Effects of silencing survivin gene by RNA interference on cell apoptosis using ultrasound targeted microbubble destruction techniques / 肿瘤

Objective:To transfect genes using ultrasound targeted microbubble destruction (UTMD) techniques and observe the effects of RNA interference on cervical cancer (HeLa) cell line in silencing survivin gene and inducing apoptosis. Methods: Recombinant expression plasmid of short hairpin RNA (shRNA) targeting survivin gene was constructed. It was co-treated with microbubbles and transfected to cultured HeLa cells followed by exposure to ultrasound (P+UTMD group). Moreover, blank control group (C), plasmid group (P), ultrasound exposure group (US), plasmid and ultrasound exposure group (P+US), plasmid+ Lipofectamine group (P+L) were used as controls, respectively. Transfection efficacy was evaluated by observing the red fluorescence in the cells by fluorescent microscopy and flow cytometry(FCM). Ultrasound intensity and exposure time were optimized. Cell apoptosis was investigated using flow cytometry analysis, Hoechst staining, and DNA ladder method. Expression of survivin mRNA was assessed by RT-PCR. Results: Restrictive enzyme digestion and sequencing analysis verified that the recombinant plasmid was successfully constructed. UTMD significantly increased gene transfection efficacy in cultured HeLa cells (P<0.01). Gene transfer was the most prominent at ultrasound intensity of 1.0 W/cm2 and exposure time of 3 min (P<0.01). RT-PCR showed that the expression of survivin mRNA in P+UTMD group was inhibited by (83.33±2.73)%. The differences were significant compared with any other groups (P<0.01). FCM analysis showed that the apoptosis ratio in P+UTMD group was significantly increased as compared with other groups (P<0.01). Hoechst staining and DNA ladder showed that apparent apoptosis and DNA ladder were detected only in P+UTMD and P+L groups. Conclusions:UTMD effectively enhances the transfection efficacy of expression plasmid. It is a novel and effective non-viral gene transfer system and has promising foreground. UTMD mediates RNA interference silenced survivin gene and induces significant cell apoptosis, which provides a new method for tumor research and gene therapy.

Study of expression of RASSF1A and Survivin genes in non-small cell lung cancer by tissue microarray / 肿瘤研究与临床

Objective To investigate the association between the expression of RASSF1A and Survivin proteins and clinicopathological characteristics in non-small cell lung cancer (NSCLC) and their clinical significance. Methods Expression of RASSF1A and Survivin proteins in the NSCLC tissue microarrays was detected by S-P Immunohistochemistry. Results The positive expression of RASSF1A in NSCLC (46.8 %) was significantly lower than that of in the normal lung tissues (92.9 %) (P < 0.001), but the positive expression of Survivin in NSCLC (78.5 %) was significantly higher than that of in the normal lung tissues (0%) (P<0.001). The percentage of RASSFI A protein expression in the stage Ⅰ and stage Ⅱ of NSCLC was evidently higher than that of in the stage Ⅲ (P<0.001, respectively), however, the percentage of Survivin protein expression in the stage Ⅰ and stage Ⅱ of NSCLC was significantly lower than that of in the stage Ⅲ (P=0.003, P=0.001). The percentage of RASSF1A in NSCLC with lymph node metastasis was observably lower than that of in cases without lymph node metastasis (P<0.05). Furthermore, there was observably negative correlation between expression of RASSF1A protein and that of Survivin protein in NSCLC (r=-0.780, P<0.001). Conclusion The loss expression of RASSF1A protein, over expression of Survivin protein and loss balance of expression of both RASSF1A and Survivin proteins in NSCLC might play important roles in the development and progression of NSCLC; RASSF1A and Survivin proteins might be acted as one helpful molecular marker to predict the lymph node metastasis and the prognosis of NSCLC.

Expression and Clinical Significance of Survivin in Acute Leukemia / 中国医师杂志

Objective To study the expression and clinical significance of survivin in acute leukemia. Methods By using semi-quantitative RT-PCR technique, survivin gene expression was detected in 43 acute leukemia(AL). Results Survivin gene expression rate in the cells of AL patients at diagnosis was 69.77%(30/43). Survivin mRNA expression rate in both 18 acute lymphocytic leukemia (ALL) and 25 acute nonlymphocytic leukemia(ANLL) (72.22% and 68.0% respectively) was signficantly higher than that in control group (33.33%,P0.05). Conclusions Survivin gene was highly expressed in AL patients and could be a potential target for treatment of AL.

Inducible effect of antisurvivin oligonucleotide on apoptosis in osteosarcoma / 中国病理生理杂志

AIM: Osteosarcoma OS-732 cell line was used to investigate the effect of antisense oligonucleotide (ASODN) on survivin expression and its inducible effect on tumor cells apoptosis. METHODS: ASODN of specific target survivin was designed, synthesized and then transfer to OS-732 cell line with different concentrations and time points. At the same time blank control group, sense oligonucleotide (SOND) group were set up for comparison. Reverse tanscriptionase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry were used to detect the expressions of survivin mRNA and protein in each OS-732 cell line group. Acridine orange /ethidium bromide (AO/EB) staining and flow cytometry were used to detect the apoptosis level and morphologic change. Mononuclear cell direct cytotoxicity assay (MTT) was used to estimate cell growth suppression. Kinase activity assay method was used to estimate the activity of caspase-3 in the cells. RESULTS: Compared with control group and SOND group, in ASODN groups, the expression of survivin mRNA and protein were obviously weaken, apoptosis rate and caspase-3 activity apparently increased, cells growth was inhibited. In each ASODN group, the effect above-mentioned has time- and concentration-dependent manner. There was no obviously difference of each index in each SODN and blank control groups. CONCLUSION: ASODN down-regulated the expression of survivin gene in OS-732 cell line specifically, and activated apoptosis effectively. It plays an important role in inducing tumor apoptosis and suppressing cell proliferation.

Construction of recombinant adenovirus vector containing human survivin gene and expression in fibroblast / 解放军医学杂志

Objective To construct the recombinant adenovirus vector which contained human survivin, then being transfected into dendritic cells. Methods Full length survivin cDNA was obtained from recombinant plasmid pCITE survivin by PCR, the PCR product was double digested with restrictive endonucleases Kpn Ⅰ and Xhol Ⅰ, then being inserted directionally into pAdTrack CMV. The plasmid of pAdTrack survivin was lined with PmeⅠ, the fragment containing survivin was reclaimed and transfected into E coli BJ5183. After having been screened by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the green fluorescence protein (GFP) and by PCR method. At the time, the culture media containing virus was transfected into fibroblast, and the expression of survivin was proved by observing the GFP and the Western blot method. Results The recombined adenovirus survivin was constructed successfully and the titer was about 1 65?10 8 pfu/ml. A band was observed by Western bolt and its relative molecular mass was about 16 5kD. Conclusion A recombinant adenovirus vector containing human survivin was constructed successfully, and the survivin protein was expressed by fibroblast. It lays the foundation for further study on survivin as a target antigen for gene therapy