Expression And Clinical Significance Of Basic Transcription Factor 3 In Colorectal Cancer And Its Influences Of Proliferation And Apoptosis In HCT116 Cell Line

Objective:1.In this study we aimed to identify the BTF3 expression in colorectal tissues,to evaluated the relationship between BTF3 expression and clinic pathological parameters.Besides,we also aimed to identify the role of BTF3 expression on colorectal cell,and tried to find out the correlation between BTF3 and clinical features of colorectal cancer,and analyze the relationship between BTF3 expression and survival prognosis.2.Study the influencesof BTF3 on proliferation,apoptosis and cloning of colon cancer cell line HCT116 and explore the relationship between BTF3 and the development of colon cancer cells,which provide new targets and new ideas for the treatment and prognosis of colorectal cancer.Methods:Part Ⅰ: The protein expression levels of BTF3 in 90 colorectal cancer tissues and their matched colorectal cancer normal tissues were detected by immunohistochemical staining for colorectal cancer and their matched normal tissues adjacent to colorectal cancer tissues.We analyzed the differential expression of BTF3 in colorectal cancer tissues and adjacent tissues,and then we explored the relationship between the expression of BTF3 and survival prognosis and clinicopathological factors in patients with colorectal cancer.Part Ⅱ: 1.Lentiviral transfection to construct a BTF3 silenced colorectal cancer cell line HCT116 model.The qRT-PCR method verified the expression level of BTF3 mRNA after transfection.2.The experimental was designed into three groups:Control group,LV-BTF3-siRNA group and LV-negative-siRNA group.3.MTT assay was used to detect cell proliferation in BTF3-silenced colorectal cancer cell line HCT116.Cell apoptosis and cycle detection were analyzed by flow cytometry.4.Plate cloning assay The clonality of cells in the BTF3 silenced colorectal cancer cell line HCT116 was examined.Western blotting and qRT-PCR were used to detect theprotein and mRNA expression levels of BTF3 down-regulated colorectal cancer cell line HCT116.Results:1.Immunohistochemical staining showed that BTF3 was localized in the nucleus and cytoplasm of colonic epithelial cells.The relative immunoreactivity score of BTF3 in colorectal cancer(CRC)tissues was 2.61±0.07(measured from the intensity of immune response).The relative immunoreactivity score of BTF3 in Adjacent noncancerous tissues(ANCT)samples was 1.90 ± 0.03(from the intensity of immune response),and the two groups of data were analyzed by independent sample t test(P <0.01),which concluded that BTF3 is up-regulated in colorectal cancer tissues compared to normal tissues adjacent to the cancer.2.The up-regulation of BTF3 expression in colorectal cancer was related to TNM stage and lymph node metastasis of colorectal(P<0.05).But there was no significant difference between gender,age and pathological grade(P>0.05).3.Overexpression of BTF3 in colorectal cancer and prognosis of colorectal cancer were analyzed by Kaplan-Meier survival curve.Log-Rank test was statistically significant(P<0.05),indicating abnormal expression of BTF3 and Kaplan-Meier survival analysis,but there was no significant difference in the recurrence-free survival of ANCT between overexpressing BTF3 and lower expressing BTF3.4.Three groups of siRNAs were designed according to the sequence of BTF3.The knockdown efficiency of siRNA-1 and siRNA-2 on BTF3 was statistically significant compared with the control group,but siRNA-1 was better than siRNA-2.The results of qRT-PCR showed that the BTF3 mRNA expression level of HCT116 cells in BTF3-siRNA group was down-regulated compared with BTF3-N-siRNA group,and the difference was statistically significant(P<0.05).5.MTT assay showed that knockdown of BTF3 could inhibit the proliferation of colorectal cancer cell line HCT116 and promote early cell apoptosis.Flow cytometry analysis showed that G2/M cell cycle arrested during BTF3 knockout and induced early apoptosis.6.The results of plate colony formation experiments showed that knockdown of BTF3 using siRNA interference technology can inhibit the colony formation of cells in colorectal cancer cell HCT116.Conclusions:BTF3 is overexpressed in colorectal cancer tissues,and its expression is associated with TNM staging,tumor lymph node metastasis,but not with pathological grade,and gender of patients.BTF3 is an independent prognostic factor for the prognosis of colorectal cancer patients.Its high expression is closely related to the poor prognosis of colorectal cancer patients.It has reference value for the molecular mechanism of colorectal cancer development and may be the guiding target for colorectal cancer treatment.