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目的解析比较蛋白质组学筛出的差异蛋白S100A4的生物学功能,验证其是否在肝癌转移复发中发挥作用。方法用逆转录聚合酶链反应(RTPCR)、蛋白免疫印迹(WB)及细胞免疫化学法分别对差异蛋白S100A4在转移潜能不同人肝癌细胞系中的表达情况进行再验证;然后,用构建S100A4反义表达质粒,转染高转移潜能人肝癌细胞系MHCC97H,通过系列与侵袭转移有关的检测,观察S100A4表达降低对MHCC97H细胞生物学行为的影响。结果验证结果均证实,S100A4在有转移潜能人肝癌细胞系MHCC97L、MHCC97H中呈高表达。转染S100A4反义重组表达质粒后的侵袭试验显示,穿过人工基底膜细胞数分别是:5.0±1.4[MHCC97H/pcDNA3.1(+)ASS100A4],16.4±1.6[MHCC97H/pcDNA3.1(+)],16.9±1.5(MHCC97H);运动试验穿过上室底膜的细胞数为:11.1±3.3[MHCC97H/pcDNA3.1(+)ASS100A4],18.9±2.0[MHCC97H/pcDNA3.1(+)],21.9±3.4(MHCC97H);细胞培养上清液中MMP9的定量结果依次为18.2[MHCC97H/pcDNA3.1(+)ASS100A4]、28.0[MHCC97H/pcDNA3.1(+)]、31.9ng/ml(MHCC97H);MMP2定量结果依次为29.8[MHCC97H/pcDNA3.1(+)ASS100A4]、26.4[MHCC97H/pcDNA3.1(+)]、26.5ng/ml(MHCC97H)。明胶酶谱分析细胞培养上清液基质金属蛋白酶(MMP)显示,S100A4表达降低的MHCC97H细胞分泌的MMP9活性明显弱于对照组。结论pcDNA3.1(+)ASS100A4转染MHCC97H细胞后,细胞的运动与侵袭潜能均明显降低。细胞MMP9分泌量也显著降低。而细胞分泌MMP2的量则变化不明显。提示差异蛋白S100A4通过增强肝癌细胞的运动侵袭能力和上调MMP的分泌参与肝癌细胞的侵袭转移过程。
Objective To compare and analyze the biological function of differential protein S100A4 screened by proteomics to verify whether it plays a role in the metastasis and recurrence of liver cancer. Methods Reverse transcriptase polymerase chain reaction (RTPCR), Western blotting (WB) and immunocytochemistry were used to verify the expression of S100A4 protein in metastatic potential human hepatocarcinoma cell lines. Then, the expression of S100A4 The recombinant plasmid was transfected into human hepatocellular carcinoma cell line MHCC97H with high metastatic potentiality. The effects of reduced expression of S100A4 on the biological behavior of MHCC97H cells were observed by serial detection of invasion and metastasis. The results verify that S100A4 was highly expressed in MHCC97L and MHCC97H cell lines with potential metastatic potential. Transfection of S100A4 antisense recombinant plasmids showed that the number of cells penetrating artificial basement membrane was 5.0 ± 1.4 [MHCC97H / pcDNA3.1 (+) ASS100A4], 16.4 ± 1.6 [MHCC97H / pcDNA3.1 (+ )] And 16.9 ± 1.5 (MHCC97H). The number of cells passing through the basement membrane of the upper chamber during exercise test was 11.1 ± 3.3 [MHCC97H / pcDNA3.1 (+) ASS100A4], 18.9 ± 2.0 [MHCC97H / pcDNA3.1 ], 21.9 ± 3.4 (MHCC97H). The quantitative results of MMP9 in cell culture supernatants were 18.2 [MHCC97H / pcDNA3.1 (+) ASS100A4], 28.0 [MHCC97H / pcDNA3.1 (+)], 31.9 ng / ml (MHCC97H). The quantitative results of MMP2 were 29.8 [MHCC97H / pcDNA3.1 (+) ASS100A4], 26.4 [MHCC97H / pcDNA3.1 (+)] and 26.5ng / ml (MHCC97H). Gelatin zymography analysis of cell culture supernatant matrix metalloproteinase (MMP) showed that the MMP9 activity of MHCC97H cells with reduced S100A4 expression was significantly weaker than that of the control group. Conclusion After transfection with pcDNA3.1 (+) ASS100A4, the cell motility and invasion potential of MHCC97H cells were significantly decreased. Cell secretion of MMP9 was also significantly reduced. The amount of cells secreting MMP2 did not change significantly. These results suggest that S100A4 is involved in the invasion and metastasis of hepatocellular carcinoma cells by enhancing the invasion and migration of hepatocellular carcinoma cells.