RESUMO
OBJECTIVE: To construct eukaryotic expression vectors by using the pSilencer3. 1-H1 neo vector for inhibiting human survivin gene by RNA interference, and to detect the effect of the silenced survivin gene on PC-3 cells. METHODS: Three target gene segments were synthesized and cloned into the pSilencer3. 1-H1 neo vector respectively to construct three recombinant eukaryotic expression vectors: pSilencer3. 1-SVV1, pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3, which were identified by enzyme digestion analysis and DNA sequencing. Then the PC-3 cells were transfected with the recombinant vectors and the interference effect detected by RT-PCR, Western blot and immunohistochemical staining. The apoptosis index of the PC-3 cells was detected by flow cytometry and their proliferation detected by MTT method. RESULTS: Enzyme digestion analysis and DNA sequencing showed that three target segments were cloned into pSilencer3. 1-H1-neo vectors. The results of RT-PCR, Western blot and immunohistochemical staining indicated that pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors could knock down the transcription and expression of survivin gene. After transfected with pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors, the apoptosis index of the PC-3 cells was increased by 10% - 15% and their growth obviously slowly down. CONCLUSION: The transcription and expression of survivin gene were inhibited effectively by the recombinant eukaryotic expression vectors (pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3) in the prostate cancer cell line PC-3. After transfected with pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors, the apoptosis index of the PC-3 cells was increased and their growth inhibited.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias da Próstata/metabolismo , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Apoptose , Linhagem Celular Tumoral , Regulação para Baixo , Vetores Genéticos , Humanos , Proteínas Inibidoras de Apoptose , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/patologia , Survivina , TransfecçãoRESUMO
AIM: To explore the expressions of Cyclin E2 in nasopharyngeal carcinoma (NPC) and clinic opathological parameters, and to investigate the role of Cyclin E2 in oncogenesis, progress, invasion and metastasis of NPC. METHODS: Sixty patients with NPC and twenty-one noncancerous nasopharyngeal epithelial samples were retrospectively studied from year 2000 to 2006, The protein and mRNA expressions of Cyclin E2 was detected by immunohistochemical staining and RT-PCR. RESULTS: The protein expressions rates of Cyclin E2 were 75% and 9.5% in nasopharyngeal samples and noncancerous nasopharyngeal epithelial samples, It was significantly higher in NPC than in normal controls (P<0.01). Furthermore, the protein expression was significantly higher in NPC patients with clinical staging and cervical lymph node or poor prognosis than in NPC patients without(P<0.01). The expression in NPC was not correlated with gender or age (P>0.05). The mRNA expression of Cyclin E2 was significantly higher in NPC than in normal controls (P<0.01). CONCLUSION: These findings demonstrate that Cyclin E2 may play important roles in the invasion, metastasis, and poor prognosis of NPC.