The Role Of Gene MEIS2 In Regulating Stemness And Drug Resistance Of Colorectal Cancer Cells And The Molecular

Colorectal cancer(CRC)was currently one of the most common gastrointestinal malignancies.The standardized morbidity and mortality rates of colorectal cancer had been increasing in the past few years,which were mainly attributed to recurrence and distant organ metastasis,and the main cause of recurrence was chemoresistance,which leaded to a decrease in survival.Some studies had reported that the development of chemoresistance was strongly related to the stem cell-like properties of tumor cells and insensitivity of chemotherapy.The first-line chemotherapy regimen recommended by the 2019 NCCN guidelines was FOLFOX and Cape OX regimens containing oxaliplatin,which some patients were still clinically insensitive,The research on the possible molecular mechanisms of colorectal cancer that leaded to recurrence due to the formation of stemness and drug resistance could provide clinical treatment of colorectal cancer and reversal of drug resistance so that achieved precision chemotherapy.MYELOID ECTOPIC VIRAL INTEGRATION SITE(MEIS)proteins include MEIS1,MEIS2,and MEIS3,which were genome-wide proteins that were considered to be cofactors of HOX proteins,that form DNA-protein complexes,which in turn affected their role in the cell.Among them,MESI2 had been shown to be involved in the pathogenesis of human cancers,and abnormalities in MEIS2 protein expression and function had been associated with a variety of cancers,and the oncogenic and oncogenic effected they exert depend on different organ sites;for example,high expression of MEIS2 was associated with improved prognosis in ovarian cancer patients,while in neuroblastoma,prostate cancer,and bladder cancer,its high expression promoted tumorigenesis and associated with its development of metastatic disease.However,the functional role of MEIS2 in CRC remained unclear.In our group,we concluded that this gene may inhibit tumor cell proliferation and affected its stem expression in colorectal cancer by WGCNA and other methods,and was associated with patient’s disease prognosis.In this study,we will investigate the expression of MEIS2 in CRC by combining our group’s previous studies on MEIS2,and examine its effect on the stemness and other malignant phenotypes of CRC cells by constructing overexpression and knockdown of MEIS2 colorectal cancer cell lines to explore the possible molecular mechanisms and provide a sufficient theoretical basis for the prevention and targeted treatment of colorectal cancer.Based on the above objectives,we divided the study into three parts as follows.Part Ⅰ Expression and significance of MEIS2 in colorectal cancer cells and tissuesObjective: To explore the expression and significance of MEIS2 in colorectal cancer cells and tissues.Methods :MEIS2 m RNA expression levels were detected in five different colorectal cancer cell lines and normal colorectal cancer epithelial cells NCM460,and one colorectal cancer cell line each with relatively high and relatively low expression of MEIS2 was derived by q RT-PCR assay.Cancer tissues and normal tissues from five pairs of colorectal cancer patients were sampled for immunohistochemical analysis;the expression levels of TCGA and other databases in colorectal cancer and their clinical significance were analyzed by bioinformatics.Results:The immunohistochemical results showed that MEIS2 was mainly expressed in the cytoplasm and nucleus in normal colorectal cancer patients,and was low or not expressed in colorectal cancer tissues;the expression levels of colorectal cancer cell lines were all lower than those of normal colorectal epithelial cells by q RT-PCR experiments;the disease-free survival of the high MEIS2 expression group was higher than that of the low expression group,and the differences were statistically significant.Conclusion :The expression level of MEIS2 is lower in colorectal cancer cells and tissues than in normal colorectal cells and tissues,and its expression is negatively correlated with the prognosis of colorectal patients.Part Ⅱ MEIS2 regulates stemness and oxaliplatin chemotherapy resistance in colorectal cancer cellsObjective :To initially explore MEIS2 in colorectal cancer cells to regulate the formation of stemness and oxaliplatin chemoresistance..Methods :Stable cell lines were constructed by lentiviral transfection with overexpression and knockdown of the gene,and the transfection efficiency and expression level of the lentiviral vector were detected by Western Blot assay and q RT-PCR assay;cell sphere-forming assay was performed to detect the sphere-forming ability of different cell lines;the relative expression of its stemness marker at protein level was explored by Western blot assay;the relative expression of MEIS2 at protein level was detected by CCK-8 and plate cloning assays were used to assess the proliferation ability of overexpressed and knockdown cell lines,respectively,and the effect on apoptosis was analyzed by flow cytometry;the cell survival