The Mechanism Research Of Intracellular AGR2 Promoting Colorectal Cancer Metastasis

Part I Intracellular AGR2(i AGR2)promotes epithelial-mesenchymal transition and migration of colorectal cancer cells via modulating expression of SNAIL and SLUGObjective: This part aims to evaluate the impact of AGR2 on epithelial-mesenchymaltransition of CRC cells and the effects of intracellular AGR2(i AGR2)on expression of SNAIL/SLUG.Methods: The expression of AGR2 in six human colorectal cancer(CRC)cell lines(Lo Vo,SW480,SW48,HCT116,DLD-1 and HT-29)was detected by semi-quantitative PCR(RTPCR)and Western blot.Accordingly,the Lo Vo /SW480 cell lines were chosen for stably overexpressing AGR2 and the DLD-1/HT-29 cell lines were chosen for stably knocking down AGR2.The AGR2 knock out HT-29(HT-29AGR2-KO)and control cells(HT-29Ctrl)were generated by CRISPR/Cas9 genomic editing technology.Transwell migration assay was used to detect the effects of AGR2 on migratory abilities of the above cells.Quantitative real-time PCR(q RT-PCR),Western blot and immunofluorescence staining were used to detect the impact of AGR2 on expression of EMT related genes including E-cadherin,N-cadherin,CLDN1 and ZO-1 in AGR2 stably overexpressing and knockdown CRC cells.Quantitative real-time PCR(q RT-PCR)was used to screen EMT related transcription factors accounting for AGR2 induced EMT in AGR2 stably overexpressing Lo Vo/SW480 cells and knockdown DLD-1/HT-29 cells.And the expression of SNAIL and SLUG was further detected by Western blot.Transwell migration assay and Western blot were used to analyze the role of SNAIL and SLUG in AGR2-induced cell migration and EMT process in AGR2 stably overexpressing cells transfected with sh SNAIL or sh SLUG.Conditioned medium of Lo Vo and SW480 cells stably overexpressing AGR2 was collected and the protein level of extracellular AGR2(e AGR2)was detected by Western blot.The cellular location of intracellular AGR2(i AGR2)was evaluated in Lo Vo cells co-transfected with AGR2-EGFP and ER labeling peptide(Dsred-ER)plasmids.Wild-type Lo Vo or SW480 cells were treated with conditioned medium(CM)from control or AGR2 stable overexpressing Lo Vo and SW480 cells for 24 h.The expression of SNAIL and SLUG was detected by Western blot and migratory abilities of cells were analyzed by Transwell assays.Control or AGR2 stable overexpressing Lo Vo and SW480 cells were treated with AGR2 neutralizing antibody(10 μg/m L)for 24 h.The expression of SNAIL and SLUG was detected by Western blot andmigratory abilities of cells were analyzed by Transwell assays.AGR2 stable overexpressing SW480 cells were treated with a Golgi stop agent(monensin)for 16 h.The expression of SNAIL,SLUG,e AGR2 and i AGR2 was detected by q RT-PCR and Western blot.Conclusions: Intracellular AGR2 promotes epithelial-mesenchymal transition and migration of colorectal cancer cells via modulating expression of SNAIL and SLUG.Part Ⅱ Intracellular AGR2(i AGR2)promotes the expression of SNAIL and SLUG by regulating REL and histone acetylation through KDELR-Gs-PKA signaling pathwayObjective: This part aims to explore the mechanism involved in i AGR2’s regulation of SNAIL and SLUG.Methods: Lo Vo and SW480 cells were transfected with vector Ctrl,wild-type AGR2,signal peptide truncated AGR2(△S)or KTEL motif truncated AGR2(△KTEL)expressing plasmids for 48 h.The expression of SNAIL and SLUG was detected by q RT-PCR and Western blot.Migratory abilities of cells were analyzed by Transwell assays.Coimmunoprecipitation(CO-IP)was used to detect the interaction of AGR2 and KDEL receptor(KDELR1/2/3)in 293T、DLD-1 and HT-29 cells.q RT-PCR was used to detect the effect of AGR2 on KDELRs expression in Lo Vo and SW480 AGR2 stably overexpressing cells.AGR2 stable overexpressing Lo Vo cells were transfected with si RNAs or sh RNAs against KDELR1,or KDELR2 or KDELR3 respectively.The expression of SNAIL and SLUG was detected by q RT-PCR and Western blot.Migratory abilities of cells were analyzed by Transwell assays.Control or AGR2 stable overexpressing Lo Vo cells stably transfected with sh NC or sh KDELR1 were injected into the tail veins of Balb/c-nude mice.Numbers of mice that developed liver metastasis in each group and numbers of liver metastatic foci in each group were counted.Kaplan-Meier method was used to analyze overall survival rate of mice.AGR2 stably overexpressing Lo Vo cells were transfected with Gs or Gq si RNAs for 48 h,ortreated with PKA inhibitor(H 89)for 24 h.The expression of SNAIL and SLUG was detected by q RT-PCR and Western blot.Transwell migration assay was used to detect the migratory abilities of cells.SNAIL and SLUG promoter luciferase reporters were used to detect the effect of i AGR2 on the transcription activation of SNAIL