The Role And Mechanism Of ¹²⁵I Seed In Inhibiting The Growth Of Cholangiocarcinoma By Down Regulating AGR2

As a malignant tumor in the biliary system,cholangiocarcinoma is the second largest primary liver malignancy after hepatocellular carcinoma,accounting for about 10%-20%of all primary liver malignancies.Cholangiocarcinoma can be divided into two subtypes:intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma according to different origins.Extrahepatic cholangiocarcinoma is divided into hilar cholangiocarcinoma and distal cholangiocarcinoma.The incidence and mortality of cholangiocarcinoma are increasing year by year.Because cholangiocarcinoma is usually asymptomatic in the early stage,60%-70%of the patients with newly diagnosed cholangiocarcinoma are diagnosed in the advanced stage and receive palliative treatment,especially chemotherapy.In the past decade,although the diagnosis and treatment of cholangiocarcinoma have made progress,the prognosis of patients with cholangiocarcinoma has not improved significantly.The 5-year survival rate(7-20%)and recurrence rate after tumor resection are still disappointing.For unresectable cholangiocarcinoma,the treatment effect of gemcitabine combined with cisplatin and other chemotherapy regimens is not ideal.However,most of the targeted therapy and immunotherapy for cholangiocarcinoma are in the clinical trial stage,and the clinical efficacy is not clear.With the continuous innovation and development of interventional technology,interventional therapy has brought more choices to patients with cholangiocarcinoma due to its advantages of minimally invasive and high efficiency.125I radioactive seeds belong to low-energy radiation source,which can be inserted into the tissues to directly irradiate the tumor to play a therapeutic role.In recent years,125I implantation has been widely used in the treatment of a variety of cancers due to its advantages of accurate positioning,small trauma,high target dose,less exposure to normal tissues,uniform distribution in tissues,long half-life,fewer complications,and local tumor reduction effect.Moreover,125I particle brachytherapy,as a feasible treatment method,can be used to inhibit the growth of cholangiocarcinoma and improve local symptoms of malignant biliary obstruction,and prolong patient survival.Although 125I seeds have many advantages in the treatment of cholangiocarcinoma,due to the differences in individual radiosensitivity and the limitation of radiation dose,some patients may have tumor recurrence after radiotherapy,and the treatment effect is not good.For those cholangiocarcinoma patients with poor radiosensitivity or radiation resistance,improving the effect of 125I seeds is an important way to prolong the survival of patients with cholangiocarcinoma.Therefore,it is particularly important to clarify the biological effect and potential molecular mechanism of 125I seeds in cholangiocarcinoma,andfurther improve the therapeutic effect of 125I seeds.We first evaluated the biological effects of 125I seeds on cholangiocarcinoma cells through in vivo and in vitro experiments.Subsequently,transcriptome sequencing combined with bioinformatics analysis was used to explore themechanism of 125I seeds in cholangiocarcinoma.It was found that AGR2 might be a target of 125I seeds in inhibiting cholangiocarcinoma.Next,meta-analysis was used to evaluate the relationship between AGR2 expression and the prognosis of cancer patients.Meanwhile,the expression of AGR2 in cholangiocarcinoma tissue samples and the clinicopathological parameters of patients with cholangiocarcinoma were evaluated to analyze the relationship between AGR2 and the prognosis of patients with cholangiocarcinoma.Then,the role of AGR2 in cholangiocarcinoma and its possible mechanism were further verified by vitro experiments.Finally,in vivo and in vitro experiments were conducted to verify whether knockdown of the target gene AGR2 can synergistically increase the inhibitory effect of 125I seeds on cholangiocarcinoma.Part Ⅰ:The biological effect of 125I