BCL6B抑制结直肠癌细胞HCT116增殖、迁移及其可能机制研究

Inhibition of B cell lymphoma 6 member B on proliferation and migration of colorectal carcinoma cell line HCT116 and study of its possible mechanism

目的:探讨B细胞淋巴瘤6B(B cell lymphoma 6 member B,BCL6B)对人结直肠癌细胞HCT116增殖、迁移的影响及机制。方法:采用定量聚合酶链反应(quantitative polymerase chain reaction,q PCR)和Western blot检测和比较人正常肠上皮细胞FHC及人结直肠癌细胞HCT116中BCL6B的内源性表达;用脂质体法将pc DNA3.1-BCL6B转入HCT116细胞,同时设转入空载体的对照组;运用MTT法、流式细胞术、划痕愈合及Transwell试验检测BCL6B对癌细胞增殖、周期及迁移能力的影响;采用Western blot检测细胞中β-连环蛋白(β-catenin)、磷酸化的糖原合成酶激酶-3β(p-GSK3β)、细胞周期蛋白(Cyclin D1)、基质金属蛋白酶-7(matrix metalloproteinase-7,MMP-7)和E-钙黏蛋白(E-cadherin)的表达。结果:q PCR和Western blot结果显示,与正常肠上皮细胞FHC相比,BCL6B在结直肠癌细胞HCT116中呈明显低表达(P=0.017);转染pc DNA3.1-BCL6B后,细胞内BCL6B表达水平明显升高(P=0.000)。与阴性对照组相比,BCL6B组HCT116细胞4 d时的增殖活性降低41.79%(P=0.040),且G1期细胞百分比增加62.09%(P=0.001);同时,BCL6B组HCT116细胞48 h划痕愈合率及穿膜细胞数均明显减少(P=0.001)。Western blot结果表明,BCL6B组HCT116细胞内β-catenin、p-GSK3β、Cyclin D1和MMP-7的表达水平较阴性对照组分别降低65.66%(P=0.028)、62.03%(P=0.001)、60.87%(P=0.021)和50.88%(P=0.030),E-钙黏蛋白的表达升高63.64%(P=0.018)。结论:BCL6B可抑制结直肠癌细胞HCT116的增殖及迁移,其机制可能涉及Wnt/β-catenin通路的抑制。

Objective：To investigate the effect of B cell lymphoma 6 member B（BCL6B） on the proliferation and migration of human colorectal cancer cell line HCT116, and to explore its possible mechanism. Methods：Quantitative polymerase chain reaction （qPCR） and Western blot were used to detect and compare the endogenous expression of BCL6B in FHC and HCT116 cells. Liposome method was used to transfer the pcDNA3.1-BCL6B to HCT116 and the control group with empty vector was set. MTT assay, flow cytometry, wound healing assay and transwell chamber assay were used to detect the influence on proliferation,cycle and migration ability of cancer cell. Western blot was adopted to detect the expression of 13-catenin,p-GSK3β, CyclinD1, MMP-7 and E-cadherin. Results： According to qPCR and Western blot, BCL6B expression was notably repressed in HCT116 cells as compared with FHC cells （P= 0.017）, and expression of BCL6B was significantly increased in HCT116 ceils after transfection with pc DNA3.1-BCL6B for 48 h（P= 0.000）. Compared with those in the control group, the OD492 value of HCT116 cells at the 4d time-point was decreased by 41.79% （P=0.040）, and the percentage of cells in G1 phase was increased by 62.09%（P=-0.001）,48 h wound healing rate and transmembrane cells of HCT116 cells in the BCL6B group were significantly reduced（P=0.001 ）. The protein levels of β-catenin, p-GSK3β, CyclinD1 and MMP-7 of HCT116 cells in the BCL6B group were dropped by 65.66% （P=0.028）, 62.03% （P=0.001）, 60.87% （P=0.021） and 50.88% （P=0.030）, respectively. E-cadherin was increased by 63.64% （P=0.018）. Conclusion ： BCL6B inhibits the proliferation and migration of HCT116 cells,and its mechanism may involve in the inhibitior/of Wnt/β-catenin pathway.