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.     

Construction of short hairpin RNA lentiviral vector targeting MAG gene and its silencing effect on MAG gene

AIM：To construct lentiviral vector-based short hairpin RNA (shRNA) targeting myelin-associated glycoprotein (MAG) gene and to evaluate its inhibitory effect on the expression of MAG gene. METHODS：Three shRNA fragments targeting MAG gene (shRNA1, shRNA2 and shRNA3) were designed and cloned into lentiviral vector pWPI. The three recombinant plasmids were identified by enzyme digestion and sequencing. Positive plasmids were co-transfected with pCDNA3-MAG-FLAG into 293T cells, and the most effective shRNA for knockdown of MAG gene was screened by Western blotting. Cells transfected with empty pWPI served as a control. Oligodendrocytes were infected with recombinant lentivirus that was produced by 293T packaging cells co-transfected with the most effective shRNA, pAX2 and pMD2G. After 48 h, the expression of MAG protein was measured by Western blotting. RESULTS：The MAG shRNA lentiviral vectors were confirmed by double enzyme digestion and sequencing, and shRNA2 showed the highest inhibitory efficacy among the three shRNA fragments. Recombinant lentivirus carrying shRNA-2 markedly decreased the expression of MAG protein in oligodendrocytes. CONCLUSION：Lentiviral vector-based shRNA targeting MAG gene specifically knocks down the gene expression, which provides a useful tool for investigating the role of MAG-specific shRNA in regulating myelination of central nerve system.     

Effect of estrogen on the gene expression of caveolin-1 in rat vascular smooth muscle cells

AIM: To assess the effect of estrogen on the gene expression of caveolin-1 in rat vascular smooth muscle cells (VSMCs). METHODS: Wistar rats were ovariectomized and subjected to subcutaneous implantation of placebo pellets (OVX+V group) or estradiol pellets (OVX+E group). 2 weeks after implantation, the expression of caveolin-1 gene in endothelium-denuded aortic tissue was examined by RT-PCR. Furthermore, Northern blotting was used to analyze the mRNA expression of caveolin-1 in cultured rat VSMCs. RESULTS: RT-PCR showed that expression of caveolin-1 gene was significantly higher in OVX+E group than that in OVX+V group. Northern blot analysis showed that the mRNA expression of caveolin-1 was higher in VSMCs pretreated with 17β-estradiol (17β-E₂) than that in VSMCs without 17β-E₂ pretreatment. CONCLUSION: Estrogen up-regulates the gene expression of caveolin-1 in the vascular wall, partially indicating the cardiovascular effect of estrogen.     

Construction of mouse ESC line stably expressing Pdx1 by Tet-On system

AIM To construct the mouse embryonic stem cell （ESC） line with stable pancreatic and duodenal homeobox 1 （Pdx1） expression by Tet-On system, which may lay a foundation for further research on the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells. METHODS The Pdx1-overexpressing lentiviral vector with green fluorescent protein marker and puromycin resistance was constructed by Tet-On system and was used to infect the mouse ESC. The cells were divided into 3 groups： blank control group （ESC group）, empty lentivirus control group （PDX1^- ESC group） and Pdx1 lentivirus transfection group （PDX1⁺ ESC group）. Flow cytometry was used to detect the transfected cells after screening by doxycycline （DOX）. The function of Tet-On system and the expression of Pdx1 gene were detected. The transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were sorted by flow cytometry, and constructed ESC line with stable expression of Pdx1 and negative control ESC line were verified. RESULTS （1） The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were 90.72% and 94.01% after screening by DOX, respectively. The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group was 97.84% and 98.13% after sorting by flow cytometry, respectively. （2） With DOX, green fluorescence was observed in PDX1^- ESC group and PDX1⁺ ESC group. The mRNA and protein expression of Pdx1 was significantly increased in PDX1⁺ ESC group （P<0.05）. Without DOX, no green fluorescence was observed in the cells of the 3 groups, and no significant difference in the mRNA and protein expression of Pdx1 was observed （P>0.05）. （3） After 3 months of cryopreservation, the cell lines still survived in resuscitation culture and were regulated by DOX. CONCLUSION Using Tet-On system, the mouse ESC line with inducible Pdx1 expression were successfully established and could be used as an effective cell model to research the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells.     

