Fetal bovine serum (FBS) and RPMI 1640 were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Trypsin was obtained from HyClone (GE Healthcare, Logan, UT, USA). RNase and propidium iodide (PI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). pGEM-T vector was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). The lentiviral expression vector pLenti, and lentivirus packaging plasmids pLP1 and pLP/VSVG were a generous gift from by Dr Huijun Zhi (Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, National Institutes of Health, Bethesda, MA, USA). pCMV-SPORT6-ITGα5 and pCMV-SPORT6-ITGβ1 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA). The plasmid pRNAT-U6.2/Lenti was obtained from GenScript (Piscataway, NJ, USA). Restriction enzymes BamHI, XhoI, KpnI and MluI were purchased from Promega Corp. (Madison, WI, USA). Lipofectamine^®2000 was purchased from Invitrogen (Thermo Fisher Scientific). ITGα5 mouse monoclonal antibody (cat. no. SC-166681), ITGβ1 monoclonal antibody (cat. no. SC-71386), GAPDH monoclonal antibody (cat. no. SC-137179) and horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (cat no. SC-2005) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All antibodies were diluted at a 1:300 ratio and all incubations were performed for 1 h at room temperature. ITGα5 and ITGβ1 reverse-transcription polymerase chain reaction (RT-PCR) kits and primers were purchased from Qiagen China (Beijing, China). The other reagents used in the present study were of analytical grade.

The 293FT cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were maintained in RPMI 1640 supplemented with 10% FBS. Human aortic VSMCs (no. PCS-100-021) were purchased from the American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI 1640 supplemented with 10% FBS and antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin; all from Sigma-Aldrich) at 37°C in a 5% CO₂ atmosphere.

The entire cDNA sequences of ITGα5 and ITGβ1 were amplified by PCR of pCMV-SPORT6-ITGα5 and pCMV-SPORT6-ITGβ1. The upstream and downstream primers both contained KpnI and MluI endonuclease sites. The cDNA was then ligated with the pGEM-T vector. The ligation products were transfected into Escherichia coli DH5α cells (Beijing Hua Yueyang Biotechnology Co. Ltd., Beijing, China), which were maintained at 37°C. The positive recombinant clones pGEM-T-ITGα5 and pGEM-T-ITGβ1 were then selected and maintained in LB medium for 4 h at 37°C. Subsequently, 1 µl bacterial medium was used as a template and PCR was performed using the EconoTaq PLUS 2X Master Mix (Lucigen, Madison, WI, USA) with T7 (5′-TAATACGACTCACTATAGGGAGA-3′) and SP6 (5′-CATACGATTTAGGTGACACTATAG-3′) primers according to manufacturer's instructions. The positive clones were subjected to DNA sequencing by Shanghai Sengong Biotech (Shanghai, China). The cloning vector and the lentivirus were cut using KpnI and MluI restriction endonucleases, following which they were ligated and transfected. Enzyme analysis and gene sequencing analysis were used to verify the accuracy of the recombinant vectors pLenti-ITGα5 and pLenti-ITGβ1.

According to the nucleotide sequence of the ITGα5 and ITGβ1 genes in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and the principles of siRNA design, two segment sequences were selected from each: 735–753 nt and 970–988 nt, and 600–618 nt and 1,283–1,301 nt, respectively. The effective siRNA sequences targeting ITGα5 and ITGβ1 were then designed using the siRNA Designer web tool from Promega Corp. and synthesized by Sengong Biotech. The full list of siRNAs and their sequences used in the present study is shown in Table I. Both ends of the hairpin target sequences contained BamHI and XhoI endonuclease sites. The cDNA containing both the sense and antisense strands of the targeting sequence was designed, synthesized and cloned into the pRNAT-U6.2/Lenti vector, which contained the H1 promoter and green fluorescent protein (GFP). The resulting lentiviral vectors containing ITGα5 or ITGβ1 siRNA were named pRNAT-U6.2/Lenti-si ITGα5-1, pRNAT-U6.2/Lenti-siITGα5-2, pRNAT-U6.2/Lenti-siITGβ1-1 and pRNAT-U6.2/Lenti-siITGβ1-2. Restriction endonuclease digestion and DNA sequencing were conducted to confirm the generation of the recombinant vectors. PCR and gene sequencing analysis were used to verify the accuracy of the recombinant vectors.

Lentivirus packaging plasmid mixtures containing Lentivirus-ITGα5 or Lentivirus-ITGβ1, or pRNAT-U6.2/Lenti-siITGα5-1, pRNAT-U6.2/Lenti-siITGα5-2, pRNAT-U6.2/Lenti-siITGβ1-1 or pRNAT-U6.2/Lenti-siITGβ1-2 were co-transfected into the 293FT cells. All transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The titer of the viral stock solutions was assessed via quantification of the expression levels of GFP as previously described (8).

