Construction of miR-196b sponge lentiviral vector and its expression in bone marrow stromal cell line ST2 / 天津医药

Objective To construct miR-196b sponge lentiviral vector,and laid the foundation for studying the function of miR-196b in bone marrow stromal cells.Methods Based on the miR-196b mature sequence,a sequence consisting of 6 tandem repeats of the complementary sequence of miR-196b was designed,and which was cloned into pUC19 plasmid by using reverse PCR.Then the six-repeat sequence was cut and subcloned into pLVX-shRNA2 lentiviral vector.The lentivirus was packaged using 293T cells,and titer determination was done.The pLVX-shRNA2 lentivirus was used as the control group,and the 196b-sponge-pLVX lentivirus was the experimental group.Then ST2 cells were infected with the viruses,and the infection efficiency was calculated.The protein level of forkhead box O1 (FoxO1) was detected by Western blot assay.Results The identity of the sponge sequence was verified by sequencing.The titer of the sponge virus was 1 × 108 PFU/mL,and the infection efficiency reached 80％.Compared with the control group,the expression level of FoxO 1 protein was significantly increased (P ＜ 0.05).Conclusion The miR-196b sponge lentiviral vector is successfully constructed,and which has the capability to inhibit endogenous miR-196b.

The effect of transforming growth factorβreceptor typeⅠon adipocyte differentiation / 天津医药

Objective To investigate the effect of transforming growth factorβreceptor typeⅠ(TGFBRⅠ) on adipocyte differentiation by using a small interference RNA (siRNA). Methods The siRNA targeting TGFBRⅠwas synthesized as experimental group, and negative control siRNA was used as control group. The efficiency of TGFBRⅠdepletion and the expression levels of adipocyte-specific transcription factors CCAAT enhancer binding protein α (C/EBPα),peroxisome proliferator-activated receptor gamma (PPARγ) and adipocyte marker gene fatty acid binding protein 4 (FABP4) were detected by quantitative real-time PCR. After treating with adipocyte differentiation agents for 5 days, the cells were stained with oil red O, and the staining of adipocyte was observed and photographed by laser confocal microscope. In addition, with isopropanol extracted oil red O, optical density values of oil red O were measured at a wavelength of 520, and which were compared between groups. Results After transfection of TGFBRⅠ siRNA, gene expression levels of TGFBRⅠwere significantly reduced in ST2 cells, the number of differentiated adipocytes was significantly increased, and the mRNA levels of adipocyte specific transcription factor C/EBPαand PPARγand adipocyte marker gene FABP4 were enhanced compared with those of control group. After treating with adipocyte differentiation agents for 5 days,the number of lipid droplets of cells with transfection of TGFBRⅠsiRNA was increased than that of cells with transfection of control siRNA. The value of optical density was higher in cells with transfection of TGFBRⅠsiRNA than that of control siRNA group. Conclusion TGFBRⅠsiRNA can effectively facilitate adipocyte formation, which suggests that TGFBRⅠis an important regulator of adipogenic differentiation from progenitor cells.

Construction of Nfic gene 3′UTR dual luciferase reporter vector and targeting verification between Nfic and miR-20a / 天津医药

