| Purpose:To detect the expression of micro RNA-375(miR-375)in colorectal cancer and its relationship with disease progression,and to construct colorectal cancer cell HCT116,with overexpression of miR-375 with lentiviral vector to study the relationship between miR-375 overexpression and CIP2A/PP2A-Akt/p-Akt signal pathway,as well as its effect on cell morphological processes such as invasion,migration,proliferation and apoptosis of colorectal cancer.Methods: The expression of miR-375 in 30 clinical colorectal cancer tissues and paracancerous tissues,three colorectal cancer cell lines(HCT116,SW480,LOVO)and one normal colorectal cell(FHC),were detected by q RT-PCR.The overexpression group of miR-375 was constructed by transfecting HCT116 with lentiviral vector,and the empty vector control group and blank control group were set up.q RT-PCR technique was used to detect the expression of miR-375 in SW480,LOVO,FHC,miR-375 overexpression group,empty vector control group,blank control group and 30 cases of colorectal cancer and para-cancerous tissues.q RT-PCR technique was used to detect the m RNA expression of HCT116 in overexpressed CIP2 A group,empty vector control group and blank control group.The protein expression of CIP2A/PP2 A and AKT/p-AKT signal pathway was detected by western blot.The Trans-well chamber model reflected the invasion ability(glial layer)and migration ability(no glial layer)of the three groups of cells.The scratch test confirmed the migration ability of the three groups of cells.The proliferation ability of the three groups was evaluated by Cell Counting Kit-8 cell counting,and the number of apoptotic cells in each group was counted by flow cytometry.The experimental results were statistically processed by Prism 5.0,and the measurement data were compared by single factor analysis of variance(ANOVA).The results of Western blot bands were scanned by Image J software,and the statistics were made according to the ratio of grayscale value to internal reference.P < 0.05 indicates that the difference is statistically significant.Results: The expression of miR-375 in colorectal cancer cells was lower than that in normal colorectal cells,and the expression level decreased with the progression of cancer,and the difference was statistically significant(P < 0.05).The construction of overexpressed HCT116 cells was completed,and q RT-PCR detection showed that the transcription and translation of CIP2 A were decreased in overexpressed group.In the miR-375 overexpression group,the number of cells passing through the superior chamber of Trans well(containing matrix glue)decreased significantly,and its invasive ability was weaker than that in empty vector group and blank group.At the same time,the experimental results of the chamber without matrix glue are similar,indicating that the migration ability is reduced.There was no significant difference in the ability of invasion and migration between the empty vector group and the blank group,but the scratch healing of the miR-375 up-regulated group was relatively poor after 48 hours of scratch experiment,which confirmed the decrease of the migration ability.Using cck-8 to detect the cell proliferation ability of the three groups indicates that the cell proliferation ability of the three groups detected by cck-8 indicates that the proliferation ability of the overexpression group is decreased.Apoptosis detected by flow cytometry showed that the percentage of apoptotic cells,late apoptotic cells and naked nuclear cells increased significantly in miR-375 overexpression group.The expression of CIP2A/PP2 A and AKT/pAKT signaling pathway protein was detected by western blot.The expression of CIP2 A decreased in the up-regulated group,p-AKT and MYC decreased significantly when the expression of AKT was unchanged.Conclusion:1.Low expression of miR-375 in colorectal cancer cells,and further decreased with tumor progression;2.Overexpression of miR-375 can probably inhibit invasion,migration,proliferation and promote apoptosis of colorectal cancer cells through the signal pathway from CIP2A/PP2 A to Akt/p-Akt. |
