MRGBP Promotes Invasion And Metastasis Of Colorectal Cancer Via DKK1/Wnt And NF-kB/p65 Mediated EMT

Research background and purposeColorectal cancer(CRC)is one of the most common malignancies with high morbidity and high mortality,and is a multi-step process involving progressive destruction of epithelial cell proliferation,apoptosis,differentiation,and survival mechanisms in China in recent years.The incidence of CRC continues to rise.Despite significant clinical advances in diagnosis and treatment,the prognosis of distant metastatic CRC patients remains poor,and the molecular mechanism of CRC metastasis remains unclear.Therefore,there is an urgent need to identify important molecules in the progression of CRC that can be used to develop new diagnostic strategies and drugs for these markers.The occurrence of colorectal cancer is closely related to the activation of various oncogenes and tumor suppressor genes.The investigation of the changes in genes involved in the development of colorectal cancer may indicate the process of the development mechanism of colorectal cancer.It is well known that the growth,proliferation,differentiation and death of cells are an orderly phenomenon.The formation of tumors is the process of abnormal proliferation of cells,and the genes involved are unusually numerous.Previous research group found that MIER3 is lowly expressed in colorectal cancer and is a protective factor affecting colorectal cancer.Subsequently,MIER3 interacting protein MRGBP was screened by immunoprecipitation and verified experimentally.The MRGBP gene is located on human chromosome 20(20q13.33)and is regulated by RNA polymerase II promoter transcription.MRGBP(MRG/MORF4L binding protein,MRGBP)is an acetylated protein complex and is an important component of the complex NuA4.The acetylated histones H4 and H2A participate in transcriptional regulation of the gene.When DNA damages,it also plays an important role in DNA damage repair.Recent studies have found that MRGBP is up-regulated in colorectal cancer tissues and has a high mutation rate,suggesting that it is closely related to the occurrence and development of colorectal cancer.However,the specific functions and related mechanisms of action of MRGBP have not yet been clarified.In this study,our group first detected the expression of MGRBP in colorectal cancer cell lines and tissues,and explored the relationship between MRGBP and colorectal cancer,and further explored its role in the development of colorectal cancer both in vivo and in vitro.Role and preliminary exploration of possible mechanisms of action.Methods1.Immunofluorescence assay was used to detect the localization of MRGBP protein in colorectal cancer cells.Immunohistochemical method was used to detect the expression of MRGBP in paraffin tissue of clinical colorectal cancer with follow-up data.QPCR was used to detect different colorectal cancer cell lines and matched colorectal cancer cells.Expression of mRNA in MRGBP in paraffin-embedded tissue;Western blot analysis was used to detect the expression of MRGBP protein in 7 colorectal cancer cell lines and 16 pairs of colorectal cancer and paracancerous tissues,and analyze the relationship between their expression levels and clinicopathological factors.And its prognostic significance.2.Lentivirus infection method was used to construct cell lines that stably interfered with and overexpressed MRGBP.CCK8,plate clony formation experiments were used to detect cell proliferation,transwell experiments and scratch experiments to detect cell invasion and metastasis,nude mice subcutaneous tumorigenesis experiments and Planted to observe the ability of tumor formation and metastasis in nude mice.3.Western blot was used to detect the expression of DKK1 after interference and overexpression of MRGBP.The interaction between the two was detected by nuclear plasma separation and immunofluorescence co-localization.Simultaneously,the changes of EMT-related marker proteins were detected.The acetylation inhibitor TH1834 was used to detect p65.The level of acetylation,to explore the possible molecular mechanism of the role of MRGBP in the development of colorectal cancer.4.Use SPSS 13.0 statistical software for data statistics.Two-sample T-test was used to test the fluorescence quantitative PCR.The relationship between the expression of immunohistochemical MRGBP and clinicopathological parameters was analyzed by Pearson Chi-square test.Kaplan-Meier method was used for survival analysis,and Log-Rank was used to test the difference.The P<0.05 difference was considered statistically significant.Transwell cell migration assays,scratch healing assays,and plate colony formation assays were used to compare the mean between two groups.Two independent sample t-tests should be used.One-way analysis of variance(One-way ANOVA)is used to compare the mean of multiple groups.Way ANOVA).Levene’s method should be used to test for variance homogeneity before analysis of variance.When variance is equal,multiple comparisons should be preceded by LSD method(LeaSt.significant difference method).When variance is uneven,Dunnett’s T3 method is used.Results1,MRGBP in colorectal cancer tissue expression and clinical pathological factors and prognosis analysisImmunohistochemistry results showed that the MRGBP protein was located in the nucleus of colorectal cancer cells.Meanwhile,the positive rate of MRGBP expression in paraffin tissue of colorectal cancer was 72%(108/150).Statistical