BACKGROUND: Survivin is known to be overexpressed in various human malignancies, including pancreatic cancer, and mediates cancer cell proliferation and tumor growth, so the regulation of this molecule could be a new ...BACKGROUND: Survivin is known to be overexpressed in various human malignancies, including pancreatic cancer, and mediates cancer cell proliferation and tumor growth, so the regulation of this molecule could be a new strategy for treating pancreatic cancer. In this study, short hairpin RNAs (shRNAs) specific to survivin were introduced into human pancreatic cancer Patu8988 cells to investigate the inhibitory effects on survivin expression and cell proliferation in vitro and in vivo. METHODS: Three kinds of shRNA specific to the survivin gene were designed and cloned into eukaryotic expression plasmid pGenesil-1 vector. Subsequently the recombinant plasmids were transfected into human pancreatic cancer Patu8988 cells with lipfectamine (TM) 2000 reagent. The mRNA and protein expressions of survivin in the transiently transfected Patu8988 cells were determined by RT-PCR, flow cytometry, and Western blotting analysis. The proliferation inhibition rates of stably transfected Patu8988 cells were determined by MTT assay. The antitumor activities of the three kinds of survivin-shRNA plasmids were evaluated in BALB/c nude mice inoculated with Patu8988 cells and bearing human pancreatic cancer. RESULTS: The three survivin-shRNA plasmids named pGenesil-1-survivin-1, pGenesil-1-survivin-2 and pGenesil-1-survivin-1+2 (with double interfering RNA sites) were successfully constructed, and were confirmed by restriction enzyme cutting and sequencing. At 48 hours after transfection, the expression of survivin mRNA and protein was inhibited in Patu8988 cells transfected with pGenesil-1-survivin-1, pGenesil-1-survivin-2, and pGenesil-1-survivin-1+2 when compared with that of either pGenesil-1-NC (with scrambled small interfering RNA) transfected cells or control cells (P<0.05). The MTT results showed that the proliferation rates of Patu8988 cells stably transfected with survivin-shRNA plasmids were reduced when compared with that of either pGenesil-1-NC transfected cells or control cells (P<0.01). Furthermore, when Patu8988 cells st展开更多
Tomographic particle image velocimetry was used to quantitatively visualize the three-dimensional co- herent structures in the logarithmic region of the turbulent boundary layer in a water tunnel. The Reynolds number ...Tomographic particle image velocimetry was used to quantitatively visualize the three-dimensional co- herent structures in the logarithmic region of the turbulent boundary layer in a water tunnel. The Reynolds number based on momentum thickness is Reo = 2 460. The in- stantaneous velocity fields give evidence of hairpin vortices aligned in the streamwise direction forming very long zones of low speed fluid, which is flanked on either side by high- speed ones. Statistical support for the existence of hairpins is given by conditional averaged eddy within an increasing spanwise width as the distance from the wall increases, and the main vortex characteristic in different wall-normal re- gions can be reflected by comparing the proportion of ejec- tion and its contribution to Reynolds stress with that of sweep event. The pre-multiplied power spectra and two-point cor- relations indicate the presence of large-scale motions in the boundary layer, which are consistent with what have been termed very large scale motions （VLSMs）. The three dimen-sional spatial correlations of three components of veloc- ity further indicate that the elongated low-speed and high- speed regions will be accompanied by a counter-rotating roll modes, as the statistical imprint of hairpin packet structures, all of which together make up the characteristic of coherent structures in the logarithmic region of the turbulent boundary layer （TBL）.展开更多
RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression o...RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms, strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed.展开更多
