ABSTRACT
Objective To evaluate the effect of exogenous wild PTEN gene stable transfected into human ovarian cancer cell line HO-8910 on phosphatidyl inositol 3-kinase( PI3K)/protein kinase B (Akt)siganal pathway and cells proliferation. Methods Wild-type PTEN recombinant eukaryotic expression plasmid was constructed and then was transfected into HO-8910 cells by lipofectamine 2000. The expression of PTEN, Akt1, Akt2, PI3K mRNA and protein of PTEN were tested by reverse transcription( RT)-PCR and Western blot. The proliferation of HO-8910 after wild PTEN gene transfected was measured by methyl thiazolyl tetrazolium(MTT). Results Wild-type PTEN gene was successfully transfected into HO-8910 cells. The results of RT-PCR and western bolt showed that there were the significant expression high level of PTEN mRNA and protein after infected by wild-PTEN plasmid than those in the control[ ( 17 372 ±23)vs.(39±1 )vs. (78 ±4)copies/ml,P ＜0. 05 ]. While the expression of mRNA of Akt1, Akt2 and PI3K were decreased clearly than those in the control [ (28 ± 2 ) vs. ( 115 ± 5 ), (7 ± 1 ) vs. ( 18 ± 2), (61 ± 2 ) vs.(84 ± 2)copies/ml , all P ＜ 0. 05 ]. The proliferation rate of HO-8910 cells was obviously slower than those in the control (90 158 ±47 vs. 148 251 ±65 vs. 250 115 ±62, P＜0.05). Conclusion Transfection of PTEN may increase the expression of PTEN and inhibit the proliferation of HO-8910 cells, in which PI3K/ Akt siganal pathway is inhibit significantly.
ABSTRACT
Objective To analyze the regulation of NEF3 expression in SH - SY5Y after RA induction. Methods The expression of NEF3 and Brg1 genes was individually detected by realtime RT - PCR and Western blot analyses. Then, the CAT reporter driven by 5' flanking region ofNEF3 gene (pBLCAT3 -2. 8k - NEF3) was co - transfccted with eukaryotic expression plasmids of pBJ5 - Brgl or pBJ5 - Brg1 - NTP into SH - SY5Y respectively. Promoter activity of the NEF3 gene was detected by competitive RT - PCR assay. Results Similar changes in the mRNA expression of NEF3 and Brg1 and that of the protein expression of Brgl were detected in Sh - SY5Y cells af-ter RA treatment for 12 -24h. Wild type Brgl can promote the expression of pBLCAT3 -2.8k -NEF3 promoter after RA induction, but not mutant Brg1. Conclusion Brgl helpes regulating the expression of NEF3 gene possibly via changes in chromatin conformation in the SH -SY5Y cells after RA induction.