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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 PCDGF in human nasopharyngeal carcinoma and to investigate the effect of small interfering RNA (siRNA) on the proliferation of nasopharyngeal carcinoma HNE-1 cells influences. Methods Immunohistochemistry was used to detect the expression of PCDGF protein in 34 cases of nasopharyngeal carcinoma and 20 cases of chronic nasopharyngeal inflammation. Two siRNAs were designed and synthesized according to the PCDGF gene, and the HNE-1 cells were transfected with LipofectamineTM2000. The expression of PCDGF mRNA and protein in transfected cells was detected by PT-PCR, Western blot and fluorescent immunocytochemistry. The growth and proliferation of transfected cells were detected by MTT assay and the cell cycle distribution by FCM. Results The positive rate of PCDGF in 34 cases of nasopharyngeal carcinoma was 85.3% (29/34). The positive rate of PCDGF in 20 cases of nasopharyngeal chronic inflammation was 15.0% (3/20), the difference was statistically significant P <0.01). Both recombinant plasmids could specifically inhibit the expression of PCDGF mRNA and protein, of which siRNA-1 had the highest silencing efficiency. After 48 h of transfection, the expression of PCDGF mRNA and protein in HNE-1 cells were down-regulated by 64.7% and 69.8%, respectively. The rate of cell proliferation inhibition was (37.07 ± 12.4)%. The number of cells in G0 / The number of cells was significantly decreased (P <0.05), and the cell cycle was arrested in G1 phase. Conclusion Specific siRNA can effectively silence the expression of PCDGF gene and significantly inhibit the proliferation of HNE-1 cells.