Recombinant hFGF-10 adenovirus promotes proliferation of kerotinocytes

AIM:To construct the recombinant adenoviral vector containing human fibroblast growth factor 10 (hFGF-10) gene, and to study the effect of the recombinant adenovirus on the proliferation of kerotinocytes. METHODS:HFGF-10 gene was amplified by PCR and ligated with shuttle vector pAdTrack-CMV to get the recombinant plasmid pAdTrack-CMV-hFGF-10, which was linearized with PmeI and transferred into Escherichia coli BJ5183 containing the adenoviral bone plasmid pAdEasy-1 for homologous recombination to obtain the recombinant adenoviral plasmid pAdEasy-hFGF-10. The recombinant adenoviral plasmid was then transfected into HEK-293 cell line to package and amplify the recombinant adenovirus. The expression of hFGF-10 in HaCat cells infected with the recombinant adenovirus was detected by Western blotting. The influence of the recombinant adenovirus on the proliferation of kerotinocytes was checked by MTT. RESULTS:The recombinant adenovirus containing hFGF-10 gene was successfully constructed, which effectively infected HaCat cells. The result of Western blotting showed that a protein in culture media of the infected HaCat cells reacted with hFGF-10 antibody. The recombinant adenovirus stimulated the proliferation of kerotinocytes. CONCLUSION:HaCat cells infected with the recombinant adenovirus expresses and secrets hFGF-10 protein, which promotes the proliferation of HaCat cells.     

Effect of adipose differentiation-related protein on proliferation and apoptosis in H9c2 cells

AIM: To construct an adipose differentiation-related protein (ADRP) eukaryotic expression vector and to explore the effect of ADRP on apoptosis of H9c2 cells induced by palmitic acid (PA). METHODS: The ADRP gene obtained by the method of RT-PCR was cloned into pEGFP-C1 plasmid. The recombinant plasmid was transformed into E.coli DH5α for amplification. The recombinant plasmid was extracted from E.coli DH5α and transfected into H9c2 cells by Lipofectamine^TM2000. The stable transformants were selected by G418 screening. Expression of green fluorescent protein was observed under fluorescence microscope and the ADRP expression was identified by RT-qPCR and Western blotting analysis. The effect of PA on the proliferation of H9c2 cells was detected by MTT assay. The apoptotic percentage of H9c2 cells caused by PA was determined by flow cytometry. RESULTS: The eukaryotic expression vector pEGFP-C1-ADRP was successfully constructed. Green fluorescent was observed in the cells transfected with pEGFP-C1 or pEGFP-C1-ADRP under fluorescence microscope. RT-qPCR and Western blotting analysis showed that recombinant cells exhibited high mRNA and protein levels of ADRP. After treated with PA at different concentrations, the apoptosis rates and the proliferation inhibition of recombinant cells were both lower than those of the other two cells. CONCLUSION: The transfected H9c2 cells with stable ADRP expression were successfully established. The over-expression of ADRP prevents the cells from apoptosis and inhibition of proliferation caused by PA, indicating that ADRP plays a protective role in H9c2 cells.     

Construction of eukaryotic expression vector of fusion gene CD80-IgG and its expression in Chinese hamster ovary cells

AIM: To construct the eukaryotic expression vector CD80-IgG by fusing the cDNA encoding extracellular portion of murine CD80 to the 5'-terminus of cDNA encoding Fc fragment of murine immunoglobulin G1 and to express the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The two cDNAs was amplified by PCR respectively from plasmid pcDNA/B7 containing the full-length cDNA of murine CD80 from murine spleen cells, and cloned to the eukaryotic expression vector pcDNA3.0 by directional cloning. The resultant recombinant plasmid pcDNA/CD80-IgG was transfected into CHO cells with liposome transfection reagent. The stably expressing cells were obtained by G418 screening. Western blot, Dot ELISA, and flow cytometry were used to detect the expression of the fusion protein and its immunological activity. RESULTS: DNA sequencing verified the correction of the construction of recombinant plasmid pcDNA/CD80-IgG. The expressed fusion protein was detected in the supernatant of transfected CHO cells and the molecular weight of the protein was similar to what we expected. Its immunological activity was also established. CONCLUSION: The recombinant plasmid pcDNA/CD80-IgG was successfully constructed and it expressed the fusion protein CD80-IgG.     