rate after treatment with different concentrations of oxaliplatin was examined by MTT assay;bioinformatics analysis was performed.Results :The cell lines SW620-MEIS2 and DLD1-MEIS2,which stably overexpressed MEIS2,and the cell lines SW620-sh RNA-MEIS2 and DLD1-sh RNA-MEIS2,which knocked down MEIS2,and their paired control cell lines SW620-NC and DLD1-NC,were successfully constructed and firstly observed by fluorescence microscopy after 24 h.The transfection efficiency of the lentiviral vectors was detected by Western Blot and q RT-PCR assays,respectively,and the expression levels of the gene were significantly increased in the overexpression cell lines and decreased in the knockdown cell lines;the sphericity rate of SW620 with higher MEIS2 expression level was significantly lower compared to that of DLD1 with lower MEIS2 expression level;the sphericity rate of DLD1 cells with higher MEIS2 expression level was significantly lower compared to that of DLD1 cells with higher MEIS2 expression level.The sphericity rate of SW620 cells with higher MEIS2 expression was significantly lower compared to that of DLD1 cells with lower MEIS2 expression;sphericity was reduced in MEIS2 overexpressing cell lines and the corresponding protein expression levels of CD133 and Lgr5 were reduced,while in MEIS2 knockdown cell lines of sphericity was relatively increased and the corresponding protein expression levels of CD133 and Lgr5 were relatively increased.Cell proliferation in the knockdown and overexpression cell lines was assessed using CCK-8 assay and plate cell cloning assay,respectively,to detect cell viability,which was significantly decreased in the overexpression cell lines SW620-MEIS2 and DLD1-MEIS2 compared to the matched control cell lines SW620-NC and DLD1-NC,respectively,while the knockdown cell lines SW620 sh RNA-MEIS2 and DLD1-sh RNA-MEIS2 proliferation viability was relatively increased compared to their paired control cell lines SW620-NC and DLD1-NC.At the apoptotic level,the proportions of SW620-MEIS2 and DLD1-MEIS2 were significantly lower and the proportions of SW620-sh RNA-MEIS2 and DLD1-sh RNA-MEIS2 were significantly higher when compared with their paired controls,respectively.It was concluded by MTT assay that the survival rate of MEIS2 high expression group was lower than that of paired control group and the cell survival rate of knockdown group was higher than that of paired group under the effect of oxaliplatin at the same concentration;bioinformatics analysis showed that the relative expression of MEIS2 was significantly lower in L-OHP resistant tissues compared with L-OHP sensitive tissues,and the results were all statistically significant(P<0.05).Conclusion:In in vitro experiments,high expression of MEIS2 inhibites the stem cell-like properties,proliferative capacity,and apoptotic capacity of colorectal cancer cells and correlated with L-OHP chemoresistance in that its high expression can enhance their chemo-sensitivity and decrease expression in the chemoresistant group.Part Ⅲ MEIS2 regulates the stemness of colorectal cancer cells and the possible molecular mechanism of oxaliplatin chemotherapy resistanceObjective:To initially explore the possible molecular mechanisms of MEIS2 in colorectal cancer cells regulating the formation of stemness and resistance to chemotherapy,in order to provide reversal of drug resistance to achieve precision chemotherapy.METHODS: To verify the downstream mechanism of MEIS2 in regulating apoptotic pathway by bioinformatics analysis of possible upstream mechanisms and Western blot assay as well as Caspace-3 activity assay.RESULTS: One of the pathways involved in the regulation of MEIS2 in colorectal cancer is the elevated level of methylation at the promoter region Cp G island at the DNA level,which prevents the promoter from binding to transcription factors and down-regulates mRNA expression,which in turn silences MEIS2 expression;MEIS2 has a 1.7% missense mutation in colorectal cancer,and the presence of this fraction leads to a decrease in MEIS2 The negative correlation between MEIS2 gene copy number and MEIS2 expression;a significant increase in caspase-3 activity was detected in MEIS2 overexpressing colorectal cancer cell lines compared to paired controls,while caspase-3 activity was significantly reduced in colorectal cancer cell lines with knockdown of MEIS2;the two groups of cell lines that underwent MEIS2 overexpression compared to paired controls,at the protein level,with a decrease in Bcl-2 and an increase in Bax,and conversely,both cell lines with MEIS2 knockdown had an increase in Bcl-2 and a decrease in Bax compared to paired controls,consistent with the results of their bioinformatics analysis being statistically significant(P<0.05).Conclusion: The upstream mechanisms of MEIS2 include hypermethylation,missense mutations,and DNA copy number abnormalities;hypermethylation of MEIS2 in the promoter region causes its expression to be downregulated in colorectal cancer;the downstream pathways of MEIS2 include anti-apoptotic pathways.