AIM: To investigate the effects of Annexin A2 (ANXA2) silencing on invasion, migration, and tumorigenic potential of hepatoma cells. METHODS: Human hepatoma cell lines [HepG2, SMMC-7721, SMMC-7402, and MHCC97-H, a nov...AIM: To investigate the effects of Annexin A2 (ANXA2) silencing on invasion, migration, and tumorigenic potential of hepatoma cells. METHODS: Human hepatoma cell lines [HepG2, SMMC-7721, SMMC-7402, and MHCC97-H, a novel human hepatocellular carcinoma (HCC) cell line with high metastasis potential] and a normal hepatocyte cell line(LO2) were used in this study. The protein and mRNA expression levels of ANXA2 were analysed by western blotting and real-time polymerase chain reaction, re-spectively. The intracellular distribution profile of ANXA2 expression was determined by immunofluorescence and immunohistochemistry. Short hairpin RNA target-ing ANXA2 was designed and stably transfected into MHCC97-H cells. Cells were cultured for in vitro analy-ses or subcutaneously injected as xenografts in mice for in vivo analyses. Effects of ANXA2 silencing on cell growth were assessed by cell counting kit-8 (CCK-8) as-say (in vitro ) and tumour-growth assay (in vivo ), on cell cycling was assessed by flow cytometry and propidium iodide staining (in vitro ), and on invasion and migration potential were assessed by transwell assay and wound-healing assay, respectively (both in vitro ). RESULTS: The MHCC97-H cells, which are known to have high metastasis potential, showed the highest lev-el of ANXA2 expression among the four HCC cell types examined; compared to the LO2 cells, the MHCC97-H expression level was 8-times higher. The ANXA2 expres-sion was effectively inhibited (about 80%) by ANXA2-specific small hairpin RNA (shRNA). ANXA2 expression in the MHCC97-H cells was mainly localized to the cel-lular membrane and cytoplasm, and some localization was detected in the nucleus. Moreover, the proliferation of MHCC97-H cells was obviously suppressed by shR-NA-mediated ANXA2 silencing in vitro , and the tumour growth inhibition rate was 38.24% in vivo . The per-centage of MHCC97-H cells in S phase dramatically de-creased (to 27.76%) under ANXA2-silenced conditions. Furthermore, ANXA2-silenced MHCC97-H cells showed lower invasiveness展开更多
To investigate the influence of osteopontin （OPN） short hairpin RNA （shRNA） on the proliferation and activity of rat vascular smooth muscle cells （VSMCs）, the expressing vector of shRNA targeting OPN was constru...To investigate the influence of osteopontin （OPN） short hairpin RNA （shRNA） on the proliferation and activity of rat vascular smooth muscle cells （VSMCs）, the expressing vector of shRNA targeting OPN was constructed and transferred into the rat VSMCs. After amplification and purification, pGenesil-1/OPNshRNA1 （PG1）, pGenesil-1/OPNshRNA2 （PG2） and pGenesil-1/OPNshRNAHK （PGH） were transfected into the cultured rat VSMC by LipofectamineTM 2000. Transfected cells were visualized by using an inverted fluorescent microscope. VSMCs transfected by optimal recombined plasmid was selected by culturing in G418 48 h later. Nude cells and cells transfected by PGH were used as control. The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. The OPN of VSMCs was suppressed by transfection of optimal recombined plasmid, and the changes in cell proliferation, adhesion and motility were evaluated by MTT, adhesion test and transwell chamber test. Levels of type I and Ⅲ collagen were measured with ELISA kit. Our results showed that VSMCs stably transfected by OPN shRNA accounted for over 50% of total cells. OPN mRNA and protein were reduced by 81% and 67% （P〈0.01） by PG1, 73% and 52% （P〈0.01） by PG2, respectively while no change was found in PGH and non-treated VSMCs. PG1 significantly suppressed the proliferation, adhesion, mobility of VSMCs and reduced the amount of type Ⅰ and Ⅲ collagen. It is concluded that recombinant plasmid can be success-fully transfected into VSMCs by LipofectamineTM 2000 and inhibit the expression of OPN. The proliferation, adhesion and mobility of VSMCs can be inhibited by knocking down OPN expression. Moreover, the transferring capability of cells is attenuated, and the secretion of type Ⅰ and Ⅲ collagen is inhibited aftter knocking-down of OPN expression. The study provides experimental evidence for clinical prevention of restenosis after percutaneous coronary intervention （PCI） by RNA interference （RNAi） technology展开更多