Effect of PRL-2 gene transfection on proliferation and cell cycle in hepatocellular cell line

AIM: To observe the changes of proliferation and cell cycle after PRL-2 gene effectively expressed in human hepatocellular cell line.METHODS: The PRL-2 vector was transfected into CL1 cell with lipofectamine reagent,the stable expression clones were screened by G418.The expression of PRL-2 mRNA was detected by real-time PCR.The expressive protein was identified by Western blotting.The subcellular localization was demonstrated by immunochemistry.The cell cycle was measured by flow cytometry.The population doubling time (TD) was analyzed by MTT assay.The expressions of cyclin A,cyclin D1,cyclin E,p16,p21Waf1 and p27Kip1 were detected by Western blotting.The p21Waf1 mRNA was determined by real- time PCR.RESULTS: The full length ORF of PRL-2 gene was inserted into the vector pcDNA3.1 (+),transfected into CL1 cells,and expressed successfully.Real-time PCR showed stable expression of PRL-2 mRNA.Western blotting confirmed the overexpression of PRL-2 protein.The subcellular localization of PRL-2 was in the plasmid.The proportion of cells in S-phase was increased.The population doubling time was reduced (P<0.01),a significant decrease was observed both in the mRNA and the protein expression of the p21Waf1 in comparison with untransfected or vector- transfected control cells (P<0.05).The expressions of cyclin D1,cyclin E,cyclinA,p16 and p27Kip1 were not appreciably different between the control and PRL-transfected cell lines.CONCLUSION: Eukaryocytic expression vector of PRL-2 has been successfully constructed,which shows stable and effective expression in CL1 cell line.PRL -2 increases cell proliferation by stimulating progression from G1 into S phase,which is primarily associated with decreased p21Waf1.     

Effects of TSG101 siRNA on the growth and drug sensitivity of SH-SY5Y cells

AIM:To investigate the effects of TSG101 siRNA on the growth and drug sensitivity of human neuroblastoma cell line SH-SY5Y.METHODS:The small interfering RNA eukaryotic expression vector specific to human TSG101 gene was constructed by gene recombination,then transfected into SH-SY5Y cells.Stable transfectants were obtained by G418 screening and further identified by RT-PCR and Western blotting analysis.The growth curve was made using MTT assay.Cell cycle distribution of the transfected cells was studied by flow cytometry and the proliferative indexes were calculated.The apoptosis after CDDP treatment was detected by DNA ladder and Annexin V/propidium iodide binding analyses.The expression of Bcl-2,Bax,P-gp and MRP were analyzed by Western blotting.RESULTS:mU6pro-TSG101 siRNA was successfully constructed and transfected into SH-SY5Y cells.As detected by MTT and flow cytometry,down-regulation of TSG101 significantly suppressed the proliferation of SH-SY5Y cells with a G1 cell cycle arrest,compared with that in control (P<0.05).As detected by DNA ladder and Annexin V/propidium iodide binding analyses,down-regulation of TSG101 significantly enhanced the sensitivity of SH-SY5Y cells to CDDP-induced apoptosis,compared with that in control (P<0.05).The expression of P-gp and Bcl-2 in transfected cells were decreased as compared with that in the control,while MRP and Bax were not.CONCLUSIONS:Down-regulation of TSG101 suppresses the proliferation of SH-SY5Y cells,and enhances the sensitivity of SH-SY5Y cells to conventional chemotherapeutic agents to a degree,suggesting TSG101 may be useful for gene therapy in the future.     

Transition of WT1 isoforms inhibits proliferation and promotes apoptosis of human leukemia cell line HL-60

AIM: To study the potential effects of exogenous Wilms tumor 1 (WT1) isoforms on the proliferation and apoptosis of human leukemia cell line HL-60. METHODS: WT1 (17AA－/KTS－) gene obtained by RT-PCR was cloned into a PCDH1-MCS1-EF1-copGFP plasmid. The recombinant plasmid was confirmed by enzyme digestion and sequencing, and was transfected into HL-60 cells by LipofectamineTM 2000. The stable transformants were selected by G418 screening. WT1(17AA－/KTS－) expression was identified by real-time fluorescence quantitative RT-PCR and Western blotting. The proliferation of the cells was measured by MTT assay. The apoptosis of the cells was determined by morphological observation and flow cytometry analysis. RESULTS: The eukaryotic expression vector PCDH1-MCS1-EF1-copGFP-WT1 (17AA－/KTS－) was successfully constructed. The recombinant cells exhibited high mRNA and protein levels of WT1(17AA－/KTS－). The growth of recombinant cells was slower than that of HL-60 cells transfected with control vector and normal HL-60 cells. After exposed to As2O3 at 2 μmol/L for 48 h, both recombinant cells and control cells exhibited the morphological characteristics of apoptosis, but the former was more typical than the latter. The apoptosis was enhanced in the recombinant cells after the cells were exposed to As2O3 for 24 h. CONCLUSION: Exogenous WT1(17AA-/KTS-) isoform inhibits the proliferation and promotes the apoptosis of leukemic cells.     

