The Expression And Significance Of IFI6 In Colorectal Cancer And The Regulatory Mechanism Of Oxaliplatin Resistance In The Treatment Of Colorectal Cancer

Part One Expression and significance of IFI6 in colorectal cancerObjective: To detect the difference of IFI6 expression in colorectal cancer tissues and adjacent tissues by Real-time PCR,so as to understand the relationship between the expression level of IFI6 and clinicopathological results of colorectal patients.In addition,the expression of IFI6 in colorectal cancer tissues was compared between the recurrence and metastasis group and the nonrecurrence and metastasis group,so as to understand the possible role of IFI6 in predicting the prognosis of colorectal cancer patients.Methods:1.Eighteen colorectal cancer tissue and paracancer tissue samples were selected from the biobank of the Third Hospital of Hebei Medical University.2.The total RNA of each sample was extracted,and the concentration of RNA in each sample was decided by ultraviolet spectrophotometer NANO2000.3.Reverse transcription of the above obtained RNA samples was performed by PCR instrument to obtain corresponding c DNA.4.Fluorescence quantitative PCR was used to detect the expression level of IFI6 in each specimen.5.The total protein in each sample was extracted and the protein concentration was detected by BSA method.6.The total protein obtained was detected by the above process and analyzed by Gel-Pro-Analyzer software.7.The possible correlation between the expression level of IFI6 and the clinicopathological benign and malignant colorectal tissues and the prognosis of patients with colorectal cancer was further analyzed statistically.Results:1.The expression level of IFI6 in colon cancer tissues was significantly higher than that in paracancer tissues.2.Real-time PCR results showed that the expression level of IFI6 in colorectal cancer tissues was significantly higher than that in paracancer tissues,and the difference was statistically significant(P<0.05).3.The expression level of IFI6 in the recurrence and metastasis group was significantly higher than that in the non-recurrence and metastasis group.4.Real-time PCR detection results showed that compared with the group without recurrence and metastasis,the expression level of IFI6 in the recurrence and metastasis group was significantly increased,and the differ-ence was statistically significant(P<0.05).5.Western Blot analysis showed that the expression of IFI6 was significantly higher in the recurrent group than in the non-recurrent group(P<0.05).Summary: Compared with paracancer tissues,the expression level of IFI6 in colorectal cancer tissues was significantly increased,and in the recurrence and metastasis group,the expression level of IFI6 was significantly increased compared with that in the non-recurrence and metastasis group.These results suggest that IFI6 may be used as a marker to predict the presence of colorectal cancer and the prognosis of patients with colorectal cancer.Part Two IFI6 downregulation reverses oxaliplatin resistance of colo-rectal cancer cells by activating the ROS-induced p38MAPKsignaling pathwayObjective: The effects of interferon-inducible protein 6(IFI6)on oxaliplatin(OXA)sensitivity,cell proliferation and apoptosis of colorectal cancer cells were investigated by constructing oxaliplatin resistant colorectal cancer cell lines and overexpressing or knocking out IFI6.The relationship between IFI6 and ROS-induced p38 MAPK signaling pathway was also investigated.Methods:1.Screening of oxaliplatin resistant colorectal cancer cell lines1)Oxaliplatin resistant colorectal cancer cell lines HCT116 and SW620 were selected and named as HCT116/OXA and SW620/OXA.Normal HCT116 and SW620 cells were used as controls.2)After screening,the above cells were treated with oxaliplatin at different concentrations for 48 h.CCK-8 assay was used to detect cell activity in each group,and IC50 was calculated: IC50-1(HCT116),IC50-2(HCT116/ OXA),IC50-3(SW620)and