[The effect of Na(+)/H(+) exchanger-1 (NHE-1) antisense expression vector on NHE-1 gene expression in human lung adenocarcinoma cells and its biological significance].
OBJECTIVE
To explore the inhibitory effect of Na(+)/H(+) exchanger-1 (NHE-1) antisense expression vector on NHE-1 gene in human lung neoplasm cells and to observe its biological effect.


METHODS
NHE-1 antisense vector (pNHE-1) was introduced into human lung adenocarcinoma cell line A549 cells with cationic liposome transfection methods. Semi-quantification RT-PCR was performed to analyze the expression level of NHE-1 mRNA in A549 cells and transfected A549 cells. Intracellular pH (pHi) values were measured with fluorescence spectrophotometer. The growth characteristics of the cells was observed. Tumor cell apoptosis was observed with in situ apoptosis assessment.


RESULTS
PCR verified the integration of antisense vector with the genomic DNA of transfected cells in positive clones. Semi-quantification RT-PCR showed that the NHE-1 gene expression level was significantly lower in transfected A549 cells (0.42 +/- 0.06) than in those untransfected (0.71 +/- 0.08, P < 0.01) and those transfected with pLXSN (0.69 +/- 0.16, P < 0.01). Compared with A549 cells, pHi values decreased significantly in transfected A549 cells at 12 h, 24 h and 48 h (6.990 +/- 0.005, 6.840 +/- 0.005 and 6.750 +/- 0.005 respectively vs 7.150 +/- 0.004, 7.140 +/- 0.007 and 7.120 +/- 0.008 respectively, P < 0.001). The growth of transfected A549 cells was significantly slower (P < 0.01), the double time was longer (3.20 vs 4.97) and the apoptosis rate was markedly higher [(24.50 +/- 3.62)% vs (1.50 +/- 0.55)%, P < 0.01].


CONCLUSIONS
The antisense vector was successfully transfected to A549 cells with liposome and integrated with transfected cell genome, and the expression of NHE-1 mRNA of A549 cells was inhibited, thereby resulted in a decrease of the pHi value by means of reduction of Na(+)/H(+) exchanging activity, with the inhibition of proliferation and growth of A549 cells and increased of A549 cell apoptosis.
Wu
Guo-ming
GM
Institute of Respiratory Medicine, People's Liberation Army, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.
Qian
Gui-sheng
GS
Huang
Gui-jun
GJ
Li
Shu-ping
SP
Yao
Wei
W
Lu
Jun-yu
JY
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D017209:Apoptosis; D002455:Cell Division; D045744:Cell Line, Tumor; D005822:Genetic Vectors; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D016372:RNA, Antisense / Q000235:genetics* / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D016372:RNA, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers