AIM: To investigate the inhibitory effect of a specific small survivin interfering RNA (siRNA) on cell proliferation and the expression of survivin in human gastric carcinoma cell line SGC-7901. METHODS: To knockdown ...AIM: To investigate the inhibitory effect of a specific small survivin interfering RNA (siRNA) on cell proliferation and the expression of survivin in human gastric carcinoma cell line SGC-7901. METHODS: To knockdown survivin expression, a small interfering RNA targeting against survivin was synthesized and transfected into SGC-7901 cells with lipofectamineTM2000. The downregulation of survivin expression at both mRNA and protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Cell proliferation inhibition rates were determined by methyl thiazolyl tetrazolium (MTT) assay. The effect of survivin siRNA on cell cycle distribution and cell apoptosis was determined by flow cytometry (FCM). RESULTS: RNA interference could efficiently suppress the survivin expression in SGC-7901 cells. At 48 h after transfection, the expression inhibition rate was 44.52% at mRNA level detected by RT-PCR and 40.17% at protein level by Western blot analysis. Downregulation of survivin resulted in significant inhibition of tumor cell growth in vitro. The cell proliferation inhibition rates at 24, 48 and 72 h after survivin siRNA and non-siliencing siRNA transfection, were 34.06%, 47.61% and 40.36%, respectively. The apoptosis rate was 3.56% and the number of cells was increased in G0/G1 phase from 38.2% to 88.6%, and decreased in S and G2/M phase at 48 h after transfection. CONCLUSION: Downregulation of survivin results in significant inhibition of tumor growth in vitro. The inhibition of survivin expression can induce apoptosis of SGC-7901 cells. The use of survivin siRNA deserves further investigation as a novel approach to cancer therapy.展开更多
AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 c...AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.展开更多
BACKGROUND: T he second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) was recently identified as a protein that is released from mitochondria in resp...BACKGROUND: T he second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) was recently identified as a protein that is released from mitochondria in response to apoptotic stimuli and promotes apoptosis by antagonizing inhibitor of apoptosis proteins. Furthermore, Smac/DIABLO plays an important regulatory role in the sensitization of cancer cells to both immune- and drug-induced apoptosis. However, little is known about the clinical significance of Smac/DIABLO in various cancers including hepatocellular carcinoma (HCC). This study was undertaken to investigate the expression of Smac and Survivin and their relationship with the apoptosis in primary HCC. METHODS: The expression of Smac and Survivin proteins was evaluated by immunohistochemistry. The mRNA expression of Smac and Survivin was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) in HCC tissues of 50 patients, para-carcinoma tissues of 20 patients, and normal liver tissues of 15 patients. RESULTS: Smac mRNA was detected by RT-PCR in HCC tissues of 21 (42.0%) of the 50 patients, para-carcinoma tissues of 19 (95.0%) of the 20 patients, and normal liver tissues of 15 (100%) of the 15 patients. Survivin mRNA was found in HCC tissues of 46 of the 50 patients, para- carcinoma tissues of 2 of the 20 patients, and normal liver tissues of 0 of 15 patients. Immunohistochemistry revealed Smac protein in HCC tissues of 20 patients (40.0%), in para-carcinoma tissues of 18 patients (90.0%), and normal liver tissues of 15 patients (100.0%). The expression of Smac was significantly different in HCC tissues and non- HCC tissues. Survivin protein was found in HCC tissuesin 45 patients, para-carcinoma tissues in 2 patients, and normal liver tissues in none of the patients. The expression of Survivin was significantly different in HCC tissues and non-HCC tissues. CONCLUSION: Smac inhibits apoptosis of HCC cells by suppression of Survivin, and the two genes probably form an important link in the展开更多
