| Background and SignificanceColorectal cancer is a common malignant tumor of digestive system,which seriously threatens human health.The existing treatment methods of colorectal cancer have different therapeutic effects among patients with colorectal cancer,and the metastasis of colorectal cancer is an important reason for the poor prognosis of patients with colorectal cancer.Although the development of new chemotherapeutic agents and targeted therapies have improved clinical outcomes in patients with metastatic colorectal cancer,a large number of patients still experience treatment failure and metastasis.Therefore,the identification of new prognostic factors and the study of pathogenesis of colorectal cancer are of great significance for the specific diagnosis and precise treatment of colorectal cancer.Endoplasmic reticulum stress(ER)is a disorder of the function in which misfolded and unfolded proteins accumulate in the lumen and Ca2+imbalance occurs under the action of injury factors.Endoplasmic reticulum stress will stop cell proliferation,in the presence of stress will start the apoptosis process.Although various cellular stress factors are prevalent in solid tumor tissues,it suggests that the ER evolution of tumor cells has a protective mechanism to ensure protein synthesis and correct folding and assembly under stress conditions.It is of great significance to study the effect and mechanism of colon cancer cell-mediated endoplasmic reticulum stress in resisting apoptosis.Endoplasmic reticulum oxidoreductin1α(ERO1α)is a key protein that regulates protein synthesis and folding in the endoplasmic reticulum lumen.Studies have shown that ERO1αoverexpression plays a key role in tumor biology,and its expression level is consistent with Tumor deterioration was positively correlated.Studies have also found that under hypoxic stress conditions,the expression level of ERO1α in liver cancer cells can increase rapidly.Knockdown of ERO1α can significantly promote the apoptosis of liver cancer cells,suggesting that ERO1α is most likely to initiate the protective mechanism of the endoplasmic reticulum stress response.One of the upstream regulatory molecules.There are few research reports about the relationship between EROla expression and endoplasmic reticulum stress on the progression of colon cancer.As an evolutionarily conserved non-coding RNA molecule,microRNA(miRNA)plays an important role in post-transcriptional gene regulation.It can regulate cell differentiation,proliferation,apoptosis,growth,migration and survival.More and more evidences show that abnormal changes in miRNA levels are closely related to the occurrence and development of cancer.It has been reported that miR-101 is a promoter of apoptosis in a variety of tumor cells,and it plays a role in promoting apoptosis mainly by inhibiting the expression of its target protein EZH2.EZH2 is a member of the polycomb protein family.It acts as a histone methyltransferase by catalyzing-the trimethylation of histone H3 lysine 27(H3-K27).It plays an important role in maintaining gene silencing and participates in the proliferation of a variety process of tumors.Our preliminary experiments found that under the conditions of endoplasmic reticulum stress,the up-regulation of ERO1α expression in colon cancer cells can inhibit the expression of miR-101 and at the same time up-regulate the level of EZH2.For this reason,we propose the research ideas that ERO1α,miR-101 and EZH2 interact to regulate cells apoptosis on signal transduction pathways mediating the occurrence and development of colon cancer.Studies have shown that Wnt/β-catenin signaling pathway plays an important role in the occurrence and progression of colon cancer.Studies have reported that EZH2 can activate Wnt/β-Catenin pathway to promote osteogenic differentiation of human bone marrow mesenchymal stem cells,Loss expression of miR-101 in colon cancer cells promotes the activation of Wnt/β-catenin signaling pathway.Therefore,it is proposed that miR-101 regulates the activation of Wnt/β-catenin signaling pathway by EZH2 as a potential mechanism of ERO1α-mediated endoplasmic