analysis showed that in 150 colorectal cancer patients,the expression of MRGBP was not significantly different from the tumor size,age,gender,and location of the tumor(P>0.05),and the depth of tumor invasion(P=0.004)Lymph node metastasis(P=0.013)and distant metastasis(P=0.001)were significantly correlated.Using the Kaplan-Meier method to analyze the 5-year survival curve,the average survival time of colorectal cancer patients with high expression of MRGBP was 61.421 months,and the average survival time of colorectal cancer patients with low expression of MRGBP was 75.065 months,indicating the survival rate of patients with high MRGBP expression group.Lower than the MRGBP low expression group,the difference was statistically significant(P<0.05).The results of qPCR experiments showed that the expression of MRGBP in 20 colorectal cancer tissue samples was significantly higher than that in the corresponding paired adjacent normal tissues(P<0.001).Western blot results showed that the expression of MRGBP protein in 16 pairs of colorectal cancer tissue samples was significantly Higher than normal tissue next to the cancer.2.Expression of MRGBP in colorectal cancer cell linesReal-time PCR and western blot experiments on colorectal cancer cell lines showed that the expression levels of MRGBP protein in different cell lines were different.3.Interfering with the expression of MRGBP inhibits the proliferation and migration of SW620 and HCT116 cellsLentivirus infection method was used to transfect SW620 and HCT116 cells with the target-interfering MRGBP expression fragment and screened with puromycin to obtain stable interference with MRGBP and control cell lines.CCK8 and plate colony formation experiments showed that compared with the control group,the in vitro proliferation ability of S W620/shMRGBP and HCT116/shMRGBP cells was significantly decreased.Invasion experiments and scratch experiments showed that the invasiveness and migration of SW620/shMRGBP and HCT 116/shMRGBP cells were significantly reduced.Subcutaneous tumorigenesis and orthotopic implantation experiments in nude mice have shown that interference with MRGBP inhibits tumorigenicity and metastasis in vivo.4.Overexpression of MRGBP promotes proliferation and migration of SW480 and DLD1 cells.Lentivirus infection method was used to transfect the fragment with targeted interference with MRGBP into SW480 and DLD1 cells and screened with puromycin to obtain stable interference with MRGBP and control cell lines.CCK8 and plate colony formation experiments showed that compared with the control group,the in vitro proliferation of SW480/MRGBP and DLD1/MRGBP cells was significantly enhanced.Invasion experiments and scratch experiments showed that the invasion and migration of SW480/MRGBP and DLD1/MRGBP cells were significantly enhanced.Subcutaneous tumorigenesis and orthotopic implantation experiments in nude mice have shown that overexpression of MRGBP promotes tumorigenicity and metastasis in vivo.5,MRGBP and DKK1,p65 relationshipWestern blot analysis of DKK1 expression in cell lines that interfered with and overexpressed MRGBP revealed that DKK1 protein expression was significantly reduced in the cell lines that interfered with MRGBP compared to the control group,whereas DKK1 was overexpressed in the MRGBP cell line.Protein expression was significantly elevated.COIP experiments verified the interaction between DKK1 and MRGBP.Among the cell lines overexpressing MRGBP,immunofluorescence experiments revealed that DKK1 nuclear import increased,DKK1 protein total decreased,beta-catnin nuclear increase,and wnt downstream target.Gene c-myc,CyclinD1,MMP7 expression increased,wnt pathway activation.By adding the tip60 acetylation inhibitor,the level of acetylation of p65 was found to be reduced.6.The effect of MRGBP on EMT in colorectal cancer cellsInverted fluorescence microscopy revealed that the shape of the colorectal cancer cells exhibited fusiform shape after overexpression of MRGBP,and the expression of Slug,N-cadherin,snail,beta-catenin,and Vimentin was increased after MRGBP was overexpressed in western blot.The expression of E-cadherin was decreased.The expression of N-cadherin,snail,beta-catenin and Vimentin was decreased and the expression of E-cadherin was increased after adding acetylase inhibitors.Conclusion1.MRGBP is located in the nucleus of colorectal cancer;High expression of MRGBP in colorectal cancer cell lines;MRGBP was expressed in tumor tissues higher than that of adjacent normal tissues.The relationship between MRGBP and colorectal tumor was preliminarily established.2.MRGBP can promote the proliferation,invasion and metastasis of colorectal cancer cells.3.MRGBP promotes the activation of WNT pathway and the acetylation of nf-kb p65 by interacting with DKK1 to promote the metastasis of colorectal cancer cells.4.MRGBP may be a new molecular marker for the progression and prognosis of colorectal cancer patients,thus providing a new molecular target for the prognosis and treatment of colorectal cancer.The innovation of this research.(1)The function and molecular mechanism of MRGBP have not been reported yet.Our group has initially determined its role in colorectal cancer through in vitro and in vivo experiments.(2)MRGBP may promote the activation of WNT by interacting with DKK1,acetylated NF-kb P65 mediates the occurrence of EMT and then participates in the regulation of invasion and metastasis of colorectal cancer cells.