| ObjectiveTo investigate the expression and significance of DUSP8 in patients with colorectal carcinoma(CRC)and the relationship with microsatellite insability(MSI);to explore the effects of DUSP8 on the proliferation and apoptosis of CRC cells with MSI and related machanism.Methods1.Immunohistochemistry was used for detecting of DUSP8 expression in human CRC TMA cancer tissues and para-cancer tissues.Based on Histochemical Scoring(H-Score)of DUSP8 expression and K-S normality test,patients were divided into two groups: DUSP8 high expression group and DUSP8 low expression group.The correlation between DUSP8 expression and clinical significance were performed by Chi-Sqr test,and Kaplan-Meier method was used to analyze the 5-year survival rate of the two groups.Then,univariate and multivariate analyses were utilized to evaluate the total factors affecting the 5-year survival rate of patients with CRC.Further analysis of relationship between DUSP8 expression and clinical indexes was referred from TCGA database.Then,immunohistochemistry detected the expression of mismatch repair proteins MLH1,MSH2 and MSH6 in human CRC TMA,and patients were divided into MSS group MSI-H group.Kaplan-Meier method was used to analyze the 5-year survival rate of the two groups.Furthermore,the clinical significance of DUSP8 expression in MSI-H and MSS group also was analyzed by DUSP8 H-Score,and univariate and multivariate analyses were utilized to evaluate the total factors affecting the 5-year survival rate of patients with CRC.Immunofluorescence and Western Blot were performed to detect the endogenous expression of DUSP8,the expression of mismatch repair proteins MLH1,PMS2,MSH2 and MSH6 in FHC,HCT116,SW480 and SW620 cells.2.Immunofluorescence and Western Blot were used to detect the expression of endogenous DUSP8 in FHC,HCT116,SW480 and SW620 cells.The lipofectamine 3000 nano-liposome was used to transiently transfect DUSP8 overexpression plasmid and p-EGFP control plasmid into HCT116 and SW620 cells in vitro.The cells were harvested 48 hours later.The transfection efficiency was observed by inverted fluorescence microscope.The cell proliferation was detected by CCK8 assay at 24 h,48,72,96 h and 120 h after transfection.Immunofluorescence was performed to detect Ki-67 expression,TUNEL in situ apoptosis,alteration in mitochondrial membrane potential JC-1 and production of mitochondrial ROS.Flow cytometry was used to detect the apoptosis ratio and cell cycle.3.HCT116 and SW620 human CRC xenograft models were established.Then,model mice were divided into HCT116 group(n=12)and SW620 group(n=16)respectively.Next,HCT116 group was randomly divided into p-EGFP group(n=6)and p-DUSP8 groups(n=6).And SW620 group was randomly divided into the p-EGFP group(n=8)and the p-DUSP8 group(n=8).In the tumor tissues,12.5 ?g of p-EGFP and 12.5 ?g of p-DUSP8 plasmid were transfected with Entranster in vivo by injection,and injected once every 3 days for 3 times,and the tumor volume was measured every 3 days.Three days after the last injection,mice were sacrificed,and the tumor tissues and lung tissues were collected.The efficiency of local plasmid transfection and the metastasis of tumor cells were observed by in vivo imaging.Immunofluorescence was performed to detect the expression of DUSP8,Ki-67 and TUNEL.The histomorphological changes of tumor tissues,lung tissues and important organs such as heart,liver,spleen,kidney and brain tissue were detected by HE staining.4.The p-DUSP8 plasmid and p-EGFP control plasmid were transiently transfected into HCT116 and SW620 cells in vitro,respectively.After 48 h,the whole protein was lysis and Western blot was used to detect the expression of DUSP8 and possible dephosphorylation substrates p-JNK1/2,p-ERK1/2,p-p38,and the proliferation and apoptosis-related proteins Cyclin D1,CDK4,p53,p21,and p-Rb,and p-JNK1/2,p-ERK1/2 and p-p38 kinase-activated related substrates c-Fos,c-Jun,p-c-Fos,p-c-Jun,p-p53 and c-Myc.Finally,STRING database was performed to predict the interacting proteins with DUSP8,which integrated into the ZDOCK Server database,and the selected interacting proteins were rigidly docked with DUSP8 to determine the interaction relationship,and the interaction between the target proteins and DUSP8 was further verified by Co-IP assay.Results1.Human CRC TMA data showed that DUSP8 was expressed in 93 cases of cancer tissues and 87 cases of