Effect of estrogen receptor 2 on the cell proliferation in prostate cancer cell line PC-3M

AIM: To construct the recombination plasmid pcDNA3.1-hERβ with the human estrogen receptor 2 (ESR2) full length cDNA and transfect it into hormone-independent prostate cancer PC-3M cell line, and to study the effects of ESR2 on proliferation in transfected cells. METHODS: The complete cDNA of ESR2 was obtained from human ovary tissue by RT-PCR technique and cloned into eukaryotic expression vector pcDNA3.1 by using gene recombination technique to construct the pcDNA3.1-hERβ recombination plasmid. The plasmid was detected by endonuclease digestion and DNA sequencing and was transfected into PC-3M cells. MTT and FCAS assay were used to test the effects of ESR2 on the ability of proliferation in PC-3M cells. RT-PCR and Western blotting were used to detect the expressions of cyclinD1 and P21Cip1. RESULTS: The results of sequencing and endonuclease digestion demonstrated that the construction of pcDNA3.1-hERβ recombination plasmid was successful. The sequence analysis suggested that the ESR2 sequence detected by PCR was identical to that published in GenBank, and the product of endonuclease was as long as the complete human ESR2 gene. 48 h after transfected the pcDNA3.1-hERβ into PC-3M cells, the expression of ESR2 mRNA and protein levels increased significantly detected by RT-PCR and Western blotting. Compared to the cells transfected with vector as control, the PC-3M cells transfected with pcDNA3.1-hERβ showed that cell population decreased and proliferation activity degraded. FCAS showed that the cells in G0/G1 stage increased and in S stage or G2/M stage decreased. RT-PCR and Western blotting showed that the expression of cyclinD1 gene reduced and expression of P21Cip1 increased. CONCLUSION: The recombination of plasmid pcDNA3.1-hERβ is constructed and transfected into the PC-3M cells successfully. The activity of cell proliferation is inhibited after pcDNA3 transfection.1-hERβ. It is possible that ESR2 inhibits cell proliferation by the expression of proliferation related genes cyclinD1 and P21Cip1.     

Construction of FAK-shRNA retroviral vector and screening of stable HCC-LM3 cell line with persistent knockdown of FAK

AIM: To construct a recombinant retroviral vector of short interfering RNA targeting focal adhesion kinase (FAK) gene and to establish a cell line with stable knockdown of FAK.METHODS: The oligonucleotides that transcribed to short hairpin RNA (shRNA) targeting FAK gene were synthesized in vitro, cloned into retroviral vector pSuper.retro and transfected into Phoenix cell line. The stable clones were screened and high-titer virus was produced. The human hepatocellular carcinoma cell line HCC-LM3 was infected with the virus-rich supernatant. The stable LM3 cell line, which showed significantly to silence FAK and associated proteins, was selected by puromycin.RESULTS: The recombinant retroviral vector was successfully constructed. Persistent knockdown of FAK in the LM3 cell line infected with the supernatant containing the retrovirus was confirmed by Western blotting. Down-regulation of FAK resulted in the inhibition of p-Akt and p-MAPK1/2 expression and led to decreased migration and invasion of the cells. The cell cycle was blocked at G₀/G₁ phase, and apoptosis was increased. The proliferation rate also decreased significantly.CONCLUSION: FAK-shRNA virus generated by recombinant retroviral vector pSuper-FAK can inhibit the protein expression of FAK and phosphorylation of Akt and MAPK1/2 in HCC-LM3 cells. Down-regulation of FAK shows a significant impact on biological behaviors of tumor cells.     