ATPase activities and Ca2+/calmodulin alterations in hippocampi of rats with PTSD-like behaviors

AIM: To explore the pathophysiological bases in the pathogenesis of the lasting emotional behavioral disorders following posttraumatic stress disorder(PTSD). METHODS: 240 male Wistar rats were divided randomly into 3 groups. Group SE(n =96) for rats with PTSD-like behavior by constant pulsating current of 100 μA with intratrain frequencies of 16 Hz, pulsating duration of 1 ms, train duration of 10 s and interstimulus interval of 7 min for 5 days with 8 times per day. Group CE(n =96) for control with electrode implanted in hippocampus without stimulation, and Group NC(n =48) for normal control. The activities of Na⁺-K⁺-ATPase and Ca²⁺ -ATPase, levels of intracellular calcium and free calmodulin(CaM), and the total CaM expression were detected in hippocampi of experimental rats. RESULTS: The activities of Na⁺-K⁺-ATPase and Ca²⁺ -ATPase in mitochondria of hippocampal cells in Group SE rats were significantly decreased at 48 h and 72 h after the last stimulation, respectively. The intracellular free calcium levels were increased, and the mean channel fluorescence of intracellular free CaM decreased remarkably at 72 h poststimulation, while the expression of total CaM was significantly elevated at 48 h after the last stimulation in hippocampi of Group SE rats. CONCLUSION: The lasting increased levels of intracellular free calcium and expression of Ca²⁺ -CaM in hippocampus, as well as the dysfunction of Na⁺-K⁺ pump and Ca²⁺ -ATPase in mitochondria may play important roles in the long-term neuropsychological sequelae in PTSD.     

Role of ANX A2 in APL-induced expression of tissue factor in THP-1 cells

AIM: To construct a lentiviral vector harboring RNAi sequence targeting human annexin A2 (ANX A2) and to investigate the functions of ANX A2 in antiphospholipid antibody (APL)-induced tissue factor (TF) expression in monocytes. METHODS: Four different short hairpin RNAs (shRNA) targeting ANX A2 gene were constructed and cloned into the pGCSIL-GFP vector. After identification with PCR and sequencing, the effective interference sequence was further determined by Western blotting analysis. The recombinant lentivirus LV-RNAi-ANX A2 harvested from 293T cells was transferred into THP-1 cells and the ANX A2 mRNA and protein expression on THP-1 cells were examined. The transferred-THP-1 cells were stimulated by APL/β2GPI compound, and the TF mRNA and TF activity was assayed. RESULTS: The RNAi sequences targeting the human ANX A2 gene were successfully inserted into the lentiviral vector and the high-performance RNA interference sequence was sieved out. The recombinant lentivirus was harvested from 293T cells with a viral titer of 3×1012 TU/L. THP-1 cells infected with LV-RNAi-ANX A2 showed almost lockout of ANX A2 both at mRNA and protein levels. The TF expression was also significantly decreased in the transferred-THP-1 cells induced by APL/β2GPI compound. CONCLUSION: The lentiviral vector constructed in the present study blocks the ANX A2 expression in THP-1 cells and further inhibits the TF expression induced by APL/β2GPI compound, which indicates that ANX A2 do play an important role in APL-induced TF expression on monocytes.     

