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目的肿瘤干细胞是肿瘤复发和转移的原因。探讨CD133基因RNA干扰(RNA interference,RNAi)的重组质粒,对人肝癌细胞株MHCC-H的CD133基因表达、上皮间质化和侵袭能力的影响。方法通过免疫磁珠分选MHCC-H细胞中的CD133阳性细胞,设计并合成特异性靶向CD133的小分子干扰RNA(small interfering RNA,siRNA)片段,并构建pSuper retro GFP-neo-siRNA-CD133表达质粒,将其转入MHCC-H-CD133+细胞,并设空白对照组、阳性对照组。通过G418筛选出稳定株。通过粘附实验、Boyden小室实验和软琼脂克隆形成实验观察各细胞株侵袭能力的变化。采用明胶酶法测定肝癌细胞的基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)和MMP-9的含量,蛋白质印迹法检测上皮间质化相关基因的变化。结果通过免疫磁珠分选后的MHCC-H细胞中CD133表达率为83.5%。qRT-PCR结果显示,shCD133组相对空白对照组的CD133表达量下降了80%,P<0.05。蛋白质印迹法检测结果显示,shCD133组的CD133表达明显下降,约为空白对照组的12%和shNC组的10%,均P<0.05。shCD133组的粘附能力为86.9±12.4,较空白对照组的568.5±53.2和shNC组的538.8±35.6明显下降,P<0.05。Boyden小室实验发现,shCD133组穿膜细胞数为(80.6±11.4)个,较空白对照组的(228.5±33.2)个和shNC组的(230.8±32.9)个明显减少,P<0.05。软琼脂克隆形成率的检测发现,shCD133的细胞克隆形成(60±5)个,比空白对照组的(178±23)个和shNC组的(168±25)个明显下降,P<0.05。明胶酶法检测发现,shCD133组MMP-2和MMP-9相对活性为0.4±0.14和0.6±0.16,较shNC组的1.03±0.19和1.3±0.16明显下降,P<0.05。蛋白质印迹法检测结果表明,shCD133组N-cadherin蛋白表达为36.3±4.5,Snail为53.6±6.7,Slug为41.63±5.6,Twist为39.4±3.9,均明显低于空白对照组的87.6±8.6、80.6±7.5、81.9±9.2和83.9±9.1,也明显低于shNC组的89.4±9.6、83.5±8.9、85.1±8.7和87.6±9.3,均P<0.05;而shCD133组E-cadherin表达为88.4±9.2,较空白对照组的57.6±8.7和shNC组的53.9±8.9明显上调,P<0.05。结论沉默MHCC-H细胞CD133基因的表达能有效抑制其上皮间质化和侵袭能力,CD133基因可能成为肝癌基因治疗的有效靶点。
Purpose Cancer stem cells are the cause of tumor recurrence and metastasis. To investigate the effect of CD133 RNA interference (RNAi) recombinant plasmid on CD133 gene expression, epithelial-mesenchymal transition and invasion of human hepatocellular carcinoma cell line MHCC-H. Methods CD133 positive cells in MHCC-H cells were sorted by immunomagnetic beads. Small interfering RNA (siRNA) fragment specific for CD133 was designed and synthesized. The pSuper retro GFP-neo-siRNA-CD133 The plasmid was transfected into MHCC-H-CD133 + cells, and a blank control group and a positive control group were set up. Stable strains were screened by G418. Adhesion experiments, Boyden chamber experiments and soft agar colony formation assay were used to observe the changes of invasiveness of each cell line. The content of matrix metalloproteinase-2 (MMP-2) and MMP-9 in hepatocellular carcinoma cells was detected by gelatinase method. The changes of epithelial-mesenchyme related genes were detected by Western blotting. Results The positive rate of CD133 in MHCC-H cells sorted by immunomagnetic beads was 83.5%. The results of qRT-PCR showed that the CD133 expression of shCD133 group was decreased by 80% compared with the blank control group (P <0.05). The results of Western blotting showed that CD133 expression in shCD133 group was significantly decreased, which was about 12% in blank control group and 10% in shNC group, both P <0.05. The adhesive ability of shCD133 group was 86.9 ± 12.4, which was significantly lower than 568.5 ± 53.2 of blank control group and 538.8 ± 35.6 of shNC group, P <0.05. Boyden chamber assay showed that the number of transmembrane cells in shCD133 group was (80.6 ± 11.4), which was significantly lower than that in control group (228.5 ± 33.2) and (230.8 ± 32.9) in shNC group (P <0.05). The results of soft agar colony formation assay showed that shCD133 cell clones formed (60 ± 5) cells, which was significantly lower than that of control group (178 ± 23) and shNC group (168 ± 25), P <0.05. Gelatinase assay showed that the relative activities of MMP-2 and MMP-9 in shCD133 group were 0.4 ± 0.14 and 0.6 ± 0.16, which were significantly lower than those in shNC group (1.03 ± 0.19 and 1.3 ± 0.16, P <0.05). The result of Western blot showed that the expression of N-cadherin protein in shCD133 group was 36.3 ± 4.5, the expression of Snail was 53.6 ± 6.7, the Slug was 41.63 ± 5.6, and the Twist was 39.4 ± 3.9, which were significantly lower than 87.6 ± 8.6,80.6 in blank control group ± 7.5,81.9 ± 9.2 and 83.9 ± 9.1, respectively, which were also significantly lower than 89.4 ± 9.6,83.5 ± 8.9,85.1 ± 8.7 and 87.6 ± 9.3 in shNC group, both P <0.05; while the expression of E-cadherin in shCD133 group was 88.4 ± 9.2 , 57.6 ± 8.7 in blank control group and 53.9 ± 8.9 in shNC group, P <0.05. Conclusion The expression of CD133 gene in MHCC-H cells silenced effectively inhibits the epithelial-mesenchymal transition and invasion. CD133 gene may be a potential therapeutic target for gene therapy of hepatocellular carcinoma.