IC50-4(SW620/OXA).3)After the cells were treated with oxaliplatin at different concentrations for 48 h,Annexin V/PI staining and flow cytometry were performed to detect the apo Ii.Expression detection of IFI6 in oxaliplatin resistant colorectal cancer cell lines2.Expression detection of IFI6 in oxaliplatin resistant colorectal cancer cell lines1)The m RNA level of IFI6 in each group was detected by Real-time PCR.2)The protein level of IFI6 in each group was detected by Western-blot.3.Effect of IFI6 on oxaliplatin mediated chemotherapy sensitivity in colorectal cancer1)IFI6 overexpression plasmid was purchased and transfected into normal tumor cells with no load as control;If I6-targeting sh RNA and control sh-NC were purchased and transfected into tumor resistant cells.real-time PCR and western-blot assay were performed 48 h after transient transfection.2)48 h after transient transfection,cells in each group were treated with oxaliplatin at different concentrations for 48 h.CCK-8 was used to detect cell activity,and IC50 of cells in each group was calculated.3)Oxaliplatin was treated 48 h after transfection,and the following experiments were performed 2 w or 48 h later:a.Detection of clone formation(2 w);b.Apoptosis was detected by flow cytometry with Annexin V/PI staining(48 h);c.Expression levels of apoptosis-related proteins,cleaved caspase-3,cleaved caspase-7,cleaved PARP(48 h),were detected by Western-blot analysis.4.To detect whether IFI6 regulates oxaliplatin mediated chemotherapy sensitivity in colorectal cancer through p38 MAPK signaling pathway.1)Oxaliplatin was treated 48 h after transfection,and the following experiments were performed 48 h later: The levels of p-p38Thr180/Tyr182 and p38 were detected by Western blot.2)p38 inhibitor SB203580 was added 48 h after transfection,oxaliplatin was treated 1 h later,and the following tests were performed 48 h later:a.The levels of p-p38Thr180/Tyr182 and p38 were detected by Western blot;b.Cell viability was detected by CCK-8 assay;c.Apoptosis was detected by flow cytometry with Annexin V/PI staining.5.To detect whether IFI6 regulates oxaliplatin mediated chemotherapy sensitivity in colorectal cancer through ROS-dependent p38 MAPK signaling pathway.1)Oxaliplatin was treated 48 h after transfection,and the following experiments were performed 48 h later: ROS content in cells was detected with H2DCF-DA kit(fluorescence image and quantitative analysis);2)48h after transfection,3 m M ROS scavher NAC(CAS: 616-91-1)was added.30 min later,oxaliplatin was treated.48 h later,the following tests were performed:a.ROS content in cells detected with H2DCF-DA kit(flow cytometry)b.The levels of p-p38Thr180/Tyr182 and p38 were detected by Western blot.6.To detect whether the knockdown of IFI6 caused the changes of MRP1,GST,BCL2,MDR.1)The resistant cell lines HCT116/OXA and SW620/OXA were transfected with si-NC and si-IFI6.MRP1,GST,BCL2 and MDR were detected by RTPCR and Western blot.Results:1.The apoptosis rate of oxaliplatin resistant cells HCT116/OXA and SW620/OXA was significantly lower than that of their parental cells.2.IFI6 is significantly upregulated in OXA-resistant CRC cells.3.Repressed IFI6 enhances the chemosensitivity of CRC cells to OXA.4.Repressed IFI6 activates the p38 MAPK signaling pathway in CRC cells.5.ROS induced p38 MAPK signaling pathway participates in IFI6-regulated OXA resistance.6.The expression of MRP1,GST and MDR decreased and the expression of BCL-2 increased in the resistant cell lines HCT116/OXA and SW620/OXA transfected with si-IFI6.Summary: Inhibition of IFI6 reduces oxaliplatin resistance in colorectal cancer cells,and may be achieved by activation of p38 MAPK signaling pathway.Conclusions:1.IFI6 may have important significance in judging the existence of colorectal cancer and the prognosis of patients with colorectal cancer.2.IFI6 is closely related to the resistance of colorectal cancer cells to oxaliplatin,and its process may be related to p38 MAPK signal pathway.