A total of 3 New Zealand rabbits (age, 2 years) were purchased from the Experimental Animal Center of Xinjiang Medical University (Ürümqi, China). Escherichia coli (E. coli) DH5α and 293T cells had been preserved in the research center. The lentivirus-negative control (NC) and pGLV-H1-green fluorescent protein (GFP)+Puro vector were purchased from Zimmer Medical International Trading Co., Ltd. (Shanghai, China). The restriction enzymes, BamHI and EcoRI, were obtained from MBI Fermentas (Amherst, NY, USA). The transfection reagent, Lipofectamine 2000, as well as TRIzol reagent and Dulbecco’s modified Eagle’s medium/F12 culture medium were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The Taq enzyme and plasmid extraction kit were obtained from Takara Bio, Inc. (Shiga, Japan), while the uPA antibody was purchased from Abcam (Cambridge, MA, USA). All animals were maintained in the Animal Facility of the Shihezi University School of Medicine. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Shihezi University, Shihezi, China.

Primers for uPA and the β-actin reference were designed using Oligo 6.0 software (Molecular Biology Insights, Inc. Cascade, CO, USA), which was conducted by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The sequences of the primers were as follows: uPA upstream, 5′-ACTACATTG TCTACCTGGGTCGGTC-3′ and downstream, 5′-ATGCAA GATGAGTTGCTCCACTTC-3′ (amplification length, 86 bp); β-actin upstream, 5′-CAGGTCATCACCATCGGCAAC-3′ and downstream, 5′-GGATGTCCACGTCGCACTTCA-3′ (amplification length, 133 bp).

Cartilage tissue was obtained from the knee-joint of a two-year-old New Zealand rabbit. The cartilage cells were collected from cartilage tissue by repeated digestion for four times, then cultured at flask with culture medium at 37°C in a 5% CO₂ humidified incubator. The medium was changed every two days. Cells were observed under an inverted microscope and images and observations were recorded with regard to the cellular morphology and adherence conditions. Further experiments were conducted when the cells are 85–90% in a confluent state for later use.

Using GenBank, the uPA gene sequence in rabbits was identified (NM-001082011). Referring to the design principles of the targeted point of siRNA, four targeted points were selected and designed using GenePharma siRNA designer v 3.0 software (Shanghai GenePharma, Shanghai, China), which were P1, P2, P3 and P4 (Table I). A pervasive disturbing sequence was also designed as the NC. The structure of the small hairpin RNA (shRNA) chain was positive-sense strand-loop-antisense strand. Each group of sequences were designed and synthesized according to the hairpin structural model. Restriction enzyme digestion was conducted at the two ends of the molecules, thus, the specific gene sequence was inserted into the vector directly following digestion (Table II). The sequencing fragments were synthesized by Zimmer Medical International Trading Co., Ltd (Shanghai, China).

As shown in Table II, following the dilution of the oligonucleotide fragments, double stranded DNA fragments were formed in the annealing reaction system. The pGLV-HI-GFP+Puro vector was linearized by BamHI and EcoRI restriction enzyme digestion (Fig. 1). Pure linearized vector fragments, double stranded DNA fragments and vector fragments were collected and combined together during a 12-h reaction. The recombinant vector loop was then transformed into the freshly prepared E. coli competent cells. Following E. coli cell culture for 16 h at 37°C, bacterial colonies were selected randomly as polymerase chain reaction (PCR) templates. Verification of the positive clones was conducted using PCR technology. The sequencing fragments were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.

Using uPA genes, RNAi lentiviral vectors and pMD2.G plasmids were tranfected together in 293T cells. After 8 h, the culture medium was changed to complete medium. The supernatant was concentrated and collected after culturing for 48 h. The virus titer of the 293T cells was determined using the dilution gradient method and calculated as follows: Virus titer (TU/ml) = (counted fluorescent cells/corresponding dilution times)/0.01. The transfected cells were stored in a −80°C refrigerator for later use.

Packaged recombinant lentivirons were classified into three groups based on multiplicity of infection (MOI) values (1, 10 or 100), and were transfected into the cartilage cells separately. The culture medium was changed to complete medium at 6 h following transfection. Cells were cultured for 3 days, following which the expression level of GFP in the cells was observed using an inverted fluorescence microscope. The transfection rate was also analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Total RNA was extracted at day 4 following transfection using TRIzol reagent. Reverse transcription of the RNA to cDNA was conducted using the RNA as a template, and then PCR-amplification of the uPA gene was performed using the cDNA as a template. The primers were designed by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. The qPCR conditions were as follows: Primary degeneration for 15 sec at 95°C, degeneration for 5 sec at 95°C and annealing for 30 sec at 60°C, which was repeated 40 times. Light absorption values were assayed at each extension stage. Following PCR, degeneration of the PCR product was conducted for 1 min at 95°C, which was then cooled-down to 55°C in order for the DNA double strands to combine together fully. A melting curve was constructed using the light absorption values.

Total protein was extracted at day 4 following transfection and gel-electrophoresis was conducted using 10% SDS-PAGE following protein quantification. Next, the proteins were transferred to a nitrocellulose membrane and then incubated with primary antibodies including PLAU (1:5,000 dilute, GeneTex Inc., Irvine, CA, USA) and β-actin (1:10,000 dilute. Abcam Inc., Shanghai, China) overnight at 4°C followed with anti-rabbit second antibodies (1:10,000 dilute, GeneTex Inc.) at room-temperature for one hour. Enhanced chemiluminescence was used for detection. β-actin was used as the internal reference.

All the results are expressed as the mean ± standard deviation. Data were analyzed by single factor analysis of variance and the t-test using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The size of the PCR amplification product, which was from the positive clone of the inserted shRNA sequence, was 384 bp according to the primer size. By contrast, the size of the PCR amplification product for the empty vector clone was 322 bp (Fig. 2). These results demonstrated that all five random clones were positive clones. The sequencing results of the shRNS carrier vector revealed the expected sequence, indicating that the shRNA vector was constructed successfully. DNA sequencing further demonstrated that the sequence was correct and showed no mutations.

293T cells were transfected using the successfully constructed uPA RNAi lentiviral vectors and pMD2.G coated plasmids. Significant expression of GFP was observed under the fluorescence microscope after 40 h (Fig. 3), and the biological titer for the collected and concentrated virus was 1×10⁸ TU/ml, as determined by the hole dilution method.

Transfection rates of the four lentiviral vectors in 293T cells and cartilage cells were observed under the various MOI values. The results demonstrated that with increasing MOI, the lentivirus transfection rate also improved. When the MOI was 100, the transfection rate was >85% (Fig. 4), which was which was used for the following experiments.

Relative qPCR was performed with β-actin as the internal reference and the results were analyzed using the standard curve method. The results indicated that the mRNA expression level of uPA decreased significantly following transfection of P1, P2, P3 or P4 plasmids into the cartilage cells (Fig. 5). In addition, the corresponding rates for the empty plasmid and NC increased, further demonstrating that these four plasmids exhibited a significant inhibiting influence on uPA (Fig. 6). Furthermore, the knockout effectiveness for the P2 RNAi plasmid was better compared with the other three plasmids. This observation was confirmed by western blot analysis. The four plasmids all inhibited the protein expression of uPA, however, the knockout effectiveness for P2 and P3 RNAi plasmids was better compared with the P1 and P4 RNAi plasmids (Fig. 7; P<0.05). Therefore, the P2 plasmid may have the most efficient inhibiting effect on uPA.