Primary liver cancer is the fifth most common malignan- cy in the world and is a leading cause of cancer-related mortality.Available treatment for hepatocellular carcino- ma(HCC),the commonest primary liver cancer,is ...Primary liver cancer is the fifth most common malignan- cy in the world and is a leading cause of cancer-related mortality.Available treatment for hepatocellular carcino- ma(HCC),the commonest primary liver cancer,is rarely curative and there is a need to develop therapy that is more effective.Specific and powerful gene silencing that can be achieved by activating RNA interference(RNAi) has generated enthusiasm for exploiting this pathway for HCC therapy.Many studies have been carried out with the aim of silencing HCC-related cellular oncogenes or the hepatocarcinogenic hepatitis B virus(HBV)and hepatitis C virus(HCV).Proof of principle studies have demonstrated promising results,and an early clinical trial assessing RNAi-based HBV therapy is currently in progress.Although the data augur well,there are several significant hurdles that need to be overcome before the goal of RNAi-based therapy for HCC is realized.Particu- larly important are the efficient and safe delivery of RNAi effecters to target malignant tissue and the limitation of unintended harmful non-specific effects.展开更多
AIM:To explore the effect of silencing of signal transducer and activator of transcription 3(STAT3) expression by RNA interference(RNAi) on growth of human hepatocellular carcinoma(HCC) in tumor-bearing nude mice in v...AIM:To explore the effect of silencing of signal transducer and activator of transcription 3(STAT3) expression by RNA interference(RNAi) on growth of human hepatocellular carcinoma(HCC) in tumor-bearing nude mice in vivo.METHODS:To construct the recombinant plasmid of pSilencer 3.0-H1-STAT3-siRNA-GFP(pSH1-siRNA-STAT3) and establish the tumor-bearing nude mouse model of the HCC cell line SMMC7721,we used intratumoral injection together with electroblotting to transfect the recombinant plasmid pSH1-siRNA-STAT3 into the transplanted tumor.The weight of the nude mice and tumor volumes were recorded.STAT3 gene transcription was detected by semi-quantitative reverse transcription polymerase chain reaction(RTPCR).Level of protein expression and location of STAT3 were determined by Western blotting and immunohistochemical staining.STAT3-related genes such as survivin,c-myc,VEGF,p53 and caspase3 mRNA and protein expression were detected in tumor tissues at the same time.The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay was used to detect apoptosis of tumor cells.RESULTS:The weight of the treated nude mice increased,and the tumor volume decreased markedly compared with those of the mock-treated and negative control groups(P < 0.01).The results of RT-PCR and Western blotting showed that mRNA and protein levels of STAT3 declined markedly in the treated group.The change in STAT3-related gene expression in tumor tissues at the mRNA and protein level also varied,the expression of survivin,VEGF and c-myc were obviously reduced,and expression of p53 and caspase3 increased(P < 0.01).Most of the tumor tissue cells in the treated group developed apoptosis that was detected by TUNEL assay.CONCLUSION:Silencing of STAT3 expression by RNAi significantly inhibits expression of STAT3 mRNA and protein,and suppresses growth of human HCC in tumor-bearing nude mice.The mechanism may be related to down-regulation of survivin,VEGF and c-myc and up-regulation of p53 and caspase3 expression.Accordingly,the STAT3 gene may act as an important and effective target in gene therapy of HCC.展开更多
Objective: To explore the inhibition of the expression of c-Myc in human hepatocellular carcinoma via vector-based RNA interference (RNAi). Methods: Two RNA interference DNA templates targeting c-Myc oncogene were des...Objective: To explore the inhibition of the expression of c-Myc in human hepatocellular carcinoma via vector-based RNA interference (RNAi). Methods: Two RNA interference DNA templates targeting c-Myc oncogene were designed via online software and synthesized. By ligation, the fragments were inserted into pSilencer 1.0-U6 to construct the recombinant siRNA expressing plasmids. The identified recombinants were introduced into BEL-7402 cells with Lipofactamine. The inhibition of c-Myc expression, together with the expression of CDK4, hTERT and Gadd45β in c-Myc down-regulated BEL-7402 cells, were analyzed by semi-quantitative RT-PCR. Results: Two recombinant plasmids pSic-myc-1 and pSic-myc-2, which direct the yields of siRNAs targeting c-Myc in cells, were constructed. Among which, pSic-myc-2 was shown to trigger a RNAi-mediated inhibition of expression of c-Myc in BEL-7402 by up to 90%. In c-Myc knockdown BEL-7402 cells, the expression of CDK4 and hTERT were down-regulated with a ratio of 85% and 57%, respectively, while the expression of Gadd45β was up-regulated by up to 110%. Conclusion: The expression of c-Myc in BEL-7402 could be suppressed by vector-based RNA interference successfully. The knockdown of c-Myc in turn resulted in the changes of expression of genes related to cell proliferation and apoptosis. Thus, our study provided a preliminary data in searching of a c-Myc-targeted RNAi therapy of human hepatocellular carcinoma.展开更多
