AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

RNA干扰技术建立人PARP-1缺陷细胞株

目的建立并鉴定聚ADP核糖聚合酶1(hPARP1)缺陷细胞株,用于研究hPARP1基因的作用机制及其缺陷与DNA损伤发生的关系。方法将用已经构建的pEGFPC1shRNA(包含两个PARP1基因及一个阴性对照)转染支气管上皮细胞(16HBE),使之在16HBE中表达,转染后命名为16HBEP1、16HBEP2及16HBEN。用蛋白免疫印迹法鉴定转染细胞中hPARP1基因的表达水平。结果pEGFPC1shRNA在真核细胞成功表达;16HBEP1和16HBEP2的蛋白表达水平分别下降了84.3%及63.7%。结论hPARP1缺陷细胞株的成功建立和鉴定为hPARP1基因功能研究提供了一种有效手段。

靶向沉默巨噬细胞中 Mcl-1基因 shRNA表达质粒的构建与鉴定

目的研究髓样细胞白血病-1(myeloid cell leukemia-1,Mcl-1)基因在小鼠巨噬细胞Raw264.7和人巨噬细胞THP-1中的表达情况,筛选出表达含量高的细胞株作为实验用细胞,根据筛选的结果构建靶向小鼠Mcl-1基因的短发夹RNA(shRNA)真核表达质粒进行转染,最后筛选出沉默Mcl-1基因效果最明显的shRNA表达质粒。方法利用半定量RT-PCR和蛋白质免疫印迹(Western blotting)分别检测两种巨噬细胞中Mcl-1mRNA和蛋白表达情况。采用小分子干扰RNA(siRNA)软件设计3个针对Mcl-1基因不同位点的shRNA片段,由公司构建携带此shRNA片段的真核表达质粒(Mcl-1shRNA1-3),然后通过脂质体将真核表达质粒载体转染到小鼠巨噬细胞株Raw264.7中,24、48h后通过倒置荧光显微镜观察转染效果,并分别采用实时定量PCR和Western blot检测Mcl-1mRNA和蛋白表达情况。结果小鼠巨噬细胞Raw264.7中Mcl-1的mRNA及蛋白质的表达显著高于人巨噬细胞,差异有统计学意义(P<0.05);构建的shRNA表达载体在24、48h均能降低Raw264.7细胞内mcl-1mRNA和蛋白水平,尤其在48h沉默效果最为明显;转染48h后与正常组、脂质体组和阴性对照组相比,差异有统计学意义(P<0.05);与Mcl-1shRNA1和Mcl-1shRNA2相比,Mcl-1shRNA3对Mcl-1mRNA和蛋白的沉默作用最强。结论成功筛选出了实验所用细胞Raw264.7及对小鼠巨噬细胞Raw264.7内Mcl-1具有明显沉默效果的靶向Mcl-1shRNA3真核表达质粒。

pSUPER-IL-11-shRNA表达载体的构建与鉴定

目的:构建及测序鉴定重组质粒p SUPER-IL-11-shRNA,检测其对甲状腺未分化癌(anaplastic thyroid carcinoma,ATC)细胞株sw579 IL-11基因的沉默效应。方法:根据Genebank中IL-11 c DNA序列,设计并合成3对两端有酶切位点的特异编码IL-11shRNA序列的寡核苷酸链,退火成互补双链后与p SUPER.retro.puro质粒载体连接,转至大肠杆菌DH-5α菌株,挑取单个抗性克隆,提取质粒进行1%琼脂糖凝胶电泳鉴定及测序分析,脂质体介导重组质粒转染ATC细胞株sw579,real-time PCR及ELISA检测其对靶基因IL-11的表达影响。结果:重组质粒p SUPER-IL-11-shRNA酶切产物凝胶电泳结果显示,3个样本均为阳性重组质粒,测序鉴定3种重组质粒p SUPER-IL-11-shRNA序列正确,转染3种重组质粒后sw579细胞裂解液中IL-11 mRNA和细胞培养上清液中IL-11蛋白显著降低。结论:本研究成功构建重组质粒p SUPER-IL-11-shRNA,为进一步探索IL-11在ATC进展中的作用奠定了基础。

Identification of the Interaction between P-Glycoprotein and Anxa2 in Multidrug-resistant Human Breast Cancer Cells

