Construction and verification of the targeted uPA‑shRNA lentiviral vector and evaluation of the transfection and silencing rate

Wang,Wei‑Shan; Guo,Feng‑Jing; Li,Chang‑Jun; Zhang,Zhen‑Dong; Shi,Chen‑Hui

doi:10.3892/etm.2014.1741

August-2014 Volume 8 Issue 2

Full Size Image

Journals

International Journal of Molecular Medicine

International Journal of Molecular Medicine is an international journal devoted to molecular mechanisms of human disease.

International Journal of Oncology

International Journal of Oncology is an international journal devoted to oncology research and cancer treatment.

Molecular Medicine Reports

Covers molecular medicine topics such as pharmacology, pathology, genetics, neuroscience, infectious diseases, molecular cardiology, and molecular surgery.

Oncology Reports

Oncology Reports is an international journal devoted to fundamental and applied research in Oncology.

Experimental and Therapeutic Medicine

Experimental and Therapeutic Medicine is an international journal devoted to laboratory and clinical medicine.

Oncology Letters

Oncology Letters is an international journal devoted to Experimental and Clinical Oncology.

Biomedical Reports

Explores a wide range of biological and medical fields, including pharmacology, genetics, microbiology, neuroscience, and molecular cardiology.

Molecular and Clinical Oncology

International journal addressing all aspects of oncology research, from tumorigenesis and oncogenes to chemotherapy and metastasis.

World Academy of Sciences Journal

Multidisciplinary open-access journal spanning biochemistry, genetics, neuroscience, environmental health, and synthetic biology.

International Journal of Functional Nutrition

Open-access journal combining biochemistry, pharmacology, immunology, and genetics to advance health through functional nutrition.

International Journal of Epigenetics

Publishes open-access research on using epigenetics to advance understanding and treatment of human disease.

Medicine International

An International Open Access Journal Devoted to General Medicine.

August-2014 Volume 8 Issue 2

Full Size Image

Article

Construction and verification of the targeted uPA‑shRNA lentiviral vector and evaluation of the transfection and silencing rate

Authors:
- Wei‑Shan Wang
- Feng‑Jing Guo
- Chang‑Jun Li
- Zhen‑Dong Zhang
- Chen‑Hui Shi
View Affiliations / Copyright

Affiliations: Department of Orthopedics, Medical College of Shihezi University, Shihezi, Xinjiang 832008, P.R. China, Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China
Pages: 435-441
|
Published online on: May 28, 2014

https://doi.org/10.3892/etm.2014.1741
Expand metrics +

Abstract

Urokinase‑type plasminogen activator (uPA) receptors, which are released by the synovial tissue, are responsible for the activation of cartilage‑breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). RNA interference (RNAi) technology has emerged as a potent tool to generate cellular knockdown phenotypes of a desired gene. The aims of the present study were to investigate the effect of siRNA specific to the uPA gene on chondrocytes and to investigate the possible mechanisms of OA. Firstly, four types of small hairpin RNA (shRNA) sequence (P1, P2, P3 and P4) were obtained from the targeted uPA gene of the New Zealand rabbit, based on siRNA theory. The sequences were designed, constructed and subjected to restriction enzyme digestion, transformation, polymerase chain reaction (PCR) identification, positive clone sequencing and lentivirus packaging. Secondly, primary culturing cartilage cells from the New Zealand rabbit were transfected with P1, P2, P3 or P4 to observe the transfection rate under a fluorescence microscope. The mRNA expression levels of uPA were analyzed in cartilage cells using quantitative PCR, while protein expression levels were analyzed in the cartilage cells using western blot technology. Four types of uPA‑shRNA lentiviral vectors were constructed successfully, which were all able to be transfected into the primary culturing cartilage cells. The transfection rate was as high as 85% when the multiplicity of infection was 100, which demonstrated that P1, P2, P3 and P4 were all capable of inhibiting the mRNA and protein expression of uPA in cartilage cells. In addition, among the four sequences, the P2 sequence exhibited the highest silencing rate of 70%. Statistical significance (P<0.05) was observed when analyzing the silencing rate of P2 compared to the other three groups. The most efficient targeted uPA‑shRNA sequence was identified following screening. The results strongly verified that siRNA lentiviral vectors can be transfected into cartilage cells to further inhibit the expression of the uPA gene efficiently and steadily. Thus, the results provide the foundation for further research on the role of uPA in the pathogenesis of OA.