Background The relationship between signal transduction and tumors has become one of the loci in cancer research. Signal transducer and activator of the transcription 6 （STAT6） signaling pathway is found to be activ...Background The relationship between signal transduction and tumors has become one of the loci in cancer research. Signal transducer and activator of the transcription 6 （STAT6） signaling pathway is found to be activated in some cancer cells. But the function of the pathway in cancer cells is unknown. This study was undertaken to investigate the effect of the Stat6 signaling pathway on apoptosis in human colon cancer cells （HT-29 cells） and the possible mechanism of Stat6 by RNA interference techniques.展开更多
Background Overexpression of breast cancer-specific gene 1 （SNCG） is associated with poor prognosis in advanced breast cancer patients. This study aimed to determine the effects of SNCG knockdown in breast cancer ce...Background Overexpression of breast cancer-specific gene 1 （SNCG） is associated with poor prognosis in advanced breast cancer patients. This study aimed to determine the effects of SNCG knockdown in breast cancer cells by using small hairpin RNA （shRNA）.Methods Four different SNCG shRNA oligonucleotides were designed and chemically synthesized to construct mammalian expression vectors. These vectors were then stably transfected into a breast cancer MCF-7 cell line to knockdown SNCG expression. After SNCG knockdown was confirmed, the stable cell lines were inoculated into nude mice. SNCG mRNA and protein expressions were analyzed by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry, respectively in both the stable cell lines and xenografts.Results All four SNCG shRNA constructs significantly reduced SNCG mRNA and protein levels in MCF-7 cells, as compared to the unrelated sequence control shRNA and the liposome control mice （P〈0.05）. SNCG-knockdown MCF-7cells formed significantly smaller tumor masses than cells expressing the unrelated sequence control or the liposome control mice （P〈0.05）.Conclusion SNCG shRNA effectively suppressed breast cancer cell formation in vivo and may be a useful clinical strategy to control breast cancer展开更多
AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vecto...AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method.RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control.Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively.CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).展开更多
A mouse model of viral encephalitis was induced by intracranial injection of a Coxsackie virus B3 suspension. Quantitative real-time reverse transcription-PCR and western blot assay were applied to detect mRNA and pro...A mouse model of viral encephalitis was induced by intracranial injection of a Coxsackie virus B3 suspension. Quantitative real-time reverse transcription-PCR and western blot assay were applied to detect mRNA and protein expression of intelectin-2 and nuclear factor-kappa B in the viral encephalitis and control groups. Nuclear factor-kappa B and intelectin-2 mRNA and protein expression were significantly increased in mice with viral encephalitis. After intraperitoneal injection of Shuanghuanglian at a dose of 1.5 mg/kg for 5 successive days, intelectin-2 and nuclear factor-kappa B protein and mRNA expression were significantly decreased. To elucidate the relationship between intelectin-2 and nuclear factor-kappa B, mice with viral encephalitis were administered an intracerebral injection of 107 pfu recombinant lentivirus expressing intelectin shRNA. Both protein and mRNA levels of intelectin and nuclear factor-kappa B in brain tissue of mice were significantly decreased. Experimental findings suggest that Shuanghuanglian injection may downregulate nuclear factor-kappa B production via suppression of intelectin production, thus inhibiting inflammation associated with viral encephalitis.展开更多
The generation of a hairpin vortex from near-wall streamwise vortices is studied via the direct numerical simulation（DNS） of the streak transient growth in the minimal channel flow at Re_τ- 400.The streak profile i...The generation of a hairpin vortex from near-wall streamwise vortices is studied via the direct numerical simulation（DNS） of the streak transient growth in the minimal channel flow at Re_τ- 400.The streak profile is obtained by conditionally averaging the DNS data of the fully developed turbulent channel flow at the same Reynolds number.The near-wall streamwise vortices are produced by the transient growth of the streak which is initially subjected to the sinuous perturbation of the spanwise velocity.It is shown that the arch head of the hairpin vortex first grows from the downstream end of the stronger streamwise vortex and then connects with the weaker,opposite-signed streamwise vortex in their overlap region,forming a complete individual hairpin structure.The vorticity transport along the vortex lines indicates that the strength increase and the spatial expansion of the arch head are due to the stretching and the turning of the vorticity vector,respectively.The hairpin packets could be further produced from the generated individual hairpin vortex following the parent-offspring process.展开更多
The relationship between the in the logarithmic law （log-law） region of bursting event and the low/high-speed streak a turbulent boundary layer is investigated. A tomographic time-resolved particle image velocimetry...The relationship between the in the logarithmic law （log-law） region of bursting event and the low/high-speed streak a turbulent boundary layer is investigated. A tomographic time-resolved particle image velocimetry （TRPIV） system is used to measure the instantaneous three-dimensional-three-component （3D-3C） velocity field. The momentum thickness based Reynolds number is about 2 460. The topological information in the log-law region is obtained experimentally. It is found that the existence of the quadrupole topological structure implies a three-pair hairpin-like vortex packet, which is in connection with the low/high-speed streak. An idealized 3D topological model is then proposed to characterize the observed hairpin vortex packet and low/high-speed streak.展开更多
We present dynamic mode decomposition (DMD) for studying the hairpin vortices generated by hemisphere protuberance measured by two-dimensional (2D) time-resolved (TR) particle image velocimetry (PIV) in a water channe...We present dynamic mode decomposition (DMD) for studying the hairpin vortices generated by hemisphere protuberance measured by two-dimensional (2D) time-resolved (TR) particle image velocimetry (PIV) in a water channel. The hairpins dynamic information is extracted by identifying their dominant frequencies and associated spatial structures. For this quasi-periodic data system, the resulting main Dynamic modes illustrate the different spatial structures associated with the wake vortex region and the near-wall region. By comparisons with proper orthogonal decomposition (POD), it can be concluded that the dynamic mode concentrates on a certain frequency component more effectively than the mode determined by POD. During the analysis, DMD has proven itself a robust and reliable algorithm to extract spatial-temporal coherent structures.展开更多
RNA interference （RNAi） is an ancient intra-cellular mechanism that regulates gene expression and cell function. Large-scale gene silencing using RNAi highthroughput screening （HTS） has opened an exciting frontier...RNA interference （RNAi） is an ancient intra-cellular mechanism that regulates gene expression and cell function. Large-scale gene silencing using RNAi highthroughput screening （HTS） has opened an exciting frontier to systematically study gene function in mammalian cells. This approach enables researchers to identify gene function in a given biological context and will provide considerable novel insight. Here, we review RNAi HTS strategies and applications using case studies in cancer biology and virology.展开更多
Diagnostic C9orf72 hexanucleotide repeat expansions(C9-HRE)is essential for the early and accurate diagnosis of amyotrophic lateral sclerosis(ALS)and will provide support for the prognosis and gene therapy of ALS.In t...Diagnostic C9orf72 hexanucleotide repeat expansions(C9-HRE)is essential for the early and accurate diagnosis of amyotrophic lateral sclerosis(ALS)and will provide support for the prognosis and gene therapy of ALS.In the present study,by combining catalytic hairpin assembly(CHA)with Mycobacterium smegmatis porin A(MspA)nanopore,a new nanopore-based strategy for the detection of C9-HRE was reported.Less than 30 repeats of C9-HRE could be detected via this method,and the results have the potential to help distinguish between patients and healthy individuals.Moreover,the method demonstrated its great specificity for C9-HRE by identifying other repeat expansions.Given the high selectivity,this approach had been successfully used to detect C9-HRE in cell and blood samples with high accuracy.This detection strategy is user-friendly and has a strong anti-interference ability,thus providing a powerful tool for clinical diagnosis.展开更多