Objective To explore the interaction of Anxa2 with P-Glycoprotein （P-gp） in the migration and invasion of the multidrug-resistant （MDR） human breast cancer cell line MCF-7/ADR. Methods A pair of short hairpin RNA （shRNA） targeting P-gp was transfected into MCF-7/ADR cells, and monoclonal cell strains were screened. The expression of P-gp was detected by Western blot. Transwell chambers were used to observe the cell migration capacity and invasion ability. The interaction between P-gp and Anxa2 was examined by immunoprecipitation and immunofluorescence confocal microscopy analyses. Results P-gp expression was significantly knocked down, and there were notable decreasing trends in the migration and invasion capability of MDR breast cancer cells （P〈0.05）. There was a close interaction between Anxa2 and P-gp. Conclusions MCF-7/ADR is an MDR human breast cancer cell line with high migration and invasion abilities. The knockdown of P-gp notably impaired the migration and invasion abilities of the tumor cells. The interaction of Anxa2 with P-pg may play an important role in time enhanced invasiveness of MDR human breast cancer cells.

Construction of a lentiviral vector for RNA interference of human VIM gene and its silencing effect in pancreatic cancer cells

Objective： To construct a lentiviral expression vector for RNA interference （RNAi） of human VIM gene; and assess its gene silencing effect in pancreatic cancer cell line Panc-1. Methods： Three pairs of human VIM gene short hairpin RNA（shRNA） sequences were designed using a software available on-line and one pair came from document. After synthesis and annealing, four double-stranded oligonucleotides （dsOligo） were cloned into the pGCL-GFP/U6 plasmid, which were subsequently confirmed by polymerase chain reaction （PCR） and DNA sequencing analysis. Real-time PCR and Westemblotting were used to screen the effective pGCL-GFP-shRNA plasmid in 293T cells, then the most effective one was packed into the recombinant lentivirus Lv-VIM-shRNA with lentiviral packing materials pHelper 1.0 and pHelper 2.0 in 293T cells. The titer of lentivirus was determined by hole-by-dilution titer assay. The silencing effect of Lv-VIM-shRNA in Panc-1 calls were validated by real-time PCR and Western-blotting. Results： An effective Lv-VIM-shRNA was successfully constructed. The titer of lentivirus was determined on 2× 10^9TU/mL. The expressions of VIM mRNA and vimentin were down-regluated in the Panc-1 cells infected with Lv-VIM-shRNA. Conclusion： An effective Lv-VIM-shRNA could inhibit the expression of VIM gene in Panc-1 cells in vitro, which provides a tool for investigating the role of VIM gene in the signaling pathway involved in tumorigenesis and progression of pancreatic cancer and searching new therapeutic targets.

Role of ADAM10 and ADAM17 in CD16b Shedding Mediated by Different Stimulators

Objective To investigate the main proteinases responsible for CD16b shedding under different stimulators. Methods HEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then overexpressed with a disintegrin and metalloproteinase 10 （ADAM10） or ADAM17, sup- pressed with short hairpin RNA of ADAM10 or ADAM I 7, and reconstituted with ADAM 10 or ADAM17, respectively. After each treatment, the cell line was stimulated with ionomycin or phorbol 12-myristate- 13-acetate （PMA） for 12 hours. The soluble CD 16b released from cell membrane was detected by immuno- precipition and immunoblot. Quantitation was then implemented to compare the amount of soluble CD 16b in cell supernatant after stimulation. Results HEK293 cell line stably expressing CD16b was successfully established. When CDI6b ex- pressing cell line was overexpressed with ADAM 10, shedding of CD 16b was increased after stimulation with ionomycin but not PMA; when the cell line overexpressed with ADAM I7, shedding of CDI6b was increased after stimulation with PMA but not ionomycin. Similarly, when ADAM10 was suppressed by short hairpin RNA, CD 16b shedding was decreased after stimulation with ionomycin; when ADAM 17 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with PMA. The shedding of CD16b was increased again when CD16b expressing cell line was reconstituted with ADAM10 and stimulated by ionomycin or reconstituted with ADAM 17 and stimulated by PMA. Conclusions Both ADAM10 and ADAM17 could shed CD16b, but they possess differed prefer- ences. ADAM10 is the main sheddase under stimulation of ionomycin, while ADAM17 is the main sheddase under stimulation of PMA.

