胆管细胞性肝癌组织CSNK1α1的表达及其与肿瘤转移和血管生成的关系

Expression of CSNK1α1 in cholangiocarcinoma and its relationship with tumor metastasis and angiogenesis

目的:探究酪蛋白激酶1α1(caseinkinase1alpha1,CSNK1α1)在胆管细胞性肝癌(intrahepatic cholangiocarcinoma, ICC)组织中的表达,及其对肿瘤细胞转移及血管生成的影响。方法:收集新疆医科大学附属肿瘤医院50例ICC组织及癌旁组织样本,实时荧光定量PCR(qRT-PCR)法和免疫组化染色法检测CSNK1α1表达水平;将ICC细胞系HUCCT-1培养后随机分为对照组、si-NC组、si-CSNK1α1组,通过脂质体介导法将靶向CSNK1α1基因的短发夹RNA序列转染至HUCCT-1细胞,qRT-PCR和蛋白质免疫印迹法(Western blotting)测定细胞内CSNK1α1mRNA与蛋白的表达变化,以鉴定转染效果;细胞划痕实验和Transwell实验检测细胞的迁移与侵袭能力变化,细胞免疫荧光染色观察细胞内上皮间质转化标志物钙黏附蛋白E(E-cadherin)和波形蛋白(Vimentin)的表达强度,血管生成实验检测肿瘤血管生成情况,Western blotting检测细胞中血管内皮生长因子(vascular endothelial growth factor, VEGF)、基质金属蛋白酶(matrix metallopeptidase, MMP)2(MMP-2)及MMP-9的蛋白表达变化。结果:ICC组织CSNK1α1 mRNA相对表达量显著高于癌旁组织,且ICC组织CSNK1α1阳性率也显著高于癌旁组织(P<0.01);与对照组和si-NC组比较,si-CSNK1α1组HUCCT-1细胞中CSNK1α1 mRNA相对表达量和蛋白相对表达量均下调,细胞划痕愈合率降低,细胞迁移与侵袭数目均减少,E-cadherin荧光密度值下降而Vimentin荧光密度值升高,细胞管样形成数目减少,细胞内VEGF、MMP-2及MMP-9的蛋白相对表达量均显著下调,差异均具有统计学意义(P<0.05)。结论:CSNK1α1在ICC组织内呈高表达,靶向下调ICC细胞CSNK1α1表达后可抑制细胞迁移、侵袭及上皮间质转化过程,并减少血管生成,从而控制肿瘤进展。

Objective: To investigate the expression of casein kinase 1 alpha1(CSNK1α1) in intrahepatic cholangiocarcinoma(ICC) tissues and its effect on tumor cell metastasis and angiogenesis. Methods: Fifty ICC tissue samples and adjacent tissue samples from Affiliated Tumor Hospital of Xinjiang Medical University were collected, and the expression level of CSNK1α1 was detected by real-time quantitative PCR(qRT-PCR) and immunohistochemical staining. The ICC cell line HUCCT-1 was cultured and randomly divided into control group, si-NC group and si-CSNK1α1 group. The short hairpin RNA sequence targeting CSNK1α1 gene was transfected into HUCCT-1 cells by liposome-mediated method, the expression changes of CSNK1α1 mRNA and protein in cells were measured by qRT-PCR and Western blotting to identify the transfection effect. Cell scratch assay and Transwell assay were used to detect changes in cell migration and invasion abilities, cell immunofluorescence staining was used to observe the expression intensity of intracellular epithelial-mesenchymal transition markers E-cadherin and Vimentin, angiogenesis assays was used to detect tumor angiogenesis, the protein expression changes of vascular endothelial growth factor(VEGF), matrix metallopeptidase(MMP) 2(MMP-2) and MMP-9 in cells were detected by Western blotting. Results: The relative expression of CSNK1α1 mRNA in ICC tissue was significantly higher than that in adjacent tissue, and the positive rate of CSNK1α1 in ICC tissue was also significantly higher than that in adjacent tissue(P<0.01). Compared with the control group and si-NC group, the relative expression of CSNK1α1 mRNA and protein in HUCCT-1 cells of si-CSNK1α1 group were down-regulated, the wound healing rate of cells decreased, the number of cell migration and invasion decreased, the fluorescence density value of cadherin decreased while the fluorescence density value of Vimentin increased, and the number of cell tube-like formation decreased, at the same time, the relative proteins expressions of VEGF, MMP-2 and MMP-9 in cells were significantly down-regulated, the differences were statistically significant(P<0.05). Conclusion: CSNK1α1 is highly expressed in ICC tissues, targeted down-regulation of CSNK1α1 expression in ICC cells can inhibit cell migration, invasion and epithelial-mesenchymal transition, and reduce angiogenesis, thereby inhibiting tumor progression.