Objective: The effects on cell-cycle and p53 expression in hepatoma cell line SK-Hep-1 were explored by transfecting exogenous p53 small double stranded RNA (dsRNA) into the SK-Hep-1 cells. Methods: p53 dsRNA and EGFP...Objective: The effects on cell-cycle and p53 expression in hepatoma cell line SK-Hep-1 were explored by transfecting exogenous p53 small double stranded RNA (dsRNA) into the SK-Hep-1 cells. Methods: p53 dsRNA and EGFP dsRNA were synthesized. SK-Hep-1 (wtp53) cell line was transfected with 200 ng and 400 ng p53 dsRNA or EGFP and EGFP+EGFP dsRNA (as positive control) or 9% NaCl (as blank control) by liposome transfection technique. Flow cytometry was adopted to measure the effects of p53 dsRNA on cell cycle. Expression of p53 protein was detected by Western-Blotting at 48 h after transfecting p53 dsRNA. Results: The number of G0-G1 phase SK-Hep-1 cells, which were transfected with 200 ng p53 dsRNA, was decreased by 52.53% comparing with the control, and decreased by 50.29% (P<0.05) comparing with the positive control cells transfected with same dosage of EGFP+EGFP dsRNA. The number of S phase cells, which were transfected with 200 ng p53 dsRNA, was increased by 146.8% comparing with the control, and increased by 128.62% (P<0.05) comparing with the positive control cells transfected with same dosage of EGFP+EGFP dsRNA. The number of G2-M phase cells, which were transfected with 200 ng p53 dsRNA, was increased by 30.56% (P<0.05) comparing with the control, and increased by 21.63% (P>0.05) comparing with the positive control cells transfected with same dosage of EGFP+EGFP dsRNA. After 48 h, p53 protein expression was not detected in the SK-Hep-1 cells transfected with p53 dsRNA. Conclusion: p53 dsRNA can obviously improve the proliferation of SK-Hep-1 cells, and suppress p53 protein expression of SK-Hep-1 cells, the former may be related to of the latter.展开更多
Background Both survivin and lung resistance related protein (LRP) are hepatocellular carcinoma (HCC). But the relationship between survivin and LRP is investigate the effects of down-regulation of survivin on LRP...Background Both survivin and lung resistance related protein (LRP) are hepatocellular carcinoma (HCC). But the relationship between survivin and LRP is investigate the effects of down-regulation of survivin on LRP expressions and the both in vitro and in vivo. related to the chemoresistances in ndefinite. The aim of this study was to reversal of chemoresistances in HCC Methods The expressions of survivin were detected by RT-PCR and Western blotting in HCC cell line SMMC-7721 and SMMC-7721/ADM. The sensitivities of these two cell lines to ADM were evaluated by MTT assays. SiRNA which targeted survivin was transfected into SMMC-7721/ADM cells, then the sensitivity of SMMC-7721/ADM cells to ADM and the expressions of survivin and LRP were detected respectively. SMMC-7721/ADM cells were transplanted subcutaneously into nude mice to establish xenograft tumors. Antitumor activities of RNA interference (RNAi) targeting survivin, various doses of ADM and combination therapies were observed respectively. Possible toxicities were evaluated. LRP expression changes were tested. Student's ttest was used for evaluating statistical significance. Results The expressions of survivin in SMMC-7721/ADM cell line showed significant elevation compared to those in SMMC-7721 cell line (P 〈0.05). Positive siRNA down-regulated the expressions of survivin significantly (P 〈0.05). SiRNA targeting survivin could sensitize SMMC-7721/ADM cells to ADM and down-regulate the expressions of LRP significantly (P 〈0.05). Growths of the tumors were significantly inhibited in positive siRNA group as compared with those in the control group from the 8th day (P 〈0.05). Combination therapies caused significant tumor inhibitions compared with tumors of nude mice in the other three groups respectively (P 〈0.05). No toxicities were found in nude mice treated by siRNA and combination therapies. The expressions of LRP were markedly reduced in tumors treated with siRNA targeting survivin (P 〈0.05). Conclusions Down regulation of survivin gene by RNAi can increase chemosensitivity of HCC both in vitro and in vivo. The reversal of drug resistance may be reduced through the inhibitions of LRP.展开更多
Background The relationship between apoptosis and tumors is a major focus in cancer research. RNA interference (RNAi) technology has emerged as a very potent tool to generate cellular knockdown phenotypes of a desi...Background The relationship between apoptosis and tumors is a major focus in cancer research. RNA interference (RNAi) technology has emerged as a very potent tool to generate cellular knockdown phenotypes of a desired gene. The aim of this study was to explore the effect of siRNA specific to the protein casein kinase 2α (CK2α) on apoptosis of laryngeal carcinoma cells and to explore possible mechanisms.Methods An siRNA expression plasmid specific to CK2α, psiRNA-hH1neo-CK2α, and a non-specific siRNA expression plasmid, psiRNA-hH1neo-cont, were constructed and transfected into Hep-2 cells by a lipofectamine method. The mRNA and protein levels of CK2α in transfected cells were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analysis. Apoptosis was measured by Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) double-staining methods. The morphological changes to Hep-2 cells were observed under transmission electron microscopy (TEM). The levels of Bcl-2 and Bax proteins were measured by Western blotting analysis.Results Levels of CK2α mRNA and protein were significantly decreased in the psiRNA-hH1neo-CK2α group compared to the other groups (P 〈0.05). The apoptotic rate of the psiRNA-hH1neo-CK2α transfected group was significantly higher compared to that in the untransfected group and the siRNA-hH1neo-cont transfected group (25.66%±0.83%, 3.66%±0.43%, and 5.18%±0.22%) (P 〈0.05). Compared with the untransfected group and the siRNA-hH1neo-cont transfected group, the psiRNA-hH1neo-CK2α transfected group presented with classical ultrastructural features of apoptosis, such as karyopyknosis, chromatic agglutination adjacent to the nuclear membrane, and apoptotic bodies. Compared with the other two groups, the level of Bcl-2 protein in the psiRNA-hH1neo-CK2α transfected group was decreased (0.20±0.09 vs. 0.72±0.16, 0.56±0.11, P 〈0.01), while the Bax protein level was increased (0.81±0.17 vs. 0.26±0.12, 0.33±0.17, P 〈0.01) and the ratio of Bcl-2 to Bax was decreased (0.25±0.05 vs. 2.76±0.21, 1.70±0.22, P 〈0.01).Conclusions The siRNA expression plasmid specific to CK2α could suppress CK2α expression and induce the apoptosis of laryngeal carcinoma cells. This is possibly by decreasing the Bcl-2/Bax ratio. CK2α may provide a potential therapeutic target against human laryngeal carcinoma.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 Sa/I 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-avidinbiotin-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
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
Objective:To knockdown the C-erbB2 gene in salivary gland adenoid cystic carcinoma SACC-83 cells using RNA interference,and determine the effect of silencing C-erbB2 on cell proliferation.Methods:C-erbB2-siRNA was t...Objective:To knockdown the C-erbB2 gene in salivary gland adenoid cystic carcinoma SACC-83 cells using RNA interference,and determine the effect of silencing C-erbB2 on cell proliferation.Methods:C-erbB2-siRNA was transfected into SACC-83 cells.RT-PCR and immunohistochemistry were used to detect C-erbB2 expression in SACC-83 cells.Cell proliferation was measured by the MTT assay and gene knockdown was achieved by RNA interference.Apoptosis was analyzed by flow cytometry.Results:Compared with the control,C-erbB2 mRNA expression was decreased in the C-erbB2-siRNA transfection group,and immunohistochemical analysis indicated that C-erbB2 protein expression was decreased.After C-erbB2-siRNA was transfected for 48 h,absorbance at 570 nm (MTT)was 0.185±0.021 compared with 0.354±0.034,0.299±0.053,and 0.314±0.049 in the blank control,liposome control and negative control siRNA groups,respectively.The differences were statistically significant (P〈0.05)between the C-erbB2-siRNA group and the control groups.Following the C-erbB2 knockdown,the percentage of apoptotic cells was 5.63%compared with 2.04%,2.85%,and 2.98%in the three control groups,respectively.Proliferation of SACC-83 cells was inhibited,and early apoptotic cells were increased.Conclusion: RNA interference can effectively silence C-erbB2 gene expression and inhibit growth of SACC-83 cells,which indicates the potential of targeting this gene as a novel gene therapy approach for the treatment of salivary gland adenoid cystic carcinoma.展开更多
Objective:To study the inhibition effect of siRNA on the expression of Wisp-1 gene in Hca-F of mouse hepatocellular carcinoma cells strain and also its effect on the proliferation,migration and adhesion of hepatocellu...Objective:To study the inhibition effect of siRNA on the expression of Wisp-1 gene in Hca-F of mouse hepatocellular carcinoma cells strain and also its effect on the proliferation,migration and adhesion of hepatocellular carcinoma cells.Methods:Three expression vectors of siRNA were constructed.Lipo2000 was employed to transfect Hca-F cells and Western blot was used to detect the inhibition effect of siRNA on the expression of Wisp-1 gene.Afterwards,CCK8 was adopted to detect the effect of Wisp-1 siRNA on the proliferation of Hca-F cells;Annexin V-FTTC/PI double staining flow cytometry was used to detect the effect of Wisp-1 siRNA on the apoptosis of Hca-F cells;Transwell was used to detect the effect of Wisp-1 siRNA on the migration of Hca-F cells.The in vitro cell adhesion kit was used to detect of Wisp-1 siRNA on the change in the components of extracellular matrix to which Hca-F cells adhered.Western blot was used to detect the activation of protein kinase B(AKT)/glycogen synthase kinase-3(3pathway and the expression of downstream target protein p53 and matrix metal!oproteinases-2.Results:The siRNA showed interference effect on the expression of Wisp-1 gene.Compared with the control group,after being transfected to cells,Wisp-1 siRNA could significantly inhibit the proliferation,migration and adhesion of Hca-F cells and also promote the cell apoptosis,which was related to the down-regulated phosphorylation of AKT and glycogen synthase kinase-3β and the expression of p53 and matrix metalIoproteinases-2(P<0.05).Conclusions:The inhibition of Wisp-1 expression can reduce the proliferation,migration and adhesion of mouse hepatocellular carcinoma cells,which is related to the AKT/ glycogen synthase kinase-3 β pathway.Wisp-1 gene may be the potential target to cure the hepatocellular carcinoma.展开更多