基金supported by grants from the Social Bureau Foundation of Suzhou (SZD0614)the Foundation of Health Bureau of Jiangsu Province (Z200622)
文摘BACKGROUND: Survivin is known to be overexpressed in various human malignancies, including pancreatic cancer, and mediates cancer cell proliferation and tumor growth, so the regulation of this molecule could be a new strategy for treating pancreatic cancer. In this study, short hairpin RNAs (shRNAs) specific to survivin were introduced into human pancreatic cancer Patu8988 cells to investigate the inhibitory effects on survivin expression and cell proliferation in vitro and in vivo. METHODS: Three kinds of shRNA specific to the survivin gene were designed and cloned into eukaryotic expression plasmid pGenesil-1 vector. Subsequently the recombinant plasmids were transfected into human pancreatic cancer Patu8988 cells with lipfectamine (TM) 2000 reagent. The mRNA and protein expressions of survivin in the transiently transfected Patu8988 cells were determined by RT-PCR, flow cytometry, and Western blotting analysis. The proliferation inhibition rates of stably transfected Patu8988 cells were determined by MTT assay. The antitumor activities of the three kinds of survivin-shRNA plasmids were evaluated in BALB/c nude mice inoculated with Patu8988 cells and bearing human pancreatic cancer. RESULTS: The three survivin-shRNA plasmids named pGenesil-1-survivin-1, pGenesil-1-survivin-2 and pGenesil-1-survivin-1+2 (with double interfering RNA sites) were successfully constructed, and were confirmed by restriction enzyme cutting and sequencing. At 48 hours after transfection, the expression of survivin mRNA and protein was inhibited in Patu8988 cells transfected with pGenesil-1-survivin-1, pGenesil-1-survivin-2, and pGenesil-1-survivin-1+2 when compared with that of either pGenesil-1-NC (with scrambled small interfering RNA) transfected cells or control cells (P<0.05). The MTT results showed that the proliferation rates of Patu8988 cells stably transfected with survivin-shRNA plasmids were reduced when compared with that of either pGenesil-1-NC transfected cells or control cells (P<0.01). Furthermore, when Patu8988 cells st
基金supported by the National Natural Science Foundation of China (10832001 and 10872145)the State Key Laboratory of Nonlinear Mechanics,Institute of Mechanics,Chinese Academy of Sciences
文摘Tomographic particle image velocimetry was used to quantitatively visualize the three-dimensional co- herent structures in the logarithmic region of the turbulent boundary layer in a water tunnel. The Reynolds number based on momentum thickness is Reo = 2 460. The in- stantaneous velocity fields give evidence of hairpin vortices aligned in the streamwise direction forming very long zones of low speed fluid, which is flanked on either side by high- speed ones. Statistical support for the existence of hairpins is given by conditional averaged eddy within an increasing spanwise width as the distance from the wall increases, and the main vortex characteristic in different wall-normal re- gions can be reflected by comparing the proportion of ejec- tion and its contribution to Reynolds stress with that of sweep event. The pre-multiplied power spectra and two-point cor- relations indicate the presence of large-scale motions in the boundary layer, which are consistent with what have been termed very large scale motions （VLSMs）. The three dimen-sional spatial correlations of three components of veloc- ity further indicate that the elongated low-speed and high- speed regions will be accompanied by a counter-rotating roll modes, as the statistical imprint of hairpin packet structures, all of which together make up the characteristic of coherent structures in the logarithmic region of the turbulent boundary layer （TBL）.
基金RFCID, No 01030152, RGC, CUHK4428/06M, ITF ITS091/03 of Hong Kong Government, and Faculty Direct Fund of the Chinese University of Hong Kong
文摘RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms, strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed.