seeds on cholangiocarcinomaObjectiveTo explore the effects of 125I seeds on the biological functions of cholangiocarcinoma cells.Methods1.First,the 125I seeds irradiation model was established,and then the logarithmically growing cells were inoculated in 6-well plates or petri dishes with a diameter of 35mm.The plate clone formation assay was used to detect the effect of 125I seeds on the proliferation of cholangiocarcinoma cells.Then,flow cytometry was used to detect the effect of 125I seeds on the cycle and apoptosis of cholangiocarcinoma cells.Finally,the wound healing assay and Transwell assay were used to detect the effect of 125I seeds on the migration and invasion of cholangiocarcinoma cells.2.Five-week-old female BALB/c nude mice weighing 16-18g were randomly divided into the control group and the 125I seeds treatment group,and the logarithmic growth phase cells were inoculated into the right groin of the nude mice.The tumor growth was observed every day.After the tumor volume reached 60mm3,the control group was given blank seed implantation,and the experimental group was given 1251 seed implantation treatment.The body weight and tumor volume of nude mice were measured every three days.3.Statistical analysis was performed using GraphPad Prism 7.0,and measurement data were expressed as mean ± standard deviation.The independent sample t test was used for comparison between the two groups,and the one-way analysis of variance(ANOVA)was used for the comparison of three or more groups.P<0.05 indicates that the difference is statistically significant.Results1.The results of the plate cloning assays showed that compared with the control group,the cumulative dose of 125I seeds irradiation at 2Gy could reduce the clonal formation rate of bile duct cancer cells QBC939(P<0.01),and the cumulative dose of 125I seeds irradiation at 4Gy could inhibit the clonal formation of QBC939 cells more significantly(P<0.001).Similarly,compared with the control group,the cumulative dose of 2Gy could also reduce the rate of RBE cell clone formation(P<0.01),and the cumulative dose of 4Gy could inhibit the RBE cell clone formation more significantly(P<0.01).2.The results of apoptosis and cycle assays showed that compared with the control group,the apoptotic rate of QBC939 cells was significantly increased when the cumulative dose of 125I seeds irradiation was 2Gy(P<0.01),and the apoptotic rate of QBC939 cells was more significantly increased when the cumulative dose was 4Gy(P<0.001).Compared with the control group,when the cumulative dose was 2Gy,the proportion of G0/G1 phase of QBC939 cells decreased,but the difference was not statistically significant,and the proportion of G2/M phase increased(P<0.05).When the cumulative dose was 4Gy,the proportion of G0/G1 phase of QBC939 cells decreased(P<0.05),and the proportion of G2/M phase increased significantly(P<0.01).Similarly,compared with the control group,the apoptotic rate of RBE cells increased when the cumulative dose of 125I seeds irradiation was 2Gy(P<0.05),and the apoptotic rate of RBE cells increased more significantly when the cumulative dose was 4Gy(P<0.001).Compared with the control group,the proportion of G0/G1 phase increased(P<0.01),the proportion of S phase decreased(P<0.05),and the proportion of G2/M phase had no significant change.When the cumulative dose was 4Gy,the proportion of G0/G1 phase increased more significantly(P<0.001),the proportion of S phase decreased(P<0.05),and the proportion of G2/M phase decreased slightly,but the difference was not statistically significant.3.The results of the wound healing assays showed that compared with the control group,the healing rate of QBC939 cells was reduced(P<0.01)by 125I seeds irradiation,and the healing rate of RBE cells was also slowed down after 125I seeds irradiation(P<0.05).Transwell assays results also showed that compared with the control group,the cumulative dose of 2Gy can inhibit the migration(P<0.01)and invasion(P<0.05)of QBC939 cells,and the cumulative dose of 4Gy could more significantly inhibit