Mercury ion(Hg^(2+)),a highly noxious of heavy metalion,has detrimental effects on the ecological environment and human health.Herein,we have developed an exonuclease III(Exo III)assisted catalytic hairpin assembly fo...Mercury ion(Hg^(2+)),a highly noxious of heavy metalion,has detrimental effects on the ecological environment and human health.Herein,we have developed an exonuclease III(Exo III)assisted catalytic hairpin assembly formation of a trivalent G-quadruplex/hemin DNAzyme for colorimetric detection of Hg^(2+).A hairpin DNA(Hr)was designed with thymine-Hg^(2+)-thymine pairs that catalyzed by Exo III is prompted to happen upon binding Hg^(2+).A released DNA fragment triggers the catalytic assembly of other three hairpins(H1,H2,and H3)to form many trivalent G-quadruplex/hemin DNA enzymes for signal output.The developed sensor shows a dynamic range from 2 pM to 2μM,with an impressively low detection limit of 0.32 pM for Hg^(2+)detection.Such a sensor also has good selectivity toward Hg^(2+)detection in the presence of other common metal ions.This strategy shows the great potential for visual detection with portable type.展开更多
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 stably transfected with survivin-shRNA were injected into BALB/c nude mice, tumor growth was dramatically lower and the tumor was smaller than that of either pGenesil-1-NC transfected cells or control cells (P<0.01). The inhibitory effect of pGenesil-1-survivin-1 was the best among the three kinds of survivin-shRNA plasmids, but no combination of inhibitory effects was found in pGenesil-1-survivin-1+2. CONCLUSIONS: shRNAs specific to survivin have gene silencing effects and inhibit pancreatic cancer cell proliferation. shRNA activity against survivin could be of potential value in gene therapy for pancreatic cancer. However, shRNAs with double combining sites did not significantly enhance the interference compared with single site shRNAs, therefore further studies on this are needed.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
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
BACKGROUND Hepatocellular carcinoma(HCC) is one of the most common malignant tumors with high mortality-to-incidence ratios. Nuclear factor erythroid 2-like 3(NFE2 L3), also known as NRF3, is a member of the cap ‘n&#...BACKGROUND Hepatocellular carcinoma(HCC) is one of the most common malignant tumors with high mortality-to-incidence ratios. Nuclear factor erythroid 2-like 3(NFE2 L3), also known as NRF3, is a member of the cap ‘n' collar basic-region leucine zipper family of transcription factors. NFE2 L3 is involved in the regulation of various biological processes, whereas its role in HCC has not been elucidated.AIM To explore the expression and biological function of NFE2 L3 in HCC.METHODS We analyzed the expression of NFE2 L3 in HCC tissues and its correlation with clinicopathological parameters based on The Cancer Genome Atlas(TCGA) data portal. Short hairpin RNA(shRNA) interference technology was utilized to knock down NFE2 L3 in vitro. Cell apoptosis, clone formation, proliferation, migration,and invasion assays were used to identify the biological effects of NFE2 L3 in BEL-7404 and SMMC-7721 cells. The expression of epithelial-mesenchymal transition(EMT) markers was examined by Western blot analysis.RESULTS TCGA analysis showed that NFE2 L3 expression was significantly positively correlated with tumor grade, T stage, and pathologic stage. The qPCR and Western blot results showed that both the mRNA and protein levels of NFE2 L3 were significantly decreased after shRNA-mediated knockdown in BEL-7404 and SMMC-7721 cells. The shRNA-mediated knockdown of NFE2 L3 could induce apoptosis and inhibit the clone formation and cell proliferation of SMMC-7721 and BEL-7404 cells. NFE2 L3 knockdown also significantly suppressed the migration, invasion, and EMT of the two cell lines.CONCLUSION Our study showed that shRNA-mediated knockdown of NFE2 L3 exhibited tumor-suppressing effects in HCC cells.展开更多
