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目的:探讨体外沉默CCL18基因的表达对卵巢上皮癌SKOV3细胞侵袭和迁移的影响。方法:化学合成3对靶向CCL18的siRNA(CCL18-siRNA61、CCL18-siRNA127、CCL18-siRNA224),体外转染至CCL18阳性的SKOV3细胞,RT-PCR检测SKOV3细胞中CCL18 mRNA的表达,选取其中干扰效率最好的序列,构建靶向CCL18的干扰质粒pSilencer4.1-CCL18-siRNA61。pSilencer4.1-CCL18-siRNA61质粒转染SKOV3细胞后,MTT法测定SKOV3细胞的增殖,流式细胞术检测细胞的细胞周期,采用Transwell法、Migration法以及Fibronectin黏附法分别测定细胞的体外侵袭、迁移、黏附能力。结果:3对靶向CCL18的siRNA中CCL18-siRNA61干扰效果最好,进而成功构建靶向CCL18的pSilencer4.1-CCL18-siRNA61干扰质粒,pSi-lencer4.1-CCL18-siRNA61质粒转染可显著下调SKOV3细胞中CCL18 mRNA的表达。pSilencer4.1-CCL18-siRNA61质粒转染不影响SKOV3细胞的增殖,但pSilencer4.1-CCL18-siRNA61质粒转染组SKOV3细胞中(S+G2+M)期细胞比例明显低于对照质粒pSilencer4.1-Ctrl-siRNA转染组[(19.71±4.4)%vs(26.45±7.91)%,P<0.05];且与pSilencer4.1-Ctrl-siRNA质粒转染相比,pSilencer4.1-CCL18-siRNA61质粒转染可有效抑制SKOV3细胞的侵袭、迁移和黏附能力[(9.91±3.41)%vs(23.75±6.81)%,(16.80±8.71)%vs(31.74±11.23)%,(6.73±4.33)%vs(17.53±6.54)%;均P<0.05]。结论:siRNA沉默CCL18的表达可抑制卵巢上皮癌细胞株SKOV3的侵袭、迁移和黏附能力。
Objective: To investigate the effect of CCL18 gene silencing in vitro on the invasion and migration of ovarian epithelial carcinoma SKOV3 cells. METHODS: Three CCL18-targeting siRNAs (CCL18-siRNA61, CCL18-siRNA127 and CCL18-siRNA224) were chemically synthesized and transfected into CCL18-positive SKOV3 cells in vitro. The expression of CCL18 mRNA in SKOV3 cells was detected by RT-PCR. The most efficient sequence was constructed to construct CCL18-targeted interfering plasmid pSilencer4.1-CCL18-siRNA61. The proliferation of SKOV3 cells was determined by MTT assay after transfected with pSilencer4.1-CCL18-siRNA61. The cell cycle was detected by flow cytometry. The cell invasion and migration were determined by Transwell assay, Migration assay and Fibronectin adhesion assay respectively , Adhesion. Results: 3 CCL18 siRNA targeting CCL18 in CCL18-siRNA61 best interference, and then the successful construction of targeting CCL18 pSilencer4.1-CCL18-siRNA61 interference plasmid, pSi-lencer4.1-CCL18-siRNA61 plasmid transfected significantly reduced CCL18 mRNA expression in SKOV3 cells. The transfection of pSilencer4.1-CCL18-siRNA61 plasmid did not affect the proliferation of SKOV3 cells, but the percentage of cells in (S + G2 + M) phase of SKOV3 cells transfected with pSilencer4.1-CCL18-siRNA61 was significantly lower than that of control plasmid pSilencer4.1 (19.71 ± 4.4)% vs (26.45 ± 7.91)%, P <0.05]. Compared with pSilencer4.1-Ctrl-siRNA plasmid transfection, pSilencer4.1-CCL18-siRNA61 plasmid Transfection could effectively inhibit the invasion, migration and adhesion of SKOV3 cells [(9.91 ± 3.41)% vs (23.75 ± 6.81)%, (16.80 ± 8.71)% vs (31.74 ± 11.23)%, (6.73 ± 4.33)% vs (17.53 ± 6.54)%, all P <0.05]. Conclusion: The silencing of CCL18 by siRNA can inhibit the invasion, migration and adhesion of ovarian epithelial cancer cell line SKOV3.