Studies Of DUOX2 On Its Expression,Function And Mechanisms In Human Colorectal Cancer

With the aging of the population and the change of lifestyle,the incidence rate of colon cancer is increasing worldwide.About 60%of patients lose the chance of radical treatment,because of their intermediate and advanced stage at first diagnosis.And once recurrence and metastasis occur in patients with radical resection,the survival of patients will be seriously threatened.Exploring the mechanism of pathogenesis and metastasis of colon cancer have become research hotspots in recent years.Deep exploration new recurrence and metastasis-related biomarkers will help to further optimize the individualized and precise treatment strategy for colon cancer patients.In this study we screened out the oncogene associated with lymph node metastasis of colon cancer:Dual oxidase 2（DUOX2）,which is located on human chromosome 15.The protein encoded by the gene is one of the important members of the NADPH（nicotinamide adenine dinucleotide phosphate）oxidase family,which participates in the process of thyroxine synthesis.The mutation of DUOX2 can lead to disorders of thyroid hormone production.The role of DUOX2 in the development of cancers has attracted wide attention in recent years.However,the expression and function of DUOX2 in colon cancer remain unclear.Our study has shown that the expression of DUOX2 mRNA in colon cancer tissues was significantly higher than that in adjacent normal tissues.High expressions of DUOX2 protein in colon cancer tissues and metastatic lymph nodes were associated with poor prognosis.The effects of DUOX2 on the malignant biological behaviors of colon cancer cells were studied by DUOX2 knockdown.We found that silencing DUOX2 could significantly inhibit the migration and invasion of HCT116 and SW480.In order to further explore the mechanism of the effect of DUOX2 on the migration and invasion of colon cancer cells,we performed Next Generation Sequencing（NGS）on colon cancer cells with DUOX2 knockdown.After knocking down of DUOX2,the differential genes were enriched in pathways in cancer,MAPK,PI3K-AKT,HTLV-1 infection,RAS and other signaling pathways.The data has been uploaded to GEO database（GSE 139918）.In addition,through immunoprecipitation（IP）and mass spectrum analysis（MS）,the candidate interaction proteins of DUOX2 in natural state were found:RPL3,TUBB4A4,HADHB,BTN1A1.RPL3 scored highest and were consistent with the subcellular localization of DUOX2.Co-immunoprecipitation（co-IP）confirmed the interaction between RPL3 and DUOX2 protein.Then we analyzed the correlation between DUOX2 and RPL3 at mRNA and protein levels.After knockdown of DUOX2,the protein expression of RPL3 was significantly up-regulated,but the mRNA level had no change.Therefore,the regulation of DUOX2 on RPL3 is at the post-transcriptional level.Since the half-life of RPL3 protein is very short,about 4 hours,knockdown of DUOX2could slow down the degradation of RPL3 protein.It was further speculated that the degradation of RPL3 protein was mediated by ubiquitin proteasome pathway.In further IP test,multiple ubiquitin chains were detected in RPL3immune complex,while the total number of multiple ubiquitin chains was significantly reduced after silencing DUOX2.Therefore,DUOX2 might affect the expression of RPL3 by affecting its ubiquitination.Studies have shown that the increased expression of RPL3 was related to the increased drug sensitivity of tumor cells,and the decreased expression of RPL3 might cause the failure of chemotherapy drugs.Since there was interaction between RPL3 and DUOX2,and the protein of RPL3 could be regulated by DUOX2,DUOX2 might affect the drug sensitivity of colon cancer cells.Based on this hypothesis,experiments were designed to explore the effect of DUOX2 on the sensitivity of 5-fluorouracil（5-FU）in colon cancer cells.The results showed that knockdown of DUOX2 enhanced the inhibitory effect of 5-FU on the proliferation of colon cancer cells.The result has also been verified in the nude mouse model.This study was divided into the following four parts.Part One The Screening of DUOX2 and its expression in coloractal cancer and its correlation with clinicopathological featuresObjective:To screen the oncogenes associated