Objective： Recurrent ovarian cancer is often resistant to drugs such as paclitaxel. Short hairpin RNA （shRNA） targeting MDRI, a gene involved in the process of drug resistance, may be a promising strategy to overco...Objective： Recurrent ovarian cancer is often resistant to drugs such as paclitaxel. Short hairpin RNA （shRNA） targeting MDRI, a gene involved in the process of drug resistance, may be a promising strategy to overcome drug resistance. Methods： Construction and identification of eukaryotic expression plasmid was transiently transfected into human ovarian cancer ce plasmid of shRNA targeting on MDR1 gene. The ne A2780/Taxol. Apoptosis was determined by flow cytometry using annexin V-FITC/PI double labeling. Expression of MDRI mRNA was detected by quantitative polymerase chain reaction （qPCR） and P-glycoprotein expression was detected using Western blot. Results The IC50 of paclitaxel in MDR1 shRNA-transfected group was significantly reduced （1.986±0.153）μmol/ml as compared with that in negative control （5.246±0.107）μmol/ml and empty vector-transfected group （5.212±0.075）μmol/ml （P〈0.05）. The percent of the relative reverse sensitivity to paclitaxel on A2780/Taxol cells was 67.1%, and the apoptotic rate was significantly increased [（6.977±0.333）%] compared with control [（1.637±0.111）%] and empty vector-transfected group [（1.663±0.114）%] （P〈0.05）. Expressions of MDR1 mRNA and P-glycoprotein were significantly reduced compared with control （P〈0.05）. Conclusion： The present study demonstrated that the eukaryotic expression plasmid of shRNA targeting on MDRI inhibited the expression of MDRI effectively, thus enhance the sensitivity of A2780/Taxol cells to paclitaxel.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 influence of short hairpin RNA(shRNA)-mediated osteopontin(OPN)gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated.Four types of OPN shRNA recombinant plasmids were c...The influence of short hairpin RNA(shRNA)-mediated osteopontin(OPN)gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated.Four types of OPN shRNA recombinant plasmids were constructed and RT-PCR assays were used to screen the most highly functional shRNA recombinant plasmids,which were transferred into the cultured ACHN cells by LipofectamineTM 2000.The cells transfected by shRNA expression vectors(ACHN/OPN)were visualized under an inverted microscope and screened...展开更多
AIM: To study the effectiveness and mechanisms of anti-human vascular endothelial growth factor (hVEGF) hairpin ribozyme on angiogenesis,oncogenicity and tumor growth in a hepatocarcinoma cell line and a xenografted m...AIM: To study the effectiveness and mechanisms of anti-human vascular endothelial growth factor (hVEGF) hairpin ribozyme on angiogenesis,oncogenicity and tumor growth in a hepatocarcinoma cell line and a xenografted model. METHODS: The artificial anti-hVEGF hairpin ribozyme was transfected into hepatocarcinoma cell line SMMC-7721 and,subsequently,polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were performed to confirm the ribozyme gene integration and transcription. To determine the effects of ribozyme ,VEGF expression was detected by semiquantitative RT-PCR and enzyme liked immunosorbent assay (ELISA). MTT assay was carried out to measure the cell proliferation. Furthermore,the transfected and control cells were inoculated into nude mice respectively,the growth of cells in nude mice and angiogenesis were observed. RESULTS: VEGF expression was down-regulated sharply by ribozyme in transfected SMMC-7721 cells and xenografted tumor. Compared to the control group,the transfected cells grew slower in cell cultures and xenografts,and the xenograft formation was delayed as well. In addition,the microvessel density of the xenografted tumor was obviously declined in the transfected group. As demonstratedby microscopy,reduction of VEGF production induced by ribozyme resulted in a significantly higher cell differentiation and less proliferation vigor in xenografted tumor. CONCLUSION: Anti-hVEGF hairpin ribozyme can effectively inhibit VEGF expression and growth of hepatocarcinoma in vitro and in vivo. VEGF is functionally related to cell proliferation,differentiation and tumori-genesis in hepatocarcinoma.展开更多
AIM:To evaluate whether recombinant complement factor B(CFB)short hairpin RNA(sh RNA)reduces laserinduced choroidal neovascularization(CNV)in rats.METHODS:Laser-induced rat CNV model was established,and then the anima...AIM:To evaluate whether recombinant complement factor B(CFB)short hairpin RNA(sh RNA)reduces laserinduced choroidal neovascularization(CNV)in rats.METHODS:Laser-induced rat CNV model was established,and then the animals underwent fundus fluorescence angiography(FFA)and hematoxylin and eosin(HE)staining.On day 3 and 7 after photocoagulation,the expression of CFB and membrane attack complex(MAC)was detected by immunhischemistry.A recombinant CFBsh RNA plasmid was constructed.CFB and scrambled sh RNA plasmids were intravenous injected into rats via the tail vein on the day of laser treatment,respectively.On day 7,the incidence of CNV