The Expression And Regulation Mechanism Of ClC-3 Chloride Channel Protein In Gastric Cancer

Background Gastric cancer(GC)is one of the most common tumors with the increasing morbidity and mortality world-wide.GC is characterized by insidious onset and high incidence of recurrence and metastasis.GC development is a complicated multistep process,influenced by the interaction between host genetic susceptibility and environmental factors.The most common genetic abnormalities in GC mainly include genomic instability,microsatellite instability(MSI)and Cp G island methylation(CIMP).In response to the genetic changes in pathogenesis of GC,several potential molecular targets have been identified to guide the prognosis of GC patients in recent years.However,current clinical trials have found that only therapies targeting HER2 and VEGFR2 are effective.To date,the prognosis of advanced GC patients remains poor.Therefore,achieving a detailed understanding of the molecular pathogenesis associated with GC and exploring more potential targets of GC for therapeutic purposes is imperative.The identification of novel tumor molecular markers has great clinical value in the effective treatment of GC.Chloride Voltage-Gated Channel 3(Cl C-3)is one of the members of chloride channel family which has attracted much attention recently.It is a multifunctional protein with roles in the regulation of extra-and intracellular ion homeostasis,acidification of intracellular vesicle and membrane excitability.But recent studies have revealed that Cl C-3 is closely related to multiple processes of carcinogenesis,including apoptosis,cell cycle and tumor multidrug resistance.However,the role and expression of Cl C-3 in digestive tract cancers is rarely reported,including GC.The molecular mechanisms by which Cl C-3 is regulated in GC are unclear.Whether Cl C-3 is a potential therapeutic molecular target and biomarker for GC patients is unknown.In this study,we identify XRCC5 as a promoter-binding protein of Cl C-3 in human gastric cancer cells by using Cl C-3 promoter probe.Moreover,XRCC5 can regulate the expression of Cl C-3 as a transcriptional co-activator.X-Ray Repair Cross Complementing 5(XRCC5)is one of the most important life cycle regulatory proteins with high focuses recently.It plays an important role in DNA damage repair and recombination,chromosome stability maintenance and gene expression regulation.Recent studies have found that XRCC5 has elevated expression in multiple tumor types,implicated in the key process of tumorigenesis.However,the role and mechanism of XRCC5 in malignant tumors has not been elucidated.Whether XRCC5 can affect the expression of Cl C-3,which may be the key molecular event in the pathogenesis of GC,and thus participate in the GC development is still unclear.The aim of this study is to investigate the expression,biological function and related pathways of Cl C-3 in GC,and explore the gene regulation mechanism and clinical significance of Cl C-3 expression,thus providing theoretical basis for the study of GC pathogenesis,and further expanding the space of molecular targeted therapy in GC treatment.Methods 1、The expression of Cl C-3 in human gastric cancer tissues and human gastric cancer cell lines was detected by immunohistochemistry and western blot.The main biological functions and related signaling pathways of Cl C-3 in gastric cancer cells were enriched by transcriptome sequencing and then verified.2、A 5’-biotin-labeled Cl C-3 promoter probe was designed and synthesized to pull down Cl C-3 promoter-binding proteins in gastric cancer cell lines by using BSBP(Biotin-Streptavidin-Beads Pull-down)assay.Subsequently,the target promoter-binding protein XRCC5 was identified by mass spectrometry analysis.By using specific antibodies against XRCC5,we performed pull-down assay and Ch IP(Chromatin Immunoprecipitation)assay to verify the interaction between XRCC5 and Cl C-3 promoter.In human gastric cancer specimens,the correlation and clinical prognosis of the expression between Cl C-3 and XRCC5 were analyzed using immunohistochemistry.3、The stable XRCC5-knockdown cell lines,stable XRCC5-overexpression cell lines,corresponding rescue model cell lines and corresponding reverse model cell lines were established by transfecting lentivirus.Through western blot,MTS,clone formation,wound scratch and transwell assays,we tested whether the expression of Cl C-3 could be regulated by XRCC5 in vitro,and explored the effects of this regulation mechanism on the main biological functions and related signaling pathways of Cl C-3 in gastric cancer.4、In molecular mechanistic studies,the gene expression regulation mode of Cl C-3 regulated by XRCC5 was explored by western blot and real-time fluorescence quantitative PCR.By constructing truncated plasmids of each fragment of Cl