Effects of three kinds of calcium activator on the proliferation of cultured vascular smooth muscle cells in rats

AIM: To investigate the effects of intracellular free calcium ([Ca²⁺]i) from different resources on the proliferation mediated by mitogen activated protein kinase (MAPK) in vascular smooth muscle cells (VSMCs). METHODS: Cultured VSMCs were used in all experiments. Calcium influx was stimulated by angiotension Ⅱ(Ang Ⅱ). The release of intracellular calcium stores was induced by inositol trisphosphate (IP₃) and ryanodine (RY). MAPK activity was measured by [γ-³²P]-ATP incorporation MAPK protein expression by western blot, VSMCs proliferation by [³H]-Leucine ([³H]-Leu) and [³H]-Thymidine ([³H]-TdR) incorporation. RESULTS: Compared to the control VSMCs, Ang Ⅱ, IP₃ and RY significantly increased [Ca²⁺]i concentration activity of MAPK and its protein content in VSMCs. The promotion of [³H]-Leu and [³H]-TdR incorporation in VSMCs was also observed (P<0.01). CONCLUSION: The study indicated that calcium activator-induced increase in the activity and protein content of MAPK was involved in the proliferation of VSMCs, which was closely related to the [Ca²⁺]i concentration but independent to its origin.     

Analgesic effect of angiotensin angiotensin Ⅱ type 2 receptor antagonist EMA401 on neuropathic pain in rats and its mechanism

AIM: To explore whether angiotensin Ⅱ type 2 receptor antagonist EMA401 decreases neuropathic pain and the expression of growth-associated protein-43 (GAP-43), protein kinase C (PKC) and calmodulin (CaM) in dorsal root ganglia (DRG) during chronic constriction injury (CCI) in rats. METHODS: SD rats were used to establish CCI model and randomly divided into 4 groups. The rats in model group were given equal volume of normal saline by intragastric administration. The rats in low dose (LD) group were given 5 mg/kg EMA401 by intragastric administration. The rats in middle dose (MD) group were given 10 mg/kg EMA401 by intragastric administration. The rats high dose (HD) group were given 20 mg/kg EMA401 by intragastric administration. The rats in sham operation group received equal volume of normal saline by intragastric administration. Thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were measured before operation and 7 d, 14 d and 28 d after CCI. After behavioral test, DRG of lumbar spinal was obtained from each group, and was used to determine Ca²⁺ concentration by o-cresolphthalein complexone microplating method, and the expression of GAP-43, PKC and CaM at mRNA and protein levels by Western blotting and RT-PCR. RESULTS: Compared with model group, EMA401 significantly increased the TWL and MWT (P<0.05). Meanwhile, EMA401 significantly reduced Ca²⁺ concentration and the expression of GAP-43, PKC and CaM at mRNA and protein levels in the DRG (P<0.05). CONCLUSION: EMA401 may attenuate neuropathic pain of CCI by inhibiting Ca²⁺ concentration and the expression of GAP-43, PKC and CaM.     

Lentivirus-mediated RNA interference targeting vascular endothelial growth factor gene enhances the sensitivity of K562 cells to STI571

AIM: To investigate the effects of RNA interference (RNAi) inhibiting the expression of vascular endothelial growth factor (VEGF) gene mediated by lentiviral vector on the proliferation and apoptosis of K562 leukemic cell line. METHODS: A lentiviral vector containing short hairpin RNA (shRNA) targeting VEGF was constructed and cotransfected with the packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real-time quantitative RT- PCR, Western blotting and ELISA. Cellular proliferation was determined by trypan blue dye exclusion and MTT assay. STI571 (imatinib mesylate)-induced apoptosis was analyzed by flow cytometry. RESULTS: The lentiviral shRNA vector targeting VEGF was successfully constructed and transfected into K562 cells. The expressions of VEGF mRNA and protein in K562-shVEGF cells transfected with pRNAT-shRNA were significantly inhibited when compared with those of K562 and K562-con cells (mock transduction). The proliferation rate of K562-shVEGF cells slowed down. After STI571 treatment, the percentages of apoptotic cells in K562-shVEGF cells increased more significantly than those of K562 and K562-con cells (P<0.05). CONCLUSION: Inhibition of VEGF by lentivirus-mediated RNAi effectively inhibits proliferation and increases the sensitivity of K562 cells to STI571.     