reticulum stress induced colon cancer cell apoptosis.This study validated the proposed scientific mechanism,revealed the relationship between endoplasmic reticulum stress-induced apoptosis and the occurrence and development of colon cancer,and confirmed that the ERO1αmolecule,the miR-101/EZH2 axis and the Wnt/β-catenin signaling pathway are of the important role played in this process,enriches the understanding of the molecular mechanisms related to colon cancer cell apoptosis and provides new target treatment ideas for colon cancer treatment strategies.ObjectiveTo explore the role of ERO 1α in endoplasmic reticulum stress induced apoptosis of colon cancer cells,reveal the relationship between endoplasmic reticulum stress mediated apoptosis and the progression of colon cancer,and confirm that ERO 1α,miR-101/EZH2 axis and Wnt/β-catenin signaling pathway are the potential mechanisms of this process.This study provides theoretical basis for the study of the molecular mechanism related to the apoptosis of colon cancer cells and the development of colon cancer,and expands the new ideas of target therapy for colon cancer.Methods1.The expression levels of ERO 1α,miR-101 and EZH2 were detected by RT-qPCR.The expression levels of ERO 1α were detected by immunohistochemistry.The correlation between ERO 1α protein expression and clinicopathological factors of colon cancer patients was analyzed by χ2 test.2.Culture human normal colon NCM460 cells,human colon cancer RKO and HT-29 cells in vitro,and detect the expression of ERO 1α,miR-101 and EZH2 by RT-qPCR.3.In RKO and HT-29 cells,THG induces endoplasmic reticulum oxidative stress.The expression of ERO 1α,miR-101 and EZH2 were detected by RT-qPCR.The cells proliferation ability was detected by CCK-8 and bromodeoxyuridine(BrdU)immunofluorescence labeling.Flow cytometry to detect cell apoptosis.Western Blot to detect the expression of apoptosis-related proteins Bax,caspase-3 and caspase-9 and the expression of c-MYC and Cyclin D1 downstream target genes of Wnt/β-catenin pathway.4.Short hairpin RNA technology knocks down ERO1α(sh-ERO1α),constructs lentiviral vectors miR-101 and anti-miR-101,constructs pcDNA-EZH2 overexpression vector,THG induces endoplasmic reticulum oxidative stress in RKO and HT-29 cells Under conditions,transfect sh-ERO1α,co-transfect sh-ERO1α+anti-miR-101,transfect miR-101,co-transfect pcDNA-EZH2+miR-101,and simultaneously transfect the corresponding negative control group,CCK-8 and BrdU was used to detect cell proliferation ability,flow cytometry was used to detect cell apoptosis,and Western Blot was used to detect the expression of apoptosis-related proteins Bax,caspase-3 and caspase-9.5.Under THG-induced oxidative stress in RKO and HT-29 cells,transfect sh-ERO1α,co-transfect sh-ERO1α+anti-miR-101,transfect miR-101,co-transfect pcDNA-EZH2+miR-101,Simultaneous transfection corresponds to the negative control group,and Western Blot detects the expression of Wnt/β-catenin signaling pathway related proteins Wnt3a,Wnt5a and β-catenin and the expression of c-MYC and Cyclin D1 downstream target genes of Wnt/β-catenin pathway.6.Nude mice were subcutaneously injected with colon cancer cell suspension,THG-induced oxidative stress colon cancer cell suspension,and THG-induced and knocked down sh-ERO1α colon cancer cell suspension to construct a nude mouse colon cancer subcutaneous tumor model,and observe the tumor size of nude mice;RT-qPCR detected the expression of ERO1α,miRNA-101 and EZH2 in tumor tissues,the expression of ERO1α and EZH2 was detected by immunohistochemistry,and the expression of apoptosis-related proteins Bax,caspase-3 and caspase-9 were detected by Western Blot and the expression of Wnt3a、Wnt5a、β-catenin、c-MYC、Cyclin D1 related to Wnt/β-catenin pathway.Results1.Compared with adjacent tissues,ERO1α and EZH2 are significantly up-regulated in colorectal cancer tissues,and miR-101 is significantly down-regulated in colorectal cancer tissues.The difference is statistically significant(P<0.01).Correlation analysis showed that the expression of EROla is correlated with the degree of differentiation of colorectal