AIM: Survivin is a novel antiapoptotic gene in which three splicing variants have been recently cloned and characterized. Survivin has been found to be abundantly expressed in a wide variety of human malignancies, whe...AIM: Survivin is a novel antiapoptotic gene in which three splicing variants have been recently cloned and characterized. Survivin has been found to be abundantly expressed in a wide variety of human malignancies, whereas it is undetectable in normal adult tissues. We aimed to study the expression of three survivin splicing variants in gastric cancer, and to evaluate the prognostic significance of the expression of survivin variants in gastric cancer.METHODS: Real time quantitative RT-PCR was performed to analyze the expression of survivin variants in 79 paired tumors and normal gastric mucosa samples at the mRNA level. Proliferative and apoptotic activity was measured using Ki-67 immunohistochemical analysis and the TUNEL method, respectively. RESULTS: All the cases tested expressed wild-type survivin mRNA, which was not only the dominant transcript, but also a poor prognostic biomarker (P=0.003). Nonantiapoptostic survivin-2B mRNA was correlated with tumor stage (P=0.001), histological type (P=0.004), and depth of tumor invasion (P=0.041), while survivin-△Ex3 mRNA showed a significant association with apoptosis (P=0.02). CONCLUSION: Wild-type survivin mRNA expression levels are of important prognostic value and significant participation of survivin-2B and survivin-△Ex3 is suggested in gastric cancer development.展开更多
AIM: To evaluate the effects of survivin on cell proliferation and apoptosis in liver cancer. METHODS: MTT assay was used to generate and optimize phosphorothioate antisense oligonucleotides (ODNs)-LipofectamineTM2000...AIM: To evaluate the effects of survivin on cell proliferation and apoptosis in liver cancer. METHODS: MTT assay was used to generate and optimize phosphorothioate antisense oligonucleotides (ODNs)-LipofectamineTM2000 (LiP) compound by varying ODNs (μg): LiP (μL) ratios from 1:0.5 to 1:5. Then, liver cancer cells (HepG2) were transfected with the compound. By using RT-PCR and Western blot, the expression levels of survivin mRNA and proteins were detected in HepG2 cells treated with antisense compounds (ODNs:LiP=1:4), and compared with those treated with sense compounds (1:4) as control. MTT assay was applied to the determination of cell proliferation in HepG2 cells. Active caspase-3 was evaluated by flow cytometric analysis. The morphological changes were assessed by electron microscopy. Laser scanning confocal microscopy was performed to detect the subcellular localization of survivin proteins in treated and untreated cells. RESULTS: Antisense compounds (1:4) down-regulated survivin expression (mRNA and protein) in a dose-dependent manner with an IC50 of 250 nmol/L. Its maximum effect was achieved at a concentration of 500 nmol/L, at which mRNA and protein levels were down-regulated by 80%. The similar results were found in MTT assay. Antisense compound (l:4)-treated cells revealed increased caspase-3-like protease activity compared with untreated cells. Untreated cells as control were primarily negative for the presence of active-caspase-3. As shown by transmission electron microscopy, treated cells with antisense compounds (1:4) resulted in morphological changes such as blebbing and loss of microvilli, vacuolization in the cytoplasm, condensation of the cytoplasm and nuclei, and fragmented chromatin. Immunofluorescence analysis confirmed the presence of survivin protein pool inside the cytoplasm in untreated cells. Labeled-FITC immunofluorescence staining of survivin clearly showed that survivin was distributed mainly in a spotted form inside the cytoplasm. Whereas cells treated with antisense compounds were rare and weak inside the cytoplasm. CONCLUSION: Down-regulation of survivin expression induced by the antisense compounds reduces tumor growth potential, promotes apoptosis and affects the localization of survivin proteins in HepG2 cells. Furthermore, survivin protein is a key molecule associated with proliferation and apoptosis, and antisense oligonucleotides targeting survivin have a bright prospect in the therapy of liver cancer.展开更多
基金Supported by the National Natural Science Foundation of China,No.30271483Grant-in-Aid from the Japanese Ministry of Education,Culture,Sports,Science,and Technology of Japan,No.13470262
文摘AIM: Survivin is a novel antiapoptotic gene in which three splicing variants have been recently cloned and characterized. Survivin has been found to be abundantly expressed in a wide variety of human malignancies, whereas it is undetectable in normal adult tissues. We aimed to study the expression of three survivin splicing variants in gastric cancer, and to evaluate the prognostic significance of the expression of survivin variants in gastric cancer.METHODS: Real time quantitative RT-PCR was performed to analyze the expression of survivin variants in 79 paired tumors and normal gastric mucosa samples at the mRNA level. Proliferative and apoptotic activity was measured using Ki-67 immunohistochemical analysis and the TUNEL method, respectively. RESULTS: All the cases tested expressed wild-type survivin mRNA, which was not only the dominant transcript, but also a poor prognostic biomarker (P=0.003). Nonantiapoptostic survivin-2B mRNA was correlated with tumor stage (P=0.001), histological type (P=0.004), and depth of tumor invasion (P=0.041), while survivin-△Ex3 mRNA showed a significant association with apoptosis (P=0.02). CONCLUSION: Wild-type survivin mRNA expression levels are of important prognostic value and significant participation of survivin-2B and survivin-△Ex3 is suggested in gastric cancer development.
基金Supported by the National Natural Science Foundation of China, No.30171059
文摘AIM: To evaluate the effects of survivin on cell proliferation and apoptosis in liver cancer. METHODS: MTT assay was used to generate and optimize phosphorothioate antisense oligonucleotides (ODNs)-LipofectamineTM2000 (LiP) compound by varying ODNs (μg): LiP (μL) ratios from 1:0.5 to 1:5. Then, liver cancer cells (HepG2) were transfected with the compound. By using RT-PCR and Western blot, the expression levels of survivin mRNA and proteins were detected in HepG2 cells treated with antisense compounds (ODNs:LiP=1:4), and compared with those treated with sense compounds (1:4) as control. MTT assay was applied to the determination of cell proliferation in HepG2 cells. Active caspase-3 was evaluated by flow cytometric analysis. The morphological changes were assessed by electron microscopy. Laser scanning confocal microscopy was performed to detect the subcellular localization of survivin proteins in treated and untreated cells. RESULTS: Antisense compounds (1:4) down-regulated survivin expression (mRNA and protein) in a dose-dependent manner with an IC50 of 250 nmol/L. Its maximum effect was achieved at a concentration of 500 nmol/L, at which mRNA and protein levels were down-regulated by 80%. The similar results were found in MTT assay. Antisense compound (l:4)-treated cells revealed increased caspase-3-like protease activity compared with untreated cells. Untreated cells as control were primarily negative for the presence of active-caspase-3. As shown by transmission electron microscopy, treated cells with antisense compounds (1:4) resulted in morphological changes such as blebbing and loss of microvilli, vacuolization in the cytoplasm, condensation of the cytoplasm and nuclei, and fragmented chromatin. Immunofluorescence analysis confirmed the presence of survivin protein pool inside the cytoplasm in untreated cells. Labeled-FITC immunofluorescence staining of survivin clearly showed that survivin was distributed mainly in a spotted form inside the cytoplasm. Whereas cells treated with antisense compounds were rare and weak inside the cytoplasm. CONCLUSION: Down-regulation of survivin expression induced by the antisense compounds reduces tumor growth potential, promotes apoptosis and affects the localization of survivin proteins in HepG2 cells. Furthermore, survivin protein is a key molecule associated with proliferation and apoptosis, and antisense oligonucleotides targeting survivin have a bright prospect in the therapy of liver cancer.