Effects of levcromakalim on pulmonary arterial endothelial cells and smooth muscle cells exposed to hypoxia

AIM:To explore the effects of levcromakalim(Lev) on pulmonary arterial endothelial cells (PAEC) and smooth muscle cells (PASMC) exposed to hypoxia and the mechanisms involved.METHODS:The effects of Lev on [Ca²⁺]_i, and expression of PKC_α, eNOS, iNOS and PDGF-B mRNA and protein levels were observed. The nitrite (NO₂^-) and entothelin-1(ET-1) concentrations in supernatant in cultured PAEC and PASMC were measured. The proliferation and apoptosis of PASMC was also detected.RESULTS:When PASMC were exposed to hypoxia, Lev reduced concentration of ET-1 in cultured cell supernatant, lowed the expression of PKC_α, iNOS and PDGF-B both at mRNA and protein levels, decreased [Ca²⁺]_i concentration, increased proliferation and promoted the apoptosis in PASMC. However, in the presence of Lev, the [Ca²⁺]_i concentration was not changed in the hypoxic PAEC. The NO₂^- concentration in cultured cell supernant and expression of eNOS at mRNA and protein levels in hypoxic PASMC and PAEC were also unchanged. The downregulated ET-1 activity in PASMC and PAEC and proliferation in PASMC involved in the inhibition of PKC_α signaling pathway.CONCLUSIONS:Lev reduce some disadvatage effect of hypoxia on PASMC and PAEC. The mechanism of Lev action may partly involve in the downregulation of PKC_α signaling functions.     

Effects of lentivirus-mediated RWDD3 silencing on proliferation and invasion of human glioma U251 cells

AIM: To investigate the effect of RWDD3 gene silencing on the biological characteristics of human glioma U251 cells.METHODS: A lentiviral vector expressing RWDD3 shRNA was constructed and transfeeted into the U251 cells. The expression of RWDD3 at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The cell activity was determined by MTT assay. The colony formation ability was detected by the colony formation assay. The cell proliferation ability was detected by BrdU incorporation assay. The cell invasion and migration were evaluated by Transwell assay. Flow cytometry was used to monitor the changes of cell cycle distribution and apoptosis.RESULTS: Recombinant lentivirus was successfully transfected into U251 cells. Compared with the cells transfected with the scrambled shRNA and control cells, the cell activity, colony formation ability, and the invasive and migratory activities were inhibited, the cell cycle was arrested in G₀/G₁ phase, and the apoptosis was increased in the U251 cells transfected with RWDD3 shRNA(P<0.05).CONCLUSION: RWDD3 plays a vital role in proliferation and invasion of glioma cells. It may serve as a potential target of gene therapy for glioma.     

Effect of CREB-shRNA on mitochondrial morphology and cell apoptosis in OGD/R-induced cortical neurons

AIM: To construct recombinant lentiviral vector with short hairpin RNA (shRNA) of CREB gene, and to investigate the effect of CREB gene silencing on mitochondrial morphology and cell apoptosis in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cortical neurons. METHODS: Three lentiviral vectors pLentiLox3.7 (PLL) inserted shRNA fragments targeting CREB gene were co-transfected with the packaging plasmids psPAX2 and pMD2.G to the 293T cells, and the virus particles, which was infected with the primary cortical neurons, was encapsulated. The protein expression of CREB was detected by Western blot. The mitochondrial morphology, cell apoptosis and the expression of Bcl-2 and Bax were evaluated by the methods of MitoTracker red, TUNEL and Western blot in OGD/R induced cortical neurons after CREB gene silencing. RESULTS: The pLL-CREB-shRNA1 was the most effective shRNA, which inhibited 80% CREB gene expression in the cortical neurons. The mitochondrial was appeared dot and fragment morphology in OGD/R induced cortical neurons with transfected pLL-CREB-shRNA1 plasmid. In addition, the expression of Bcl-2 was decreased, the expression of Bax, and the apoptosis of the neurons were increased by tranfected with pLL-CREB-shRNA1. CONCLUSION: CREB shRNA recombinant lentiviral vector specifically inhibits the expression of CREB gene. CREB gene silencing promotes the cell apoptosis and mitochondrial morphological changes in the cortical neurons induced by OGD/R.     