基金Supported by The Society Development of Nantong,No.HS2012034the Jiangsu Health Projects,No.BL2012053 and No.K201102+1 种基金the Priority Academic Program Development of Jiangsuthe International S and T Cooperation Program of China,No.2013DFA32150
文摘AIM: To investigate the effects of Annexin A2 (ANXA2) silencing on invasion, migration, and tumorigenic potential of hepatoma cells. METHODS: Human hepatoma cell lines [HepG2, SMMC-7721, SMMC-7402, and MHCC97-H, a novel human hepatocellular carcinoma (HCC) cell line with high metastasis potential] and a normal hepatocyte cell line(LO2) were used in this study. The protein and mRNA expression levels of ANXA2 were analysed by western blotting and real-time polymerase chain reaction, re-spectively. The intracellular distribution profile of ANXA2 expression was determined by immunofluorescence and immunohistochemistry. Short hairpin RNA target-ing ANXA2 was designed and stably transfected into MHCC97-H cells. Cells were cultured for in vitro analy-ses or subcutaneously injected as xenografts in mice for in vivo analyses. Effects of ANXA2 silencing on cell growth were assessed by cell counting kit-8 (CCK-8) as-say (in vitro ) and tumour-growth assay (in vivo ), on cell cycling was assessed by flow cytometry and propidium iodide staining (in vitro ), and on invasion and migration potential were assessed by transwell assay and wound-healing assay, respectively (both in vitro ). RESULTS: The MHCC97-H cells, which are known to have high metastasis potential, showed the highest lev-el of ANXA2 expression among the four HCC cell types examined; compared to the LO2 cells, the MHCC97-H expression level was 8-times higher. The ANXA2 expres-sion was effectively inhibited (about 80%) by ANXA2-specific small hairpin RNA (shRNA). ANXA2 expression in the MHCC97-H cells was mainly localized to the cel-lular membrane and cytoplasm, and some localization was detected in the nucleus. Moreover, the proliferation of MHCC97-H cells was obviously suppressed by shR-NA-mediated ANXA2 silencing in vitro , and the tumour growth inhibition rate was 38.24% in vivo . The per-centage of MHCC97-H cells in S phase dramatically de-creased (to 27.76%) under ANXA2-silenced conditions. Furthermore, ANXA2-silenced MHCC97-H cells showed lower invasiveness
基金supported by a grant from the Science and Technology Foundation of Hubei Province (No.2006AA-301C18)
文摘To investigate the influence of osteopontin （OPN） short hairpin RNA （shRNA） on the proliferation and activity of rat vascular smooth muscle cells （VSMCs）, the expressing vector of shRNA targeting OPN was constructed and transferred into the rat VSMCs. After amplification and purification, pGenesil-1/OPNshRNA1 （PG1）, pGenesil-1/OPNshRNA2 （PG2） and pGenesil-1/OPNshRNAHK （PGH） were transfected into the cultured rat VSMC by LipofectamineTM 2000. Transfected cells were visualized by using an inverted fluorescent microscope. VSMCs transfected by optimal recombined plasmid was selected by culturing in G418 48 h later. Nude cells and cells transfected by PGH were used as control. The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. The OPN of VSMCs was suppressed by transfection of optimal recombined plasmid, and the changes in cell proliferation, adhesion and motility were evaluated by MTT, adhesion test and transwell chamber test. Levels of type I and Ⅲ collagen were measured with ELISA kit. Our results showed that VSMCs stably transfected by OPN shRNA accounted for over 50% of total cells. OPN mRNA and protein were reduced by 81% and 67% （P〈0.01） by PG1, 73% and 52% （P〈0.01） by PG2, respectively while no change was found in PGH and non-treated VSMCs. PG1 significantly suppressed the proliferation, adhesion, mobility of VSMCs and reduced the amount of type Ⅰ and Ⅲ collagen. It is concluded that recombinant plasmid can be success-fully transfected into VSMCs by LipofectamineTM 2000 and inhibit the expression of OPN. The proliferation, adhesion and mobility of VSMCs can be inhibited by knocking down OPN expression. Moreover, the transferring capability of cells is attenuated, and the secretion of type Ⅰ and Ⅲ collagen is inhibited aftter knocking-down of OPN expression. The study provides experimental evidence for clinical prevention of restenosis after percutaneous coronary intervention （PCI） by RNA interference （RNAi） technology
基金This study was supported by grants from the National Science Foundation of China (No. 30470981) and the National Science Foundation of Hubei Province (No. 2005ABA103).
