The Role Of Endoplasmic Reticulum Stress-regulated ARHGEF2 In Angiogenesis And Sensitivity To Lenvatinib In Hepatocellular Carcinoma

Background:The mortality rate of Hepatocellular carcinoma(HCC)ranks third among all malignancies.Due to the high invasiveness and metastasis of HCC,most patients lose the opportunity for surgical treatment,and systemic therapy is the preferred treatment option for them.Molecular targeted therapies,such as lenvatinib,are first-line treatments for advanced HCC.Although lenvatinib has extended the median overall survival(OS)of inoperable patients with advanced HCC to 13.6 months,its long-term survival still remains unsatisfactory for the majority of patients.The efficacy of molecular targeted drugs in HCC is significantly limited by the inherent and acquired drug resistance generated by targeted therapy.Therefore,the current treatment situation of HCC remains severe.Endoplasmic reticulum(ER)stress is widely involved in the process of tumor invasion and metastasis,inducing resistance of HCC cells to various chemotherapeutic and molecular targeted drugs.However,the mechanism of ER stress-induced drug resistance involves multiple signaling pathways,which cannot be fully explained at present.Currently,it is considered that blocking the downstream regulatory pathways and action targets of ER stress could be an effective strategy to treat ER stress-related drug resistance.Hence,exploring ER stress-related target genes in HCC can provide a new research direction and theoretical basis for the therapeutic resistance of molecular targeted drugs.Object:To explore the key target genes of ER stress that promote cancer in HCC and to discover new therapeutic targets for ER stress-related molecular targeted drug resistance.Methods:(1)The intersection of differentially expressed genes was conducted by analyzing RNA sequencing,CHIP sequencing,and ATAC sequencing data of ER stress model cells and control HCC cells to identify key target genes.Western Blot,q RT-PCR,and immunofluorescence were used to verify whether ER stress regulated the m RNA and protein levels of ARHGEF2.(2)Jaspar database was utilized to predict upstream transcription factors of ARHGEF2,and three possible promoter binding sites were designed.Dual luciferase assay was employed to identify the binding sites of transcription factor ZNF263 and ARHGEF2.HCC cells were transfected with small interfering RNA(si RNA)and overexpressed plasmids targeting ZNF263,and Western blot and q RT-PCR were performed to verify the regulatory effect of ZNF263 on ARHGEF2.(3)Immunohistochemistry(IHC),Western Blot,and q RT-PCR were employed to detect the expression of ARHGEF2 in liver cancer and adjacent tissues.The correlation between ARHGEF2 and clinical stage,histological grade,and prognosis was analyzed based on clinicopathological data.Public data from the TCGA database were used to verify the correlation between ARHGEF2 and clinicopathological features.(4)The ARHGEF2 stable interference and ARHGEF2 stable overexpression cell lines were constructed by lentivirus technique.The effect of ARHGEF2 on cell proliferation in vitro and in vivo was detected by CCK-8,colony formation assay and tumor formation experiment in nude mice.The role of ARHGEF2 in angiogenesis was determined by HUVECs tube formation assay,Transwell assay,wound-healing assay and Chicken chorioallantoic membrane(CAM)assay.(5)The role of ARHGEF2 in the sensitivity of lenvatinib was determined by CCK-8,colony formation assay and flow cytometry.HUVECs tube formation assay,CAM,CCK-8 and colony formation assay were used to verify the role of ER stress in inducing angiogenesis and treatment sensitivity to lenvatinib.After knockdown of ARHGEF2,the ER stress-induced angiogenesis and the ER stress-related resistance to lenvatinib were detected by CCK-8,colony formation assay,flow cytometry and angiogenesis related experiments.The animal experiments were administered to test whether the interference of ARHGEF2 combined with lenvatinib could achieve better therapeutic effect.(6)Transcriptome sequencing was performed to search for key downstream effectors of ARHGEF2.Western Blot and q RT-PCR were used to verify whether the downstream target gene EDN1 was regulated by ZNF263/ARHGEF2.Small interfering RNA technology was used to interfere with EDN1 in HCC cells,and the role of EDN1 in angiogenesis was detected by tube formation assay,Transwell assay,wound healing assay and CAM assay.EDN1 was knocked down in HCC cells overexpressing ARHGEF2,and angiogenesis related experiments were used to detect whether ARHGEF2-induced angiogenesis was mediated by EDN1.Result:(1)ER stress upregulated the expression of ARHGEF2 in HCC cells.We analyzed the RNA-sequencing,ATAC-sequencing,and CHIP-sequencing data to find the differentially expressed genes.Here,we list the five upregulated genes including ARHGEF2,TRIB3,NMNAT2,HKDC1 and CREB5.Combined with the sequencing results and verification of the differential genes,the upregulated gene,ARHGEF2,was screened.Western Blot,q RT-PCR and immunofluorescence experiments all proved that the ER stress up-regulated the expression of ARHGEF2 protein and m RNA,while the ER stress inhibitor 4-PBA down-regulated the expression of ARHGEF2 in HCC cells.