with lymph node metastasis,detect the expression level of DUOX2 in different tissues of colon cancer patients,and explore the correlation between the expression of DUOX2and clinicopathological parameters and survival.Methods:1.The previous microarray data（GES104836）of our research group were analyzed again to screen the oncogenes associated with lymph node metastasis.24 pairs of colon cancer tissues and matched normal tissues were collected to verify the candidate oncogenes by q RT-PCR.2.The sample size was expanded to 89 cases.The cancer tissues and matched adjacent normal tissues were collected from patients with colon cancer.q RT-PCR was used to detect the expression of DUOX2 mRNA in colon cancer and adjacent normal tissues and the clinicopathological parameters were collected.3.The paraffin specimens were collected from 50 patients with metastatic colorectal cancer,including normal margin tissue,colon cancer tissue,metastatic lymph node tissue and metastatic liver tissue.The expression of DUOX2 protein in different tissues was detected by immunohistochemistry.4.T-test and chi-square test were used to analyze the correlation between the expression of DUOX2 and clinicopathological parameters in colon cancer patients.All the patients were followed up for survival.Kaplan Meier method was used to analyze the correlation between DUOX2 protein expression and survival.Results:1.After analyzing the previous microarray data,nine oncogenes related to lymph node metastasis were screened out:CCDC113、PODXL2、PDZK1IP1、SYNE4、SHH、MAGEA3、DUOX2、DUOXA2 and NPFFR1.q RT-PCR results showed that the expressions of CCDC113、PODXL2、SYNE4、DUOX2、DUOXA2 in colon cancer tissues were higher than adjacent normal tissues significantly.There were no significant differences in the expression of PDZK1IP1、SHH and MAGEA3 between colon cancer and adjacent normal tissues.NPFFR1 could not be successfully amplified,which might be due to the relatively low expression in colon cancer.The expression of DUOX2 was the highest in colon cancer and was used for further study.2.qRT-PCR was used to detect the relative expression of DUOX2.The results showed that the expression of DUOX2 in colon cancer tissues was up-regulated compared with adjacent normal tissues significantly（P<0.05）.The expression of DUOX2 in colon cancer with positive lymph node metastasis（n=37）was significantly higher than that in colon cancer with negative lymph node metastasis（n=52）,and the difference was statistically significant（P<0.05）.3.The relationship between the expression of DUOX2 mRNA and clinicopathological parameters of colon cancer was analyzed.The results showed that the high expression of DUOX2 mRNA was significantly correlated with male,T3+T4,N+,stage III+IV（P<0.05）,but there was no significant correlation found between DUOX2 mRNA and other clinicopathological parameters（P>0.05）.4.Immunohistochemistry（IHC）was used to detected the expression of DUOX2 protein in different tissues.Compared with normal margin tissues,the expression of DUOX2 protein in colon cancer tissues,metastatic lymph node tissues and metastatic liver tissues were significantly up-regulated（P<0.001）.The expression of DUOX2 protein in metastatic lymph node tissues and metastatic liver tissues were significantly higher than that in colon cancer tissues（P<0.001）.5.Statistical analysis of follow-up data showed that the survival time of patients with DUOX2 protein“+”,“++”,“+++”in colon cancer tissues were37.00months,23.00 months and 20.00 months,respectively,P<0.05;the survival time of patients with DUOX2 protein“+/++”,“+++”in metastatic lymph node tissues were 28.75 months,21.50 months,respectively,P<0.05;the survival time of patients with DUOX2 protein“+/++”,“+++”in metastatic liver tissues were 23.70 months,26.00 months,respectively,P>0.05.Summary:1.The expression of DUOX2 in colon cancer tissues was significantly higher than that in adjacent normal tissues,and the expression of DUOX2 in colon cancer tissues with positive lymph node metastasis was significantly higher than that in colon cancer tissues with negative lymph node metastasis.2.The expression of DUOX2 mRNA was significantly correlated with gender,degree of invasion,lymph node metastasis and TNM stage.3.High expression of DUOX2 protein in