AIM: To select the optimal antisense accessible sites of survivin, a highly expressed gene in tumor tissues, in order to explore a novel approach to improve biological therapy of gastric cancer. METHODS: The 20 mer ra...AIM: To select the optimal antisense accessible sites of survivin, a highly expressed gene in tumor tissues, in order to explore a novel approach to improve biological therapy of gastric cancer. METHODS: The 20 mer random oligonucleotide library was synthesized, hybridized with in vitro transcribed total survivin cRNA, then digested by RNase H. After primer extension and autoradiography, the antisense accessible sites (AAS) of survivin were selected. Then RNADraw software was used to analyze and choose the AAS with obvious stem-loop structures, according to which the complementary antisense oligonucleotides (AS-ODNs) were synthesized and transferred into survivin highly- expressing gastric cancer cell line MKN-45. Survivin expression was detected by RT-PCR and Western Blotting. Cellular growth activities were assayed by tetrazolium bromide (MTT) colorimetry. Cellular ultrastructure was observed by electronic microscopy, while apoptosis was detected by annexin V-FITC and propidium iodide staining flow cytometry. RESULTS: Thirteen AAS of survivin were selected In vitro. Four AAS with stem-loop structures were chosen, locating at 207-226 bp, 187-206 bp, 126-145 bp and 44-63 bp of survivin cDNA respectively. When compared with non-tranfection controls, their corresponding AS-ODNs (AS-ODN1, AS-ODN2, AS-ODN3 and AS-ODN4) could reduce Survivin mRNA levels in MKN-45 cells by 54.3±±1.1% (t= 6.12, P<0.01), 86.1±±1.0% (t= 5.27, P<0.01), 32.2±±1.3% (t= 7.34, P<0.01) and 56.2±±0.9% (t = 6.45, P<0.01) respectively, while survivin protein levels were decreased by 42.2±±2.5% (t = 6.26, P<0.01), 75.4±±3.1% (t= 7.11, P<0.01), 28.3±±2.0% (t= 6.04, P<0.01) and 45.8±±1.2% (t = 6.38,P<0.01) respectively. After transfection with 600 nmol/L AS-ODN1-AS-ODN4for 24 h, cell growth was inhibited by 28.12±±1.54% (t= 7.62, P<0.01), 38.42±±3.12% (t= 7.75, P<0.01), 21.46±±2.63% (t= 5.94, P<0.01) and 32.12±1.77% (t = 6.17, P<0.01) respectively. Partial cancer cells presented the characteristic morphological changes of apopt展开更多
文摘AIM: To investigate the inhibitory effect of a specific small survivin interfering RNA (siRNA) on cell proliferation and the expression of survivin in human gastric carcinoma cell line SGC-7901. METHODS: To knockdown survivin expression, a small interfering RNA targeting against survivin was synthesized and transfected into SGC-7901 cells with lipofectamineTM2000. The downregulation of survivin expression at both mRNA and protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Cell proliferation inhibition rates were determined by methyl thiazolyl tetrazolium (MTT) assay. The effect of survivin siRNA on cell cycle distribution and cell apoptosis was determined by flow cytometry (FCM). RESULTS: RNA interference could efficiently suppress the survivin expression in SGC-7901 cells. At 48 h after transfection, the expression inhibition rate was 44.52% at mRNA level detected by RT-PCR and 40.17% at protein level by Western blot analysis. Downregulation of survivin resulted in significant inhibition of tumor cell growth in vitro. The cell proliferation inhibition rates at 24, 48 and 72 h after survivin siRNA and non-siliencing siRNA transfection, were 34.06%, 47.61% and 40.36%, respectively. The apoptosis rate was 3.56% and the number of cells was increased in G0/G1 phase from 38.2% to 88.6%, and decreased in S and G2/M phase at 48 h after transfection. CONCLUSION: Downregulation of survivin results in significant inhibition of tumor growth in vitro. The inhibition of survivin expression can induce apoptosis of SGC-7901 cells. The use of survivin siRNA deserves further investigation as a novel approach to cancer therapy.
文摘AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.