Objective To construct a luciferase reporter vector containing the 3′untranslated region (3′UTR) of nuclear factor I-C (nuclear factor I-C, Nfic), and apply dual luciferase reporter gene system to determine the association between microRNA-20a (miR-20a) and its potential target gene Nfic. Methods The potential complementary binding sites of miR-20a and Nfic were predicted by Targetscan. The 3′UTR of Nfic fragment amplified by PCR was cloned into luciferase reporter vector MIR- Report Luciferase. The luciferase reporters containing 3′ UTR of Nfic and miR- 20 mimics (experimental group) or NC mimics (control group) were co-transfected into 293-AD cells. Cells were collected, and then dual-luciferase reporter assay was performed to detect the luciferase activity of the two groups of cells, consequently the relationship between miR-20a and Nfic was identified. The miR-20a mimics and NC mimics were transfected into marrow stromal cell line ST2 respectively. The total cell lysates were collected, and the expression level of NFIC was detected by Western blotting assay. Results Results of double enzyme digestion and DNA sequencing showed that sequence of luciferase reporter vector was correct. miR-20a specificity bounded to Nfic 3′UTR and inhibited the luciferase activity of the reporter construct (P<0.05). Western blotting assay showed that the NFIC protein level was obviously down-regulated in ST2 cells after the transfection of miR-20a mimics compared with that of control. Conclusion The luciferase reporter vector containing the 3′UTR of Nfic is constructed successfully, which confirms that miR-20a can direct effect on Nfic3′UTR and repress its luciferase activity.

Effect of AsperosaponinⅥon adipocyte differentiation in ST-2 cells and its underlying mechanisms / 天津医药

Objective The effect of Asperosaponin Ⅵ(ASAⅥ)on adipocyte differentiation and the involvement of Wnt signal pathway was investigated. Methods The murine bone marrow stromal cell line ST-2 were divided into 6 groups:control group, adipocyte differentiation group, and 4 different doses of ASAⅥgroups. Control group was exposed to the vehicle, adipocyte differentiation group was exposed to adipogenic reagent, and those 4 ASAⅥgroups were treated with different concentration(10-7, 10-6, 10-5, 10-4 mol/L)of ASAⅥafter adipocyte differentiation induction. 5 days later, oil red O staining was performed to calculate adipocyte rate. Then mRNA transcription levels of PPARγ, FABP4 genes andβ-catenin that were Wnt/β-catenin signaling pathway proteins were examined by FQ-PCR. Then Wnt pathway inhibitor DKK1 was supplemented into ST-2 cells treated with 10-4 mol/L ASAⅥfor 5 days. After that FQ-PCR was used to detect whether tran?scription levels of PPARγ, FABP4 andβ-catenin in ST-2 cells were changed. Results Compared with adipocyte differenti?ation group 10-5 mol/L and 10-4 mol/L ASAⅥtreatments greatly down-regulated the number of lipid droplets and markedly inhibited transcription levels of adipocyte characterization transcription factors included PPARγ, FABP4 while up-regulat?ed transcription level ofβ-catenin in ST-2 cells. DKK1 can reverse the inhibitory effect of ASAⅥon adipocyte differentia?tion in ST-2 adipocyte. The transcription levels of PPARγand FABP4 were up-regulated significantly while transcription level ofβ-catenin was inhibited. Conclusion ASAⅥblocks adipocyte differentiation in ST-2 cells which might be medi?ated through activating Wnt/β-catenin signaling pathway.

The impact of miR-320-3p on adipocyte differentiation / 天津医药

Objective To study the role of miR-320-3p in adipocyte differentiation. Methods Marrow mesenchymal stem cells were isolated from mice and cultured then induced with adipogenic agents for 3 days. The transcription level of miR-320-3p was examined by qRT-PCR. Stromal ST2 cells were transfected with miR-320-3p, followed by adipogenic treatment. Oil-red O staining and qRT-PCR were employed to assess the differentiation of adipocytes induced by miR-320-3p. Results The expression level of miR-320-3p increased in MSCs after adipogenic treatment (P < 0.01). Addition of miR-320-3p in ST2 cells promoted the formation of oil-red O positive adipocytes and up-regulated the expression levels of adipogenic transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein-α(C/EBPα) and the marker gene adipocyte fatty acid binding protein 4 (FABP4),compared to cells that transfected with miR-320-3p mimics (P<0.05). Conclusion miR-320-3p promotes ST2 cells differentiation into adipocytes.