Construction of a lentiviral vector for RNA interference of human EGFL7 gene and its stable expression in human laryngeal carcinoma cells

AIM：To investigate the role of epidermal growth factor-like domain 7 (EGFL7) in the pathogenesis and progress of laryngeal carcinoma via constructing a lentiviral expression vector for RNA interference (RNAi) of human EGFL7 gene and assessing the gene-silencing effect of the vector in human laryngeal epidermoid carcinoma (HEp-2) cells. METHODS：Specific RNAi target sequences were designed focused on human EGFL7 gene sequence. The double-stranded oligonucleotides were cloned into the pcDNA6.2-GW/EmGFP-miR plasmid after synthesis and annealing. A positive clone was subcloned into the pLenti6.3-MCS/V5-DEST vector after sequence analysis. The recombinant lentivirus was harvested from 293T cells co-transfected with the positive recombinant plasmid and lentiviral packing materials. HEp-2 cells were infected with the recombinant lentivirus and the cells with stable EGFL7 knockdown were screened by blasticidin selection. EGFL7 mRNA expression in the cells was determined by real-time fluorescence quantitative PCR. RESULTS：A recombinant lentiviral vector expressing short hairpin RNAs (shRNAs) against EGFL7 gene was obtained and confirmed by DNA sequencing. The virus titer was 5×1011 TU/L, and the silencing efficiency was 97%. CONCLUSION：A lentiviral vector targeting human EGFL7 gene, capable of stable EGFL7 gene knockdown in HEp-2 cells, has been successfully constructed, which provides a basis for further study of the relationship between human laryngeal carcinoma and EGFL7 protein.     

Effect of OX-LDL and simvastatin on PKC activity and cytosolic free Ca2+ in cultured rat aortic smooth muscle cells

AMI:To clarify whether OX-LDL and simvastatin can induce the changes of PKC activity and cytosolic free Ca²⁺ in rat aortic smooth muscle cells (ASMC). METHODS:PKC activity and cytosolic free Ca²⁺ were measured by its ability to transfer phosphate from [³²P]ATP to lysine-rich histone and flow cytometric analysis after loading with the Ca²⁺dye fluo-3/Am, respectively. RESULTS:OX-LDL increased PKC total activity in a dose-dependent manner and induced translocation of PKC from the cytosolic to membrane, while OX-LDL induced biphasic [Ca²⁺]i responses including the rapid initial transient phase and the sustained phase. When simvastatin was added, the translocation of PKC was markedly decreased and simvastatin did not impair the initial peak response to OX-LDL but significantly reduced the subsequent plateau phase. CONCLUSSION:OX-LDL can induce dynamic changes of signal transduction of PKC and cytosolic free Ca²⁺ in ASMC and these two events are closely linked.     

Nicotinic acid promotes lysosomal free cholesterol efflux via LXRα/NPC1 pathway in macrophages