and SLUG promoters in AGR2 stably overexpressing Lo Vo cells.Bioinformatics analysis was conducted to predict transcription factors responsible for i AGR2-mediated upregulation of SNAIL and SLUG.Lo Vo cells were co-transfected with SNAIL or SLUG promoter luciferase reporter vectors and dose-increased c-Rel(REL)expressing or control plasmids for 48 h and luciferase activity was measured.The expression of c-Rel(REL)was detected by Western blot in control or AGR2 overexpressing Lo Vo cells stably transfected with sh KDELR1 or treated with PKA inhibitor H 89.The expression of SNAIL,SLUG and REL in control or AGR2 overexpressing Lo Vo cells stably transfected with sh REL was detected by Western blot.Transwell migration assay was used to detect the impact of REL on i AGR2 promoted cell migration.Lo Vo cells were treated with Panobinostat,a pan histone deacetylase(HDAC)inhibitor for 24 h,and the expression of SNAIL and SLUG was detected by q RT-PCR.Western blot was used to detect the expression of acetylated histones(H3K9/14/18/27/56;H4K5/8/12;H2AK5 and H2BK5)in AGR2 stably overexpressing Lo Vo and SW480 cells.AGR2 stable overexpressing Lo Vo cells were transfected with si RNAs against KDLER1/2/3 or Gs,Gq for 48 h,or treated with PKA inhibitor H 89 for 24 h,the expression of H3K9 ac was detected by Western blot.Chromatin immunoprecipitation(CHIP)assay was used to determine the binding of H3K9 ac to SNAIL and SLUG promoters.q RT-PCR was used to screen the histone acetylases(GCN5,CBP,P300,PCAF)and deacetylases(SIRT1,SIRT2,SIRT6)accounted for i AGR2-KDELR upregulated H3K9 ac in Lo Vo AGR2 stable overexpressing cells transfected with sh KDELR1.Western blot and Transwell migration assay were used to detect the effects of histone acetylase PCAF and deacetylase SIRT2 on i AGR2 promoted SNAIL,SLUG,H3K9 ac expression and cell migration.Conclusions: Intracellular AGR2(i AGR2)promotes the expression of SNAIL and SLUG byregulating REL and histone acetylation through KDELR-Gs-PKA signaling pathway.Part Ⅲ Intracellular AGR2(i AGR2)responses to macrophages derived PGE2 stimuliObjective: This part aims to explore the relationship between i AGR2 and PGE2 in the tumor microenvironment and provide a novel therapeutic strategy for inhibiting tumor metastasis.Methods: Lo Vo and HT-29 cells were treated with PGE2 at different concentrations(0,50,100,200 ng/m L)for 24 h.The expression of AGR2 was detected by Western blot and cell migration was analyzed by Transwell assay.Lo Vo,SW480,HT-29,CT26 and HT-29AGR2-KO cells were treated with 200 ng/m L PGE2 for 24 h,the expression of EMT related genes,including E-cadherin,N-cadherin,ZO-1 and SANIL was detected by Western blot.Lo Vo and HT-29 cells were transfected with si AGR2 for 24 h,followed by treating with 200 ng/m L PGE2 for another 24 h,cell migration was analyzed by Transwell assay.Blood samples of colorectal cancer patients and healthy volunteers were collected and peripheral blood mononuclear cells(PBMCs)were isolated by Ficoll density gradient centrifugation.The conditioned medium was collected after 12 h in vitro culture,and the PGE2 content was detected by an ELISA kit.Lo Vo and HT-29 cells were treated with conditioned medium(CM)from CRC patients derived PBMCs pre-treated with or without COX2 inhibitor celecoxib(CXB)for 24 h.The cell migratory ability was detected by Transwell migration assay and the expression of AGR2 was detected by Western blot.The bone marrow derived macrophages(BMDMs)were isolated from mice and polarized into M2 type by IL-4 treatment.CT26 cells were treated with conditioned medium(CM)from BMDMs pre-treated with or without CXB for 24 h.The cell migratory ability was detected by Transwell migration assay and the expression of AGR2 was detected by Western blot.Lo Vo and HT-29 cells were transfected with si AGR2 for 24 h,followed by treating with conditioned medium(CM)from CRC patients derived PBMCs for 24 h and harvested for Transwell migration assays.Theexpression of AGR2 was detected by Western blot in Lo Vo/HT-29 cells stably transfected with sh EP4 or treated with AKT inhibitor MK2206,followed by treating with PGE2(200 ng/m L).Control or Agr2 silenced CT26 cells were injected into the spleens of Balb/c mice,followed by treating with celecoxib(CXB)or Ctrl(0.5% Sodium carboxymethyl acid solution)by gavage.Numbers of mice that developed liver metastasis in each group and numbers of liver metastatic foci in each group were counted.Kaplan-Meier method was used to analyze overall survival rate of mice.The heart,spleen,lung and kidney tissues were imaged by H&E staining to observe toxicity.Conclusions: AGR2 is upregulated by PGE2 through EP4-PI3K-AKT signal and mediates PGE2’s modulation on EMT and migration of CRC cells.