Establishment of miR-223 knockdown lentivirus / 天津医药

Objective To construct miR-223 knockdown lentivirus vector and provide a tool for further study of the function of miR-223. Methods According to the Invitrogen miR-RNAi online design tool, a pair of complementary oligo?nucleotides encoding miR-223 mature sequence was designed, annealed and ligated with pcDNA6.2-GW/EmGFP-miR vec?tor. Then miR-RNAi expression cassette was cut and subcloned into lentiviral pCDH-CMV-MCS-EF1-copGFP vector. The lentiviruses were packaged and titered, and then ST2 cells were infected with viruses. The efficiency of infection was calcu?lated, and the knockdown of endogenous miR-223 was detected by using real-time RT-PCR. Results Restriction enzyme digestion and sequencing results showed that miR-223 lentivirus construct was successfully made. Lentivirus that knock?down miR-223 expression packaged and infected of target cells. The expression of GFP green fluorescent protein accounted for 80%-90%and the virus titer was 1×109 PFU/mL. The infection efficiency reached 90%. Compared with negative control virus, miR-223 knockdown lentivirus significantly down-regulated the expression of miR-223 in ST2, and was 31%(n=3, t=15.091, P<0.05). Conclusion miR-223 knockdown lentivirus is successfully made. It provides a tool for further studying the function of miR-223.

Parathyroid hormone(1-34) regulates the expression of osteoclast inhibitory lectin via multiple signaling pathways in UMR106 osteoblast-like cells / 中华骨科杂志

Objective To investigate the regulation of parathyroid hormone(1-34) on mRNA expression of osteoclast inhibitory lectin (OCIL) gene in UMR106 osteoblastic-like cells and involved signaling pathway.Methods Rat UMR106 osteoblastic-like cells were cultured and treated with various concentration of PTH(1-34) and specific agonists or inhibitors of PKA,PKC,Ca2+/calmodulin-dependent protein kinase (CaMK) and mitogen-activated protein kinase (MAPK) signal pathways for indicated time intervals.Then the cells were gathered at indicated time points and total RNA were extracted.OCIL mRNA expression was analyzed using real-time PCR technique.Results PTH(1-34) stimulated OCIL mRNA expression in a time-and dose-dependentmanner.A dose of 10 nmol/L PTH(1-34) started to induce OCIL mRNA from 6 h,with a highest increase of about 2.8-fold vs.control group (without PTH treatment) at 24 h.The up-regulation of OCIL mRNA began and reached maximum later than RANKL induction and OPG suppression effected by PTH(1-34).Protein Kinase A (PKA) signaling activators forskolin(FSK) and dibutyryl cAMP (db-cAMP),as well as calcium ionophore A23187 all up-regulated OCIL mRNA with the maximal induction of about 4.2-fold,4.5-fold and 5.1-fold.Protein Kinase C (PKC) activator phorbol-12-myristate-13-acetate(PMA) reduced OCIL mRNA expression at the early stage(2-6 h),with the highest down-regulation of 50％ at 6 h.However,the inhibitory effect on OCIL mRNA turned into slightly stimulatory effect later (24 h).PKA inhibitor KT5720,calmodulin antagonist W-7,CaMK Ⅱ inhibitor KN-62 and mitogen-activated protein kinase (MAPK) inhibitor PD98059 all blocked PTH(1-34)-induced OCIL mRNA expression by the maximal reduction of 56％,61％,63％ and 50％ respectively.There also exist cross-talks between different signal pathways.MAPK inhibitor PD98059 blocked the expression of OCIL mRNA which was stimulated by PKA activators FSK or db-cAMP,with the reduction of 98％ and 63％ respectively,while the OCIL mRNA expression stimulated by A23187 remained unaffected.Conclusion PTH(1-34) increased OCIL mRNA expression in vitro through cAMP/PKA,Ca2+/CaMK and MAPK signaling pathways.