文摘Background The relationship between signal transduction and tumors has become one of the loci in cancer research. Signal transducer and activator of the transcription 6 （STAT6） signaling pathway is found to be activated in some cancer cells. But the function of the pathway in cancer cells is unknown. This study was undertaken to investigate the effect of the Stat6 signaling pathway on apoptosis in human colon cancer cells （HT-29 cells） and the possible mechanism of Stat6 by RNA interference techniques.
文摘Background Overexpression of breast cancer-specific gene 1 （SNCG） is associated with poor prognosis in advanced breast cancer patients. This study aimed to determine the effects of SNCG knockdown in breast cancer cells by using small hairpin RNA （shRNA）.Methods Four different SNCG shRNA oligonucleotides were designed and chemically synthesized to construct mammalian expression vectors. These vectors were then stably transfected into a breast cancer MCF-7 cell line to knockdown SNCG expression. After SNCG knockdown was confirmed, the stable cell lines were inoculated into nude mice. SNCG mRNA and protein expressions were analyzed by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry, respectively in both the stable cell lines and xenografts.Results All four SNCG shRNA constructs significantly reduced SNCG mRNA and protein levels in MCF-7 cells, as compared to the unrelated sequence control shRNA and the liposome control mice （P〈0.05）. SNCG-knockdown MCF-7cells formed significantly smaller tumor masses than cells expressing the unrelated sequence control or the liposome control mice （P〈0.05）.Conclusion SNCG shRNA effectively suppressed breast cancer cell formation in vivo and may be a useful clinical strategy to control breast cancer
基金Supported by the National Natural Science Foundation of China, No. 30371270 the Major Program of Department of Science and Technology of Zhejiang Province, No. 2003C13015
文摘AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method.RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control.Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively.CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).
基金funded by the Health Research Fund from the Health Department of Shanxi Province, China, No.04015
文摘A mouse model of viral encephalitis was induced by intracranial injection of a Coxsackie virus B3 suspension. Quantitative real-time reverse transcription-PCR and western blot assay were applied to detect mRNA and protein expression of intelectin-2 and nuclear factor-kappa B in the viral encephalitis and control groups. Nuclear factor-kappa B and intelectin-2 mRNA and protein expression were significantly increased in mice with viral encephalitis. After intraperitoneal injection of Shuanghuanglian at a dose of 1.5 mg/kg for 5 successive days, intelectin-2 and nuclear factor-kappa B protein and mRNA expression were significantly decreased. To elucidate the relationship between intelectin-2 and nuclear factor-kappa B, mice with viral encephalitis were administered an intracerebral injection of 107 pfu recombinant lentivirus expressing intelectin shRNA. Both protein and mRNA levels of intelectin and nuclear factor-kappa B in brain tissue of mice were significantly decreased. Experimental findings suggest that Shuanghuanglian injection may downregulate nuclear factor-kappa B production via suppression of intelectin production, thus inhibiting inflammation associated with viral encephalitis.