colon cancer and metastatic lymph nodes is associated with poor prognosis.Part Two The effect of DUOX2 on biological function and 5-fluorouracil sensitivity of colon cancer cellsObjective:To study the effect of DUOX2 expression on proliferation,cloning,apoptosis and migration on human colon cancer cell lines,and to explore the effect of DUOX2 on the drug sensitivity of colon cancer cell line to 5-FU.Methods:1.Western blot was used to detect the expression of DUOX2 protein in SW480,SW620,HCT116,HT29 and DLD-1 cell lines as well as NCM-460.2.Small interferingRNA（si1-DUOX2,si2-DUOX2,si3-DUOX2）and negative controlRNA（si-NC）were designed to infect the target cells.The knockdown efficiency of DUOX2 was detected by Western blot and q RT-PCR.3.The ability of cell clone and proliferation were detected by plate clone formation test and MTS test.4.Scratch test and Transwell test were used to detect the migration and invasion of tumor cells.5.CCK-8 assay was used to detect the inhibitory effect of 5-FU on the proliferation of colon cancer cells.6.Flow cytometry was used to detect the apoptosis rate and cell cycle distribution of colon cancer cells.Results:1.The expression of DUOX2 in different colon cancer cell lines was detected by Western blot.We found that the expression of DUOX2 protein in SW480,SW620,HCT116,HT29 and DLD-1 cell lines was significantly higher than that in NCM-460 cells.In this study,HCT116 and SW480 with moderate DUOX2 expression level were selected for further study.2.In HCT116 and SW480 cells,the expression levels of DUOX2 mRNA and protein in si1-DUOX2 and si2-DUOX2 groups were significantly lower than those in the negative control group（si-NC）,while the mRNA and protein expressions of DUOX2 in si3-DUOX2 group had no significant changes.Therefore,si1-DUOX2 and si2-DUOX2 were selected for further study.3.In MTS experiment,the cell viability（OD value）was detected at 0,24,48 hours.The results showed that in HCT116 and SW480 cells,DUOX2knockdown caused no significant change in OD value（P>0.05）.Compared with si-NC group,there was no significant change in the clone ability of si1-DUOX2 and si2-DUOX2 groups（P>0.05）.4.Transwell test and scratch test showed that the number of cells penetrating the membrane in si1-DUOX2 and si2-DUOX2 groups were significantly reduced compared with si-NC group（P<0.05）,and the wound healing ability was significantly inhibited（P<0.05）,indicating that silencing DUOX2 can significantly inhibit the migration and invasion of HCT116 and SW480 colon cancer cell lines.5.CCK-8 assay showed that after treated with different concentrations of5-FU,the IC₅₀ values of sh-DUOX2 groups in HT29 and HCT116 cells were significantly lower than in sh-Control groups[HT29:（31.43±3.92）vs（48.42±6.23）μg/ml,P<0.01;HCT116:（15.5±4.42）vs（29.3±7.56）μg/ml,P<0.01].Therefore,knockdown of DUOX2 can significantly reduce IC₅₀ of5-FU.6.Flow cytometry showed that the apoptosis rates of HT29 and HCT116cells in sh-DUOX2+5-FU group were significantly higher than those in sh-Control+5-FU group and sh-Control group.Cell cycle analysis showed that the G0/G1 arrest ability of sh-DUOX2 cells was enhanced by 5-FU compared with sh-Control cells.Summary:1.Transfection of siRNA could significantly inhibit the expression of DUOX2 in HCT116 and SW480 colon cancer cell lines.2.Down regulation of DUOX2 had no significant effect on the proliferation of HCT116 and SW480 colon cancer cell lines.3.Down regulation of DUOX2 expression can significantly inhibit the migration and invasion of HCT116 and SW480 cells.4.Knockdown of DUOX2 can significantly enhance the apoptosis of colon cancer cells induced by 5-FU and enhance the cell cycle arrest induced by 5-FU.Part Three The mechanism underlying the effect of DUOX2 on biological function of human colon cancer cellsObjective:To explore the interaction proteins and the downstream signaling pathways of DUOX2 in colon cancer cells,and to further reveal the mechanism of DUOX2 affecting the biological function of colon cancer cells.Methods:1.Collected the protein lysate of SW480 cells.The interaction protein of DUOX2 was detected by immunoprecipitation（IP）and protein mass spectrometry（MS）.The interaction protein RPL3 and DUOX2 were verified by co-immunoprecipitation（co-IP）.2.DUOX2 was knockdown