para-cancer tissues.However,the expression level of DUSP8 was not significantly different between cancer tissues and para-cancer tissues.The range of DUSP8 H-Score was 0.125~86.92.According to H-Score,the K-S normality test showed that the expression of DUSP8 in cancer tissues and para-cancer tissues did not conform to the normal distribution(P<0.05).Based on the median H-Score of DUSP8(value is 41.32),patients were divided into DUSP8 high expression group(46 cases)and DUSP8 low expression group(47 cases).Further analysis showed that there were no significant difference on DUSP8 expression between the two groups regarding age,gender,histological differentiation,pathological type,tumor size,TN stage and clinical stage respectively(P>0.05).In M stage,there were 4 cases of M1 stage in DUSP8 low expression group and 0 cases in DUSP8 high expression group.The difference between the two groups was statistically significant(P<0.05).Kaplan-Meier analysis showed that there was no significant difference on the 5-year survival rate between the two groups(P>0.05).Cox regression analysis showed that the tumor size,TNM stage and lymph node metastasis were associated with 5-year survival rate in patients with CRC(P<0.05),and the tumor size and TNM stage were independent factors affecting 5-year survival rate(P<0.05).The TCGA database analysis showed that there was no remarkable difference in the expression of DUSP8 between cancer tissues and para-cancer tissues in 327 patients with CRC(P>0.05).Moreover,there were also no significant difference on DUSP8 expression regarding ages,genders,clinical stages,races and body weights of CRC patients(P>0.05).In term of MSI aspect,Human CRC TMA analysis revealed that 17 of 93 patients had MLH1 or MSH2 and MSH6 expression loss,belonging to MSI-H CRC,and 76 cases were MSS CRC.Kaplan-Meier analysis found that the 5-year survival rate of MSI-H CRC patients was significantly longer than that of MSS CRC patients(P<0.05).The expression of DUSP8 mRNA in MSI-H CRC patients was significantly lower than that of MSS CRC patients(P<0.05).In MSI-H CRC,the expression of DUSP8 was correlated with tumor size and survival status(P<0.05),but not with TNM stage and clinical stage(P>0.05).In MSS CRC,the expression of DUSP8 was closely related to the tumor size,NM stage,clinical stage and survival rate of patients(P<0.05),but not related to the patient's T stage(P>0.05).Western Blot and immunofluorescence data showed that HCT116 cells were MSI-H type;FHC,SW480 and SW620 cells were MSS type.Furthermore,in MSS CRC,Cox regression analysis showed that the expression level of DUSP8,TNM stage and distant metastasis were associated with 5-year survival rate in patients with CRC(P<0.05),and the DUSP8 expression,tumor size and TNM stage were independent factors affecting 5-year survival rate(P<0.05).The endogenous protein expression of DUSP8 was significantly higher in HCT116 cells compared to other cells.And the endogenous protein expression of p-JNK1/2,p-ERK1/2 and p-p38 was higher in SW480 cells than other cells.2.The p-DUSP8 plasmid and p-EGFP control plasmid were transiently transfected into HCT116 and SW620 cells in vitro,respectively.After 48 h,the cells were collected.EGFP was highly expressed in the cells.CCK8 and colony formation assay showed that compared with the p-EGFP group,the proliferation and colony formation ability of HCT116 cells in the p-DUSP8 group were significantly accelerated(P<0.05),conversely,the proliferation and clone formation ability of SW620 cells were significantly weakened(P<0.05).Immunofluorescence data showed that compared with p-EGFP group,the expression of Ki-67 in HCT116 cells of p-DUSP8 group obviously increased(P<0.05),while cell apoptosis was significantly decreased(P<0.05).Moreover,mitochondrial membrane potential JC-1 Red fluorescence increased obviously,and mitochondrial ROS production decreased significantly.In contrast,in SW620 cells,the expression of Ki-67 was clearly downregulated(P<0.05),while cell apoptosis increased significantly(P<0.05).And mitochondrial membrane potential JC-1 enhanced green fluorescence,and mitochondrial ROS production was significantly upregulated.The flow cytometry analysis showed that compared with p-EGFP group,the apoptosis rate of HCT116 cells in p-DUSP8 group significantly decreased(P<0.05),while the apoptosis rate of SW620 cells definitely increased(P<0.05).Cell cycle analysis