C-3 promoter reporter gene,combined with double luciferase reporter gene system and Ch IP experiment,we investigate the effect of XRCC5 on the activity of Cl C-3 promoter,and their possible specific binding sites in the promoter region of Cl C-3.To further explore the regulation mechanism of Cl C-3 gene expression,the possible transcription factor interacted with XRCC5 in the promoter region of Cl C-3 was identified and verified by co-immunoprecipitation,pull-down and immunofluorescence assays.5、Through animal model,we validated whether the expression and biological function of Cl C-3 were regulated by XRCC5 in the subcutaneous transplanted tumors of nude mice.The expression of Cl C-3 chloride channel protein and its specific regulatory mechanism were revealed in vivo and in vitro.Results 1、Cl C-3 was overexpressed in human GC tissues and GC cell lines.The GC patients with high expression of Cl C-3 had deep invasion depth,more lymph node metastasis,late TNM stage,and short overall survival time.2、In the gastric cancer cell model,knockdown of Cl C-3 significantly inhibited the proliferation and migration of GC cells,and down-regulate the expression of P-PI3 K and P-AKT in PI3K/AKT signaling pathway.3、In the gastric cancer cell model,the promoter binding protein of Cl C-3 was identified as XRCC5 by BSBP and protein spectrum analysis.By pull-down and CHIP assay,we found that XRCC5 could specifically bind to the promoter region of Cl C-3.4、In clinical samples of human gastric cancer patients,the expression of Cl C-3 and XRCC5 in gastric cancer tissues was higher than that in adjacent normal tissues,and the expression of Cl C-3 was positively correlated with XRCC5 in gastric cancer tissues Patients with high expression of Cl C-3 or high expression of XRCC5 had worse prognosis,while patients with high expression of both Cl C-3 and XRCC5 had the shortest survival time and the worst prognosis.5、In the gastric cancer cell model,knockdown of XRCC5 significantly reduced the expression of Cl C-3 and inhibited the proliferation,clonogenicity,migration and invasion of GC cells,while this inhibition effects could be rescued by Cl C-3 overexpression.In contrast,overexpression of XRCC5 significantly increased the expression of Cl C-3 and promoted the proliferation,clonogenicity,migration and invasion of GC cells,while this promotion effects could be reversed by Cl C-3 knockdown.6、In the gastric cancer cell model,Ch IP assays indicated that the binding of XRCC5 to the Cl C-3 DNA was suppressed after XRCC5 knockdown.Dual-luciferase reporter assays revealed that knockdown of XRCC5 impaired the promoter activities of the p GL4.10-Cl C-3-248 and p GL4.10-Cl C-3-538 reporter plasmids.In addition,the RNA level of Cl C-3 was inhibited after XRCC5 knockdown and increased after XRCC5 overexpression,and knockdown of XRCC5 down-regulate the expression of P-PI3 K and P-AKT in downstream PI3K/AKT signaling pathway of Cl C-3.7、In the gastric cancer cell model,PARP1 was identified as a candidate protein specifically binding to XRCC5 in the nucleus.The interaction between XRCC5 and PARP1 was confirmed by Co-immunoprecipitation.Pull-down assay confirmed that PARP1 could also specifically bind to the DNA promoter region of Cl C-3.Immunofluorescence assay confirmed that XRCC5 and PARP1 were co-localized in the nucleus of gastric cancer cells.Moreover,the promotion effects of XRCC5 overexpression on Cl C-3 expression,cell proliferation and cell clonogenicity were also reversed by PARP1 knockdown.8、In mouse xenograft models,knockdown of XRCC5 significantly inhibited the formation ability of GC tumor xenograft,with decreased tumor weights and tumor volumes,and the expression of XRCC5 and Cl C-3 was distinctly reduced.In contrast,XRCC5 overexpression promoted the formation ability of GC tumor xenograft and Cl C-3 expression in the pathological section,combined with increased tumor weights and tumor volumes,and the promotion effects could be reversed by Cl C-3 knockdown.Conclusions 1、Overexpression of Cl C-3 is a poor prognostic biomarker for gastric cancer.2、The primary biological functions of Cl C-3 are identified as cell proliferation and migration,and PI3K/Akt signaling pathway may be the downstream signaling pathway of Cl C-3.3、The expression and function of Cl C-3 in GC can be regulated by XRCC5 in vitro and in vivo.4、As a transcriptional co-activator,XRCC5 interacts with transcription factor PARP1 to form a transcriptional regulatory complex,which binds the Cl C-3 promoter region and affects subsequent promoter activity,thus regulating Cl C-3 expression at the transcriptional level.5、Both Cl C-3 and XRCC5 are independent prognostic factors of overall survival in GC patients,and double targeting Cl C-3 and XRCC5 may provide promising diagnostic and therapeutic potential for GC treatment.