was determined by FFA,and the expression of CFB and vascular endothelial growth factor(VEGF)in retinal pigment epithelium(RPE)/choroidal tissues was detected by immunhischemistry,Western blot and/or semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR)in CFB and scrambled sh RNA groups.The possible adverse effects of CFB-sh RNA injection were assessed by transmission electron microscopy and electroretinography.RESULTS:FFA and HE results indicated that a laserinduced rat CNV model was successfully established on day 7 after photocoagulation.The expression of CFB and MAC was extremely weak in normal retina and choroid,and increased on day 3 after photocoagulation.However,it started to reduce on day 7.CFB sh RNA plasmid was successfully constructed and induced CFB knockdown in the retinal and choroidal tissues.FFA showed CFB knockdown significantly inhibited incidence of CNV in rats.Moreover,CFB knockdown significantly inhibited the expression of VEGF in RPE/choroidal tissues.CFB sh RNA caused no obvious side effects in eyes.CONCLUSION:CFB knockdown significantly inhibits the formation and development of CNV in vivo through reducing the expression of VEGF,which is a potential therapy target.The alternative pathway of complement activation plays an important role in CNV formation.展开更多
BACKGROUND Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.The identification of novel diagnostic and prognostic biomarkers for CRC is a key research imperative.Immunohistochemical analysis ...BACKGROUND Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.The identification of novel diagnostic and prognostic biomarkers for CRC is a key research imperative.Immunohistochemical analysis has revealed high expression of centromere protein K(CENPK)in CRC.However,the role of CENPK in the progression of CRC is not well characterized.AIM To evaluate the effects of knockdown of CENPK and overexpression of Cullin 4A(CUL4A)in RKO and HCT116 cells.METHODS Human colon cancer samples were collected and tested using a human gene expression chip.We identified CENPK as a potential oncogene for CRC based on bioinformatics analysis.In vitro experiments verified the function of this gene.We investigated the expression of CENPK in RKO and HCT116 cells using quantitative polymerase chain reaction(qPCR),western blot,and flow cytometry.The effect of short hairpin RNA(shRNA)virus-infected RKO cells on tumor growth was evaluated in vivo using quantitative analysis of fluorescence imaging.To evaluate the effects of knockdown of CENPK and overexpression of CUL4A in RKO and HCT116 cells,we performed a series of in vitro experiments,using qPCR,western blot,MTT assay,and flow cytometry.RESULTS We demonstrated overexpression of CENPK in human colon cancer samples.CENPK was an independent risk factor in patients with CRC.The downstream genes FBX32,CUL4A,and Yesassociated protein isoform 1 were examined to evaluate the regulatory action of CENPK in RKO cells.Significantly delayed xenograft tumor emergence,slower growth rate,and lower final tumor weight and volume were observed in the CENPK short hairpin RNA virus infected group compared with the CENPK negative control group.The CENPK gene interference inhibited the proliferation of RKO cells in vitro and in vivo.The lentivirus-mediated shRNA interference of CENPK inhibited the proliferation of RKO and HCT116 colon cancer cells,with overexpression of the CUL4A.CONCLUSION We indicated a potential role of CENPK in promoting tumor proliferation,and it may be a novel diagnostic and prognostic biomarker for CRC.展开更多
The hairpin probe using microwave resonance in plasma is applicable to high pressure 1.33 ×10^3-1.01×10^5 Pa）） as developed recently. In this work, an analytic model of the hairpin resonator probe surround...The hairpin probe using microwave resonance in plasma is applicable to high pressure 1.33 ×10^3-1.01×10^5 Pa）） as developed recently. In this work, an analytic model of the hairpin resonator probe surrounded by a thin dielectric layer and a sheath layer is proposed. The correction factor due to these surroundings is analytically found and confirmed by electromagnetic field finite difference time domain simulation, thus enabling the accurate measurement of electron density in a high-pressure non-equilibrium uniform discharge.展开更多