Lentiviral vector-mediated down-regulation of IL-17A receptor in hepatic stellate cells results in decreased secretion of IL-6

AIM: To investigate the mechanism of interleukin (IL)-6 secretion through blocking the IL-17A/IL-17A recepto (IL-17RA) signaling pathway with a short hairpin RNA (shRNA) in hepatic stellate cells (HSCs) in vitro . METHODS: HSCs were derived from the livers of adul male Sprague-Dawley rats. IL-6 expression was evalu ated using real-time quantitative polymerase chain reaction and enzyme linked immunosorbent assay. The phosphorylation activity of p38 mitogen activated pro tein kinases (MAPK) and extracellular regulated pro tein kinases (ERK) 1/2 upon induction by IL-17A and suppression by IL-17RA shRNA were examined using Western blotting.RESULTS: IL-6 expression induced by IL-17A was significantly increased compared to control in HSCs (P < 0.01 in a dose-dependent manner). Suppression of IL17RA using lentiviral-mediated shRNA inhibited IL-6 expression induced by IL-17A compared to group with only IL-17A treatment (1.44 ± 0.17 vs 4.07 ± 0.43, P < 0.01). IL-17A induced rapid phosphorylation of p38 MAPK and ERK1/2 after 5 min exposure, and showed the strongest levels of phosphorylation of p38 MAPK and ERK1/2 at 15 min in IL-17A-treated HSCs. IL-6 mRNA expression induced by IL-17A (100 ng/mL) for 3 h exposure was inhibited by preincubation with specific inhibitors of p38 MAPK (SB-203580) and ERK1/2 (PD-98059) compared to groups without inhibitors preincubation (1.67 ± 0.24, 2.01 ± 0.10 vs 4.08 ± 0.59, P < 0.01). Moreover, lentiviral-mediated IL-17RA shRNA 1 inhibited IL-17A-induced IL-6 mRNA expression compared to random shRNA in HSCs (1.44 ± 0.17 vs 3.98 ± 0.68, P < 0.01). Lentiviral-mediated IL17RA shRNA 1 inhibited phosphorylation of p38 MAPK and ERK1/2 induced by 15 min IL-17A (100 ng/mL) exposure. CONCLUSION: Down-regulation of the IL-17RA receptor by shRNA decreased IL-6 expression induced by IL-17A via p38 MAPK and ERK1/2 phosphorylation in HSCs. Suppression of IL-17RA expression may be a strategy to reduce the inflammatory response induced by IL-17A in the liver.

Expression of FOXG1 is associated with the malignancy of human glioma

Objective： Recent evidence indicates that the increased expression of FOXG1 is associated with tumor genesis. This study was designed to explore the expression and role which FOXG1 plays in human glioma. Methods： We detected the expression of FOXG1 by immunohistochemistry in glioma tissue samples. Following the down-regulation of FOXG1 in glioma cell lines by a specific short hairpin RNA, the function of FOXG1 in proliferation and apoptosis was assessed. Results： Glioma tissues exhibited notably higher expression of FOXG1 compared with control brain tissues and was positively corre- lated with histological malignancy. The down-regulation of FOXG1 in glioma cells led to a cell apoptosis in vitro. Cenclusion： The overexpression of FOXG1 is a novel glioma malignancy marker, and FOXG1 may be used as a new target in therapeutic strategies for human glioma.

Downregulation of MTDH using short hairpin RNA inhibited EMT in breast cancer cells

Objective： Metadherin （MTDH） could regulate epithelial-mesenchymal transition （EMT）, and is involved in tumor metastasis.This study was designed to observe the effect of MTDH-short hairpin RNA （shRNA） on EMT, and the role of MTDH in breast tumor metastasis. Methods： RNA interference plasmid that can express shRNA targeting MTDH or shRNA-nega- tive plasmid that does not match any known human coding mRNA was designed, constructed and named MTDH-shRNA and MTDH-shRNA-neg, which were transiently transfected into MDA-MB-231 cells using LipofectamineTM2000. After 48 h, the levels of MTDH, E-cadherin and a-SMA expression were determined by reverse transcription-polymerase chain reactin （RT- PCR）, Western blot. The invasion and immigration potential was examined by Transwell chamber invasion or migration assay. Results： Compared with MDA-MB-231, the MTDH mRNA level was down-regulated by 41.2%, the MTDH protein level was down-regulated by 40.3%. The invasion and immigration potential of MDA-MB-231 cells was decreased after transfection of MTDH-shRNA. Compared with MDA-MB-231 or MTDH-shRNA-neg, the mRNA and protein level of a-SMA was reduced and E-candherin were increased in MTDH-shRNA, with statisticat significance. Conclusion： Downregulation of MTDH increase E-candherin expression and reduced a-SMA expression, which inhibit EMT in MDA-MB-231 cells. This knockdown significantly suppresse migration and invasion in MDA-MB-231 cells.