in HCT116 and SW480 cells.The expression of RPL3 at mRNA level and protein level were detected by q RT-PCR and Western Blot.DUOX2 and RPL3 expression were checked in colon cancer specimens by q RT-PCR and IHC.3.Colon cancer cells with DUOX2 knockdown were treated with actinomycin（CHX）.Cell protein at different time points（0h,2h,4h,6h,8h,10h）were collected.The stability of RPL3 protein was measured by Western blot.Proteasome inhibitor MG132 was added to cells with DUOX2knockdown to observe the degradation of RPL3 protein.Protein lysate of HCT116 and SW480 cells were collected from the sh-control and sh-DUOX2cells,and the RPL3 antibody was used to for the IP test.The ubiquitin protein expression in different groups were detected by Western blot.4.DUOX2 overexpression plasmid（pc DNA3.1-DUOX2）,RPL3overexpression plasmid（pc DNA3.1-RPL3）and the negative control（pc DNA3.1-control）were constructed and were transfected into HCT116 and SW480 cells respectively.The effect of RPL3 on invasion and metastasis induced by DUOX2 were detected by transwell and scratch test.5.The NGS technology was used to detect the differentially expressed genes in HCT116 cells with DUOX2 knockdown（si1-DUOX2,si2-DUOX2）and control cells（si-NC）to find the pathway of differential gene enrichment.This result was further verified at the mRNA and protein levels.Results:1.The results of IP and MS showed that RPL3,TUBB4A,HADHB and BTN1A1 were the candidate downstream genes of DUOX2.Co-IP test showed that RPL3 interacted with DUOX2 protein.2.After knockdown of DUOX2 in HCT116 and SW480 cells,the mRNA level of RPL3 did not change significantly,but the protein expression of RPL3was significantly up-regulated.Tissue samples from different parts of metastatic colon cancer patients were taken for verification.The results showed that there was a significant negative correlation between DUOX2 and RPL3 at protein level,but not mRNA level,which was found in colon cancer tissues,metastatic lymph nodes and liver tissues（Kendal’s tau＿b values were-0.354,-0.323 and-0.285,respectively）.3.Knockdown of DUOX2 significantly increased the stability of RPL3protein under CHX treatment for 10 hours.Compared with sh-control group,proteasome inhibitor MG132 treatment may cause less RPL3 entering ubiquitination degradation in sh-DUOX2 group.4.Compared with the pc DNA3.1-control,DUOX2 protein level was significantly increased in pc DNA3.1-DUOX2 group,and RPL3 protein level was significantly increased in pc DNA3.1-RPL3 group.DUOX2 overex-pression may enhance the ability of cell migration and invasion and these effects were reversed by overexpression of RPL3.5.NGS results（GSE:139918）showed that si1-DUOX2 vs.si-NC differential genes were mainly enriched in Pathways in cancer,MAPK,PI3K-AKT signaling pathway and HTLV-1 infection;si2-DUOX2 vs.si-NC differential genes were mainly enriched in Pathways in cancer,PI3K-AKT,RAS,and MAPK signaling pathways,etc.In PI3K-AKT signaling pathway,EGFR,AKT1 and MYC changed most significantly.DUOX2 knockdown led to significantly decreased expression of EGFR at mRNA level,while the mRNA expressions of AKT1 and MYC was significantly increased.DUOX2overexpression may cause increased EGFR expression at protein level,and decreased AKT,p-AKT and MYC protein levels.The increase of RPL3protein level could not reverse the increase of EGFR,but could reverse the decrease of AKT,p-AKT and MYC caused by DUOX2 overexpression.Summary:1.There was a significant negative correlation between RPL3 and DUOX2 at protein level,but not at mRNA level.2.DUOX2 affects RPL3 protein expression by affecting the ubiquitination degradation process of RPL3.3.RPL3 could reverse the effect of DUOX2 on cell migration and invasion.4.After knockdown of DUOX2,lots of differential genes were enriched in PI3K-AKT signaling pathway,and some factors in the pathway could be reversed by RPL3.Part Four The effect of silencing DUOX2 on the tumorigenesis of colon cancer cells and 5-FU sensitivity in transplanted tumor in nude miceObjective:To explore the effect of DUOX2 gene silencing on subcut-aneous tumorigenesis and hepatopulmonary metastasis in nude mice by constructing subcutaneous tumor and visceral metastasis model,and to exp-lore the effect of DUOX2 on 5-FU efficacy of subcutaneous