基金supported by the National Natural Science Foundation of China(Grants 11490551,11472154, 11132005,and 11322221)
文摘The generation of a hairpin vortex from near-wall streamwise vortices is studied via the direct numerical simulation（DNS） of the streak transient growth in the minimal channel flow at Re_τ- 400.The streak profile is obtained by conditionally averaging the DNS data of the fully developed turbulent channel flow at the same Reynolds number.The near-wall streamwise vortices are produced by the transient growth of the streak which is initially subjected to the sinuous perturbation of the spanwise velocity.It is shown that the arch head of the hairpin vortex first grows from the downstream end of the stronger streamwise vortex and then connects with the weaker,opposite-signed streamwise vortex in their overlap region,forming a complete individual hairpin structure.The vorticity transport along the vortex lines indicates that the strength increase and the spatial expansion of the arch head are due to the stretching and the turning of the vorticity vector,respectively.The hairpin packets could be further produced from the generated individual hairpin vortex following the parent-offspring process.
基金Project supported by the National Natural Science Foundation of China(Nos.1332006,11272233,11202122,and 11411130150)the National Fundamental Research Program of China(973 Program)(No.2012CB720101)
文摘The relationship between the in the logarithmic law （log-law） region of bursting event and the low/high-speed streak a turbulent boundary layer is investigated. A tomographic time-resolved particle image velocimetry （TRPIV） system is used to measure the instantaneous three-dimensional-three-component （3D-3C） velocity field. The momentum thickness based Reynolds number is about 2 460. The topological information in the log-law region is obtained experimentally. It is found that the existence of the quadrupole topological structure implies a three-pair hairpin-like vortex packet, which is in connection with the low/high-speed streak. An idealized 3D topological model is then proposed to characterize the observed hairpin vortex packet and low/high-speed streak.
基金supported by the National Natural Science Foundation of China (Grant Nos. 10832001 and 10872145)the State Key Laboratory of Nonlinear Mechanics, Institute of Mechanics, Chinese Academy of Sciences
文摘We present dynamic mode decomposition (DMD) for studying the hairpin vortices generated by hemisphere protuberance measured by two-dimensional (2D) time-resolved (TR) particle image velocimetry (PIV) in a water channel. The hairpins dynamic information is extracted by identifying their dominant frequencies and associated spatial structures. For this quasi-periodic data system, the resulting main Dynamic modes illustrate the different spatial structures associated with the wake vortex region and the near-wall region. By comparisons with proper orthogonal decomposition (POD), it can be concluded that the dynamic mode concentrates on a certain frequency component more effectively than the mode determined by POD. During the analysis, DMD has proven itself a robust and reliable algorithm to extract spatial-temporal coherent structures.
文摘RNA interference （RNAi） is an ancient intra-cellular mechanism that regulates gene expression and cell function. Large-scale gene silencing using RNAi highthroughput screening （HTS） has opened an exciting frontier to systematically study gene function in mammalian cells. This approach enables researchers to identify gene function in a given biological context and will provide considerable novel insight. Here, we review RNAi HTS strategies and applications using case studies in cancer biology and virology.
基金supported by a grant from the National Key Research and Development Program of China(No.2022YFB3205600)National Natural Science Foundation of China(No.82004341)+3 种基金China Postdoctoral Science Foundation(No.2022M712286)Sichuan Science and Technology Program(No.2020JDTD0022)Sichuan Administration of Traditional Chinese Medicine(No.2023MS078)Sichuan University Postdoctoral Interdisciplinary Innovation Fund(No.JCXK2225)。
文摘Diagnostic C9orf72 hexanucleotide repeat expansions(C9-HRE)is essential for the early and accurate diagnosis of amyotrophic lateral sclerosis(ALS)and will provide support for the prognosis and gene therapy of ALS.In the present study,by combining catalytic hairpin assembly(CHA)with Mycobacterium smegmatis porin A(MspA)nanopore,a new nanopore-based strategy for the detection of C9-HRE was reported.Less than 30 repeats of C9-HRE could be detected via this method,and the results have the potential to help distinguish between patients and healthy individuals.Moreover,the method demonstrated its great specificity for C9-HRE by identifying other repeat expansions.Given the high selectivity,this approach had been successfully used to detect C9-HRE in cell and blood samples with high accuracy.This detection strategy is user-friendly and has a strong anti-interference ability,thus providing a powerful tool for clinical diagnosis.