cancer,TNM staging and lymphatic metastasis.2.Compared with human normal colon NCM460 cells,the expression of ERO1αand EZH2 was significantly up-regulated in human colon cancer RKO and HT-29 cells,while the expression of miR-101 was significantly down-regulated,and the difference was statistically significant(P<0.01).3.After THG induction,compared with the control group,the expression of EROla and EZH2 was significantly down-regulated in RKO and HT-29 cells,and the expression of miR-101 was significantly up-regulated,and the difference was statistically significant(P<0.01).At the same time,in THG-induced group,the proliferation activity of RKO and HT-29 cells were significantly decreased,and the apoptosis rate was significantly increased,and the difference was statistically significant(P<0.01).4.Compared with the control group,the proliferation activity of RKO and HT-29 cells in the sh-ERO1α transfected group was significantly decreased,and the apoptosis rate was significantly increased.The difference was statistically significant(P<0.01);Compared with the sh-ERO1α transfected group,in the co-transfected sh-ERO1α+anti-miR-101 group,the cell proliferation activity increased,and the apoptosis rate decreased,and the difference was statistically significant(P<0.05).Compared with the control group,in the miR-101 transfected group,the proliferation activity of RKO and HT-29 cells was significantly decreased,and the apoptosis rate was significantly increased.The difference was statistically significant(P<0.01),which was similar to the miR-101 transfected group.Compared with the co-transfected pcDNA-EZH2+miR-101 group,the cell proliferation activity increased,and the apoptosis rate decreased,and the difference was statistically significant(P<0.05).5.Compared with the control group,the expression of Wnt3a,Wnt5a andβ-catenin were significantly down-regulated in the sh-ERO1α transfected group,while the expression of c-MYC and Cyclin D1 downstream target genes of Wnt/β-catenin pathway were down-regulated too,and the difference was statistically significant(P<0.01).Compared with the sh-ERO1α transfected group,the expressions of Wnt3a、Wnt5a、β-catenin、c-MYC、Cyclin D1 were up-regulated in the co-transfected ERO1α+anti-miR-101 group,and the difference was statistically significant(P<0.05).Compared with the control group,the expression of Wnt3a,Wnt5a、β-catenin、c-MYC、Cyclin D1 were significantly down-regulated in the miR-101 transfected group,and the difference was statistically significant(P<0.01).Compared with the miR-101 transfected group,the expressions of Wnt3a,Wnt5a、β-catenin、c-MYC、CyclinD1 were up-regulated in the co-transfected EZH2+miR-101 group,and the difference was statistically significant(P<0.05).6.Compared with the control group,the volume of tumor formation in the THG group was reduced,the expression level of ERO1α and EZH2 were reduced,the expression level of miR-101 were increased,and the expression of apoptosis-related proteins Bax,caspase-3,caspase-9 were increased,while the expression of Wnt3a、Wnt5a、β-catenin、c-MYC、Cyelin D1 related to Wnt/β-catenin pathway were decreased;The volume of THG+sh-ERO1α tumors was further reduced,the expression levels of EROla and EZH2 were further reduced,the expression levels of miR-101 was further increased,and the expressions of Bax,caspase-3 and caspase-9 were further increased.The expression of Wnt3a,Wnt5a、β-catenin、c-MYC、Cyclin D1 related to Wnt/β-catenin pathway were further decreased,the difference.was statistically significant(P<0.01,P<0.001).At the same time,the difference between THG+sh-ERO1α group and THG group is also statistically significant(P<0.01).ConclusionsERO1α and EZH2 are up-regulated in colorectal cancer tissues and colon cancer cell lines,while miRNA-101 is down-regulated,and the induction of THG endoplasmic reticulum oxidative stress can reverse this phenomenon.ERO1αparticipates in the apoptosis and proliferation of RKO and HT-29 cells induced by endoplasmic reticulum stress,mainly by regulating the miR-101/EZH2 axis and affecting the Wnt/β-catenin signaling pathway. |
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