Effect Of Escherichia Coli On Regulating The Proliferation Activity Of Colorectal Cancer Cells Through Molecular Clock Protein CRY2

Objective: Colorectal cancer(CRC)is one of the three major malignant tumors in the world.It is a multifactorial disease involving genetic susceptibility,environmental exposure,and unhealthy lifestyles.Studies have shown that dysregulation of intestinal flora can promote the progress of colorectal cancer by damaging the intestinal mucosal barrier,stimulating chronic inflammation,secreting pro-tumor toxic metabolites,and suppressing anti-tumor cellular immune responses,and affect CRC.Invasion,migration,radiotherapy and chemotherapy efficacy and prognosis.Molecular clock rhythm disturbances participate in the occurrence and development of colorectal cancer by regulating the expression of proto-oncogenes and tumor suppressor genes,disrupting the cell cycle,and inducing inflammation.At the same time,the rhythmic shock of the intestinal flora and the imbalance of microbial metabolite regulation can also have an effect on the circadian rhythm of the host;therefore,we believe that the intestinal microorganisms have a key regulatory effect on the occurrence and development of colorectal cancer by regulating the circadian rhythm of the host;But its specific adjustment mechanism remains to be studied.This study explored the effect of Escherichia coli on the expression of molecular rhythm gene CRY2.Escherichia coli and its metabolites regulate the proliferation and apoptosis of colorectal cancer cells through the molecular clock protein CRY2 and its regulatory role in inducing antichemotherapy effects.The research work will help to clarify the interaction mechanism between E.coli and the circadian rhythm of the host,and provide a theoretical basis for the combination of intestinal microecology combined with circadian rhythm balance treatment of colorectal cancer.Methods:(1)Using liposome transfection technology to construct rhythm clock gene CRY2 overexpressing colorectal cancer cell line,using Hoechst 33342 and propidium iodide(Propidium Iodide,PI)double staining method to detect rhythm clock gene CRY2 overexpression Effect on the apoptosis and necrosis of colorectal cancer HCT-116 and RKO cells;verify the effect of circadian rhythm on the activity of colorectal cancer cells.(2)Co-culture of E.coli and its metabolites with colorectal cancer cells to detect changes in apoptosis and necrosis of colorectal cancer cells;MTS method to detect the effects of E.coli and metabolites on the proliferation activity of colorectal cancer cells;Western bolt Method to detect changes in the expression of rhythm clock protein cryptochrome 2(CRY2),autophagy protein Beclin-1,and pro-apoptosis-related proteins Casoase3 and PARP in colorectal cancer cells after intervention by E.coli and metabolites;Metabolites cause the effect of proliferative activity of colorectal cancer cells through the rhythm clock gene CRY2.(3)Use the CRY protein stabilizer KL001 to restore the negative feedback adjustment ring of the rhythm clock of colorectal cancer cells,detect the changes of apoptosis,necrosis and proliferation of colorectal cancer cells caused by E.coli and its metabolites;detect the small molecule compound KL001 by Western bolt Changes in the expression levels of rhythm clock protein cryptochrome 2(CRY2),autophagy protein Beclin-1,and pro-apoptosis-related proteins Casoase3 and PARP in colorectal cancer cells after intervention Bacillus and its metabolites cause the influence of colorectal cancer cell proliferation activity.(4)The drug KL001,E.coli and its metabolites jointly treat colorectal cancer cells.The MTS method was used to detect the chemotherapy effect of the chemotherapeutic drugs oxaliplatin and 5-fluorouracil on colorectal cancer cells.The effect of drugs on killing colorectal cancer cells.Results:(1)Verification of rhythm clock gene CRY2 overexpression of colorectal cancer HCT-116 and RKO cell lines by q RT-PCR and Western bolts;compared with blank and negative control groups,after overexpression of CRY2 gene,colorectal cancer Apoptosis and necrosis of cell lines increased significantly.(2)E.coli and its metabolites obviously inhibited the apoptosis and necrosis of colorectal cancer cells;MTS results showed that the cell proliferation activity of E.coli and its metabolites increased after treatment of colorectal cancer cells,HCT-116 cell proliferation activity was Significantly increased on days 5 and 7(P <0.01,P <0.05),and RKO cell proliferation activity was significantly increased on days 1,3,and 4(P <0.001).The intervention of Escherichia coli and its metabolites can significantly down-regulate the expression of CRY2 protein in rhythmic clock of colorectal cancer cells(HCT-116: P<0.05;RKO: P <0.001);Metabolites significantly down-regulated the expression levels of pro-apoptosis-related proteins Caspase-3 and PARP,while the expression levels of autophagy protein Beclin-1 were up-regulated.(3)After the small molecule compound KL001 stabilizes the rhythm clock CRY2 protein and restores the negative feedback regulation mechanism of colorectal cancer cell rhythm clock,it significantly reduces the proliferative activity of colorectal cancer HCT-116 and RKO cells,and this degree of inhibition increases with the concentration of the drug Increased and increased(KL001-5u M P <0.01,KL001-25 u M P <0.001);small molecule compound KL001 stabilized rhythm clock CRY2 protein has an inhibitory effect on the proliferation of colorectal cancer cells caused by E.coli and its metabolites;apoptosis The results of autophagy protein detection showed that the small molecule compound KL001 significantly up-regulated the expression levels of pro-apoptosis-related proteins Caspase-3 and PARP,while the expression level of the autophagy protein Beclin-1 was down-regulated.(4)Escherichia coli and its metabolites greatly weakened the chemotherapy effect of the chemotherapeutic drugs oxaliplatin and 5-fluorouracil on colorectal cancer cells;the small molecule compound KL001 greatly enhanced the chemotherapy drugs on colorectal cancer cells by stabilizing the CRY2 protein Chemotherapy effect;the small molecule compound KL001 stabilizes the CRY2 protein and has a certain repair effect on the resistance of E.coli and its metabolites to chemotherapyConclusion: Escherichia coli and its metabolites can reduce the apoptosis and necrosis of colorectal cancer HCT-116 and RKO cells by inhibiting the expression of molecular clock CRY2 protein,and promote the proliferation of colorectal cancer cells;The antichemotherapy effect of colorectal cancer cells;the repair rhythm clock CRY2 protein has an inhibitory effect on the proliferation activity and chemotherapy resistance of colorectal cancer cells caused by E.coli and its metabolites.This study provides a theoretical basis for the treatment of colorectal cancer with intestinal microecology combined with circadian rhythm balance.