The Study Of MiR-15a-5p Regulating Multidrug Resistance Through The Wnt/?-catenin Pathway In Colorectal Cancer

Colorectal cancer?CRC?is a common malignant tumor of the digestive tract.At present,chemotherapy is still the main adjuvant method in the treatment of advanced colorectal cancer.However,due to the occurrence of multidrug resistance,the effect of chemotherapy is not significant.Therefore,finding resistance targets for chemotherapeutics is the key to improving the anticancer clinic treatment efficacy.Multidrug resistance refers to the fact that while tumor cells are resistant to one drug,they are also resistant to other drugs with different molecular structure functioned in different mechanisms.One of the main reasons for multidrug resistance is the over-expression of P-glycoprotein?P-gp?,a member of the ATP-binding cassette?ABC?structural transporter superfamily,encoded by multidrug resistance 1 gene?MDR1?.These transporter protein can pump the chemotherapy drugs out of the cells by using the energy released by the hydrolysis of ATP.Thus,resulting in a decrease in the concentration of the chemotherapy drugs in cells.The Wnt/?-catenin pathway is closely related to the expression of P-gp.Historically,Studies have found that the inhibiting of the Wnt/?-catenin signaling pathway can down-regulate P-gp expression.micro RNA is a type of small non-coding single-stranded RNA that can regulate the expression of target genes.miR-15a-5p is a member of the miR-15 family,recent years' studies have shown that it can inhibit the translation of target m RNA or promote the degradation of target m RNA by targeting the 3?UTR of a variety of coding genes,post-transcriptional regulation of genes.However,the expression of miR-15a-5p and its correlation with multidrug resistance in colorectal cancer are few.The purpose of this study was to investigate the expression of miR-15a-5p and the mechanism of it in multidrug resistance in colorectal cancer.ObjectiveThe expression of miR-15a-5p and P-gp in colorectal cancer tissues was detected at the tissue level,the relationship between miR-15a-5p and P-gp was analyzed.To study the effect of miR-15a-5p on the expression of key proteins Wnt3 a,?-catenin and P-gp in the Wnt/?-catenin signaling pathway at the cellular level,and explore the regulation of miR-15a-5p in multidrug resistance in colorectal cancer role and mechanism.Method1.The expressions of miR-15a-5p and MDR1 m RNA in colorectal cancer tissues and adjacent normal intestinal mucosa tissues were detected by quantitative real-time PCR,and the correlation between miR-15a-5p and MDR1 m RNA expression in colorectal cancer tissues was analyzed.2.The expression of miR-15a-5p in colorectal cancer cells HCT-116 and colorectal cancer resistant cells HCT-116/LOHP were detected by quantitative real-time PCR.3.Western blot experiment was used to detect the expression of Wnt3 a,?-catenin,and P-gp in colorectal cancer cells HCT-116 and colorectal cancer resistant cells HCT-116/LOHP.4.HCT-116/LOHP cells were divided into five groups by cell transfection: blank control group,control group transfected with miR-15a-5p mimics NC,experimental group transfected with miR-15a-5p mimics,control group transfected with miR-15a-5p inhibitor NC,and the experimental group transfected with miR-15a-5p inhibitor.Quantitative real-time PCR was used to detect the expression of miR-15a-5p.The MTT test was used to detect the proliferation of cells in different concentrations of oxaliplatin and calculate its growth inhibition rate,half inhibitory concentration(IC₅₀)and drug resistance index?RI?.5.Quantitative real-time PCR was used to detect the expression of Wnt3 a,?-catenin and MDR1 m RNA in these groups after transfection,and Western blot was used to detect the expression of Wnt3 a,?-catenin,and P-gp in these groups.Result 1 Tissue level1.1 The expression of miR-15a-5p and MDR1 m RNA in colorectal carcinoma and adjacent non-tumor tissues.The expression of miR-15a-5p in colorectal carcinoma was 0.51?0.25?,which was significantly lower than that of adjacent non-tumor tissues,1.17?2.95?.The expression of MDR1 m RNA in colorectal carcinoma was 2.21?4.07?,which was significantly higher than that of adjacent non-tumor tissue 1.13?2.28?.?P<0.05?.1.2 Correlation between the expression of miR-15a-5p and MDR1 m RNA in colorectal cancer tissues.Spearman correlation analysis showed that there was a negative correlation between miR-15a-5p expression and MDR1 m RNA expression in colorectal cancer tissues?r =-0.343,P <0.05?2 Cellular level2.1 MTT method was used to verify the resistance of HCT-116/LOHP cells to oxaliplatin The IC₅₀ of oxaliplatin on HCT-116 cells and HCT-116/LOHP cells were?13.51±2.62??g/ml and?103.08±12.29??g/ml respectively.The resistance index was 7.63.2.2 The results of quantitative real-time PCR showed the relative expression of miR-15a-5p in HCT-116 group was 1.00±0.00 and HCT-116/LOHP group was?0.16±0.05??P < 0.01?,The differences were statistically significant.2.3 The result of western blot showed the relative expression of Wnt3a??