miR-203过表达对结肠癌HT-29细胞紫杉醇化疗敏感性的影响及可能机制

目的采用脂质体转染法制备miR-203过表达结肠癌HT-29细胞,探讨miR-203过表达对结肠癌HT-29细胞紫杉醇化疗敏感性的影响及可能机制。方法取对数生长期结肠成纤维细胞CCD-18Co和结肠癌HT-29细胞,采用实时荧光定量PCR法检测miR-203相对表达量。取对数生长期结肠癌HT-29细胞,分为过表达组(转染miR-203-pcDNA 3.1质粒)、空载组(转染NC-pcDNA3.1质粒)和对照组(不作任何干预),采用实时荧光定量PCR法检测3组转染48h时miR-203相对表达量。取转染48h时过表达组、空载组、对照组细胞,用1、5、10、50、100μmol/L紫杉醇处理细胞48h,采用MTT法测定细胞增殖抑制率,并计算3组紫杉醇对细胞的半数抑制浓度(half inhibitory concentration,IC₅₀)。转染48h,取对照组细胞分为对照1组和紫杉醇组,取过表达组细胞分为过表达1组和过表达+紫杉醇组,紫杉醇组和过表达+紫杉醇组应用对照组IC₅₀值(28.20μmol/L)浓度的紫杉醇处理,对照1组和过表达1组不进行处理,48h后采用流式细胞术检测4组细胞凋亡,采用Western blot法检测4组细胞磷脂酰肌醇-3激酶(phosphatidylinositol-3 kinase,PI3K)、蛋白激酶B(protein kinase B,Akt)、磷酸化Akt、caspase-3蛋白相对表达量。结果结肠癌HT-29细胞miR-203相对表达量(0.38±0.05)低于CCD-18Co细胞(1.01±0.09)(P<0.05)。转染48h,过表达组miR-203相对表达量(1.87±0.11)高于空载组(0.39±0.04)和对照组(0.40±0.05)(P<0.05),空载组与对照组比较差异无统计学意义(P>0.05)。1、5、10、50、100μmol/L紫杉醇处理48h,过表达组细胞增殖抑制率高于空载组和对照组(P<0.05),IC₅₀值低于空载组和对照组(P<0.05),空载组与对照组比较差异无统计学意义(P>0.05)。紫杉醇处理48h,过表达1组、紫杉醇组、过表达+紫杉醇组细胞凋亡率(18.25±1.70)%、(19.11±1.83)%、(57.59±4.31)%]高于对照1组(8.10±1.14)%](P<0.05),过表达+紫杉醇组高于过表达1组和紫杉醇组(P<0.05),过表达1组与紫杉醇组比较差异无统计学意义(P>0.05)。紫杉醇处理48h,过表达1组、紫杉醇组、过表达+紫杉醇组PI3K(0.81±0.05、0.83±0.05、0.32±0.04)、磷酸化Akt(0.57±0.04、0.55±0.04、0.18±0.03)、caspase-3(0.67±0.13、0.65±0.10、0.19±0.10)蛋白相对表达量低于对照1组(1.12±0.09、0.80±0.06、1.25±0.12)(P<0.05),过表达+紫杉醇组低于过表达1组和紫杉醇组(P<0.05),过表达1组与紫杉醇组比较差异均无统计学意义(P>0.05);4组Akt蛋白相对表达量比较差异均无统计学意义(P>0.05)。结论结肠癌HT-29细胞miR-203呈低表达,过表达miR-203可提高结肠癌HT-29细胞对紫杉醇的敏感性,促进HT-29细胞凋亡,其机制可能与抑制PI3K、caspase-3激活及Akt磷酸化有关。

Influence of miR-203overexpression on paclitaxel chemotherapy sensitivity to colon cancer cells and its mechanism

Objective To prepare the miR-203overexpressed colon cancer HT-29cells by liposome mediated transfection and to investigate the influence and mechanism of miR-203overexpression on paclitaxel chemotherapy sensitivity to colon cancer cells.Methods Real-time fluorescence quantitative PCR was used to detect the relative expression of miR-203in colon fibroblast cell lines and colon cancer HT-29cell lines in logarithmic growth phase.The HT-29cells in logarithmic growth phase was divided into overexpression group which was transfected with miR-203-pcDNA3.1plasmid,empty vector group which was transfected with NC-pcDNA3.1plasmid,and control group receiving no intervention.The relative expression of miR-203was detected after 48hof transfection in three groups by real-time fluorescence quantitative PCR.The cells in three groups were treated with 1,5,10,50and 100μmol/L of paclitaxel for 48h,the cell proliferation inhibition rate was measured by MTT method,and the half inhibitory concentration(IC₅₀)of paclitaxel on the cells was calculated.Control group was divided into control group 1and paclitaxel group,and overexpression group was divided into overexpression group 1and the overexpression+paclitaxel group after 48hof transfection.Paclitaxel group and overexpression+paclitaxel group were treated with 28.20μmol/L of paclitaxel(control IC₅₀value).Control group 1and overexpression group 1received no treatment.The apoptosis was detected by flow cytometry in control group,overexpression group,paclitaxel group,overexpression+paclitaxel group after the treatment with paclitaxel for 48h,and the relative expressions of phosphatidylinositol-3kinase(PI3K),protein kinase B(Akt),phosphorylated Akt and caspase-3proteins were detected in four groups by Western blot.Results The relative expression of miR-203was lower in colon cancer HT-29cells(0.38±0.05)than that in CCD-18Co cells(1.01±0.09)(P<0.05).After 48hof transfection,the relative expression of miR-203was higher in overexpression group(1.87±0.11)than that in enmpty vector group(0.39±0.04)and control group(0.40±0.05)(P<0.05),and showed no significant difference between empty vector group and control group(P>0.05).After treatment with 1,5,10,50and 100μmol/L of paclitaxel for 48h,the cell proliferation inhibition rate was higher in overexpression group than that in empty vector group and control group(P<0.05),and IC₅₀was lower in overexpression group than that in empty vector group and control group(P<0.05),while both indexes showed no significant differences between empty vector group and control group(P>0.05).After 48hof paclitaxel treatment,the apoptosis rate was higher in overexpression group 1((18.25±1.70)%),paclitaxel group((19.11±1.83)%)and overexpression+paclitaxel group((57.59±4.31)%)than that in control group 1((8.10±1.14)%)(P<0.05),was higher in overexpression+paclitaxel group than that in overexpression group 1and paclitaxel group(P<0.05),and showed no significant difference between overexpression group 1and paclitaxel group(P>0.05).After 48hof paclitaxel treatment,the relative expressions of PI3Kprotein(0.81±0.05,0.83±0.05,0.32±0.04),phosphorylated Akt protein(0.57±0.04,0.55±0.04,0.18±0.03),and caspase-3protein(0.67±0.13,0.65±0.10,0.19±0.10)were lower in overexpression group 1,paclitaxel group and overexpression+paclitaxel group than those in control group 1(1.12±0.09,0.80±0.06,1.25±0.12)(P<0.05),were lower in overexpression+paclitaxel group than those in overexpression group 1and paclitaxel group(P<0.05),and showed no significant differences between overexpression group 1and paclitaxel group(P>0.05).The relative expression of Akt protein showed no significant difference among four groups(P>0.05).Conclusion miR-203is lowly expressed in HT-29cells.The overexpression of miR-203can enhance the sensitivity of paclitaxel to colon cancer HT-29 cells and promote the apoptosis of HT-29cells,which might be correlated with the inhibition of PI3K,activation of caspase-3and Akt phosphorylation.