Inhibitory Effects of 1, 25-Dihydroxy-Vitamin D3 on Differentiation of Adipocytes / 天津医药

Objective To investigate the effect of 1, 25-dihydroxy-vitamin D3 (1, 25 (OH)2D3) on adipocyte differen-tiation and the underlying mechanism. Methods The mesenchymal stem cell line C3H10T1/2 was randomly divided into 6 groups including control group, differentiation group and 4 different doses of 1, 25(OH)2D3 groups. The control group was treated with vehicle. The differentiation group was supplemented with adipocyte differentiation reagent. And the 1,25(OH)2D3 groups were treated with adipocyte differentiation reagents and 10-9, 10-8, 10-7 and 10-6 mol/L of 25(OH)2D3. After culturing for 5 days, the cells were stained with oil red O, and the expression levels of adipocyte-specific transcription factors and Wnt/β-catenin signaling pathway related genes were examined by RT-PCR or Western blot methods. Results 1,25(OH)2D3 sig-nificantly reduced the number of differentiated adipocytes and blocked the mRNA levels of adipocyte specific transcription factor PPARγ(peroxisome proliferator-activated receptor gamma), C/EBPα(CCAAT enhancer binding proteinα) and adipo-cyte characterization factor aP2 (fatty acid binding protein 4). These were paralleled by the decreased mRNA expression of Wnt/β-catenin signaling pathway inhibitor sFRP1 (Secreted frizzled-related protein 1) and the increased level ofβ-catenin protein. Conclusion 1, 25(OH)2D3 inhibits adipocyte differentiation, which may be related to the activation of Wnt/β-catenin signaling.

Regulation of osteoclast inhibitory lectin ( OCIL ) expression by prostaglandin E2 in rat osteoblastic cells / 中华内分泌代谢杂志

To investigate the regulation of osteoclast inhibitory lectin (OCIL) mRNA expression by prostaglandin E2 ( PGE2 ) in rat osteoblastic cells and the involved signaling pathways. Rat primary osteoblasts and UMR106 osteoblast-like cells were cultured and treated with various doses of PGE2 or regulators of different signaling pathways for different periods of time, the cells were then harvested at indicated dates. Total RNA were isolated and OCIL mRNA expression were studied by real-time PCR. PGE2, Forskolin, db-cAMP, and A23187 increased OCIL mRNA by 2. 38 fold,4. 2 fold,4. 5 fold, and 5. 1 fold ( all P＜0. 01 ) respectively, while PMA downregulated OCIL mRNA expression by 50% ( P＜0. 01 ). KT-5720, verapamil, W7, and PD98059 downregulated PGE2 induced OCIL mRNA expression by 56%, 40%, 65%, and 60%, respectively( all P＜0. 0l ). While chelerythrine enhanced PGE2 induced OCIL mRNA expression by 30% ( P＜0. 05 ). PGE2 up-regulated the expression of OCIL in rat osteoblastic cells via PKA, MAPK, and Ca2+/Calmodulin signaling pathways.

Relationship between 24-hour mean pulse pressure and the cardiac function in the elderly with isolated systolic hypertension

To investigate the effect of mean pulse pressure during 24 hours on cardiac function in the elderly with isolated systolic hypertension. One hundred seventy two elderly patients with isolated systolic hypertension were enrolled to determine mean pulse pressure by monitoring 24-hour ambulatory blood pressure and to analyze the cardiac function by nuclide cardiac blood pool imaging. Twenty four hours mean pulse pressure negatively correlated with left ventricular ejection fraction [LVEF] [r = - 0.46, P < 0.01], and also with peak filling rate [RFR] [r = - 0.41, P < 0.05]. The greater the mean pulse pressure, the worse the cardiac function [P < 0.01]. The 24-hours mean pulse pressure was an important factor predicting risk for cardiac disfunction in the elderly with isolated systolic hypertension

The Effect of Staphylococcus Aureus on Expression of iNOS and IL-1β in THP- 1 Monocytes under High Glucose Concentration / 天津医药