Different discharge morphologies in atmospheric Ar and He plasmas are excited by using a pulsed microwave hairpin resonator.Ar plasmas form an arched plasma plume at the opened end of the hairpin,whereas He plumes gen...Different discharge morphologies in atmospheric Ar and He plasmas are excited by using a pulsed microwave hairpin resonator.Ar plasmas form an arched plasma plume at the opened end of the hairpin,whereas He plumes generate only a contracted plasmas in between both tips of metal electrodes.Despite this different point,their discharge processes have three similar characteristics:(i)the ionization occurs at the main electrode firstly and then develops to the slave electrode,(ii)during the shrinking stage the middle domain of the discharge channels disappears at last,and(iii)even at zero power input(in between pulses)a weak light region always exists in the discharge channels.Both experimental results and electromagnetic simulations suggest that the discharge is resonantly excited by the local enhanced electric fields.In addition,Ar ionization and excitation energies are lower than those of He,the effect of Ar gas flow is far greater than that of He gas,and the contribution of accelerated electrons only locates at the domain with the strongest electric fields.These reasons could be used to interpret the different characteristic plume morphologies of the proposed atmospheric Ar and He plasmas.展开更多
Objective:The aim of this study was to study the effect of Bcl-2 small hairpin RNA(shRNA) enhancing methotrexate(MTX)-induced apoptosis of Raji cells.Methods:Expression plasmid with Bcl-2 shRNA was transfected into Ra...Objective:The aim of this study was to study the effect of Bcl-2 small hairpin RNA(shRNA) enhancing methotrexate(MTX)-induced apoptosis of Raji cells.Methods:Expression plasmid with Bcl-2 shRNA was transfected into Raji cells by Lipofectmine 2000 and then treated with MTX.At 48 h of transfection,the expression level of Bcl-2 mRNA and protein was evaluated by RT-PCR and immunofluorescence.MTT assay was used to analyze cell proliferation at 24,48 and 72 h.Apoptosis was detected by Giemsa staining and flow cytometric cell cycle analysis.Results:After transfection with Bcl-2 shRNA,the expression levels of Bcl-2 mRNA and protein in Raji cells decreased(P < 0.05).Using Giemsa staining,cells transfected with Bcl-2 shRNA combined with MTX at 48 h displayed changes of apoptosis.MTX significantly inhibited the growth of cells after transfected with Bcl-2 shRNA(P < 0.05).Apoptotic rates of the Raji cells treated with Bcl-2 shRNA combined with MTX significantly increased(P < 0.05),compared with either control shRNA/MTX combination or MTX-treatment cells alone.Conclusion:Our results suggest the shRNA against Bcl-2 mRNA could increase MTX-induced apoptosis of Raji cells.展开更多
基金Supported by The Science and Technology Project of General Administration of Quality Supervision,Inspection and Quarantine (2015IK126)The Science and Technology Project of Changsha City of Hunan Province of China (KQ1602124).
文摘Mercury ion(Hg^(2+)),a highly noxious of heavy metalion,has detrimental effects on the ecological environment and human health.Herein,we have developed an exonuclease III(Exo III)assisted catalytic hairpin assembly formation of a trivalent G-quadruplex/hemin DNAzyme for colorimetric detection of Hg^(2+).A hairpin DNA(Hr)was designed with thymine-Hg^(2+)-thymine pairs that catalyzed by Exo III is prompted to happen upon binding Hg^(2+).A released DNA fragment triggers the catalytic assembly of other three hairpins(H1,H2,and H3)to form many trivalent G-quadruplex/hemin DNA enzymes for signal output.The developed sensor shows a dynamic range from 2 pM to 2μM,with an impressively low detection limit of 0.32 pM for Hg^(2+)detection.Such a sensor also has good selectivity toward Hg^(2+)detection in the presence of other common metal ions.This strategy shows the great potential for visual detection with portable type.
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 stably transfected with survivin-shRNA were injected into BALB/c nude mice, tumor growth was dramatically lower and the tumor was smaller than that of either pGenesil-1-NC transfected cells or control cells (P<0.01). The inhibitory effect of pGenesil-1-survivin-1 was the best among the three kinds of survivin-shRNA plasmids, but no combination of inhibitory effects was found in pGenesil-1-survivin-1+2. CONCLUSIONS: shRNAs specific to survivin have gene silencing effects and inhibit pancreatic cancer cell proliferation. shRNA activity against survivin could be of potential value in gene therapy for pancreatic cancer. However, shRNAs with double combining sites did not significantly enhance the interference compared with single site shRNAs, therefore further studies on this are needed.