AIM To explore the effects of nicotinic acid (NA) on lysosomal free cholesterol efflux in macrophages and its underlying mechanism. METHODS Macrophages induced from human monocytic leukemia cell line THP-1 by phorbol myristate acetate served as the cell model. Laser scanning confocal microscopy was applied to observe the effects of NA on lysosomal free cholesterol efflux in macrophages loaded with oxidized low-density lipoprotein (oxLDL). The influences of nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist Ned-19, Ca²⁺ chelator BAPTA, liver X receptor α (LXRα) siRNA and Niemann-Pick C1 protein (NPC1) siRNA on NA effects were also evaluated. RT-qPCR and Western blot were conducted to evaluate the influence of NA, Ned-19 and BAPTA on LXRα mRNA and NPC1 protein expression. RESULTS NA dose-dependently promoted lysosomal free cholesterol efflux in macrophages. This effect was markedly inhibited by Ned-19 and BAPTA. NA increased NPC1 protein and LXRα mRNA expression. These effects were also attenuated by Ned-19 and BAPTA remarkably. LXRα siRNA significantly inhibited the promoting effect of NA on NPC1 protein expression. Silencing of LXRα and NPC1 with siRNA remarkably abolished the effect of NA on lysosomal free cholesterol efflux. CONCLUSION NA promotes lysosomal free cholesterol efflux in macrophages. This effect may be mediated by the increased production of NAADP, which subsequently promotes Ca²⁺ release through lysosomal transient receptor potential mucolipin 1 (TRPML1) channel and finally up-regulates NPC1 protein expression via LXRα.     

Role of AT1R-CaN signaling pathway in regulation of Nav1.5 protein expression in hypertrophic ventricular myocytes from neonatal rats

AIM: To investigate the effect of angiotensin II type 1 receptor (AT₁R)-calcineurin (CaN) signaling pathway on the expression of sodium current channel Nav1.5 at mRNA and protein levels in the hypertrophic ventricular myocytes from neonatal rats.METHODS: The ventricular myocytes were isolated from the ventricles of 1-day-old neonatal Sprague-Dawley rats and were divided into 4 groups according to different drug intervention as control group, phenylephrine (PE) group, losartan (Los)+PE group and cyclosporin A (CsA)+PE group. The method of RNA interference mediated by adenovirus carrying short hairpin RNA (shRNA) was used to knock down the gene which encodes the beta subtype of CaN A subunit (CnAβ) and the cells were divided into 4 groups as Ad-Null group, Ad-Null+PE group, Ad-CnAβshRNA1 group and Ad-CnAβshRNA1+PE group. The mRNA expression of brain natriuretic peptide (BNP), β-myosin heavy chain (β-MHC) and Nav1.5 was detected by RT-qPCR. The protein levels of CnAβ and Nav1.5 in the whole-cell extracts were determined by Western blot analysis.RESULTS: Treatment of the neonatal rat ventricular myocytes with PE for 24 h increased the protein-to-DNA ratio and the mRNA expression of BNP and β-MHC. The size of the cell surface was also increased after PE treatment. Treatment of the cells with PE increased the protein expression of CnAβ, and reduced the protein expression of Nav1.5. Both Los and CsA prevented those effects of PE. The mRNA expression of Nav1.5 was reduced by PE, and no significant difference of Nav1.5 mRNA expression among PE group, Los+PE group and CsA+PE group was observed. Silencing of CnAβ in the neonatal rat ventricular myocytes using Ad-CnAβshRNA1 inhibited the ability of PE to increase the mRNA expression of BNP, and diminished the ability of PE to reduce the protein expression of Nav1.5.CONCLUSION: AT₁R-CaN signaling pathway participates in regulating protein expression of Nav1.5 in the hypertrophic ventricular myocytes from neonatal rats.     

Expression of prostate-specific membrane antigen (PSMA) after the transfection of shRNA lentivirus with PSMA construction in 293 cells

