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@#[Abstract] Objective: To investigate he effect of tetracycline- (Tet-on) mediated livin RNA interference on growth of lung carcinoma xenegrafts, and find a better regulatory way to interfere the development on lung cancer. Methods: livin shRNA lentiviral vectors were constructed; and the lung cancerA549 cells were subcutaneously injected into right upper back of nude mice to establish xenegraft model. The livin shRNAlentiviral vectors were injected into xenografts to interfere the expression of livin, then tetracycline was injected intraperitoneally for the induction. The suppressive effect of Tet-on mediated livin RNA interference efficiency was investigated and lung cancer xenograft development was observed. Results: After the induction with Tet-on, livin gene expression was significantly inhibited by livin shRNAcompared with the control group and Tet-on-NC group; the xenograft volume in Tet-on- livin shRNAgroup was significantly smaller than that in control group and Tet-on-NC group ([5.31±0.86]g vs [8.22±0.63]g and [7.17±0.54] g, P<0.05). Moreover, little body toxicity was observed and no nude mice died in this study. Conclusion: The Tet-on mediated livin shRNA could suppress the growth of lung cancer development with good targeting and controllable characteristics, which might provide a potent tool for treating lung cancer with livin protein as target.
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[ ABSTRACT] AIM:To study the effect of livin gene-modified bone marrow mesenchymal stem cells ( BM-MSCs) transplantation on the cardiac function following acute myocardial infarction in a rat model and the expression of livin , caspase-3, caspase-7 and caspase-9 in the livin gene-modified BM-MSCs.METHODS: The MSCs were obtained by the whole bone marrow culture method , and the apoptosis of the MSCs after infection with adenovirus vector carrying enhanced green fluorescent protein ( EGFP) gene and livin recombinant vector ( rAd-livin) were detected by flow cytometry .The ex-pression of livin, caspase-3, caspase-7 and caspase-9 was detected by Western blot .After permanent left anterior descend-ing artery occlusion , the rats were randomized to receive intramyocardial injection of DMEM without cells ( vehicle group ) , or containing MSCs ( MSCs group ) , MSCs ( EGFP ) ( rAd-control/MSCs group ) or MSCs ( livin ) ( rAd-livin/MSCs group).Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), the maximum in-creased rate of left ventricular pressure ( -dp/dtmax ) and the maximum decline rate of left ventricular pressure ( +dp/dtmax ) were recorded for evaluating the cardiac functions .RESULTS: The apoptosis of rAd-livin/MSCs was significantly decreased as compared with MSCs and rAd-control/MSCs (P<0.05).Meanwhile, the expression of caspase-3, caspase-7 and caspase-9 was significantly downregulated as compared with the other 2 groups ( P<0.05 ) .The cardiac function in rAd-livin/MSCs group was significantly improved as compared with DMEM group , and those in the other 2 groups got the similar results, but the function in rAd-livin/MSCs group was better improved .Meanwhile, the number of surviving cells in rAd-livin/MSCs group was significantly improved as compared with the other 2 groups .CONCLUSION:The apoptosis of MSCs is decreased after rAd-livin transfection, and the expression of caspase-3, caspase-7 and caspase-9 is also significant-ly downregulated while the expression of livin is significantly upregulated .Transplantation of livin-modified BM-MSCs by lentiviral vector results in better prognosis for treating myocardial infarction by enhancing cell survival .