the migration(P<0.001)and invasion of QBC939 cells(P<0.01).Similarly,compared with the control group,the cumulative dose of 2Gy could inhibit the migration(P<0.01)and invasion of RBE cells(P<0.01),and the cumulative dose of 4Gy could more significantly inhibit the migration(P<0.001)and invasion of RBE cells.4.The results of in vivo experiments showed that the tumor volume growth rate of nude mice in the 125I seeds treatment group was lower than that of the control group(P<0.01),and the average tumor weight was also lower than that of the control group(P<0.05).Moreover,TUNEL fluorescence staining showed that the apoptosis rate of tumor cells in xenograft tumor tissue was increased after 125I seeds treatment(P<0.001).Conclusion125I seed can inhibit the proliferation,migration and invasion,block the cell cycle and promote apoptosis of cholangiocarcinoma cells.125I seed can inhibit the growth of cholangiocarcinoma xenografts and promote apoptosis of cancer cell in vivo.Part Ⅱ:Transcriptome expression analysis of cholangiocarcinoma cells irradiated with 125I seedsObjectiveTo explore the potential target and possible mechanism of 125I seeds inhibiting the growth of cholangiocarcinoma by transcriptome sequencing and bioinformatics analysis.Methods1.Firstly,the gene expression data set of cholangiocarcinoma was downloaded from geo database,and then the original data was standardized by Normalize Between Arrays function in LIMMA package of R language for further analysis.The differential genes of each dataset were screened by LIMMA packet of R language,and the original P values were multiple corrected using Benjamini and Hochberg False Discovery Rates to obtain the adjusted P values(adj.P).The difference fold change(FC)of gene expression between cholangiocarcinoma and normal bile duct tissues was calculated,and log2 was taken for the difference FC.The screening threshold of differential mRNA was set as adj.P<0.05 and |log2FC|≥2.5.Then,the differentially expressed mRNAs in each GEO dataset were arranged in descending order according to log2 FC value,and the differentially expressed mRNAs in cholangiocarcinoma were screened out,and the heat map and volcano map were drawn.Finally,the biological function and pathway enrichment of differentially expressed mRNAs were analyzed using the Gene Ontology(GO)database and the Kyoto Encyclopedia of Genes and Genomes(KEGG)database.2.The logarithmic growth phase cells of control group and 125I seeds treatment group were selected,and three samples of each group were analyzed by transcriptome sequencing.Firstly,the differentially expressed genes among samples were screened by gene quantitative analysis,and then the differentially expressed genes were further analyzed by GO function enrichment analysis and pathway enrichment analysis.Finally,combined with the differentially expressed genes of cholangiocarcinoma in GEO database,the potential targets of 125I seeds were screened out through cross comparison and construction of Venn diagram.3.Quantitative real-time PCR(QRT-PCR)was used to detect the expression of AGR2 and DUSP1 in control group and 125I seeds treatment group.4.Western blot(WB)was used to detect the protein expression of AGR2,DUSP1,p38 MAPK,p-p38 MAPK,p53 and p-p53 in the control group and 125I seeds treatment group.5.Immunohistochemistry(IHC)was used to detect the positive expression of AGR2,DUSP1,p-p38 MAPK and p-p53 proteins in the Xenograft tumor tissues of the control group and 125I seeds treatment group.6.Statistical analysis was performed using GraphPad Prism 7.0,and measurement data were expressed as mean±standard deviation.The independent sample t test was used for comparison between the two groups,and the one-way analysis of variance(ANOVA)was used for the comparison of three or more groups.P<0.05 indicates that the difference is statistically significant.Results1.In GSE26566 dataset,there were 758 differentially expressed mRNAs in cholangiocarcinoma,of which 466 differentially expressed mRNAs were down-regulated and 292 differentially expressed