文摘BACKGROUND: T he second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) was recently identified as a protein that is released from mitochondria in response to apoptotic stimuli and promotes apoptosis by antagonizing inhibitor of apoptosis proteins. Furthermore, Smac/DIABLO plays an important regulatory role in the sensitization of cancer cells to both immune- and drug-induced apoptosis. However, little is known about the clinical significance of Smac/DIABLO in various cancers including hepatocellular carcinoma (HCC). This study was undertaken to investigate the expression of Smac and Survivin and their relationship with the apoptosis in primary HCC. METHODS: The expression of Smac and Survivin proteins was evaluated by immunohistochemistry. The mRNA expression of Smac and Survivin was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) in HCC tissues of 50 patients, para-carcinoma tissues of 20 patients, and normal liver tissues of 15 patients. RESULTS: Smac mRNA was detected by RT-PCR in HCC tissues of 21 (42.0%) of the 50 patients, para-carcinoma tissues of 19 (95.0%) of the 20 patients, and normal liver tissues of 15 (100%) of the 15 patients. Survivin mRNA was found in HCC tissues of 46 of the 50 patients, para- carcinoma tissues of 2 of the 20 patients, and normal liver tissues of 0 of 15 patients. Immunohistochemistry revealed Smac protein in HCC tissues of 20 patients (40.0%), in para-carcinoma tissues of 18 patients (90.0%), and normal liver tissues of 15 patients (100.0%). The expression of Smac was significantly different in HCC tissues and non- HCC tissues. Survivin protein was found in HCC tissuesin 45 patients, para-carcinoma tissues in 2 patients, and normal liver tissues in none of the patients. The expression of Survivin was significantly different in HCC tissues and non-HCC tissues. CONCLUSION: Smac inhibits apoptosis of HCC cells by suppression of Survivin, and the two genes probably form an important link in the
基金Supported by National Natural Science Foundation of China, No.30200284Science Foundation of Huazhong University of Science and Technology
文摘AIM: To select the optimal antisense accessible sites of survivin, a highly expressed gene in tumor tissues, in order to explore a novel approach to improve biological therapy of gastric cancer. METHODS: The 20 mer random oligonucleotide library was synthesized, hybridized with in vitro transcribed total survivin cRNA, then digested by RNase H. After primer extension and autoradiography, the antisense accessible sites (AAS) of survivin were selected. Then RNADraw software was used to analyze and choose the AAS with obvious stem-loop structures, according to which the complementary antisense oligonucleotides (AS-ODNs) were synthesized and transferred into survivin highly- expressing gastric cancer cell line MKN-45. Survivin expression was detected by RT-PCR and Western Blotting. Cellular growth activities were assayed by tetrazolium bromide (MTT) colorimetry. Cellular ultrastructure was observed by electronic microscopy, while apoptosis was detected by annexin V-FITC and propidium iodide staining flow cytometry. RESULTS: Thirteen AAS of survivin were selected In vitro. Four AAS with stem-loop structures were chosen, locating at 207-226 bp, 187-206 bp, 126-145 bp and 44-63 bp of survivin cDNA respectively. When compared with non-tranfection controls, their corresponding AS-ODNs (AS-ODN1, AS-ODN2, AS-ODN3 and AS-ODN4) could reduce Survivin mRNA levels in MKN-45 cells by 54.3±±1.1% (t= 6.12, P<0.01), 86.1±±1.0% (t= 5.27, P<0.01), 32.2±±1.3% (t= 7.34, P<0.01) and 56.2±±0.9% (t = 6.45, P<0.01) respectively, while survivin protein levels were decreased by 42.2±±2.5% (t = 6.26, P<0.01), 75.4±±3.1% (t= 7.11, P<0.01), 28.3±±2.0% (t= 6.04, P<0.01) and 45.8±±1.2% (t = 6.38,P<0.01) respectively. After transfection with 600 nmol/L AS-ODN1-AS-ODN4for 24 h, cell growth was inhibited by 28.12±±1.54% (t= 7.62, P<0.01), 38.42±±3.12% (t= 7.75, P<0.01), 21.46±±2.63% (t= 5.94, P<0.01) and 32.12±1.77% (t = 6.17, P<0.01) respectively. Partial cancer cells presented the characteristic morphological changes of apopt