-catenin and P-gp protein in HCT-116/LOHP group were significantly higher than that in HCT-116 group?P<0.05?.2.4 The results of quantitative real-time PCR showed the relative expression of miR-15a-5p in that blank control group,the control group transfected with miR-15a-5p mimics NC,the experimental group transfected with miR-15a-5p mimics,the control group transfected with miR-15a-5p inhibitor NC,and the experimental group transfected with miR-15a-5p inhibitor in HCT-116/LOHP cells were 1.00±0.00,1.08±0.10,277.01±20.81,1.07±0.12,and 0.38±0.04.The relative expression of miR-15a-5p in group which HCT-116/LOHP cells were transfected with miR-15a-5p mimics was significantly increased?F=527.82,P < 0.001?.The relative expression of miR-15a-5p in group which HCT-116/LOHP cells were transfected with miR-15a-5p inhibitor was significantly reduced.?F = 79.93,P<0.001?.2.5 The MTT assay was used to detect the resistance of oxaliplatin cells in each group after transfection.The IC₅₀ of oxaliplatin on miR-15a-5p mimics NC transfection group was?100.35±10.57??g/ml.The miR-15a-5p mimics transfection group was?40.78±2.47??g/ml,and the difference was statistically significant?P<0.01?.The IC₅₀ of oxaliplatin on miR-15a-5p inhibitor NC transfection group was?102.08 ± 5.95??g/ml,and miR-15a-5p inhibitor transfection group was?132.77 ± 7.97??g/ml,The difference was statistically significant?P<0.01?.The IC₅₀ of oxaliplatin on the blank control group was?99.98±2.63??g/ml,and there was no significant difference in HCT-116 / LOHP cells between the blank control group and the negative control group?all P>0.05?2.6 Through Western blot experiment,it was found that the expression of Wnt3 a,?-catenin and P-gp proteins in HCT-116/LOHP cells transfected with miR-15a-5p mimics was lower than that in miR-15a-5p mimics NC group?P<0.05?.The expression of Wnt3 a,?-catenin and P-gp proteins were not significantly varied in the blank control group compared with the miR-15a-5p mimics NC group?P>0.05?.2.7Through Western blot experiment,it was found that the expression of Wnt3 a,?-catenin and P-gp proteins in HCT-116/LOHP cells transfected with miR-15a-5p inhibitor was increased than that in miR-15a-5p inhibitor NC group?P<0.05?.The expression of Wnt3 a,?-catenin and P-gp proteins were not significantly varied in the blank control group compared with the miR-15a-5p inhibitor NC group?P>0.05?.2.8 The results of quantitative real-time PCR showed that the expression of Wnt3 a,?-catenin and MDR1 m RNA in HCT-116/LOHP cells which transfected with miR-15a-5p mimics were lower than the group that transfected with miR-15a-5p mimics NC?P<0.05?.The expressions of Wnt3 a,?-catenin and MDR1 m RNA in the blank control group were not significantly different from those in the transfected miR-15a-5p mimics NC group?P>0.05?.2.9 The results of quantitative real-time PCR showed that the expression of Wnt3 a,?-catenin and MDR1 m RNA in HCT-116/LOHP cells which transfected with miR-15a-5p inhibitor were higher than the group transfected with miR-15a-5p inhibitor NC group?P<0.05?.There were no significant changes in the expression of Wnt3 a,?-catenin and MDR1 m RNA between the transfected miR-15a-5p inhibitor NC group and blank control group?P>0.05?.Conclusion1.The miR-15a-5p characterized low expression in colorectal carcinoma while MDR1 showed a high expression in colorectal carcinoma,and the expression of miR-15a-5p and MDR1 showed a negative relationship.2.The expression level of miR-15a-5p in HCT-116/LOHP cells was significantly lower than that of HCT-116 cells,and the expression levels of Wnt3 a,?-catenin and P-gp in HCT-116 /LOHP cells were significantly higher than that of HCT-116 cells.The miR-15a-5p and Wnt/?-catenin pathway play an important role in the formation of multidrug resistance in colorectal cancer.3.Up-regulating the expression of miR-15a-5p can significantly enhance the sensitivity of the HCT-116/LOHP cells to chemotherapeutic drugs.While down-regulating the expression of miR-15a-5p can greatly reduce the sensitivity of the HCT-116/LOHP cells to chemotherapeutic drugs.That shows the miR-15a-5p can regulate MDR in colorectel cancer.4.Up-regulating the expression of miR-15a-5p can significantly inhibit the expression of key proteins in the Wnt/?-catenin signaling pathway,such as the expression of Wnt3 a,?-catenin and P-gp,while down-regulating the expression of miR-15a-5p can significantly increase the expression of Wnt3 a,?-catenin and P-gp downstream.That shows the miR-15a-5p can regulate MDR in colorectel cancer by influencing the activity of Wnt/ ?-catenin pathway and mediating the expression of P-gp.