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the Changzhou High-Level Medical Talents Training Project,No.2016ZCLJ002
文摘BACKGROUND Hepatocellular carcinoma(HCC) is one of the most common malignant tumors with high mortality-to-incidence ratios. Nuclear factor erythroid 2-like 3(NFE2 L3), also known as NRF3, is a member of the cap ‘n' collar basic-region leucine zipper family of transcription factors. NFE2 L3 is involved in the regulation of various biological processes, whereas its role in HCC has not been elucidated.AIM To explore the expression and biological function of NFE2 L3 in HCC.METHODS We analyzed the expression of NFE2 L3 in HCC tissues and its correlation with clinicopathological parameters based on The Cancer Genome Atlas(TCGA) data portal. Short hairpin RNA(shRNA) interference technology was utilized to knock down NFE2 L3 in vitro. Cell apoptosis, clone formation, proliferation, migration,and invasion assays were used to identify the biological effects of NFE2 L3 in BEL-7404 and SMMC-7721 cells. The expression of epithelial-mesenchymal transition(EMT) markers was examined by Western blot analysis.RESULTS TCGA analysis showed that NFE2 L3 expression was significantly positively correlated with tumor grade, T stage, and pathologic stage. The qPCR and Western blot results showed that both the mRNA and protein levels of NFE2 L3 were significantly decreased after shRNA-mediated knockdown in BEL-7404 and SMMC-7721 cells. The shRNA-mediated knockdown of NFE2 L3 could induce apoptosis and inhibit the clone formation and cell proliferation of SMMC-7721 and BEL-7404 cells. NFE2 L3 knockdown also significantly suppressed the migration, invasion, and EMT of the two cell lines.CONCLUSION Our study showed that shRNA-mediated knockdown of NFE2 L3 exhibited tumor-suppressing effects in HCC cells.
基金support from laboratory of the second hospital and medical college of Shandong university
文摘Objective： Recurrent ovarian cancer is often resistant to drugs such as paclitaxel. Short hairpin RNA （shRNA） targeting MDRI, a gene involved in the process of drug resistance, may be a promising strategy to overcome drug resistance. Methods： Construction and identification of eukaryotic expression plasmid was transiently transfected into human ovarian cancer ce plasmid of shRNA targeting on MDR1 gene. The ne A2780/Taxol. Apoptosis was determined by flow cytometry using annexin V-FITC/PI double labeling. Expression of MDRI mRNA was detected by quantitative polymerase chain reaction （qPCR） and P-glycoprotein expression was detected using Western blot. Results The IC50 of paclitaxel in MDR1 shRNA-transfected group was significantly reduced （1.986±0.153）μmol/ml as compared with that in negative control （5.246±0.107）μmol/ml and empty vector-transfected group （5.212±0.075）μmol/ml （P〈0.05）. The percent of the relative reverse sensitivity to paclitaxel on A2780/Taxol cells was 67.1%, and the apoptotic rate was significantly increased [（6.977±0.333）%] compared with control [（1.637±0.111）%] and empty vector-transfected group [（1.663±0.114）%] （P〈0.05）. Expressions of MDR1 mRNA and P-glycoprotein were significantly reduced compared with control （P〈0.05）. Conclusion： The present study demonstrated that the eukaryotic expression plasmid of shRNA targeting on MDRI inhibited the expression of MDRI effectively, thus enhance the sensitivity of A2780/Taxol cells to paclitaxel.
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supported by a grant from the Major State Basic Research Development Program of China(973 Program)(No.2002CB513100)
文摘The influence of short hairpin RNA(shRNA)-mediated osteopontin(OPN)gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated.Four types of OPN shRNA recombinant plasmids were constructed and RT-PCR assays were used to screen the most highly functional shRNA recombinant plasmids,which were transferred into the cultured ACHN cells by LipofectamineTM 2000.The cells transfected by shRNA expression vectors(ACHN/OPN)were visualized under an inverted microscope and screened...
