| Background:The occurrence of gastric carcinoma is a kind of various factors and multiple stages evolved process. Its occurrence and development are highly associationed with over-expression of COX-2/5-LOX gene in gastric cancer cell, Our previous study have found that, the expression of COX-2and5-LOX gene in stomach cancer cells can be inhibited by double selective inhibitors,and the growth of stomach cancer was inhibited too.Meanwhile,inhibition of the both genes was more effective than one of the genes separatly. RNA interference (RNAi) is a transcription gene silencing, shRNA is famous of its effective, specific, fast, and genetic advantages. Up to now, we don’t know that whether COX-2and5-LOX gene could be silenced simtiously by RNAi.Objective:To constructe the short clip RNA (shRNA) eukaryotic expression vector of5-lipoxygenase (5-LOX) gene and cyclooxygenase-2(COX-2) gene respectively, to observe the effects of human gastric adenocarcinoma cell line SGC-7901on growth by common silencing5-LOX gene and COX-2gene.Methords:1. Eukaryotic expression plasmid contained two arraies of5-LOX shRNA and COX-2shRNA were constructed respectively.2. Co-transfected into SGC-7901with Lipofectamine2000, inhibition effects of mRNA and protein were analyzed by RT-PCR and Western blot separately.3. Cell proliferation ability was detected by MTT method, apoptotic rate was examined by flow cytometry.Results:1. It was proved the eukaryotic expression vector P-sh5-LOX and P-shCOX-2were constructed successfully by gene sequencing, green fluorescent expression was observed by fluorescence microscopy in transfected groups.2.After transfected, green fluorescent expression in SGC-7901cell was detected by fluorescence microscopy, the expression of5-LOX mRNA and COX-2mRNA in PGenesil-1group was (0.58±0.02) and (0.82±0.05),in SGC-7901group was (0.68±0.02) and (0.89±0.08) respectively, there is no significant difference between the two groups (P>0.05).5-LOX mRNA expression (0.38±0.06) of Psh-5-LOX group, COX-2mRNA expression (0.57±0.04) of Psh-COX-2group and5-LOX/COX-2mRNA expression (0.35±0.06,0.50±0.02) of co-transfected group were significantly lower than those in SGC-7901group and PGenesil-1group (P<0.05).5-LOX protein and COX-2protein expression of PGenesil-1group were (0.73±0.02) and (0.81±0.02), which in SGC-7901group were (0.75±0.03) and (0.83±0.04) separately,there was no significant difference between the two groups (P>0.05).5-LOX protein (0.41±0.02) of Psh-5-LOX group,COX-2protein (0.36±0.01) of Psh-COX-2group,5-LOX protein and COX-2protein expression (0.39±0.01,0.33±0.01) of co-transfected group were significantly lower than those in SGC-7901group and PGenesil-1group(P<0.05).3. MTT result:the cell proliferation inhibition rate in P-sh5-LOX group, P-shCOX-2group and co-transfected group were (27.38±0.64)%,(29.05±0.61)%and (50.23±1.73)%, which was markedly higher than that in PGenesil-1group [(3.78±0.39)%] respectively(P<0.05).The cell proliferation inhibition rate in co-transfected group was higher than alone transfected group(P<0.05).4. Flow cytometry (FCM)result:the apoptosis rate in P-sh5-LOX group, P-shCOX-2group, and co-transfected group were obviously higher than that in control group(P<0.05),they were(15.31±1.53)%,(16.69±1.28)%,(27.27±1.18)%and (0.99±0.46)%respectively, the apoptosis rate of co-transfected group was much higher than alone transfected group (P<0.05).Conclusion:5-LOX and COX-2gene were common silenced effectively in SGC-7901, either the co-transfected group or alone transfected groups could restrain the proliferation and promot apoptosis of SGC-7901, the co-transfected group has a superior anticancer efficacy than the alone transfected groups. |
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