AIM: To obtain shRNA sequences that can stably block the expression of prostate-specific membrane antigen (PSMA) in the prostate cancer cell line LNCaP and construct the lentivirus vector. METHODS: According to genetic information, we design siRNA1, siRNA2 and siRNA3 of PSMA, the three siRNA sequences targeting the cds area of PSMA gene and then forming the corresponding four pairs of complementary single strand DNA of shRNA, including the sense strand and the antisense strand. The sequence of sense strand from 5 to 3 was: enzyme digestion site (BamHⅠ), interference sequence (19 bp), the loop-stem structure (TTCAAGAGA), the reverse complementary sequence of interference sequence (19 bp), the ending signal (TTTTT) and enzyme digestion site (EcoRⅠ). The synthetic shRNA sequence was inserted into the empty pSIH1-H1-copGFP shRNA vector, and transfected into the prostate cancer cells. The inhibitory effect of PSMA mRNA expression by transfecting different sequences was detected through real-time PCR, and the inhibitory effect of P65 protein expression was also detected by Western blotting. RESULTS: The second shRNA sequence, located in PSMA (NM_004476) 1207-1226 and its stem-loop sequence was 5-GATCC GTCTCAAAGTGCCCTACAA TTCAAGAGA TTGTAGGGCACTTTGAGAC TTTTT G-3, showing the best inhibitory effect on PSMA mRNA expression in prostate cancer cell line was 60% and the protein expression was 86%. After the transfection, the prostate cancer cell line expreesed the low level of PSMA stably. CONCLUSION: It is successful to obtain shRNA sequences that can stably block the expression of PSMA in the prostate cancer cell line LNCaP and obtain the high inhibitory rates for expression of mRNA and protein of PSMA. The construction of the lentivirus vector pSIH-PSMA-siRNA2 provides solid foundation for further experimental studies on the function of PSMA.     

Effects of p204 expression on expression of p21 and proliferation of vascular smooth muscle cells in rats

AIM: To observe the effect of interferon-inducible protein 204 (p204) on the expression of p21 and proliferation of vascular smooth muscle cells (VSMCs) in rats. METHODS: Interferon alpha (IFN-α) and small interference RNA (siRNA) targeting p204 gene ( Ifi204 ) was used to intervene cultured VSMCs in vitro instantaneously, then the cell vitality was determined by MTT assay to reflect the cell proliferation. The cell cycle was analyzed by flow cytometry. The expression of p204 and p21 at mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blotting. RESULTS: In rat VSMCs, IFN-α induced the increase in the expression of p204 at mRNA and protein levels, reduced the cell vitality and the G₁/S phase transition, and up-regulated the expression of p21 at mRNA and protein levels. Transfection of Ifi204 siRNA restrained the expression of p204 and p21, increased the cell vitality and promoted the G₁/S phase transition. CONCLUSION: The expression of p204 restrains the proliferation of rat VSMCs, probably by activating the expression of p21.     

Role of calcium ion in the cyotoxicity and DNA fragment induction in HL-60 cells by 6F isolated from Pteris semipinnata L.

AIM: To investigate the changes of cytosolic free calcium concentration([Ca²⁺]_i) and expression of Bcl-2 in HL-60 cells treated by 6F isolated from Pteris semipinnata L.(PSL), and to discuss the relations between calcium ion and cytotoxicity and DNA fragment induction effects of 6F. METHODS: HL-60 cells were used as in vitro model. [Ca²⁺]_i was measured on fluorescent spectrophotometry using Fura-2/AM as Ca²⁺ indicator. Bcl-2 expressing level was measured by flow cytometry. Tetrazolium salt(MTT) and diphenylamine staining methods were applied for cytotoxicity assay and DNA fragmentation detection, respectively. RESULTS: [Ca²⁺]_i increased obviously in a dose and time dependent manner after treated HL-60 cells with 6F. 6F decreased the expressing level of Bcl-2. Adding 2 mmol/L Ca²⁺ to the medium, or 1 mmol/L EDTA to chelate Ca²⁺, or 4 μmol/L calcium ionophore A 23187 to increase the concentration of cytosolic Ca²⁺, the DNA fragment induction by 6F was not affected, whereas the cytotoxicity of 6F was enhanced. 250 μmol/L Zn²⁺ attenuated the DNA fragment induction, and the cytotoxicity of 6F against HL-60 cells was enhanced significantly. CONCLUSION: It was speculated that the decreased expressing of Bcl-2 by compound 6F was related to increased [Ca²⁺]_i in HL-60 cells, and DNA fragment induction was possibly catalyzed by Ca²⁺ - independent DNase.