鼻咽癌畸胎瘤细胞源性生长因子表达及siRNA对HNE-1细胞株增殖的抑制

Expression of PCDGF in nasopharyngeal carcinoma and its silencing in cell proliferation

目的探讨畸胎瘤细胞源性生长因子(PC cell-derived growth factor,PCDGF)在人鼻咽癌中的表达,研究小干扰RNA(small interfering RNA,siRNA)对鼻咽癌HNE-1细胞增殖的影响。方法采用免疫组织化学的方法检测34例鼻咽癌、20例鼻咽慢性炎症组织中PCDGF蛋白的表达情况。根据PCDGF基因设计合成2条siRNA,利用LipofectamineTM2000转染鼻咽癌HNE-1细胞。通过PT-PCR、Western blot、荧光免疫细胞化学法检测转染后细胞PCDGF mRNA和蛋白表达,采用MTT检测转染后细胞的生长增殖情况,FCM法检测细胞周期分布。结果 34例鼻咽癌组织中PCDGF阳性表达率为85.3%(29/34),20例鼻咽部慢性炎症组织中阳性率为15.0%(3/20),2组间差异有统计学意义(P<0.01)。2条重组质粒均能特异性的抑制PCDGF mRNA和蛋白的表达,其中siRNA-1的沉默效率最高。转染48 h后,HNE-1细胞中PCDGF mRNA和蛋白表达水平分别下调64.7%和69.8%,细胞增殖抑制率为(37.07±12.4)%,siRNA-1组G0/G1期细胞增多,S期细胞数量明显减少(P<0.05),细胞周期被阻滞于G1期。结论特异性siRNA能够有效沉默PCDGF基因表达,并显著抑制鼻咽癌HNE-1细胞增殖。

Objective To investigate the expression of PC cell-derived growth factor（PCDGF） in nasopharyngeal carcinoma tissues and determine the effect of small interfering RNA（siRNA） against PCDGF gene on the proliferation in nasopharyngeal carcinoma HNE-1 cell line.Methods Immunohistochemical staining was used to examine the expression of PCDGF protein in 34 tissue samples of nasopharyngeal carcinoma and 20 samples of chronic nasopharyngeal inflammation.Two siRNA expression plasmids targeting PCDGF mRNA were constructed based on the nucleotide sequence of PCDGF and were respectively transferred into human nasopharyngeal carcinoma HNE-1 cells via LipofectamineTM2000.The expression of PCDGF at mRNA and protein levels were analyzed by reverse transcription polymerase chain reaction（RT-PCR）,Western blotting and immunofluorescence assay,respectively.The cell proliferation was determined by MTT assay.The distribution of cell cycle was assessed by flow cytometry.Results The positive expression rate was 85.3%（29/34） in nasopharyngeal carcinoma,while only 15.0%（3/20） in chronic nasopharyngeal inflammation,with significant difference between each other（P〈0.01）.The 2 recombinant plasmids inhibited the expressions of PCDGF,and the siRNA-1 was demonstrated to have the strongest silencing effect on PCDGF expression in HNE-1 cells at 48 h after transfection.The expression of PCDGF were reduced by 64.7% and 69.8%,respectively at mRNA and protein levels.PCDGF silence resulted in the proliferation inhibitory ratio of（37.07±12.4）% and more cells arrested at G0/G1 phase in HNE-1 cells.Conclusion Specific siRNA of PCDGF effectively inhibits the gene expression and observably suppresses cell proliferation in nasopharyngeal carcinoma HNE-1 cells.