The development of human endometrial carcinoma(HEC) is a complex pathologic process involves several oncogenes and tumor suppressor genes.The full-length paired-box gene 2(pax2),a recently discovered oncogene,promotes...The development of human endometrial carcinoma(HEC) is a complex pathologic process involves several oncogenes and tumor suppressor genes.The full-length paired-box gene 2(pax2),a recently discovered oncogene,promotes cell proliferation and growth and inhibits apoptosis of HEC cells.Here,we examined the effect of pax2 small interfering RNA(siRNA) on the growth of transplanted HEC cells in nude mice.The expression of Pax2 in 21 cases of normal endometrium and 38 cases of HEC was examined by immohistochemistry(IHC).HEC models were developed by subcutaneously transferring HEC cells into nude mice,followed by treatment with empty lentivirus vector,lentivirus vector-based pax2 siRNA,and phosphate buffered saline,respectively.Four weeks later,tumor size was measured,tumor inhibition rate was calculated,and histological analyses were conducted after staining with hematoxylin and eosin.The expression of Pax2 and Bcl-2 was detected by Western blot;proliferating cell nuclear antigen(PCNA) was detected by IHC.Significant differences were observed in the positive rate of Pax2 between normal endometrium and HEC(14.2% vs.60.5%,P<0.01).The expression index of Pax2 in well differentiated tumors was 1.88±1.68,much lower than that in tumors of moderate(3.07±1.96,P<0.05) or poor differentiation(5.45±2.76,P<0.01).Tumor necrosis increased,nuclear basophilia stain decreased,tumor growth was inhibited,and PCNA,Pax2,and Bcl-2 expression was reduced in HEC models treated with pax2 siRNA.These results indicate that Pax2 expression is related to HEC tumor biology with the increased expression of Pax2 correlated to malignancy.pax2 siRNA down-regulates Pax2 expression and inhibits tumorigenesis of HEC in nude mice,possibly due to cell apoptosis and the inhibition of tumor proliferation induced by down-regulation of Bcl-2.展开更多
Objective: To study the radiosensitivity of SiHa cells with Ku80 inhibition by RNAi. Methods: pKu80-siRNA and pNeg-siRNA were constructed and then transfected into SiHa cells. Western blot and RT-PCR were applied to m...Objective: To study the radiosensitivity of SiHa cells with Ku80 inhibition by RNAi. Methods: pKu80-siRNA and pNeg-siRNA were constructed and then transfected into SiHa cells. Western blot and RT-PCR were applied to measure the expression of Ku80. After 10 Gy irradiation with 6 MV X-ray, cells were harvested at 20 h, 28 h and 48 h, and analyzed by flow cytometry for apoptosis rate. The SF2 and α, β values of cells were acquired by clone formation array. Results: Two stable transfection cell clones SiHa/Ku80-siRNA and SiHa/Neg-siRNA were screened from the pKu80-siRNA and pNeg-siRNA transfected cells. Western blot and RT-PCR indicated that the expression of Ku80 was suppressed by the Ku80-siRNA. The apoptosis rates of SiHa/Ku80-siRNA were higher than control cells at 28 h and 48 h after X-ray irradiation (P < 0.05). The SF2 value of SiHa/Ku80-siRNA (0.422) was lower than that of SiHa/Neg-siRNA cells (0.587) or untransfected cells (0.583), and the α value of SiHa/Ku80-siRNA was 0.295 ± 0.016, higher than that of SiHa/Neg-siRNA (0.176 ± 0.010) or control cells (0.188 ± 0.011). Conclusion: Inhibition of Ku80 could enhance the radiosensitivity of SiHa, which showed that Ku80 could be a good target for molecular therapy.展开更多
OBJECTIVE To study the efficiency of silencing survivin gene expression by an anti-survivin small RNA expression plasmid and to examine its impact on apoptosis, proliferation and invasive ability of the SW480 colorec-...OBJECTIVE To study the efficiency of silencing survivin gene expression by an anti-survivin small RNA expression plasmid and to examine its impact on apoptosis, proliferation and invasive ability of the SW480 colorec-tal cancer cell line. METHODS An expression plasmid of survivin siRNA (pRNAT/sur-siR-NA) was constructed and transduced into SW480 cells. Western blot analysis and a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) were used to measure the change in expression level of the survivin protein and mRNA after transduction of the expression plasmid into the SW480. Apoptosis, proliferation, and invasive ability of the SW480 cells was evaluated by flow cytometry, MTT assay and Boy-den Chamber Assay, respectively. RESULTS The expression plasmid (pRNAT/sur-siRNA) against survivin down-regulated survivin protein and mRNA expression dramatically, with a down-regulation of 85% and 80%, respectively. After transfection of the pRNAT/sur-siRNA to the SW480 cells, the apoptotic rate of the pRNAT/ sur-siRNA/SW480 cells was 16.9%, and the proliferation rate was 37.4%, significantly different compared to the controls (P<0.01). Cell invasive studies (Boyden Chamber Assay) showed that number of cells penetrating the membrane for the pRNAT/sur-siRNA/SW480, pRNAT/SW480 and the SW480 cells was 153±66, 505±65 and 578±98, respectively (P<0.01). CONCLUSION siRNA against survivin can significantly inhibit the expression of survivin in SW480 cells, and thus promote apoptosis, inhibit proliferation and invasive ability.展开更多
