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Sirna Targeting Leptin Gene In Human Lung Adenocarcinoma A549 Cell Proliferation And Apoptosis

Posted on:2013-09-16Degree:MasterType:Thesis
Country:ChinaCandidate:K W ChengFull Text:PDF
GTID:2244330374492888Subject:Respiratory disease
Abstract/Summary:PDF Full Text Request
Objective To construct a lentiviral leptin small interfering RNA (siRNA) vector tosilence leptin gene expression,and to study its effect on proliferation and apoptosis inlung carcinoma cells.Methods Five groups of cells were established,including interference with agroup(siRNA-a), interference with b group(siRNA-b),respective negative controlgroups and blank control group(A549). Cells growth were observed underfluorescence microscope after5days and11days. Through MTT colorimetric,weassayed the growth of three groups of cells,including blank control group, siRNA-agroup and negative control group from the first day to the sixth day, and drew agrowth curve to compare the proliferations of cells. Assayed the rates of apoptosis ofthe cells by flow cytometry to observe the siRNA’s impact on apoptosis in A549cells.Expression of leptin mRNA and protein in three group of cells was examined byRT-PCR and Western bloting analysis.Results siRNA-a cells’s appearance was almost normal. After five days,thefluorescence efficiency was still high, which showed it was a stable infection.But itgrew slower than the blank and negative control groups.After48hours of siRNA-bcell’s constructing,the cell swelling, deformation and cavitation can be seen undermicroscope and after5days,the cell debris,apoptotic bodies can be seen,then11days later,siRNA-b cells was almost dead completely. In MTT test,the siRNA-a group cellsgrew slower than the other groups from the third day(P<0.01); but there were nosignificant differences among other groups. With flow cytometry, the apoptosis ratesof siRNA-a(38.7%)and siRNA-b(48.9%) groups was higher than blank(21.1%)andnegative control groups (22.1%).Expression of leptin mRNA and protein in interfencegroup was signigicantly lower than others groups as showed by RT-PCR and Westernbloting analysis.Conclusion Lentiviral leptin siRNA can decrease leptin expression in human lungadenocarcinoma A549cells,inhibit proliferation and promote apoptosis in A549cells.It is expected that siRNA targeting leptin will be a new gene therapy to lungadenocarcinoma. Objective To explore the effect of leptin gene silencing by RNA interference onxenograft of lung adenocarcinoma A549cells in BALB/C nude mice.Methods Three different BALB/C nude mice models were established bysubcutaneously inoculating differently treated A549cells into3nude mice groups:the interference group was inoculated with cells with lentivirus-delivered LeptinsiRNA, while the negative group was inoculated with cells transfected only withlipofectamine2000reagent and the blank control group was inoculated with normalA549cells. Then the growth of tumors was observed, and the volume and weight ofthe tumors were measured at different time points. The curve of tumor growth wasthen described. Leptin protein expression in the tumor tissues was determined byimmunohistochemistry. Cell apoptosis of tumor tissues was detected by TUNEL.Results Slower tumor growth, small tumor volume and lighter tumor weight wereobserved in the interference group as compared to the negative and blankgroups(P<0.05). Leptin protein expression level was significantly lower than theblank and the negative groups. The apoptosis rate in the interference group wassignificantly higher than that two control groups(P<0.05).Conclusion The lentivirus-delivered Leptin siRNA was shown to inhibit the proliferation of transplantation tumor of lung carcinoma effectively, and Leptin maybe one of the targets for the treatment of lung cancer.
Keywords/Search Tags:Leptin, RNA interference, A549cells, Proliferation, ApoptosisLeptin, Transplantation
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