tumor.Methods:1.HCT116 and HT29 cell lines with low DUOX2 expression（sh-DUOX2）and corresponding negative control（sh-Control）were constructed by lentiviral infection.Western blot was used to detect the efficiency of lentiviral transfection.2.5-week-old female BALB/C nude mice were divided into 6 groups randomly.HCT116 cells of sh-DUOX2 or sh-Control were injected subcutaneously（n=6/group,1×10⁶cells/mouse）.The same amounts of HCT116 cells of sh-DUOX2 and sh-Control groups were injected by tail vein or by tail vein（n=5/group,1×10⁶cells/mouse）.The same amounts of HT29cells of sh-DUOX2 and sh-Control groups were injected by tail vein（n=6/group,1×10⁶cells/mouse）.3.Metastatic tumors,lung and liver tissues of nude mice were taken for HE staining,and the occurrence of liver and lung metastases in different groups of nude mice was counted.4.Another 20 BALB/C nude mice were randomly divided into two groups（10 mice/group）.HT29 cells（1×10⁶cells/mouse）in sh-Control and sh-DUOX2 groups were injected subcutaneously into the middle and rear armpit of nude mice respectively.After subcutaneous nodules appeared,5mice in each group were given intraperitoneal injection of 5-FU（5mg/kg）,once per 3 days.The tumor volume was calculated by 1/2×long diameter×short diameter².The nude mice were killed 4 weeks later,the transplanted tumors were removed and weighed.Results:1.The expression of DUOX2 in HCT116 and HT29 cells were decreased significantly at mRNA and protein level with lentivirus transfection.2.Subcutaneous transplantation tumor model showed that,there was no significant difference in the weight of tumor between sh-DUOX2 group and sh-Control group.IHC results showed that there was a negative correlation between DUOX2 protein and RPL3 protein levels.3.The tail vein injection model showed that,the incidence of lung metastasis and liver metastasis in sh-DUOX2 group was significantly decreased when compared with sh-Control.In addition,cervical metastatic nodules were found both in sh-Control group and sh-DUOX2 group of nude mice injected with HT29 cells,and the weight of nodules of sh-DUOX2 group was significantly lower than that of sh-Control group.4.There was no significant difference in the volume and mass of transplanted tumor between sh-Control group and sh-DUOX2 group without5-FU treatment.The volume and mass of transplanted tumor in sh-DUOX2+5-FU group were significantly lower both than those in sh-DUOX2 group and sh-Control+5-FU group（all P<0.01）.The results showed that knockdown of DUOX2 significantly improved the therapeutic effect of 5-FU.Summary:1.Down regulation of DUOX2 expression had no significant effect on the proliferation of subcutaneous transplanted tumor in nude mice.2.Down regulation of DUOX2 expression can significantly reduce the incidence of lung metastasis and liver metastasis in nude mice3.Down regulating the expression of DUOX2 can significantly improve the sensitivity of subcutaneous xenografts to 5-FU in nude mice.Conclusions:1.Compared with adjacent normal tissues,the expression DUOX2 at mRNA level in colon cancer tissues was significantly up-regulated,and it was significantly correlated with gender,invasion degree,lymph node metastasis and TNM stage.The high expression of DUOX2 protein in colon cancer tissues and metastatic lymph node was associated with poor prognosis of patients.2.DUOX2 knockdown had no significant effect on the proliferation of HCT116 and SW480 colon cancer cells,but could significantly inhibit the migration and invasion of the cells.3.In colon cancer cells,RPL3,as the downstream interaction protein of DUOX2 protein,has a negative correlation with DUOX2 protein.DUOX2affects the expression of RPL3 protein by affecting its ubiquitination degradation process.RPL3 could reverse the effect of DUOX2 on cell migration and invasion.4.DUOX2 knockdown might lead to a large number of differential genes enriched in PI3K-AKT signaling pathway,and some factors in the pathway could be reversed by RPL3.5.DUOX2 knockdown could reduce the incidence of lung and liver metastasis in nude mice significantly.However,it had no effect on the proliferation of subcutaneous transplanted tumor in nude mice.6.DUOX2 knockdown could significantly enhance the sensitivity of colon cancer cells to 5-FU.