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Objective To explore the effects of silencing Livin gene by RNA interference mediated by lentiviral vector on colorectal cancer HT-29 cell xenograft growth and sensitivity to radiotherapy in nude mice.Methods BALB/c nude mice models were established by subcutaneously inoculating differently treated HT-29 cells into nude mice and the tumor growth situation of tumors was observed by measuring the volume of tumors and the weight of the nude mice at different time points after cell seeding.Livin expression was detected by RT-PCR and immunohistochemistry,respetively.Apoptosis rate was detected by TUNEL.Normal saline,lentivirus carring unrelated sequences,lentivirus caning Livin shRNA were injected intratumorally.All the nude mice were given 10 Gy of 6 MV X-ray irradiation.The changes of mice weight and the tumor volume were measured at different time points and the weight and tumor growth curves were drawn.Results The inhibition rate of tumor volume was(50.04 ± 0.07)％ and the tumor weight of the RNA interfering group was significantly less than that in experimental group compared to the blank and negative groups(F=4.85,P＜0.05),and the inhibition rate of tumor weight was(50.27 ±0.17)％.Relative Livin mRNA expression level in the RNA interfering experimental group was(17.75 ±0.08)％,and was significantly lower than that of the blank group(67.60 ± 0.05)％ and the negative group(68.54 ± 0.03)％(F=89.97,P＜0.01).Livin protein expression level in the RNA inferring group was also significantly lower[(36.00 ± 3.40)％ versus(85.00 ± 3.15)％,(80.33 ± 3.08)％,F=107.32,P＜0.01].The apoptosis rate in the RNA interfering experimental group was significantly higher than that in the blank and the negative groups[(23.67 ± 2.25)％ versus(5.00 ± 1.50)％,(8.33 ± 1.82)％,F=56.94,P＜0.01].Combined with radiotherapy,the tumor volume at different groups had significant difference(F=10.70,P＜0.01),and RNA interfering group was significantly less than negative group and blank group(F=7.01-9.32,P＜0.01).Conclusions Silencing of Livin gene expression by lentiviral vector-mediated RNA interference could inhibit the growth of colorectal HT-29 cell xenograft and increase the sensitivity of the transplanted tumors to radiotherapy.
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Objective To detect the expression of apoptotic inhibitor gene Livin in different esophageal epithelial lesions and to analyze the relation between the expression of Livin with pathologic characteristics. Methods The expressions of Livin mRNA were detected by real-time fluorescence quantitative PCR in 40 patients with different esophageal epithelial lesions including normal, atypical hyperplasia, carcinoma in situ and invasive carcinoma. Results The expression of Livin was progressively increased from normal to invasive carcinoma. Conclusion The expression of Livin is significantly related to the progression of esophageal cancer. The detection of Livin by real-time fluorescence quantitative PCR may be a reliable means for early diagnosis of esophageal cancer.
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Objective To investigate effects of Livin antisense oligodeoxynucleotides(ASODN) on the proliferation and apoptosis of human leukemia(HL60) cells.Methods Livin protein on HL60 cells was examined by immunohistochemistry.Specific phosphorothioate antisense oligodeoxynucleotides and missense oligodeoxynucleotides target Livin mRNA were synthesized and transfected into HL60 cells following cationic liposome.The proliferation inhibition of HL60 cells was assessed by MTT.The expression of Livin mRNA was detected by RT-PCR.Transmission electron microscope and TUNEL technology were used to detect the apoptosis and morphologic change.ResultsASODN of 600 nmol/L inhibited the HL60 cell proliferation and the expressions of Livin mRNA.The percentage of apoptosis detected by TUNEL was 38.48%?4.37%.cellar ultrastructure was markedly destroyed by Livin ASODN.A significant difference was found when compared with the control group(P
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Objective: To transfect a recombinant short hairpin RNA(shRNA)expression vector targeting Livin gene isoform(BIRC71,BIRC72)into the cervical cancer cell line(Hela cell),in an attempt to observe RNAi-mediated silen- cing on Livin gene and the induction of Hela apoptosis.Methods: Hela cells were transfected with the recombinant plas- mid pGenesil-1-BIRC71,pGenesil-1-BIRC72 and pGenesil-1-HK via Lipofectamine~(TM)2000.The expression levels of Livin was determined in Hela cells before and after transfection by fluorescence quantitative real-time PCR and Western blotting. The apoptosis rate of cells was determined by FCM 24,48 and 72h after transfection.Results: The transfection efficiency at 48h was higher than those at 24 and 72h.After transfection with pGenesil-1-BIRC71 and pGenesil-1-BIRC72,gene and protein levels of Livin were significantly reduced(P