(2)ER stress upregulated ARHGEF2 expression through the activation of ZNF263.Interfered with ZNF263 in HCC cell lines Hep G2 and Hep3 B,respectively.Western Blot and q RT-PCR showed that the depletion of ZNF263 downregulated the expression of the ARHGEF2 protein and m RNA levels.On the contrary,overexpression of ZNF263 in HCC cell lines Huh7 and MHCC97 H led to up-regulation of ARHGEF2 expression.Three potential promoter binding sites of ZNF263 were identified using the Jaspar database.The results of dual luciferase reporter assay indicate that R3(-355 /-335,AGGGGAGGGAAAAAGGGAGGG)might be the binding site for ZNF263.(3)ARHGEF2 was highly expressed in HCC and was associated with a poor prognosis.Immunohistochemical(IHC)staining was performed on the microarrays of 138 patients with HCC and adjacent tissues.The results confirmed that ARHGEF2 was highly expressed in hepatocellular carcinoma tissues compared with paracancer tissues,and correlated with clinical stage,histological grade and poor prognosis.Western Blot and q RT-PCR results also indicated that the expression of ARHGEF2 in HCC was significantly higher than that in adjacent tissues.The results from TCGA database analysis confirm this conclusion again.(4)ARHGEF2 promoted proliferation of HCC in vitro and in vivo.Stable ARHGEF2 interference and ARHGEF2 overexpression HCC cell lines were constructed.CCK-8 assay,colony formation assay and animal experiment showed that ARHGEF2 knocked down could significantly inhibit the proliferation of HCC cells in vitro and in vitro,while overexpression of ARHGEF2 significantly enhanced the proliferation and tumorigenesis of HCC cells.(5)ARHGEF2 as a new target for promoting angiogenesis.Stable ARHGEF2 interference and ARHGEF2 overexpression cell lines were constructed.Transwell assay and wound healing assay of HUVEC cells showed that interference with ARHGEF2 inhibited the migration and invasion of vascular endothelial cells.Overexpression of ARHGEF2 significantly promoted the migration and invasion of HUVECs.Tube formation and CAM experiments showed that ARHGEF2 promoted microtubule formation in vitro and increased neovascularization in vivo.(6)ARHGEF2 was involved in the resistance to lenvatinib.The results of CCK-8 and flow cytometry indicated that interference with ARHGEF2 could increase the sensitivity of Hep G2 cells to lenvatinib,which could better inhibit cell proliferation and promote cell apoptosis.On the contrary,overexpression of ARHGEF2 in MHCC97 H cells resulted in decreased sensitivity of cells to lenvatinib.Tube formation and CAM experiments showed that ER stress induced neovascularization.The involvement of ER stress in lenvatinib resistance was verified by CCK-8 and colony formation assay.However,after ARHGEF2 knocked down,ER stress-induced angiogenesis and cell resistance to lenvatinib were partially inhibited.The animal experiment showed that,compared with the control group and the lenvatinib monotherapy,lenvatinib combined with ARHGEF2 interference could obtain better tumor inhibition effect in vivo.(7)EDN1 was the downstream effector of ARHGEF2.Transcriptional sequencing was performed on ARHGEF2-knockdown cells and the control cells.A total of 272 differential genes were identified,among which EDN1 was a common gene involved in multiple pathways.Western Blot and q RT-PCR were used to verify the regulatory effect of ARHGEF2 on EDN1.It was found that ER stress /ZNF263/ARHGEF2 pathway up regulated the expression levels of EDN1 protein and m RNA in HCC cells.(8)ARHGEF2 exerted an angiogenic effect via EDN1.Transfected EDN1 with small interfering RNA in HCC cells,and cell conditioned medium was collected for the tube formation assay,CAM assay,Transwell assay and wound-healing assay.The results showed that EDN1 knocked down could significantly inhibit the tubule formation,migration and invasion ability of HUVEC cells,and suppress the number of new blood vessels in chicken embryos.Interference with EDN1 in ARHGEF2 overexpressed cell lines reversed the effect of ARHGEF2 on promoting angiogenesis.Conclusion:(1)ARHGEF2 was a key target gene for ER stress in HCC cells.(2)ER stress regulated the expression of ARHGEF2 at the transcriptional level through ZNF263.(3)ARHGEF2 was highly expressed in liver cancer,and was correlated with clinical stage,histological grade and poor prognosis.(4)ARHGEF2 promoted cell proliferation,tumor growth and angiogenesis in vivo and in vitro.(5)EDN1 was a downstream target gene of ZNF263/ARHGEF2 pathway,and ARHGEF2 exerted an angiogenic effect via EDN1.(6)ARHGEF2 was involved in the sensitivity of HCC cells to lenvatinib.ARHGEF2 knockdown reversed ER stress-induced angiogenesis and apoptotic resistance of HCC cells to lenvatinib.