mRNAs were up-regulated.In GSE76297 dataset,there were 350 differentially expressed mRNAs in cholangiocarcinoma,of which 292 differentially expressed mRNAs were down-regulated and 58 differentially expressed mRNAs were up-regulated.There were 30 up-regulated mRNAs in both GSE26566 and GSE76297 dataset,and 216 down-regulated mRNAs in both dataset.2.Transcriptome sequencing of the control group and QBC939 cells irradiated with 125I seeds revealed that several differentially expressed mRNAs were up-regulated or down-regulated,including 166 up-regulated mRNAs and 71 down-regulated mRNAs.Combined with the 30 mRNAs up-regulated and 216 mRNAs down-regulated in both GSE26566 and GSE76297 datasets,we selected the top 100 differential mRNAs obtained from transcriptome sequencing to cross-compare them and create the Venn diagram.Finally,it was found that CYP2A7,DmGDH and ITIH4 were down-regulated in cholangiocarcinoma and up-regulated after 125I seeds treatment.The expression of AGR2 was up-regulated in cholangiocarcinoma and down-regulated after 125I seeds treatment.KEGG pathway enrichment results showed that the pathways with the most differential gene enrichment were MAPK signaling pathway,AGE-RAGE signaling pathway in diabetic complications,and IL-17 signaling pathway.3.QRT-PCR results showed that the expression of AGR2 and DUSP in QBC939 and RBE cells were down-regulated in 125I seeds treatment group compared with the control group.4.WB results showed that compared with the control group,the proteinexpressions of AGR2 and DUSP1 in QBC939 and RBE cells were down-regulated,while the protein expressions of p-p38 MAPK and p-p53 were up-regulated in 125I seeds treatment group.5.IHC results also showed that compared with the control group,the positivestaining of AGR2 and DUSP1 protein was decreased,while the positive staining of p-p38 MAPK and p-p53 protein was increased in the 125I seeds treatment group.Conclusion125I seeds can inhibit the expression of AGR2 and DUSP1 incholangiocarcinoma,regulating p38 MAPK signaling pathway,and promoting the expression of p-p38 MAPK and p-p53.Part Ⅲ:Correlation analysis of the expression of AGR2 incholangiocarcinoma with pathological parameters and prognosis ObjectiveTo evaluate the correlation between the expression of AGR2 and thepathological parameters and prognosis of patients with cholangiocarcinoma.Methods1.Firstly,MEDLINE,Web of Science,Cochrane Library and EMBASEdatabases were searched by computer.The retrieval date is from the establishment date of the database to May 6,2 020.The two investigators screened the subjects and abstracts independently according to inclusion and exclusion criteria,and reviewed the full text to evaluate the eligibility of studies that correlate AGR2 expression with prognosis in cancer patients.Then,the following information was extracted from each included article:first author(year of publication),type of cancer,country of patients,number of patients(high/low expression of AGR2),median age of patients in the study,detection method of AGR2 expression,sample type,follow-up time(months),HR and 95%CI.The Newcastle Ottawa scale(NOS)was used to assess the quality of the included original studies.Finally,the expression of AGR2 in cancer patients was analyzed by meta-analysis.2.The expression of AGR2 in 46 cholangiocarcinoma and adjacent normal bile duct tissues was detected by IHC.Then,IHC analysis software Quant Center and H-score(Histochemical SCORE,which is a histological scoring method to deal with immunohistochemical results,with a value range of 0-300,the higher the value,the stronger the positive value)were used to score the depth and amount of positive staining in the tissues.Finally,the correlation between the expression of AGR2 in cholangiocarcinoma and the clinicopathological parameters and prognosis of patients with cholangiocarcinoma was analyzed.3.The Stata 14.0,SPSS 21.0,GraphPad Prism 7.0 and R language were used to analyze the data.In the statistical analysis of data in the Meta-analysis,95%CI and HR were used to evaluate the