基金The Cancer Association of South Africa,theSixth Research Framework Programme of the European UnionProject RIGHT,LSHB-CT-2004-005276South African NationalResearch Foundation and Poliomyelitis Research Foundationsupport research
文摘Primary liver cancer is the fifth most common malignan- cy in the world and is a leading cause of cancer-related mortality.Available treatment for hepatocellular carcino- ma(HCC),the commonest primary liver cancer,is rarely curative and there is a need to develop therapy that is more effective.Specific and powerful gene silencing that can be achieved by activating RNA interference(RNAi) has generated enthusiasm for exploiting this pathway for HCC therapy.Many studies have been carried out with the aim of silencing HCC-related cellular oncogenes or the hepatocarcinogenic hepatitis B virus(HBV)and hepatitis C virus(HCV).Proof of principle studies have demonstrated promising results,and an early clinical trial assessing RNAi-based HBV therapy is currently in progress.Although the data augur well,there are several significant hurdles that need to be overcome before the goal of RNAi-based therapy for HCC is realized.Particu- larly important are the efficient and safe delivery of RNAi effecters to target malignant tissue and the limitation of unintended harmful non-specific effects.
基金Supported by The Science and Technology Fund of Jilin Province,No. 200505219
文摘AIM:To explore the effect of silencing of signal transducer and activator of transcription 3(STAT3) expression by RNA interference(RNAi) on growth of human hepatocellular carcinoma(HCC) in tumor-bearing nude mice in vivo.METHODS:To construct the recombinant plasmid of pSilencer 3.0-H1-STAT3-siRNA-GFP(pSH1-siRNA-STAT3) and establish the tumor-bearing nude mouse model of the HCC cell line SMMC7721,we used intratumoral injection together with electroblotting to transfect the recombinant plasmid pSH1-siRNA-STAT3 into the transplanted tumor.The weight of the nude mice and tumor volumes were recorded.STAT3 gene transcription was detected by semi-quantitative reverse transcription polymerase chain reaction(RTPCR).Level of protein expression and location of STAT3 were determined by Western blotting and immunohistochemical staining.STAT3-related genes such as survivin,c-myc,VEGF,p53 and caspase3 mRNA and protein expression were detected in tumor tissues at the same time.The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay was used to detect apoptosis of tumor cells.RESULTS:The weight of the treated nude mice increased,and the tumor volume decreased markedly compared with those of the mock-treated and negative control groups(P < 0.01).The results of RT-PCR and Western blotting showed that mRNA and protein levels of STAT3 declined markedly in the treated group.The change in STAT3-related gene expression in tumor tissues at the mRNA and protein level also varied,the expression of survivin,VEGF and c-myc were obviously reduced,and expression of p53 and caspase3 increased(P < 0.01).Most of the tumor tissue cells in the treated group developed apoptosis that was detected by TUNEL assay.CONCLUSION:Silencing of STAT3 expression by RNAi significantly inhibits expression of STAT3 mRNA and protein,and suppresses growth of human HCC in tumor-bearing nude mice.The mechanism may be related to down-regulation of survivin,VEGF and c-myc and up-regulation of p53 and caspase3 expression.Accordingly,the STAT3 gene may act as an important and effective target in gene therapy of HCC.
基金Supported by grants from the Young Scientific and Technical InnovationFoundation of Fujian Province (No. 2004J067).
文摘Objective: To explore the inhibition of the expression of c-Myc in human hepatocellular carcinoma via vector-based RNA interference (RNAi). Methods: Two RNA interference DNA templates targeting c-Myc oncogene were designed via online software and synthesized. By ligation, the fragments were inserted into pSilencer 1.0-U6 to construct the recombinant siRNA expressing plasmids. The identified recombinants were introduced into BEL-7402 cells with Lipofactamine. The inhibition of c-Myc expression, together with the expression of CDK4, hTERT and Gadd45β in c-Myc down-regulated BEL-7402 cells, were analyzed by semi-quantitative RT-PCR. Results: Two recombinant plasmids pSic-myc-1 and pSic-myc-2, which direct the yields of siRNAs targeting c-Myc in cells, were constructed. Among which, pSic-myc-2 was shown to trigger a RNAi-mediated inhibition of expression of c-Myc in BEL-7402 by up to 90%. In c-Myc knockdown BEL-7402 cells, the expression of CDK4 and hTERT were down-regulated with a ratio of 85% and 57%, respectively, while the expression of Gadd45β was up-regulated by up to 110%. Conclusion: The expression of c-Myc in BEL-7402 could be suppressed by vector-based RNA interference successfully. The knockdown of c-Myc in turn resulted in the changes of expression of genes related to cell proliferation and apoptosis. Thus, our study provided a preliminary data in searching of a c-Myc-targeted RNAi therapy of human hepatocellular carcinoma.