Construction of recombinant adenovirus expressing BDNF and its expression in expanded rat mesenchymal stem cells in vitro

AIM:To construct recombinant adenovirus vector containing brain derived neurotrophic factor, (BDNF) gene using bacterial homogenous recombination, and investigate the expression in expanded rat mesenchymal stem cells (rMSC) in vitro.METHODS:BDNF gene and proBDNF gene were subcloned into adenovirus shuttle plasmid pAdTrack-CMV containing enhanced green fluorescent protein gene (EGFP) expression cassette, forming shuttle vector of pAdTrack-BDNF, and pAdTrack-proBDNF, and co-transformed into BJ5183 bacterial cells with adenovirus backbone vector pAdEasy-1 using chemical transformation. After the recombinant adenovirus vector was obtained, the identified recombinant adenovirus plasmid DNA was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. rMSC were infected by recombinant adenovirus and EGFP expression was detected using fluorescent microscope. Infection efficiency was assessed by flow cytometrics. Western blotting identified expression of Ad -proBDNF and Ad-BDNF in rMSC. rMSC infected with Ad -proBDNF and Ad-BDNF were induced to differentiate into neuron-like cells. rMSC infected with Ad -proBDNF and Ad-BDNF were injected into nude mice and assessd in vivo.RESULTS:We successfully constructed the recombinant adenovirus Ad -proBDNF and Ad-BDNF that expressed in expanded rMSC in vitro.CONCLUSION:Recombinant adenovirus high-effectively mediates Ad -proBDNF and Ad-BDNF expression in expanded rMSC in vitro and in vivo.     

Effect of recombinant connective tissue growth factor on the phenotype transition of rat adventitia fibroblasts

AIM:To construct pcDNA3.1(+)/connective tissue growth factor (CTGF) eukaryotic expression plasmid and to investigate its role in the promotion of phenotypic transition in adventitia fibroblasts (AF). METHODS:The expression vector pcDNA3.1(+)/CTGF was constructed by routine molecular biological method. The expression vector pcDNA3.1(+)/CTGF was confirmed by restriction enzyme digestion and sequencing method. The expression vector pcDNA3.1(+)/CTGF was transfected into AF and the exogenous expression was observed. The expression of the α-SM 〖JP+1〗actin was examined by Western blotting. RESULTS:The eukaryotic expression vector of CTGF was successfully constructed, which was transfected into AF, the expressed CTGF promoted phenotype transition in AF. CONCLUSION:The pcDNA3.1(+)/CTGF plasmid was constructed and transfected into AF, the expressed CTGF promoted phenotype transition in AF.     

Effects of STK15 overexpression on growth of human esophageal carcinoma cell line KYSE150

AIM:To investigate the effects of serine/threonine kinase 15 (STK15) overexpression on the growth of human esophageal squamous-cell carcinoma (ESCC) cell line KYSE150. METHODS:Recombinant pEGFP-C1-STK15 expression vector was transfected into KYSE150 cells using LipofectamineTM 2000 and the expression of STK15 was detected by fluorescence microscopy and Western blotting. The proliferation of the cells in vitro was measured by MTT assay. The cell cycle distribution and apoptosis were detected by flow cytometry. The proliferation of the cells in vivo was measured by tumorigenicity experiment in nude mice. RESULTS:After recombinant pEGFP-C1-STK15 expression vector was stably transfected into KYSE150 cells, GFP-STK15 fusion protein localized to centrosome and spindle. The STK15-overexpressing colonies were further confirmed by Western blotting. MTT assay showed that the proliferation of the cells in STK15 overexpression group was increased compared with control group (P<0.01). Flow cytometry analysis showed that the percentage of the cells in G0/G1 phase and the cell apoptosis in STK15 overexpression group were decreased compared with control group (P<0.01). The tumorigenicity experiment in nude mice showed that the proliferation of the cells in STK15 overexpression group was increased compared with control group (P<0.01). CONCLUSION: Overexpression of STK15 in human ESCC KYSE150 cells promotes the cell growth in vitro and in vivo, indicating that STK15 may serve as a novel therapeutic target for esophageal carcinoma.     