Objective:To investigate the effect of staphylococcus aureus on expressions of inducible nitric oxide synthase (iNOS) and interleukin-1 beta (IL-1β) in THP-1 monocytes under high glucose concentrations. Methods:THP- 1 monocytes were incubated at different surroundings with 2×2 factorial design. There were 4 experimental groups in the study, which were analyzed by univariate analysis of variance. The total RNA was distilled. The expressions of iNOS and IL-1β were examined by SQ-RT-PCR. Results:The expressions of iNOS and IL-1β were affected by high glucose concentration, bacteria and both of them together in THP-1 monocytes. The expressions of iNOS and IL-1β were weakened in high glucose concentration compared with that of control(P < 0.01). The expressions of iNOS and IL-1β were weakened in the high glucose concentration and bacterial infection compared with that of the high glucose concentration only(P < 0.01). The expressions of iNOS and IL-1β increased in bacterial infection compared with that of control(P < 0.01). The expression of IL-1β increased in high glucose concentration and bacterial infection compared with that of the high glucose concentration only(P < 0.01) but the expression of iNOS was not distinct. Conclusion:When staphylococcus aureus infected monocytes, the expressions of iNOS and IL-1β were weakened in high glucose concentration, which suggested that the depressed function of immunocyte was related with high glucose concentration.

Regulation of MCP-1 in Osteoblasts by PTHrP in Breast Cancer Cells / 天津医药

Objective:To identify the regulation of monoeyte chemoattractant protein-1(MCP-1)in osteoblasts by parathyroid hormone related protein(PTHrP) in breast cancer cells.Methods:Osteoblast-like UMR106 cells were treated with 10~(-8)mol/L PTHrP (1-34) for different times and the expression of MCP-1 was studied by semiquantitative RT-PCR.Recombinant shRNA plsmid PTH1R/pRNAT was constructed and transfected into UMR106 cells.Stable PTH1R/UMR106 cells were established,in which PTH1R expression was knocked-down.The conditioned medium(CM) of breast cancer MDA-MB231 was added to UMR 106 and PTH1R/UMR106 cultures respectively.Thereafter,mRNA of MCP-1 was examined by semiquantitative RT-PCR.Results:PTHrP induced expression of MCP-1 in UMR106 cells and the induction began at 3 h and reached the maximum at 6 h.Treatment of UMR106 cells with CM of MDA-MB231 also significantly induced mRNA of MCP-1 at 6 h(1.238 1±o.115 5 and 0.598 4±0.036 4,P＜0.05).This effect was partly blocked by the knockdown of PTH1R in UMR106 cells(0.867 2±0.045 7,P＜0.05).Conclusion:The osteoblastic MCP-1 is up-regulated in breast cancer through expression of PTHrP.

Expression Products of Chimeric BMP2 and BMP7 Induce Osteoblast Differentiation / 天津医药

Objective: To study the osteoinductive activity of chimeric molecule of bone morphogenetic protein(BMP)2 and BMP7 expressed in mammalian cells. Methods: Sequences encoding mature peptides of BMP2 and BMP7 were separately amplified by PCR and then linked by overlap-extension PCR with a DNA sequence encoding a flexible peptide (Gly_4Ser)_3 between them. The chimeric DNA sequence was cloned into secretory expression plasmid pcDNA3/sec and then the recombinant plasmid pcDNA3 -BMP2/7 was transfected into CHO-K1 cells. In the presence of G418,cells that stably expressed BMP2/7 were screened out. Thereafter, the conditioned culture medium of the transfected cells was collected and used to treat C3H10T1/2 cells. RT-PCR was employed to study the activity of the recombinant product in inducing osteoblast differentiation. Results: The expression products of chimeric BMP2/7 significantly enhanced the mRNA expression levels of osteoblast phenotype genes, such as alkaline phosphatase, osteocalcin and osteoblast specific transcription factor runt-related transcription factor 2 in C3H10T1/2 cells(P < 0.01). Conclusion: The chimeric expression products of BMP2/7 are capable of forming heterodimers and thus effectively induce non-bone derived cells to differentiate into osteoblasts.

