丝氨酸-苏氨酸激酶受体相关蛋白在肝癌细胞生物学功能中的作用

Role of serine-threonine kinase receptor-related protein in the biological function of hepatocellular carcinoma cells

目的检测丝氨酸-苏氨酸激酶受体相关蛋白(STRAP)在肝癌细胞及正常肝组织中的表达,并探索其对肝癌细胞生物学功能的影响。方法通过检测九种肝癌细胞株及正常肝组织STRAP相对表达量,并分别在肝癌细胞株HepG2、SNU449中构建STRAP过表达及敲减细胞株。使用实时定量反转录聚合酶链反应(RT-qPCR)和蛋白印迹技术检测细胞株构建后STRAP表达,用Transwell小室及含有基质胶的Transwell小室检测肝癌细胞的迁移和侵袭能力,并使用细胞计数试剂盒(CCK-8)及细胞克隆形成实验检测STRAP对肝癌细胞增殖能力的影响,并利用流式细胞学技术检测STRAP对肝癌细胞凋亡的影响。结果肝癌细胞株中STRAP表达水平明显高于正常肝组织;在肝癌细胞中STRAP敲减后,敲减组与对照组mRNA相对表达量分别为(0.17±0.19)、(2.22±1.02),敲减组表达水平明显低于对照组(P<0.05)。过表达STRAP基因后,过表达组与对照组mRNA相对表达量分别为(3.25±0.39)、(1.05±0.37),过表达组明显高于对照组(P<0.01)。STRAP基因敲减后,SNU449细胞克隆形成实验对照组和敲减组细胞克隆数目分别为(221.30±9.60)、(58.30±4.49),迁移实验对照组和敲减组细胞穿膜数分别为(0.76±0.04)、(0.44±0.06),侵袭实验对照组和敲减组细胞穿膜数分别为(1.11±0.05)、(0.60±0.05),细胞增殖实验敲减组在第72、96、120小时,细胞增殖数目较对照组下降,细胞凋亡实验对照组和敲减组细胞凋亡比例分别(4.11±0.12)、(11.73±0.40),细胞的克隆形成能力、迁移侵袭能力及增殖能力明显受抑制(P<0.05),细胞凋亡明显增多(P<0.01);SRTAP基因过表达后,HepG2细胞在第48、72、96、120小时,细胞增殖数目较对照组增多,细胞增殖能力明显增强(P<0.01),细胞凋亡实验对照组和过表达组细胞凋亡比例分别(8.87±0.12)、(8.34±0.06),细胞凋亡明显受抑制(P<0.05)。结论STRAP在肝癌细胞中呈高表达,并且起着促进肝癌细胞增殖、增强肝癌细胞迁移侵袭能力,同时抑制肝癌细胞凋亡的作用。

Objective To detect the expression of serine-threonine kinase receptor-related protein (STRAP) in hepatocellular carcinoma (HCC) cells and normal liver tissues, and to explore its biological functions. Methods The relative expression levels of STRAP in 10 HCC cell lines and normal liver tissues were detected, and STRAP overexpression and knockdown cell lines were constructed in HepG2 and SNU449, respectively. The real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to determine the expression of STRAP after the construction of cell lines. The migration and invasion ability of HCC cells was detected by Transwell chamber and Transwell chamber containing Matrigel. Cell proliferation was examined by cell counting kit-8 (CCK-8) and cell clone formation assay, and cell apoptosis was verified by flow cytometry. Results The expression level of STRAP in HCC cell lines was significantly higher than that in normal liver tissues. After STRAP knockdown, the relative expression of mRNA in knockdown group and control group was (0.17±0.19) and (2.22±1.02), respectively. The expression level of the knockdown group was significantly lower than that in the control group (P<0.05), while overexpressing STRAP, the relative expression levels of mRNA in overexpression group and control group were (3.25±0.39) and (1.05±0.37)(P<0.01). After the STRAP gene knockdown, the number of cell clones in control group and knockdown group was (221.30±9.60) and (58.30±4.49) respectively. The number of cells passing through the membrane in control group and knockdown group was (0.76±0.04) and (0.44±0.06) in cell migration assay, and (1.11±0.05) and (0.60±0.05) in cell invasion assay, respectively. In the cell proliferation assay, the number of cell proliferation in the knockdown group decreased as compared with that in the control group at 72, 96 and 120 h. The apoptosis rate in the control group and knockdown group was (4.11±0.12) and (11.73±0.40), respectively. The colony forming ability, migration invasive ability and proliferation ability of SNU449 were significantly inhibited (P<0.05), while the apoptosis was significantly increased (P<0.01). After overexpression of SRTAP gene, the proliferation of HepG2 cells increased at 48, 72, 96 and 120 h as compared with the control group (P<0.001). The cell proliferation assay indicated that the apoptosis rate in the control group and overexpression group was (8.87±0.12) and (8.34±0.06) respectively (P<0.05). Conclusion STRAP is highly expressed in HCC cells, and promotes proliferation, enhances migration and invasion, and inhibits apoptosis of HCC cells.