High expression of PDX-1 in bone marrow mesenchymal stem cells mediated by superfect

AIM: To construct a eukaryotic expression vector containing pancreatic duodenal homebox-1 (PDX-1) and to elevate the expression efficiency of exogenous gene in rat bone marrow mesenchymal stem cells (MSCs). METHODS: Recombinant vector containing PDX-1 was constructed. Flow cytometry was used to identify the cell cycle of bone marrow mesenchymal stem cells (MSCs) cultured in vitro. Recombinant vector containing PDX-1 was transfected into bone marrow MSCs using superfect in medium. After being selected by G418, RT-PCR and Western blotting were used to investigate the expression of PDX-1 in MSCs. RESULTS: Restricted enzyme analysis and sequencing showed that PDX-1 gene segment was consistent with that in GenBank. Flow cytometry showed that there were about 85.9% cells at the cell cycle of G0/G1. The whole cells transfected emitted green fluorescence under flow cytometry. The efficiency of transfection was above 40%. RT-PCR and Western blotting demonstrated that there was expression of PDX-1 in transfected bone marrow MSCs. CONCLUSION: Recombinant vector containing PDX-1 was constructed successfully. Superfect mediated expression of exogenous gene in bone marrow MSCs in a high efficiency, and bone marrow MSCs containing exogenous gene are an ideal cells for gene therapy.     

Caspase-8 small hairpin RNA attenuates apoptosis of human bone marrow mesenchymal stem cells under conditions of serum deprivation and hypoxia

AIM：To investigate the effect of caspase-8 small hairpin RNA (shRNA) on attenuating apoptosis of human mesenchymal stem cells (hMSCs). METHODS：Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed. Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR. The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid, which was linearized and transfected into HEK293 cells for packaging and amplification of the recombinant adenovirus rAd-Cap8 shRNA. The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting. Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hMSCs under the conditions of serum deprivation and hypoxia. The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR. RESULTS：The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR. The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant adenovirus (rAd)-Cap8 shRNA successfully. rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 expression in hMSCs. Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the apoptotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia, with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2. CONCLUSION：Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia.     

Biological characteristics of bone marrow mesenchymal stem cells from GFP transgenic mice transfected with human β-nerve growth factor gene

AIM:To investigate the changes of biological characteristics of GFP transgenic mouse bone marrow mesenchymal stem cells (MSCs), which were transfected with human β-nerve growth factor (β-NGF) gene in a recombinant eukaryotic expression vector. METHODS:MSCs obtained from GFP transgenic mice were isolated, cultured and purified by the whole bone marrow adherence methods. Human β-NGF gene was transfected into the MSCs by a recombinant eukaryotic expression vector. The β-NGF expression in the MSCs was detected by the method of immunocytochemistry. Hippocampal neurons from neonatal mice were cultured with culture supernatant of the MSCs transfected with pcDNA3-β-NGF and the biological characteristics of the MSCs were investigated 3 d after culture under inverted phase-contrast microscope. RESULTS:The β-NGF positive rate of MSCs in pcDNA3-β-NGF transfection group ［(37.12±2.14)%］ was significantly higher than that in MSCs control group ［(2.36±0.62)%］ and blank control group ［(1.43±0.76)%］.The neurite length of neonatal mouse hippocampal neurons cultured with culture supernatant from pcDNA3-β-NGF-transfected MSCs ［(31±3)μm］ was significantly longer than that in negative control group ［(23±4)μm］, suggesting that MSCs transfected with β-NGF gene maintained better biological characteristics. CONCLUSION: The constructed recombinant eukaryotic expression vector of human β-NGF gene can be transfected into MSCs efficiently and NGF can be effectively expressed in MSCs. MSCs transfected with β-NGF gene are capable of stable expression and secretion of β-NGF, and maintenance of better biological characteristics.     

Effects of hepatitis C virus core protein on cell cycle of HepG2 cells

AIM: To investigate the molecular mechanism of hepatitis C virus (HCV) chronic infection by studying the effect of its core protein on cell growth and the expression of cell cycle regulators such as cyclin D1 and pRb/p130 in HepG2 cells. METHODS: A eukaryotic expression vector that carried a gene encoding HCV-core-1b was constructed. The cDNA of HCV core protein was subcloned into pBabe-Flag-puro vector to generate pBabe-Flag-HCV-core-1b. The plasmid was transfected into Pheonix 293T packaging cells to produce retroviruses. The virus-containing supernatant collected from the cell culture was used to infect HepG2 cells and subsequently the cell line that stably expressed the core protein of HCV was obtained.The cell cycle was analyzed by flow cytometry. The expression of cyclin D1 and pRb/p130 was examined by Western blotting. RESULTS: The pBabe-Flag-HCV-core-1b vector was confirmed by DNA sequencing. The expression of HCV gene type 1b core protein was verified by Western blotting. The overexpression of HCV gene type 1b core protein impaired the cell cycle progression in G₀/G₁ phase and significantly reduced the levels of cyclin D1 and pRb/p130 in the cells. CONCLUSION: A eukaryotic expression plasmid that contains the cDNA of HCV core protein is successfully constructed, and a HepG2 cell line which stably expressed the core protein of HCV is also established. HCV gene type 1b core protein inhibits the cell cycle possibly through down-regulation of cyclin D1 and pRb/p130 proteins in the cells.     

