早期B细胞因子3在肝癌中的表达及对HepG2细胞株作用的研究

Expression of EBF3 in hepatocellular carcinoma and the effect of EBF3 overexpression on HepG2 cell proliferation

目的研究肝癌及远端非癌肝组织中早期B细胞因子3（EBF3）mRNA与蛋白质表达差异及临床意义，并研究EBF3-EGFP（增强型绿色荧光蛋白）融合蛋白表达对肝癌细胞株HepG2细胞增殖的影响。方法采用荧光定量PCR技术检测20例配对肝癌及远端非癌肝组织EBF3mRNA水平，Westernblot检测5例配对标本EBF3蛋白表达。将EBF3与EGFP融合蛋白表达载体pEGFP／EBF3转染HepG2细胞株，倒置荧光显微镜观察EBF3-EGFP融合蛋白表达，流式细胞术测定转染后不同时间S期细胞分数（SPF）和增殖指数（PI）。结果20例肝癌和配对远端非癌肝组织中EBF3mRNA与B：MmRNA的对数比值分别为0．55±0．12和0．22±0．23，差异有统计学意义（t=5．69，P〈0．001）；Westernblot结果表明EBF3蛋白主要表达于细胞核中，5例肝癌组织核蛋白中EBF3条带平均灰度值为26．35±14.06，与远端非癌肝组织（7．86±8．47）相比差异有统计学意义（t=2．52，P=0．036）；将pEGFP／EBF3导入HepG2细胞株24h后，可观察到EBF3-EGFP融合蛋白主要表达于核内。转染pEGFP／EBF3后48h和72h，SPF和PI均显著高于pEGFP转染细胞组。结论肝癌组织中EBF3mRNA及其蛋白表达均上调，EBF3基因导入HepG2促进细胞株增殖，EBF3在肝癌中的作用有待进一步研究。

Objective To-investigate the expression and clinical significance of early B-cell factor 3 （EBF3） mRNA and protein in hepatocellular carcinoma（HCC） and the effect of EBF3 overexpression on HepG2 cell proliferation. Methods The expression levels of EBF3 mRNA in 20 pairs surgical specimens of HCC and their distant noncancerous tissues were detected by fluorescence quantitative real-time polymerase chain reaction（FQ-RT-PCR）. Western blot was used to detect the expression levels of EBF3 protein in 5 pairs surgical specimens of HCC and distant noncancerous tissues. The fusion protein EBF3-EGFP was expressed in HepG2 cells by transfection of pEGFP/EBF3 into the synchronized HepG2 cells using lipofectAMINE 2000 regent. Expression of EBF3-EGFP fusion protein was observed under the inverted fluorescence microscope. S-phase fraction （SPF） and proliferating index （PI） were analyzed with flow cytometry （FCM）. Results The ratio of EBF3 mRNA to β2M mRNA in HCC tissues was significantly higher than that in distant liver non-cancerous tissues（0.55 ± 0.12 versus 0.22 ± 0.23, t = 5.69, P 〈 0. 001 ）. EBF3 protein in nuclear extracts of HCC tissues was about 4 fold that in distant non-cancerous tissues （26.35±14. 06 versus 7.86 ±8.47, t = 2.52, P = 0. 036）. Fluorescence microscopy revealed that 24 h after transfection of pEGFP/EBF3 into hepatoma HepG2, the fluorescence of EBF3-EGFP fusion protein was mainly observed in the nucleus. After transfcction for 24 h and 48 h, SPF and PI were markedly increased in HepG2 cells transfected by pEGFP/EBF3 as compared with that in pEGFP-N1 transfected cells. Conclusion The expression of EBF3 at both mRNA and protein levels was up-regulated in HCC tissues. EBF3 promotes HepG2 cells proliferation through DNA replication, effect of EBF3 in HCC needs to be further investigated.