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


CONCLUSIONS
Down-regulating LRIG3 gene expression can improve the proliferation of glioma GL15 cells.
Cai
Ming-Jun
MJ
Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China.
Xie
Rui-Fan
RF
Han
Lin
L
Chen
Ru-Dong
RD
Wang
Bao-Feng
BF
Ye
Fei
F
Guo
Dong-Sheng
DS
Lei
Ting
T
D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005910:Glioma / Q000473:pathology* / Q000628:therapy; D006801:Humans; D019394:Ki-67 Antigen / Q000032:analysis*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis*; D034622:RNA Interference
D019394:Ki-67 Antigen; C496406:LRIG3 protein, human; D008565:Membrane Proteins; D018809:Proliferating Cell Nuclear Antigen