showed that the proportion of G0/G1 phase of HCT116 cells in p-DUSP8 group was downregulated(P<0.05);the proportion of S phase was upregulated(P<0.05),and the proportion of G2/M phase decreased(P<0.05).While in SW620 cells,G0/G1 phase ratio in p-DUSP8 group increased obviously(P<0.05);the S-phase ratio of cells was declined(P<0.05),and the G2/M-phase ratio was upregulated(P<0.05).3.Data showed that compared with the p-EGFP control group,the tumor growth of the HCT116 xenograft model in the p-DUSP8 group significantly increased(P<0.05).The in vivo imaging analysis showed that the tumor size was proportional to the fluorescence intensity of the local enrichment,and tumor cell foci was more obvious the lung tissue.While the tumor growth of the SW620 xenograft model in the p-DUSP8 group significantly decreased slower(P<0.05).And the in vivo imaging analysis showed that the tumor size was inversely proportional to the intensity of the locally enriched fluorescent signal.Moreover,the tumor cell foci in the lung tissue decreased.Immunofluorescence data further showed that,compared with control group,DUSP8 was highly expressed in the tumor tissues of p-DUSP8 group in the HCT16 or SW620 xenograft models.Compared with the p-EGFP control group,the expression of Ki-67 in the HCT116 tumor tissues of the p-DUSP8 group obviously increased(P<0.05),and apoptotic cells also significantly decreased(P<0.05).Conversely,the expression of Ki-67 decreased obviously in SW620 tumor tissues in p-DUSP8 group(P<0.05),and the apoptotic cells increased significantly(P<0.05).Finally,HE staining data showed that there were no obvious pathological changes in the heart,liver,spleen,kidney and brain tissues between the p-DUSP8 group and the p-EGFP group in the HCT116 or SW620 xenograft models.4.The p-DUSP8 plasmid and p-EGFP control plasmid were transiently transfected into HCT116 and SW620 cells in vitro,respectively.After 48 h,the cell whole protein was collected.Western Blot data showed that in HCT116 cells,compared with those in p-EGFP group,the expression levels of DUSP8 and p-ERK1/2 in p-DUSP8 group were significantly upregulated(P<0.05)and the expression of p-JNK1/2 was obviously downregulated(P<0.05).Moreover,the expression levels of growth and apoptosis-related proteins CDK4 and p-Rb in p-EGFP group were obviously higher than those in p-EGFP group(P<0.05).However,there were no significant difference in the expression levels of p-p38,Cyclin D1 and p53,as well as the relevant substrates of p-JNK1/2,p-ERK1/2,p-p38 kinases such as c-Fos,c-Jun and p-c-Fos(P>0.05).Further analysis showed that the expression levels of p21,p-c-Jun and p-p53 were remarkably downregulated in p-DUSP group(P<0.05).However,the expression level of c-Myc was significantly upregulated(P<0.05).In SW620 cells,compared with those in p-EGFP group,the expression levels of DUSP8 in p-DUSP8 group was significantly upregulated(P<0.05)and the expression of p-JNK1/2,p-ERK1/2 and p-p38,as well as Cyclin D1 and CDK4,were obviously downregulated(P<0.05).However,there were no significant difference in the expression levels of p53,p21 and p-Rb(P>0.05).And the expression of p-JNK1/2,p-ER K1/2,p-p38 kinase-activated related substrates c-Fos,c-Jun,p-c-Jun and p-p53 also was no obvious significance(P>0.05).Further analysis showed that the expression levels of p-c-Fos and c-Myc were remarkably downregulated in p-DUSP group(P<0.05).Molecular modeling and docking analysis showed that DUSP8(PDB: 4JMK)might directedly interact with MAPK1(ERK2,PDB: 4S31),MAPK8(JNK1,PDB: 3PZ1),MAPK11(p38?,PDB: 3GP0)and MAPK14(p38?,PDB: 5ETI).However,there was no potential interaction between DUSP8 and MAPK3(ERK1,PDB: 2ZOQ).Finally,Co-IP results showed that in both HCT116 cells and SW620 cells,DUSP8 could bind to endogenous p-JNK1/2.Conclusions 1.The expression of DUSP8 is closely related to the microsatellite stability(MSS)of human CRC.2.Upregulation of DUSP8 expression promotes the proliferation and inhibits the apoptosis of MSI-H CRC in vitro and in vivo,while inhibits the growth and impetus the apoptosis of MSS CRC in vitro.3.Upregulation of DUSP8 expression in MSI-H and MSS CRC cells is closely related to the different of dephosphorylatd substrates p-JNK1/2,p-ERK1/2 and p-p38 and the inconsistency of downstream transcription factors activated by corresponding phosphorylated substrates. |
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