Expression and analysis of the extracellular domain of human glucocorticoid-induced tumor necrosis factor receptor ligand in Escherichia coli / 生物工程学报

GITRL (Glucocorticoid-induced tumor necrosis factor receptor ligand) has been recently identified as a novel inhibitor of osteoclastogenesis and hence called Osteostat. In this study, we expressed recombinant extracellular domain of GITRL protein in Escherichia coli and analyzed its bioactivity. Using an Eco31I enzyme-based restriction and ligation method, we obtained an E. coli-preferred DNA sequence coding for the extracellular domain of human GITRL. The DNA was cloned into expression vector pQE-30Xa that encodes a fusion tag of 6xHis before the insert. The resultant recombinant expression vector pQE/GITRL was subsequently transformed into E. coli strain M15[pREP4]. After induction with Isopropyl beta-D-Thiogalactoside (IPTG), the cells produced the fusion protein mainly in the form of inclusion bodies as identified by SDS-PAGE. The recombinant protein was purified by affinity chromatography through Ni-NTA column and recognized by anti-His polyclonal antibody using Western blotting analysis. Moreover, we established a simple, efficient and sensitive reporter gene-based method to detect the activity of the recombinant protein. The results showed that the target protein was biologically active.

Effects of Epimedium on the Expression of Interleukin-6 Messenger Ribonucleic Acid in Bone of Ovariectomized Rat / 中华妇产科杂志

Objective To investigate the effects of the traditional herb-epimedium on the expression of interleukin-6 (IL-6) mRNA in bone of ovariectomized rat. Methods Forty female rats were randomly allocated into 4 groups, 10 in each: ovariectomized (OVX) group, sham operation group, OVX followed by epimedium (group 3)or nilestriol (group 4) for 3 months respectively. All rats were then sacrificed,and total RNA were directly isolated from their right tibia. Interleukin-6 mRNA expression was detected by relative semiquantitative reverse transcription-polymerase chain reaction technique. Lumbar bone mineral density (BMD) was measured by dual-energy X ray absorptiometry before sacrifice. Results The BMD of epimedium group was significantly higher than that in the OVX group ( P

Cloning of hTERT promoter and its specific transcriptional activity in MCF7 breast cancer cell / 中国癌症杂志

Background and purpose:Gene therapy is a novel approach for the treatment of the patients with breast cancer. One of the effective ways is to direct transgenic expression to specific tissues or tumors with the use of tissue-specific-promoters (TSP). hTERT (human telomerase reverse transcriptase) is highly expressed in many types of cancers including breast cancer. Thus, we hypothesized that the hTERT promoter targeting with gene therapy vectors could be exploited for breast cancer. In this study, we amplified hTERT gene promoter and cloned it into the reporter vector pEGFP and pGL3-Basic. Afterwards, the specific transcription of hTERT promoter in MCF7 cells was evaluated. Methods:hTERT gene minimal promoter was PCR amplified and cloned into the reporter plasmid pEGFP-1 and pGL3-Basic.The constructs pEGFP/TERT and pGL3/TERT were transfected into MCF7 breast cancer cells and HBL100 human epithelial cells, respectively.The expression of EGFP and luciferase were investigated, respectively..Results:pEGFP/TERT and pGL3 /TERT bearing hTERT gene promoter were constructed. The specific expression of EGFP was detected in MCF7 cells while little expression of EGFP was seen in HBL100 cells.In accordance with EGFP, luciferase driven by hTERT also showed specific and high activity in MCF7 cell (RLU/U: 33784), which is 15 times higher than in HBL100 (RLU/U: 2400).Conclusions:The high transcriptional activity of hTERT gene promoter in MCF7 cell indicates its potential utility as a novel candidate for transcriptional targeting of breast cancer.