effect of AGR2 overexpression on the prognosis of cancer patients.Heterogeneity was assessed using Q test and I2 test,and low heterogeneity was considered when P>0.1 or I2<50%.The fixed effects model was used for low heterogeneity,otherwise the random effects model was used to merge the HR of each study to produce the forest plots.Sensitivity analysis was performed to check the stability of the combined results.The Begg funnel plot and Egger linear regression were used to evaluate whether there was publication bias in the included studies.The correlation between the protein expression level of AGR2 and the clinical and pathological variables was analyzed by Chi-Square or Fisher’s precise test.Kaplan-Meier curve was used to evaluate the survival time,and log-rank test was used to analyze the differences between groups.Univariate and multivariate analyses were conducted by Cox proportional risk regression model to determine whether each index could be used as an independent influencing factor for prognosis.Measurement data are expressed as mean ± standard deviation.The independent sample t test was used for comparison between the two groups.P<0.05 indicates that the difference is statistically significant.Results1.Thirteen articles(including 14 studies)were included in the meta-analysis,including 8 patient populations in Europe,2 in the United States,1 in Brazil,and 3 in China,involving a total of 2619 patients.The results of heterogeneity test showed that I2=78.5%,P=0.000,indicating high heterogeneity among studies.Therefore,random-effects model was used for meta-analysis,and the results showed that high expression of AGR2 had a negative effect on OS of cancer patients(HR=2.02,95%CI:1.40-2.91,P=0.000).The results of subgroup analysis suggested that regional differences in patient populations and different sample detection methods might be the sources of heterogeneity among studies.The results of sensitivity analysis showed that the results of Meta-analysis were robust,but there was still some publication bias.2.IHC results showed that the expression of AGR2 was high in cholangiocarcinoma and low in normal bile duct tissues,and the difference was statistically significant(P<0.001).The high expression of AGR2 was positively correlated with the age(P=0.043)and TNM stage(P=0.033)of patients with cholangiocarcinoma.The expression of AGR2 protein was high in 10/24(41.67%)of patients with Ⅰ-Ⅱ stage and 15/22(68.18%)of patients with Ⅲ-Ⅳ stage.Moreover,the results of survival analysis showed that the OS of patients with cholangiocarcinoma with high AGR2 expression was shorter than that of patients with cholangiocarcinoma with low AGR2 expression,and the difference was statistically significant(P=0.017).Compared with patients with cholangiocarcinoma with low AGR2 expression,patients with cholangiocarcinoma with high AGR2 expression also had shorter DFS,but the difference was not statistically significant(P=0.19).Univariate Cox regression analysis showed that lymph node metastasis(HR=3.977,95%CI 1.710-9.252,P=0.001)and TNM stage(HR=3.865,95%CI 1.920-7.780,P=0.000)were the factors affecting patients’ OS.Multivariate Cox regression analysis showed that TNM stage(HR=4.588,95%CI 2.011-10.467,P=0.000)was a factor affecting patients’ OS.ConclusionHigh expression of AGR2 is associated with poor prognosis of cholangiocarcinoma.Part Ⅳ:Role and mechanism of AGR2 in cholangiocarcinomaObjectiveTo evaluate the role and mechanism of AGR2 in cholangiocarcinoma.Methods1.Firstly,lentiviral transfection was used to construct the cholangiocarcinoma cell lines with stable knockdown of AGR2 gene.Then,CCK8 and plate cloning assay were used to detect the effect of AGR2 knockdown on the proliferation of cholangiocarcinoma cells QBC939 and RBE.The effects of AGR2 knockdown on the apoptosis and cell cycle of QBC939 and RBE cells were detected by flow cytometry.The effects of AGR2 knockdown on the migration and invasion of QBC939 and RBE cells were evaluated by wound healing assay and Transwell assay.Finally,the sensitivity of QBC939 