View Figures

View References

1	Eden G, Archinti M, Furlan F, Murphy R and Degryse B: The urokinase receptor interactome. Curr Pharm Des. 17:1874–1889. 2011. View Article : Google Scholar : PubMed/NCBI
2	Kwaan HC and McMahon B: The role of plasminogen-plasmin system in cancer. Cancer Treat Res. 148:43–66. 2009. View Article : Google Scholar
3	Kim KS, Lee YA, Choi HM, Yoo MC and Yang HI: Implication of MMP-9 and urokinase plasminogen activator (uPA) in the activation of pro-matrix metalloproteinase (MMP)-13. Rheumatol Int. 32:3069–3075. 2012. View Article : Google Scholar : PubMed/NCBI
4	Wang JT and Gong SS: Effects of siRNA specific to the protein kinase CK2α on apoptosis of laryngeal carcinoma cells. Chin Med J (Engl). 125:1581–1585. 2012.
5	Zeng QY, Zang CH, Li XF, Dong HY, Zhang AL and Lin L: Associated risk factors of knee osteoarthritis: a population survey in Taiyuan, China. Chin Med J (Engl). 119:1522–1527. 2006.PubMed/NCBI
6	Racine J and Aaron RK: Pathogenesis and epidemiology of osteoarthritis. R I Med J. 96:19–22. 2013.PubMed/NCBI
7	Maldonado M and Nam J: The role of changes in extracellular matrix of cartilage in the presence of inflammation on the pathology of osteoarthritis. Biomed Res Int. Aug 28–2013.(Epub ahead of print). View Article : Google Scholar
8	Sengupta K, Krishnaraju AV, Vishal AA, et al: Comparative efficacy and tolerability of 5-Loxin and Aflapin against osteoarthritis of the knee: a double blind, randomized, placebo controlled clinical study. Int J Med Sci. 7:366–377. 2010. View Article : Google Scholar
9	Iagnocco A and Naredo E: Osteoarthritis: research update and clinical applications. Rheumatology (Oxford). 51(Suppl 7): vii2–vii5. 2012. View Article : Google Scholar : PubMed/NCBI
10	Loeser RF: Aging processes and the development of osteoarthritis. Curr Opin Rheumatol. 25:108–113. 2013. View Article : Google Scholar : PubMed/NCBI
11	Haseeb A and Haqqi TM: Immunopathogenesis of osteoarthritis. Clin Immunol. 146:185–196. 2013. View Article : Google Scholar : PubMed/NCBI
12	Kapoor M, Martel-Pelletier J, Lajeunesse D, et al: Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol. 7:33–42. 2011. View Article : Google Scholar : PubMed/NCBI
13	Mobasheri A: Osteoarthritis year 2012 in review: biomarkers. Osteoarthritis Cartilage. 20:1451–1464. 2012.PubMed/NCBI
14	Koskinen A, Vuolteenaho K, Nieminen R, et al: Leptin enhances MMP-1, MMP-3 and MMP-13 production in human osteoarthritic cartilage and correlates with MMP-1 and MMP-3 in synovial fluid from OA patients. Clin Exp Rheumatol. 29:57–64. 2011.PubMed/NCBI
15	Lee AS, Ellman MB, Yan DA, et al: A current review of molecular mechanisms regarding osteoarthritis and pain. Gene. 527:440–447. 2013. View Article : Google Scholar : PubMed/NCBI
16	Murab S, Chameettachal S, Bhattacharjee M, Das S, Kaplan DL and Ghosh S: Matrix-embedded cytokines to simulate osteoarthritis-like cartilage microenvironments. Tissue Eng Part A. 19:1733–1753. 2013. View Article : Google Scholar : PubMed/NCBI
17	Yeh CC, Chang SF, Huang TY, et al: Shear stress modulates macrophage-induced urokinase plasminogen activator expression in human chondrocytes. Arthritis Res Ther. 15:R532013. View Article : Google Scholar : PubMed/NCBI
18	Yuasa T, Otani T, Koike T, Iwamoto M and Enomoto-Iwamoto M: Wnt/beta-catenin signaling stimulates matrix catabolic genes and activity in articular chondrocytes: its possible role in joint degeneration. Lab Invest. 88:264–274. 2008. View Article : Google Scholar : PubMed/NCBI
19	Kubowicz P, Żelaszczyk D and Pękala E: RNAi in clinical studies. Curr Med Chem. 20:1801–1816. 2013. View Article : Google Scholar : PubMed/NCBI
20	Shim MS and Kwon YJ: Efficient and targeted delivery of siRNA in vivo. FEBS J. 277:4814–4827. 2010. View Article : Google Scholar : PubMed/NCBI
21	Chu X, You H, Yuan X, et al: Protective effect of lentivirus-mediated siRNA targeting ADAMTS-5 on cartilage degradation in a rat model of osteoarthritis. Int J Mol Med. 31:1222–1228. 2013.PubMed/NCBI
22	Wilson RC and Doudna JA: Molecular mechanisms of RNA interference. Annu Rev Biophys. 42:217–239. 2013. View Article : Google Scholar
23	Mekkawy AH, Morris DL and Pourgholami MH: Urokinase plasminogen activator system as a potential target for cancer therapy. Future Oncol. 5:1487–1499. 2009. View Article : Google Scholar : PubMed/NCBI

Journals

International Journal of Molecular Medicine

International Journal of Oncology

Molecular Medicine Reports

Oncology Reports

Experimental and Therapeutic Medicine

Oncology Letters

Biomedical Reports

Molecular and Clinical Oncology

World Academy of Sciences Journal

International Journal of Functional Nutrition

International Journal of Epigenetics

Medicine International

Construction and verification of the targeted uPA‑shRNA lentiviral vector and evaluation of the transfection and silencing rate

This article is mentioned in:

Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Related Articles