基金Supported by the Grant from Calling for Tenders by Key Subject of Jiangsu Province, No. WK200221
文摘AIM: To study the effectiveness and mechanisms of anti-human vascular endothelial growth factor (hVEGF) hairpin ribozyme on angiogenesis,oncogenicity and tumor growth in a hepatocarcinoma cell line and a xenografted model. METHODS: The artificial anti-hVEGF hairpin ribozyme was transfected into hepatocarcinoma cell line SMMC-7721 and,subsequently,polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were performed to confirm the ribozyme gene integration and transcription. To determine the effects of ribozyme ,VEGF expression was detected by semiquantitative RT-PCR and enzyme liked immunosorbent assay (ELISA). MTT assay was carried out to measure the cell proliferation. Furthermore,the transfected and control cells were inoculated into nude mice respectively,the growth of cells in nude mice and angiogenesis were observed. RESULTS: VEGF expression was down-regulated sharply by ribozyme in transfected SMMC-7721 cells and xenografted tumor. Compared to the control group,the transfected cells grew slower in cell cultures and xenografts,and the xenograft formation was delayed as well. In addition,the microvessel density of the xenografted tumor was obviously declined in the transfected group. As demonstratedby microscopy,reduction of VEGF production induced by ribozyme resulted in a significantly higher cell differentiation and less proliferation vigor in xenografted tumor. CONCLUSION: Anti-hVEGF hairpin ribozyme can effectively inhibit VEGF expression and growth of hepatocarcinoma in vitro and in vivo. VEGF is functionally related to cell proliferation,differentiation and tumori-genesis in hepatocarcinoma.
文摘AIM:To evaluate whether recombinant complement factor B(CFB)short hairpin RNA(sh RNA)reduces laserinduced choroidal neovascularization(CNV)in rats.METHODS:Laser-induced rat CNV model was established,and then the animals underwent fundus fluorescence angiography(FFA)and hematoxylin and eosin(HE)staining.On day 3 and 7 after photocoagulation,the expression of CFB and membrane attack complex(MAC)was detected by immunhischemistry.A recombinant CFBsh RNA plasmid was constructed.CFB and scrambled sh RNA plasmids were intravenous injected into rats via the tail vein on the day of laser treatment,respectively.On day 7,the incidence of CNV was determined by FFA,and the expression of CFB and vascular endothelial growth factor(VEGF)in retinal pigment epithelium(RPE)/choroidal tissues was detected by immunhischemistry,Western blot and/or semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR)in CFB and scrambled sh RNA groups.The possible adverse effects of CFB-sh RNA injection were assessed by transmission electron microscopy and electroretinography.RESULTS:FFA and HE results indicated that a laserinduced rat CNV model was successfully established on day 7 after photocoagulation.The expression of CFB and MAC was extremely weak in normal retina and choroid,and increased on day 3 after photocoagulation.However,it started to reduce on day 7.CFB sh RNA plasmid was successfully constructed and induced CFB knockdown in the retinal and choroidal tissues.FFA showed CFB knockdown significantly inhibited incidence of CNV in rats.Moreover,CFB knockdown significantly inhibited the expression of VEGF in RPE/choroidal tissues.CFB sh RNA caused no obvious side effects in eyes.CONCLUSION:CFB knockdown significantly inhibits the formation and development of CNV in vivo through reducing the expression of VEGF,which is a potential therapy target.The alternative pathway of complement activation plays an important role in CNV formation.
基金the National Natural Science Foundation of China,No.81860416 and No.22168028Inner Mongolia Autonomous Region Grassland Talent Innovation Talent Team Fund,No.2019Inner Mongolia Natural Science Fund,No.2021MS02005.
