitative RT-PCR and western blot assay were utilized to detect the EMT-related genes,MDR-related genes and cell survival-related genes.5.Online database analysis of miRNAs:the prediction of miR-27a-5p targets and the complementary sequences of pre-miR-27a-5p and APEX1 3'UTR were adjusted for databases miRDB,miRTarBase,TargetScan,TarBase v.8,miRBase,and the cross-checking was aggregated by VENNY 2.1.The Kaplan-Meier survival curve of miR-27a-5p was plotted by Kaplan Meier plotter.The multivariate Cox proportional hazards analysis of miR-27a-5p was performed using ONCOMIR.6.Dual-luciferase reporter assay was utilized to validate the interaction between APEX1 and miR-27a-5p.7.After overexpression of miR-27a-5p in WT cells and inhibition of endogenous miR-27a-5p(by anti-miR-27a-5p)in KO cells:The cell proliferation was estimated by CCK-8 assay,and the cell mobility was detected by wound healing assay and transwell chamber assay.8.Novel GC-targeted liposomal drug delivery formulations(GP5/Lipo/DOX,GP5/Lipo/DOX/miR-27a-5p and GP5/Lipo/DOX/Anti-miRNA)were established using thin-film evaporation and ultrasonic method,ammonium sulfate gradient method(for DOX loading)and properties of cationic liposome(for miR-27a-5p encapsulation).9.The formulations were characterized for various parameters using dynamic light scattering laser particle analyzer,fluorescence spectrophotometer and SEM and TEM.The mass ratio of GP5/Lipo/DOX and miR-27a-5p was optimized and used for targeting efficiency detection by cell transfection.10.After cell transfection with GC targeted drug delivery formulations:The cell proliferation was evaluated by CCK-8 assay,colony formation assay and cell culture observation.The cell mobility was detected by wound healing assay and transwell chamber assay.The cell apoptosis was detected by flow cytometry.The cell mobility-related cytoskeletal microstructures were detected by crystal violet staining,SEM observation,TEM observation and immunofluorescence(IF)assay.The western blot assay was utilized to detect the EMT-related genes,MDR-related genes and cell survival-related genes.Results1.The expression of APEX1 and VEGF is up-regulated in GC cells and they act as oncogenes.2.The APEX1 and VEGF gene deleted cell lines were established based on CRISPR/Cas9(n)editing.3.The cell proliferation,mobility and cell mobility-related cytoskeletal microstructures were inhibited after APEX1and VEGF deletion.4.Cell cycle arrest was detected in GC cells after APEX1 and VEGF deletion,while the cell apoptosis did not show significant changes.5.The expression of EMT-related genes and MDR-related genes were significantly altered after APEX1 and VEGF deletion,suggests that APEX1 and VEGF modulate the formation of EMT,and may regulate the sensitivity of GC cells towards chemotherapeutics.6.The expression of cell survival-related genes was significantly altered after APEX1 and VEGF deletion,suggests that APEX1 and VEGF may modulate the GC progression and the sensitivity of GC towards chemotherapeutics through MAPK and AKT signaling.7.The results from the dual-luciferase reporter assay suggest that APEX1 one of the most specific targets of miR-27a-5p.8.The cell proliferation and mobility were decreased after the overexpression of miR-27a-5p in WT cells,suggests that miR-27a-5p is a potent suppressor of APEX1 in GC progression.The blockage of endogenous miR-27a-5p(Anti-miRNA)in KO cells reverses the APEX1 deletion-induced reduction of cell proliferation and mobility.9.GC specific peptide-functionalized liposomal drug delivery formulations(GP5/Lipo/DO,GP5/Lipo/DOX/miR-27a-5p and GP5/Lipo/DOX/Anti-miRNA)were well prepared and characterized,which meets the requirements of subsequent cell experiments.10.GC targeted liposomal drug delivery formulations can deliver the DOX and miR-27a-5p/Anti-miR-27a-5p effectively to SGC-7901 cells.11.KO cells were more sensitive to DOX than SGC-7901(WT)cells.12.DOX inhibits cell proliferation,mobility,mobility-related cytoskeletal microstructures,EMT-related genes,MDR-related genes and the cell survival-related genes,and enhances the cell apoptosis of GC cells,and miR-27a-5p promotes the inhibition or enhancement induced by DOX.13.miR-27a-5p and DOX play a synergistic anticancer effect in GC through negatively regulating the expression of APEX1,and the blockage of endogenous miR-27a-5p(by anti-miRNA)significantly reverses the APEX1 deletion and DOX-induced reduction of cell mobility and increase of cell apoptosis.14.The APEXl/miR-27a-5p axis may be involved in the signal pathway of GC via MAPK and AKT signaling.Conclusions1.The expression of APEX1 and VEGF is elevated in GC as oncogenes,and the deletion of APEX 1 and VEGF inhibits the GC malignancy.2.miR-27a-5p is a potent suppressor of APEX1 in GC,especially in cell proliferation and migration,and inhibition of miR-27a-5p(by anti-miRNA)reverses the APEX1 deletion-induced reduction of cell proliferation and migration.3.GC targeted liposomal drug delivery formulations(GP5/Lipo/DOX,GP5/Lipo/DOX/miR-27a-5p and GP5/Lipo/DOX/Anti-miRNA)specifically target the SGC-7901 cells and can deliver the DOX,miR-27a-5p or anti-miRNA to cells.4.KO cells were more sensitive to DOX than SGC-7901(WT)cells,suggest that APEX1 deletion increased the sensitivity of GC cells to DOX,and that APEX1 may be involved in the MDR development.5.DOX inhibits the malignant phenotypes of GC,and miR-27a-5p plays a synergistic anticancer effect with DOX in promoting such inhibition.Blockage of endogenous miR-27a-5p(by anti-miRNA)partially reverses such inhibition,indicates that miR-27a-5p produces a synergistic anticancer effect with DOX in the GC tumorigenesis,progression and MDR development through negatively regulating APEX1.6.APEX1 may be contributing to the MDR development by its involvement in the DNA repair manner,and APEX1/miR-27a-5p axis may be involved in the signal pathway of GC tumorigenesis,progression and MDR development via MAPK and AKT signaling.