Pin1 expression affects cell proliferation and apoptosis of SW620 cells in colorectal carcinoma

Objective:The aim of our study was to investigate the effects of Pin1 reduction on SW620 cell proliferation and apoptosis in human colorectal carcinoma.Methods:We constructed a plasmid of RNA interfering(shRNA) for Pin1 gene(pGenesl-1-Pin1),then the plasmid was transfected into colorectal carcinoma SW620 cells line by liposome mediation.The protein expression of Pin1 was tested by Western blotting.The proliferation rate was analyzed by MTT and the apoptotic rate of cells was tested by flow cytometry.In order to explain further the effect of Pin1 in SW620 cells,the protein level of Bcl-2 was analyzed by Western blotting.Results:pGenesil-1-Pin1 plasmid was successfully constructed and confirmed by sequencing.The protein relative levels of Pin1 were 0.06 ± 0.04 for the P-shRNA/SW620 cells,and 0.32 ± 0.09 for the P-Con/SW620 cells.The cell growth rate of SW620 cells was slower while the apoptotic rate was increased after transfection with pGenesil-Pin1 plasmid,and the apoptotic rate was 12.38% ± 1.55% for the P-shRNA/SW620 group.At the same time,we found that the protein expression of Bcl-2 was also reduced.The results were 0.13 ± 0.04 for the P-shRNA/SW620 cells,and 0.36 ± 0.08 for the P-Con/SW620 cells.Conclusion:Inhibited Pin1 expression may suppress the cell proliferation and promote apoptosis of colorectal carcinoma cells in vitro.

The influence of down-regulation of ACP1 by RNAi on the metastasis capability of osteosarcoma cell line MG-63

Objective： The aim of this study was to study the inhibition effect of small interfering RNAs （siRNA） on gene expression in MG-63 cells,and to study the inhibitory effect on metastasis of MG-63. Methods： A plasmid of a short hairpin RNA targeting acid phosphatase 1 （ACP1） was constructed and transfected into MG-63 cell line. ACP1 expression of MG-63 cells before and after transfection was detected by RT-PCR and Western blot. The capacity of adhesion, migration and inva- sion was examined by adhesion assay, migration assay and transwell assay. Results： The recombinant plasmid pGenesil-l/ ACPI-shRNA was successfully constructed, shRNA efficiently inhibited the expression of ACPI by gene and protein level and suppressed cell migration. The adhesion decreased from 96.41±8.83 to 43.38 ± 6.03 （P 〈 0.01）, invasion ability from 56.5 ± 4.8 to 36.3 ± 6.1 （P 〈 0.05）. Conclusion： Down-regulating ACP1 by shRNA reduced the capacity of metastasis of MG-63 cell,which providing a novo-approach to biotherapy of cancer.

Algal oligosaccharides ameliorate osteoporosis via up-regulation of parathyroid hormone 1-84 and vascular endothelial growth factor

OBJECTIVE: To determine whether algal oligosaccharide affects the levels of parathyroid hormone 1-84(PTH1-84) and vascular endothelial growth factor(VEGF).METHODS: An osteoporosis rat model was established via bilateral ovariectomy. The model rats were fed algal oligosaccharides(molecular weights:600-1, 200 Da) for 4 months. Bone mineral density(BMD) was then measured. MG-63 human osteoblastic cells were treated with algal oligosaccharides. The expression of PTH1-84 and VEGF was then examined. Oligosaccharide-treated cells were transfected with PTH1-84 short hairpin RNA(sh RNA), VEGF sh RNA, and PTH1-84-VEGF small interfering RNA(si RNA). The growth rates were then compared between transfected and non-transfected cells.RESULTS: Algal oligosaccharides increased the BMD of the osteoporosis rat model compared with untreated controls(P < 0.05). When MG-63 cells were treated with algal oligosaccharides, the growth rate increased by 25% compared with the control group at day 3(P < 0.05). In addition, the expression of PTH84 and VEGF was enhanced. Con-versely, when the cells were transfected with PTH84 sh RNA, VEGF sh RNA, or PTH1-84-VEGF si RNA, the growth rate was decreased by 17%, 35% and 70%, respectively, compared with controls at day 3(P < 0.05).CONCLUSION: Algal oligosaccharides ameliorate osteoporosis via up-regulation of PTH1-84 and VEGF. Algal oligosaccharides should be developed as a potential drug for osteoporosis treatment.