and RBE cells to gemcitabine and cisplatin after AGR2 knockdown was evaluated.2.QRT-PCR was used to detect the expression of AGR2 and DUSP1 in QBC939 and RBE after AGR2 knockdown.The expressions of AGR2,DUSP1,p38 MAPK,p-p38 MAPK,p53 and p-p53 protein in QBC939 and RBE were detected by WB.3.Statistical analysis was performed using GraphPad Prism 7.0,and measurement data were expressed as mean±standard deviation.The independent sample t test was used for comparison between the two groups,and the one-way analysis of variance(ANOVA)was used for the comparison of three or more groups.P<0.05 indicates that the difference is statistically significant.Results1.AGR2 knockdown cholangiocarcinoma cell lines were successfully constructed and named as QBC939-KD and RBE-KD,respectively.QBC939-NC and RBE-NC served as the cholangiocarcinoma cell lines of control group.2.The results of CCK8 assays showed that the proliferation rate of QBC939-KD and RBE-KD of cholangiocarcinoma cells after AGR2 knockdown was significantly lower than that of the control group(P<0.01).The results of plate cloning assays showed that the clonal formation rate of QBC939-KD and RBE-NC of the cholangiocarcinoma cells after AGR2 knockdown was significantly lower than that of the control group(P<0.01).3.The cell apoptosis and cell cycle assays showed that apoptosis ratio of cholangiocarcinoma cells QBC939-KD after AGR2 knockdown was increased compared with the control cells QBC939-NC(P<0.01).After AGR2 knockdown,the proportion of QBC939-KD in G0/G1 phase was decreased(P<0.01),and the proportion of G2/M phase was increased(P<0.001).In addition,the apoptosis ratio of cholangiocarcinoma cells RBE-KD after AGR2 knockdown was also increased compared with the control cells RBE-NC(P<0.05).The proportion of G0/G1 stage of RBE-KD in cholangiocarcinoma cells after AGR2 knockdown was decreased(P<0.05),and the proportion of G2/M stage was increased(P<0.01).4.The results of wound healing assays showed that the healing rate of cholangiocarcinoma cells QBC939-KD after AGR2 knockdown was slower than that of control cells QBC939-NC(P<0.01).Similarly,the healing rate of cholangiocarcinoma cells RBE-KD after AGR2 knockdown was also slowed down compared with control cells RBE-NC(P<0.01).The results of transwell assay also showed that migration(P<0.01)and invasion ability(P<0.01)of QBC939-KD cells after AGR2 knockdown were decreased compared with the control cells QBC939-NC.Similarly,the ability of migration(P<0.001)and invasion(P<0.01)of cholangiocarcinoma cells RBE-KD after AGR2 knockdown were decreased compared with control cells RBE-NC.5.The results of plate cloning assays showed that compared with the control group,the clone formation rate of QBC939-KD cell was decreased(P<0.05),and the clone formation rate of RBE-KD cell was also decreased(P<0.05)under the same drug concentration gemcitabine treatment.The results of apoptosis assay showed that compared with the control group,the apoptosis ratio of QBC939-KD cells was increased(P<0.001),and apoptosis ratio of RBE-KD cells was also increased(P<0.05)under the same 40nM gemcitabine treatment.6.The results of plate cloning assays showed that compared with the control group,the clone formation rate of QBC939-KD cell was decreased(P<0.05),and the clone formation rate of RBE-KD cell was also decreased(P<0.05)under the same drug concentration cisplatin treatment.The results of apoptosis assay showed that compared with the control group,the apoptosis ratio of QBC939-KD cells was increased(P<0.001),and apoptosis ratio of RBE-KD cells was also increased(P<0.05)under the same 8nM cisplatin treatment.7.QRT-RCR results showed that compared with the control group,the relative expression level of DUSP1 in QBC939-KD cells was decreased(P<0.05),and the relative expression level of DUSP1 in RBE-KD cells was also decreased(P<0.05).WB results showed that compared with the control group,the expression of DUSP1 protein was decreased(P<0.001),the expression of p-p38 MAPK(P<0.01),p-53(P<0.05)and p-p53(P<0.01)protein were increased