RNA干扰介导的LRIG3基因沉默对胶质瘤GL15细胞增殖及PCNA和Ki-67表达的影响

Effect of RNAi-mediated LRIG3 gene silencing on proliferation of glioma GL15 cells and expression of PCNA and Ki-67.

作者信息

Cai Ming-Jun, Xie Rui-Fan, Han Lin, Chen Ru-Dong, Wang Bao-Feng, Ye Fei, Guo Dong-Sheng, Lei Ting

机构信息

Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China.

出版信息

Ai Zheng. 2009 Jan;28(1):1-4. Epub 2009 Jan 28.

PMID:19448407

Abstract

BACKGROUND AND OBJECTIVE

Leucine-rich repeats and immunoglobin-like domains 3 (LRIG3), a member of LRIG gene family, is down-regulated in various human cancers, but its functions are still unclear. This study was to explore the effect of RNA interference (RNAi)-mediated LRIG3 gene silencing on the proliferation of glioma GL15 cells and the expression of proliferating cell nuclear antigen (PCNA) and Ki-67, and investigate possible mechanisms.

METHODS

The plasmids pGenesil2-LRIG3-shRNA1 and pGenesil2-LRIG3-shRNA2 which containing U6 promoter and LRIG3-specific short hairpin RNA (shRNA) and the plasmid pGenesil2-negative-shRNA containing unspecific shRNA were transfected into GL15 cells. Stable cell clones were selected by G418. The mRNA and protein levels of LRIG3 were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation was detected by MTT assay. The expression of PCNA and Ki-67 in GL15 cells were examined by SABC immunohistochemistry.

RESULTS

Compared with those in control cells, the mRNA levels of LRIG3 transcripts were reduced by 52.4% and 63.8% in shRNA1- and shRNA2-transfected cells, respectively; its protein levels were reduced by 50.9% and 67.4%, respectively. Cell proliferation was enhanced by LRIG3 shRNA transfection. The positive rate of PCNA was significantly higher in shRNA1- and shRNA2-transfected cells than in control cells [(72.13 +/- 5.64)% and (81.93 +/- 5.23)% vs. (35.40 +/- 5.69)%, p < 0.01]. The positive rate of Ki-67 was also significantly higher in shRNA1- and shRNA2-transfected cells than in control cells [(82.27 +/- 5.50)% and (88.67 +/- 3.52)% vs. (49.73 +/- 5.73)%, p < 0.01]. PCNA expression was positively correlated to Ki-67 expression (r =0.932, p < 0.001).

CONCLUSION

Down-regulating LRIG3 gene expression can improve the proliferation of glioma GL15 cells.

摘要

背景与目的

富含亮氨酸重复序列和免疫球蛋白样结构域3（LRIG3）是LRIG基因家族成员，在多种人类癌症中表达下调，但其功能仍不清楚。本研究旨在探讨RNA干扰（RNAi）介导的LRIG3基因沉默对胶质瘤GL15细胞增殖及增殖细胞核抗原（PCNA）和Ki-67表达的影响，并探讨其可能机制。

方法

将含U6启动子和LRIG3特异性短发夹RNA（shRNA）的质粒pGenesil2-LRIG3-shRNA1和pGenesil2-LRIG3-shRNA2以及含非特异性shRNA的质粒pGenesil2-阴性-shRNA转染至GL15细胞。用G418筛选稳定细胞克隆。采用逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法检测LRIG3的mRNA和蛋白水平。采用MTT法检测细胞增殖。用SABC免疫组织化学法检测GL15细胞中PCNA和Ki-67的表达。

结果

与对照细胞相比，shRNA1和shRNA2转染细胞中LRIG3转录本的mRNA水平分别降低了52.4%和63.8%；其蛋白水平分别降低了50.9%和67.4%。LRIG3 shRNA转染增强了细胞增殖。shRNA1和shRNA2转染细胞中PCNA的阳性率显著高于对照细胞[（72.13±5.64）%和（81.93±5.23）%对（35.40±5.69）%，p<0.01]。shRNA1和shRNA2转染细胞中Ki-67的阳性率也显著高于对照细胞[（82.27±5.50）%和（88.67±3.52）%对（49.73±5.73）%，p<0.01]。PCNA表达与Ki-67表达呈正相关（r=0.932，p<0.001）。