文摘BACKGROUND Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.The identification of novel diagnostic and prognostic biomarkers for CRC is a key research imperative.Immunohistochemical analysis has revealed high expression of centromere protein K(CENPK)in CRC.However,the role of CENPK in the progression of CRC is not well characterized.AIM To evaluate the effects of knockdown of CENPK and overexpression of Cullin 4A(CUL4A)in RKO and HCT116 cells.METHODS Human colon cancer samples were collected and tested using a human gene expression chip.We identified CENPK as a potential oncogene for CRC based on bioinformatics analysis.In vitro experiments verified the function of this gene.We investigated the expression of CENPK in RKO and HCT116 cells using quantitative polymerase chain reaction(qPCR),western blot,and flow cytometry.The effect of short hairpin RNA(shRNA)virus-infected RKO cells on tumor growth was evaluated in vivo using quantitative analysis of fluorescence imaging.To evaluate the effects of knockdown of CENPK and overexpression of CUL4A in RKO and HCT116 cells,we performed a series of in vitro experiments,using qPCR,western blot,MTT assay,and flow cytometry.RESULTS We demonstrated overexpression of CENPK in human colon cancer samples.CENPK was an independent risk factor in patients with CRC.The downstream genes FBX32,CUL4A,and Yesassociated protein isoform 1 were examined to evaluate the regulatory action of CENPK in RKO cells.Significantly delayed xenograft tumor emergence,slower growth rate,and lower final tumor weight and volume were observed in the CENPK short hairpin RNA virus infected group compared with the CENPK negative control group.The CENPK gene interference inhibited the proliferation of RKO cells in vitro and in vivo.The lentivirus-mediated shRNA interference of CENPK inhibited the proliferation of RKO and HCT116 colon cancer cells,with overexpression of the CUL4A.CONCLUSION We indicated a potential role of CENPK in promoting tumor proliferation,and it may be a novel diagnostic and prognostic biomarker for CRC.
基金supported by the National Natural Science Foundation of China (Grant No. 10835004 )the 2nd Knowledge Cluster Research Project of Japan:Tokai Region Nanotechnology Manufacturing Cluster (Innovation of Environment Friendly Highly Functional Materials and Devices)
文摘The hairpin probe using microwave resonance in plasma is applicable to high pressure 1.33 ×10^3-1.01×10^5 Pa）） as developed recently. In this work, an analytic model of the hairpin resonator probe surrounded by a thin dielectric layer and a sheath layer is proposed. The correction factor due to these surroundings is analytically found and confirmed by electromagnetic field finite difference time domain simulation, thus enabling the accurate measurement of electron density in a high-pressure non-equilibrium uniform discharge.
基金Project supported by the National Natural Science Foundation of China(Grant Nos.11575003 and 51607003)
文摘Different discharge morphologies in atmospheric Ar and He plasmas are excited by using a pulsed microwave hairpin resonator.Ar plasmas form an arched plasma plume at the opened end of the hairpin,whereas He plumes generate only a contracted plasmas in between both tips of metal electrodes.Despite this different point,their discharge processes have three similar characteristics:(i)the ionization occurs at the main electrode firstly and then develops to the slave electrode,(ii)during the shrinking stage the middle domain of the discharge channels disappears at last,and(iii)even at zero power input(in between pulses)a weak light region always exists in the discharge channels.Both experimental results and electromagnetic simulations suggest that the discharge is resonantly excited by the local enhanced electric fields.In addition,Ar ionization and excitation energies are lower than those of He,the effect of Ar gas flow is far greater than that of He gas,and the contribution of accelerated electrons only locates at the domain with the strongest electric fields.These reasons could be used to interpret the different characteristic plume morphologies of the proposed atmospheric Ar and He plasmas.
基金Supported by the grants from the Natural Science Program Foundation of Guangdong Province(No.04010446)the Overseas Chinese Affairs Office of the State Council Key Discipline Construction Fund (No.51205002)
文摘Objective:The aim of this study was to study the effect of Bcl-2 small hairpin RNA(shRNA) enhancing methotrexate(MTX)-induced apoptosis of Raji cells.Methods:Expression plasmid with Bcl-2 shRNA was transfected into Raji cells by Lipofectmine 2000 and then treated with MTX.At 48 h of transfection,the expression level of Bcl-2 mRNA and protein was evaluated by RT-PCR and immunofluorescence.MTT assay was used to analyze cell proliferation at 24,48 and 72 h.Apoptosis was detected by Giemsa staining and flow cytometric cell cycle analysis.Results:After transfection with Bcl-2 shRNA,the expression levels of Bcl-2 mRNA and protein in Raji cells decreased(P < 0.05).Using Giemsa staining,cells transfected with Bcl-2 shRNA combined with MTX at 48 h displayed changes of apoptosis.MTX significantly inhibited the growth of cells after transfected with Bcl-2 shRNA(P < 0.05).Apoptotic rates of the Raji cells treated with Bcl-2 shRNA combined with MTX significantly increased(P < 0.05),compared with either control shRNA/MTX combination or MTX-treatment cells alone.Conclusion:Our results suggest the shRNA against Bcl-2 mRNA could increase MTX-induced apoptosis of Raji cells.
