微小RNA-125b在结肠癌组织中的表达及其与肿瘤细胞耐药性的关系

Expression of microRNA - 125b in colon cancer and its correlation with tumor cell drug resistance

目的探讨微小RNA（miRNA，miR）-125b在结肠癌组织中表达及与肿瘤细胞耐药性的关系。方法收集63例择期行手术治疗的结肠癌组织及癌旁组织标本，培养及转染人结肠癌细胞株HCT8和耐长春新碱结肠癌细胞株HCTS／VCR，根据转染情况将HCT8细胞分成3组：转染miR-125b抑制物组、转染miR-阴性对照组和未转染组，将HCT8／VCR细胞分成3组：转染miR-125b模拟物组、转染miR-阴性对照组和未转染组，利用细胞计数试剂盒（CCK-8）法对细胞存活情况进行检测，利用实时荧光定量聚合酶链反应（FQ-PCR）检测miR-125b在结肠癌、癌旁组织及细胞中表达，利用Western blot检测细胞中B细胞淋巴瘤／白血病-2（bcl-2）蛋白及P-糖蛋白（P-gP）表达。结果结肠癌组织中miR-125b相对表达量（3．76±0．93）显著低于癌旁组织（5．18±1．45），差异有统计学意义（P〈0．01），miR-125b在结肠癌组织中表达与淋巴结转移、远处转移和TNM分期有关（P〈0．05）；miR-125b在HCT8细胞中相对表达量显著高于HCTS／VCR细胞，差异有统计学意义（P〈0．05），Western blot结果显示，bcl-2蛋白和P—gp蛋白在HCT8细胞中表达量显著低于HCTS／VCR细胞，差异均有统计学意义（P〈0．05）；对HCT8细胞转染miR-125b抑制物后，Western blot结果显示，bcl-2蛋白在miR-125b抑制物组HCT8细胞中表达量显著高于阴性对照组和空白对照组，差异均有统计学意义（P〈0．05）；CCK-8结果显示，miR-125b抑制物组HCT8细胞在各浓度下存活率均高于阴性对照组和空白对照组，差异均有统计学意义（P〈0．05）；对HCTS／VCR细胞转染miR-125b模拟物后，Western blot结果显示，bcl-2蛋白在miR-125b模拟物组HCT8／VCR细胞中表达量显著低于阴性对照组和空白对照组，差异均有统计学意义（P〈0．05）；CCK-8结果显示，miR-125b模拟组HCT8／VCR细胞存活率显著低于阴性对照组和空白对照组，差异均有统计学意义（P〈0．05）。结论miR-125b在结肠癌组织中呈低表达，与肿瘤细胞耐药性关系密切，可能是通过bcl-2蛋白调控的细胞凋亡路径实现。

Objective To investigate the expression of microRNA （miRNA, miR） - 125b in colon cancer and its correlation with tumor cell drug resistance. Methods Sixty - three cases of colon cancer tissues and adjacent tissues were selected from elective surgery treatment patients in Henan Tumor Hospital. The HCT8 and HCT8/VCR cells were cultured and transected. According to the HCT8 transfection situation, the HCT8 cells were divided into miR - 125b inhibitor - transfected group, transfected miR - negative control group and untransfected group. The HCT8/VCR cells were divided into transfected miR - 125b mimic group, transfected miR- negative control group and untransfected group. Cell survivals were deteced by cell counting kit -8 （CCK -8 ） method. The expression of miR - 125b in colon cancer cells and adjacent tissues was tested by real -time fluorescent quantitative polymerase chain reaction （FQ -PCR）. The expression of B cell lymphoma/leukemia - 2 （ bcl - 2） protein and Permeability glycoprotein （ P - gp） protein was detected by Western blotting. Results The relative expression of miR - 125b in colon cancer tissue was （3.76 ± 0.93）, significantly lower than （5.18± 1.45） in adjacent tissue with the difference being statistically significant （P 〈 0. 01 ）. The expression of miR- 125b was related with lymph node metastasis, distant metastasis and TNM stage （ P 〈 0. 05 ）. The relative expression of miR - 125b in HCT8 cells was significantly higher than in HCTS/VCR cells with the difference beinng statistically significant （ P 〈 0. 05 ）. Western blotting analysis showed the expression of bcl - 2 protein and P - gp protein in HCT8 cells was significantly lower than in HCTS/VCR cells with the differences being statistically significant （P 〈 0. 05）. After transfection of HCT8 cells with miR - 125b inhibitor, Western blotting showed the ex- pression of bcl - 2 protein in miR - 125b inhibitor - transfedted group was significantly higher than in the negative control group and blank control group （ P 〈 0. 05 ）. CCK - 8 assay showed the survival rate of HCT8 ceils in miR - 125b inhibitor - transfected group at each concentration was higher than the negative control group and blank control group with the differences being statistically significant （ P 〈 0. 05 ）. After transfection of HCTS/VCR cells with miR - 125b mimic, Western blotting showed the expression of bcl -2 protein in miR - 125b mimic group was significantly lower than the negative control group and blank control group with the differences being statistically significant （ P 〈 0.05 ）. CCK - 8 assay showed the survival rate of HCT8/VCR cells in miR - 125b mimic group at each concentration was lower than the negative control group and blank control group with the differences being statistically significant （ P 〈 0. 05 ）. Conclusion The expression of miR - 125b in colon cancer tissue was low, and closely related to tumor cell drug resistance. It might be implemented through apoptotic pathways regulated by bcl -2 protein.