基金This work was supported by the NationalPostdoctoral Science Foundation of China(No.2003034300)
文摘Objective: The effects on cell-cycle and p53 expression in hepatoma cell line SK-Hep-1 were explored by transfecting exogenous p53 small double stranded RNA (dsRNA) into the SK-Hep-1 cells. Methods: p53 dsRNA and EGFP dsRNA were synthesized. SK-Hep-1 (wtp53) cell line was transfected with 200 ng and 400 ng p53 dsRNA or EGFP and EGFP+EGFP dsRNA (as positive control) or 9% NaCl (as blank control) by liposome transfection technique. Flow cytometry was adopted to measure the effects of p53 dsRNA on cell cycle. Expression of p53 protein was detected by Western-Blotting at 48 h after transfecting p53 dsRNA. Results: The number of G0-G1 phase SK-Hep-1 cells, which were transfected with 200 ng p53 dsRNA, was decreased by 52.53% comparing with the control, and decreased by 50.29% (P<0.05) comparing with the positive control cells transfected with same dosage of EGFP+EGFP dsRNA. The number of S phase cells, which were transfected with 200 ng p53 dsRNA, was increased by 146.8% comparing with the control, and increased by 128.62% (P<0.05) comparing with the positive control cells transfected with same dosage of EGFP+EGFP dsRNA. The number of G2-M phase cells, which were transfected with 200 ng p53 dsRNA, was increased by 30.56% (P<0.05) comparing with the control, and increased by 21.63% (P>0.05) comparing with the positive control cells transfected with same dosage of EGFP+EGFP dsRNA. After 48 h, p53 protein expression was not detected in the SK-Hep-1 cells transfected with p53 dsRNA. Conclusion: p53 dsRNA can obviously improve the proliferation of SK-Hep-1 cells, and suppress p53 protein expression of SK-Hep-1 cells, the former may be related to of the latter.
基金This study was supported by the grants from the Program for New Century Excellent Talents in University, Ministry of Education, China (No. NCET-06-0350) and the Program for Outstanding Youth Science Foundation, Heilongjiang Province, China (No. JC200616).Acknowledgements: We thank Neal Okarter from the Department of Food Science, Comell University for reading and revising the manuscript.
文摘Background Both survivin and lung resistance related protein (LRP) are hepatocellular carcinoma (HCC). But the relationship between survivin and LRP is investigate the effects of down-regulation of survivin on LRP expressions and the both in vitro and in vivo. related to the chemoresistances in ndefinite. The aim of this study was to reversal of chemoresistances in HCC Methods The expressions of survivin were detected by RT-PCR and Western blotting in HCC cell line SMMC-7721 and SMMC-7721/ADM. The sensitivities of these two cell lines to ADM were evaluated by MTT assays. SiRNA which targeted survivin was transfected into SMMC-7721/ADM cells, then the sensitivity of SMMC-7721/ADM cells to ADM and the expressions of survivin and LRP were detected respectively. SMMC-7721/ADM cells were transplanted subcutaneously into nude mice to establish xenograft tumors. Antitumor activities of RNA interference (RNAi) targeting survivin, various doses of ADM and combination therapies were observed respectively. Possible toxicities were evaluated. LRP expression changes were tested. Student's ttest was used for evaluating statistical significance. Results The expressions of survivin in SMMC-7721/ADM cell line showed significant elevation compared to those in SMMC-7721 cell line (P 〈0.05). Positive siRNA down-regulated the expressions of survivin significantly (P 〈0.05). SiRNA targeting survivin could sensitize SMMC-7721/ADM cells to ADM and down-regulate the expressions of LRP significantly (P 〈0.05). Growths of the tumors were significantly inhibited in positive siRNA group as compared with those in the control group from the 8th day (P 〈0.05). Combination therapies caused significant tumor inhibitions compared with tumors of nude mice in the other three groups respectively (P 〈0.05). No toxicities were found in nude mice treated by siRNA and combination therapies. The expressions of LRP were markedly reduced in tumors treated with siRNA targeting survivin (P 〈0.05). Conclusions Down regulation of survivin gene by RNAi can increase chemosensitivity of HCC both in vitro and in vivo. The reversal of drug resistance may be reduced through the inhibitions of LRP.