in QBC939-KD cells.Moreover,the relative expression of DUSP1 in RBE-KD cells decreased(P<0.01),the expression of p-p38 MAPK(P<0.01)and p-p53(P<0.05)protein was increased compared with the control group.ConclusionThe knockdown of AGR2 can inhibit the proliferation,migration and invasion,promote apoptosis and block the cell cycle of cholangiocarcinoma cells,and also improve the sensitivity of gemcitabine and cisplatin to inhibit the growth of cholangiocarcinoma cells.AGR2 can inhibit the activation of p38 MAPK and p53 by regulating the DUSP1-mediated regulatory pathway.However,knockdown of AGR2 can reverse the above pathway and play an anti-cancer role.Part Ⅴ:Effect of knockdown of AGR2 expression on growth inhibition of cholangiocarcinoma by 125I seedsObjectiveTo explore whether knocking down the expression of AGR2 can increase the inhibitory effect of 125I seeds on cholangiocarcinoma cells.Methods1.The effect of AGR2 knockdown combined with 125I seeds irradiation on the proliferation ability of cholangiocarcinoma cells QBC939 and RBE were detected by plate cloning assay.Flow cytometry was used to detect the effects of AGR2 knockdown combined with 125I seeds irradiation on the apoptosis of QBC939 and RBE cells.The wound healing assays and Transwell assay were used to evaluate the effects of AGR2 knockdown combined with wound healing assays irradiation on the migration and invasion ability of QBC939 and RBE cells.2.Twenty BALB/c female nude mice aged 5 weeks and weighing 16 to 18g were randomly divided into four groups:QBC939-NC group,QBC939-KD group,QBC939-NC+125I seeds treatment group,and QBC939-KD+125I seeds treatment group.The logarithmic growth phase cells were inoculated into the right groin of nude mice.The growth of tumor was observed every day.When the tumor volume reached 60mm3,the mice were implanted with blank seeds and 125I seeds respectively.The weight of nude mice and tumor volume were measured every three days.TUNEL assay was used to detect the apoptosis of cancer cells in each group.The positive expressions of AGR2,DUSP1,p-p38 MAPK and p-p53 proteins in the xenograft tumor tissues of each group were detected by IHC staining.3.Statistical analysis was performed using GraphPad Prism 7.0,and measurement data were expressed as mean±standard deviation.The independent sample t test was used for comparison between the two groups,and the one-way analysis of variance(ANOVA)was used for the comparison of three or more groups.P<0.05 indicates that the difference is statistically significant.Results1.The results of plate cloning assays showed that compared with the control group QBC939-NC,the clone formation rate of the AGR2 knockdown group QBC939-KD(P<0.001)and the 125I seeds irradiation group(QBC939-NC/125I)(P<0.001)were reduced.Moreover,compared with the 125I seeds irradiation group(QBC939-NC/125I),the cell clone formation rate of AGR2 knockdown combined with 125I seeds irradiation group(QBC939-KD/125I)was significantly lower(P<0.01).Similarly,compared with the control group RBE-NC,the clone formation rate of the AGR2 knockdown group RBE-KD(P<0.01)and the 125I seeds irradiation group(RBE-NC/125I)(P<0.001)were reduced.Moreover,compared with the 125I seeds irradiation group(RBE-NC/125I),the cell clone formation rate of AGR2 knockdowncombined with 125I seeds irradiation group(RBE-KD/125I)was significantly lower(P<0.01).2.The results of apoptosis assays showed that compared with the control group QBC939-NC,the cell apoptosis rate of the AGR2 knockdown group QBC939-KD(P<0.01)and the 125I seeds irradiation group(QBC939-NC/125I)(P<0.05)were increased.In addition,compared with the 125I seeds irradiation group(QBC939-NC/125I),the cell apoptosis rate of AGR2 knockdown combined with 125I seeds irradiation group(QBC939-KD/125I)was also increased(P<0.001).Similarly,compared with the control group RBE-NC,the cell apoptosis rate of the AGR2 knockdown group RBE-KD(P<0.05)and the 125I seeds irradiation group(RBE-NC/125I)(P<0.001)were increased.In addition,compared with the 125I seeds irradiation