Construction of Pax-8 gene recombinant plasmid and study of its function

AIM: To investigate the effects of over-expression of Pax-8 gene on the proliferation and apoptosis of H9c2 cells(a cardiomyocyte cell line). METHODS: The full length of rat Pax-8 gene was restrictively digested by Kpn I and Not I from the pCMV sport6-Pax-8 vector, and then inserted into the eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)-Pax-8 was confirmed by restriction endonuclease digestion and sequencing. The pcDNA3.1(+)-Pax-8 was transfected into H9c2 cells. The expression of Pax-8 at mRNA and protein levels was identified after transfection by RT-PCR and Western blotting. The cell proliferation was measured by CCK-8. Cell apoptosis was induced by serum deprivation in H9c2 cells transfected with Pax-8 gene. The apoptosis rate of the cells was determined by flow cytometry with annexin V-FITC and propidium iodide double staining. The protein expression of activated caspase-3 was measured by Western blotting. RESULTS: The full length of Pax-8 gene was successfully cloned into pcDNA3.1(+) expression vector and over-expression of Pax-8 at mRNA and protein levels was observed in H9c2 cells transfected with Pax-8 gene as compared to the wild-type cells and the cells transfected with an empty vector (both P<0.05). Transfection of Pax-8 gene promoted the proliferation of the cardiomyocytes (P<0.05) and inhibited the apoptosis rates induced by serum deprivation (P<0.01). The expression level of activated caspase-3 was increased by serum deprivation and attenuated by Pax-8 transfection (P<0.01). CONCLUSION: The pcDNA3.1(+)-Pax-8 expression vector was successfully constructed and over-expression of Pax-8 gene in cardiomyocytes is obtained. Pax-8 gene acts as an anti-apoptotic factor in cardiomyocytes by promoting cell proliferation and inhibiting apoptosis.     

Construction of a lentiviral RNA interference vector targeting rat myocardin and its effect on vascular smooth muscle cell differentiation

AIM: To construct a lentiviral RNA interference(RNAi)vector targeting rat myocardin mRNA and to investigate its effect on the differentiation of vascular smooth muscle cells(VSMCs).METHODS: Three pairs of dsDNA targeting rat myocardin mRNA were designed, synthesized and cloned into lentiviral vector pGCSIL-GFP to generate pGCSIL-GFP-shMyocd lentvirus. A Flag-tagged myocardin-overexpression vector pEGFP-N1-Myocd was constructed with pEGFP-N1/X124G. After these two vectors were cotransfected into 293T cells, the flag protein was assessed by Western blotting to analyze the knockdown effect of pGCSIL-GFP-shMyocd. The expression of myocardin and SM22α was also detected by RT-PCR and Western blotting after the pGCSIL-GFP-shMyocd viruses were transfected into primary cultured rat aortal VSMCs.RESULTS: The rat myocardin lentviral RNAi vector pGCSIL-GFP-shMyocd and myocardin-overexpression vector pEGFP-N1-Myocd were successfully constructed. After these two kinds of vectors were cotransfected into 293T cells,the No.1 interfering vector displayed the highest inhibitory effect on flag expression.After the No.1 lentvirus at the titer of 1×10¹² TU/L was transfected into VSMCs, the myocardin and SM22α expression was significantly attenuated. CONCLUSION: The lentiviral pGCSIL-GFP-shMyocd RNAi vector is successfully constructed, which is useful for further study regarding the molecular mechanism of the phenotypic switching in VSMCs under special pathological conditions such as atherosclerosis. Inhibition of myocardin expression in VSMCs leads to the decrease in the expression of differentiation marker, and implies a crucial role of myocardin in VSMCs differentiation.