文摘Background The relationship between apoptosis and tumors is a major focus in cancer research. RNA interference (RNAi) technology has emerged as a very potent tool to generate cellular knockdown phenotypes of a desired gene. The aim of this study was to explore the effect of siRNA specific to the protein casein kinase 2α (CK2α) on apoptosis of laryngeal carcinoma cells and to explore possible mechanisms.Methods An siRNA expression plasmid specific to CK2α, psiRNA-hH1neo-CK2α, and a non-specific siRNA expression plasmid, psiRNA-hH1neo-cont, were constructed and transfected into Hep-2 cells by a lipofectamine method. The mRNA and protein levels of CK2α in transfected cells were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analysis. Apoptosis was measured by Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) double-staining methods. The morphological changes to Hep-2 cells were observed under transmission electron microscopy (TEM). The levels of Bcl-2 and Bax proteins were measured by Western blotting analysis.Results Levels of CK2α mRNA and protein were significantly decreased in the psiRNA-hH1neo-CK2α group compared to the other groups (P 〈0.05). The apoptotic rate of the psiRNA-hH1neo-CK2α transfected group was significantly higher compared to that in the untransfected group and the siRNA-hH1neo-cont transfected group (25.66%±0.83%, 3.66%±0.43%, and 5.18%±0.22%) (P 〈0.05). Compared with the untransfected group and the siRNA-hH1neo-cont transfected group, the psiRNA-hH1neo-CK2α transfected group presented with classical ultrastructural features of apoptosis, such as karyopyknosis, chromatic agglutination adjacent to the nuclear membrane, and apoptotic bodies. Compared with the other two groups, the level of Bcl-2 protein in the psiRNA-hH1neo-CK2α transfected group was decreased (0.20±0.09 vs. 0.72±0.16, 0.56±0.11, P 〈0.01), while the Bax protein level was increased (0.81±0.17 vs. 0.26±0.12, 0.33±0.17, P 〈0.01) and the ratio of Bcl-2 to Bax was decreased (0.25±0.05 vs. 2.76±0.21, 1.70±0.22, P 〈0.01).Conclusions The siRNA expression plasmid specific to CK2α could suppress CK2α expression and induce the apoptosis of laryngeal carcinoma cells. This is possibly by decreasing the Bcl-2/Bax ratio. CK2α may provide a potential therapeutic target against human laryngeal carcinoma.
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 Sa/I 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-avidinbiotin-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Objective:To knockdown the C-erbB2 gene in salivary gland adenoid cystic carcinoma SACC-83 cells using RNA interference,and determine the effect of silencing C-erbB2 on cell proliferation.Methods:C-erbB2-siRNA was transfected into SACC-83 cells.RT-PCR and immunohistochemistry were used to detect C-erbB2 expression in SACC-83 cells.Cell proliferation was measured by the MTT assay and gene knockdown was achieved by RNA interference.Apoptosis was analyzed by flow cytometry.Results:Compared with the control,C-erbB2 mRNA expression was decreased in the C-erbB2-siRNA transfection group,and immunohistochemical analysis indicated that C-erbB2 protein expression was decreased.After C-erbB2-siRNA was transfected for 48 h,absorbance at 570 nm (MTT)was 0.185±0.021 compared with 0.354±0.034,0.299±0.053,and 0.314±0.049 in the blank control,liposome control and negative control siRNA groups,respectively.The differences were statistically significant (P〈0.05)between the C-erbB2-siRNA group and the control groups.Following the C-erbB2 knockdown,the percentage of apoptotic cells was 5.63%compared with 2.04%,2.85%,and 2.98%in the three control groups,respectively.Proliferation of SACC-83 cells was inhibited,and early apoptotic cells were increased.Conclusion: RNA interference can effectively silence C-erbB2 gene expression and inhibit growth of SACC-83 cells,which indicates the potential of targeting this gene as a novel gene therapy approach for the treatment of salivary gland adenoid cystic carcinoma.
基金supported by Shandong Scientific and Technological Development Project Fund(No.2013GSF11825)
文摘Objective:To study the inhibition effect of siRNA on the expression of Wisp-1 gene in Hca-F of mouse hepatocellular carcinoma cells strain and also its effect on the proliferation,migration and adhesion of hepatocellular carcinoma cells.Methods:Three expression vectors of siRNA were constructed.Lipo2000 was employed to transfect Hca-F cells and Western blot was used to detect the inhibition effect of siRNA on the expression of Wisp-1 gene.Afterwards,CCK8 was adopted to detect the effect of Wisp-1 siRNA on the proliferation of Hca-F cells;Annexin V-FTTC/PI double staining flow cytometry was used to detect the effect of Wisp-1 siRNA on the apoptosis of Hca-F cells;Transwell was used to detect the effect of Wisp-1 siRNA on the migration of Hca-F cells.The in vitro cell adhesion kit was used to detect of Wisp-1 siRNA on the change in the components of extracellular matrix to which Hca-F cells adhered.Western blot was used to detect the activation of protein kinase B(AKT)/glycogen synthase kinase-3(3pathway and the expression of downstream target protein p53 and matrix metal!oproteinases-2.Results:The siRNA showed interference effect on the expression of Wisp-1 gene.Compared with the control group,after being transfected to cells,Wisp-1 siRNA could significantly inhibit the proliferation,migration and adhesion of Hca-F cells and also promote the cell apoptosis,which was related to the down-regulated phosphorylation of AKT and glycogen synthase kinase-3β and the expression of p53 and matrix metalIoproteinases-2(P<0.05).Conclusions:The inhibition of Wisp-1 expression can reduce the proliferation,migration and adhesion of mouse hepatocellular carcinoma cells,which is related to the AKT/ glycogen synthase kinase-3 β pathway.Wisp-1 gene may be the potential target to cure the hepatocellular carcinoma.