group(RBE-NC/125I),the cell apoptosis rate of AGR2 knockdown combined with 125I seeds irradiation group(RBE-KD/125I)was also increased(P<0.05).3.The results of wound healing assays showed that compared with the control group QBC939-NC,the migration distance of the AGR2 knockdown group QBC939-KD cells(P<0.001)and 125I seeds irradiation group cells(QBC939-NC/125I)(P<0.001)were shorter.Moreover,compared with the 125I seeds irradiation group(QBC939-NC/125I),the cells migration distance of the AGR2 knockdown combined with 125I seeds irradiation group(QBC939-KD/125I)was significantly reduced(P<0.01).Similarly,compared with the control group RBE-NC,the migration distance of the AGR2 knockdown group RBE-KD cells(P<0.01)and 125I seeds irradiation group cells(RBE-NC/125I)(P<0.05)were shorter.Moreover,compared with the 125I seeds irradiation group(RBE-NC/125I),the cells migration distance of the AGR2 knockdown combined with 125I seeds irradiation group(RBE-KD/125I)was significantly reduced(P<0.001).The results of transwell assays also showed that compared with control group QBC939-NC,the cell migration and invasion ability of AGR2 knockdown group QBC939-KD and 125I seeds irradiation group(QBC939-NC/125I)were decreased.Moreover,compared with the 125I seeds irradiation group(QBC939-NC/125I),the cell migration and invasion ability of AGR2 knockdown combined with 125I seed irradiation group(QBC939-KD/125I)were significantly decreased.Similarly,compared with control group RBE-NC,the cell migration and invasion ability of AGR2 knockdown group RBE-KD and 125I seeds irradiation group(RBE-NC/125I)were decreased.Moreover,compared with the 125I seeds irradiation group(RBE-NC/125I),the cell migration and invasion ability of AGR2 knockdown combined with 125I seed irradiation group(RBE-KD/125I)were significantly decreased.4.The results of vivo experiments showed that compared with the control group QBC939-NC,the growth rate of xenograft tumor in the AGR2 knockdown group QBC939-KD and the 125I seeds irradiation group(QBC939-NC/125I)were slower.Moreover,compared with the 125I seeds irradiation group(QBC939-NC/125I),the growth rate of xenograft tumor in AGR2 knockdown combined with 125I seeds irradiation group(QBC939-KD/125I)was also slower.Similarly,compared with the control group QBC939-NC,the size and weight of xenograft tumor in the AGR2 knockdown group QBC939-KD and the 125I seeds irradiation group(QBC939-NC/125I)were reduced.Moreover,compared with the 125I seeds irradiation group(QBC939-NC/125I),the size and weight of xenograft tumor in AGR2 knockdown combined with 125I seeds irradiation group(QBC939-KD/125I)was also reduced.TUNEL fluorescence staining showed that compared with the control group QBC939-NC,the cancer cell apoptosis rate of xenograft tumor in the AGR2 knockdown group QBC939-KD and the 125I seeds irradiation group(QBC939-NC/125I)were increased.In addition,compared with the 125I seeds irradiation group(QBC939-NC/125I),the cancer cell apoptosis rate of xenograft tumor in AGR2 knockdown combined with 125I seeds irradiation group(QBC939-KD/125I)was also increased.5.IHC staining results showed that compared with the control group QBC939-NC,the AGR2 and DUSP1 positive staining of xenograft tumor tissues were weakened,and the p-p38 MAPK and p-p53 positive staining of xenograft tumortissues were enhanced in the AGR2 knockdown group QBC939-KD and the 125I seeds irradiation group(QBC939-NC/125I).In addition,compared with the 125I seeds irradiation group(QBC939-NC/125I),the AGR2 and DUSP1 positive staining of xenograft tumor tissues was also weakened,and the p-p38 MAPK and p-p53 positive staining of xenograft tumor tissues was also enhanced in AGR2 knockdown combined with 125I seeds irradiation group(QBC939-KD/125I).ConclusionAGR2 gene knockdown can cooperate with 125I seeds to inhibit the proliferation,migration and invasion of cholangiocarcinoma cells,and promote the apoptosis ofcholangiocarcinoma cells.AGR2 gene knockdown combined with 125I seeds may play its anti-cholangiocarcinoma role by activating p38 MAPK and p53.