文摘The development of human endometrial carcinoma(HEC) is a complex pathologic process involves several oncogenes and tumor suppressor genes.The full-length paired-box gene 2(pax2),a recently discovered oncogene,promotes cell proliferation and growth and inhibits apoptosis of HEC cells.Here,we examined the effect of pax2 small interfering RNA(siRNA) on the growth of transplanted HEC cells in nude mice.The expression of Pax2 in 21 cases of normal endometrium and 38 cases of HEC was examined by immohistochemistry(IHC).HEC models were developed by subcutaneously transferring HEC cells into nude mice,followed by treatment with empty lentivirus vector,lentivirus vector-based pax2 siRNA,and phosphate buffered saline,respectively.Four weeks later,tumor size was measured,tumor inhibition rate was calculated,and histological analyses were conducted after staining with hematoxylin and eosin.The expression of Pax2 and Bcl-2 was detected by Western blot;proliferating cell nuclear antigen(PCNA) was detected by IHC.Significant differences were observed in the positive rate of Pax2 between normal endometrium and HEC(14.2% vs.60.5%,P<0.01).The expression index of Pax2 in well differentiated tumors was 1.88±1.68,much lower than that in tumors of moderate(3.07±1.96,P<0.05) or poor differentiation(5.45±2.76,P<0.01).Tumor necrosis increased,nuclear basophilia stain decreased,tumor growth was inhibited,and PCNA,Pax2,and Bcl-2 expression was reduced in HEC models treated with pax2 siRNA.These results indicate that Pax2 expression is related to HEC tumor biology with the increased expression of Pax2 correlated to malignancy.pax2 siRNA down-regulates Pax2 expression and inhibits tumorigenesis of HEC in nude mice,possibly due to cell apoptosis and the inhibition of tumor proliferation induced by down-regulation of Bcl-2.
文摘Objective: To study the radiosensitivity of SiHa cells with Ku80 inhibition by RNAi. Methods: pKu80-siRNA and pNeg-siRNA were constructed and then transfected into SiHa cells. Western blot and RT-PCR were applied to measure the expression of Ku80. After 10 Gy irradiation with 6 MV X-ray, cells were harvested at 20 h, 28 h and 48 h, and analyzed by flow cytometry for apoptosis rate. The SF2 and α, β values of cells were acquired by clone formation array. Results: Two stable transfection cell clones SiHa/Ku80-siRNA and SiHa/Neg-siRNA were screened from the pKu80-siRNA and pNeg-siRNA transfected cells. Western blot and RT-PCR indicated that the expression of Ku80 was suppressed by the Ku80-siRNA. The apoptosis rates of SiHa/Ku80-siRNA were higher than control cells at 28 h and 48 h after X-ray irradiation (P < 0.05). The SF2 value of SiHa/Ku80-siRNA (0.422) was lower than that of SiHa/Neg-siRNA cells (0.587) or untransfected cells (0.583), and the α value of SiHa/Ku80-siRNA was 0.295 ± 0.016, higher than that of SiHa/Neg-siRNA (0.176 ± 0.010) or control cells (0.188 ± 0.011). Conclusion: Inhibition of Ku80 could enhance the radiosensitivity of SiHa, which showed that Ku80 could be a good target for molecular therapy.
基金This work was supported by China National Foundation of Natural Science (No. 30171053).
文摘OBJECTIVE To study the efficiency of silencing survivin gene expression by an anti-survivin small RNA expression plasmid and to examine its impact on apoptosis, proliferation and invasive ability of the SW480 colorec-tal cancer cell line. METHODS An expression plasmid of survivin siRNA (pRNAT/sur-siR-NA) was constructed and transduced into SW480 cells. Western blot analysis and a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) were used to measure the change in expression level of the survivin protein and mRNA after transduction of the expression plasmid into the SW480. Apoptosis, proliferation, and invasive ability of the SW480 cells was evaluated by flow cytometry, MTT assay and Boy-den Chamber Assay, respectively. RESULTS The expression plasmid (pRNAT/sur-siRNA) against survivin down-regulated survivin protein and mRNA expression dramatically, with a down-regulation of 85% and 80%, respectively. After transfection of the pRNAT/sur-siRNA to the SW480 cells, the apoptotic rate of the pRNAT/ sur-siRNA/SW480 cells was 16.9%, and the proliferation rate was 37.4%, significantly different compared to the controls (P<0.01). Cell invasive studies (Boyden Chamber Assay) showed that number of cells penetrating the membrane for the pRNAT/sur-siRNA/SW480, pRNAT/SW480 and the SW480 cells was 153±66, 505±65 and 578±98, respectively (P<0.01). CONCLUSION siRNA against survivin can significantly inhibit the expression of survivin in SW480 cells, and thus promote apoptosis, inhibit proliferation and invasive ability.