Recombinant lentiviruses were trans-fected into the VSMCs in order to establish the following cell lines with upregulated and downregulated ITGα5 and ITGβ1 gene expression: Lentivirus-ITGα5, Lentivirus-ITGβ1, pRNAT-U6.2/Lenti-siITGα5-1, pRNAT-U6.2/Lenti-siITGα5-2, pRNAT-U6.2/Lenti-siITGβ1-1, pRNAT-U6.2/Lenti-siITGβ1-2, pLentiGFP empty vector and pRNAT-U6.2/Lenti empty. Lipofectamine 2000 (Invitrogen) was used for all transfections according to the manufacturer's instructions. Screening with G418 (Sigma-Aldrich) was used to obtain stably transfected VSMCs. The transfected VMSC cell lines were named: ITGα5-overexpressing cell line (EX-ITGα5), ITGβ1-overexpressing cell line (EX-ITGβ1), ITGα5-knockdown cell line (si-ITGα5), ITGβ1-knockdown cell line (si-ITGβ1), pLentiGFP empty vector-transfected cell line (Con-Ex) and pRNAT-U6.2/Lenti empty vector-transfected cell line (Con-si), respectively. Post-transfection, lentivirus-ITGα5 was trans-fected into the EX-ITGβ1 cell line in order to generate a cell line overexpressing both ITGα5 and ITGβ1 - this cell line was named D-EX. In addition, pRNAT-U6.2/Lenti-siITGα5-1 was transfected into the si-ITGβ1-2 cells in order to generate a cell line exhibiting both ITGα5 and ITGβ1 knockdown - this cell line was named D-si. Quantitative (q-PCR) and western blotting were used to detect the changes to ITGα5 and ITGβ1 gene and protein expression levels in all of the stably transfected cell lines (4,9). Cell growth was observed using microscopy (Inverted microscope IX83; Olympus, Japan).

All experiments were conducted using the nine cell lines: EX-ITGα5, EX-ITGβ1, D-EX, si-ITGα5, si-ITGβ1, D-si, Con-Ex, Con-si and normal VSMCs. The nine cell lines were divided into three groups: Overexpressing groups, including EX-ITGα5, EX-ITGβ1 and D-EX; knockdown groups, including si-ITGα5, si-ITGβ1 and D-si; and control groups, including Con-Ex, Con-si and normal VSMCs.

All of the cell lines were cultured in 96-well microtiter plates (Corning, Inc., Corning, NY, USA) at 1×10⁴ cells/well and incubated for five days at 37°C. Subsequently, 20 µl MTT (5 mg/ml; Jiemei Gene Pharmaceutical, Shanghai, China) was added to each well and incubated for 4 h at 37°C. Following removal of the supernatant, 150 µl dimethyl sulfoxide (Jiemei Gene Pharmaceutical) was added to each well, and the absorbance values were measured using a microplate reader (iMark™ Microplate Absorbance reader; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 490 nm (9).

The migration assay was performed using a Transwell system, which allows cells to migrate through an 8-µm pore polycarbonate membrane in a millicell (10,11). Briefly, the cells were serum-starved for 24 h and then plated (1×10⁴ cells/well) in serum-free medium (Gibco) in the upper chamber of a 24-well Transwell plate (Corning). The lower chamber was filled with 1.5 ml medium supplemented with 500 µl chemotactic factor (Jiemei Gene Pharmaceutical). After 48 h, the cells that remained on the upper surface of the membrane were removed using a cotton swab, and the cells on the lower surface of the membrane were fixed with cold methanol for 15 min and stained with 0.2% crystal violet (Jiemei Gene Pharmaceutical). The cells that had migrated to the bottom of the membrane were visualized and counted using an inverted microscope. For each repetition, cells in four randomly selected fields were counted and averaged. Results were expressed as a ratio of the untreated group.

The cells were cultured in six-well culture plates for 24 h and incubated for a further 24 h in serum-free medium. The cells were then incubated in medium containing serum for another 48 h. The cell cycle was assessed according to the intensity of PI staining, as described previously (12). Briefly, ethanol-fixed cells were centrifuged and washed twice with phosphate-buffered saline. Subsequently, cells were treated with 100 µl RNase (Promega) for 30 min at 37°C, followed by the addition of 100 µl propidium iodide dye (1 µg/m;l Sigma-Aldrich) and incubation for 30 min in the dark. Cell cycle analysis was performed using a flow Cytometer (FACS Aria II; BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using Modfit LT software, version 3.2 (BD Biosciences).

Total RNA was isolated using a Total RNA Extraction kit (Qiagen, Hilden, Germany). Total RNA was then reverse-transcribed in a 20-µl reaction solution containing Revert AID™ First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific). The subsequent RT-qPCR, used to determine the expression levels of ILK and FAK, was conducted as described previously (13,14). The sequences of the individual pairs of ILK, FAK, ITGα5, ITGβ1 and GAPDH primers (Qiagen) are presented in Table II. The PCR reactions were performed using the ABI StepOne Plus PCR system (Applied Biosystems; Thermo Fisher Scientific) in 96-well reaction plates. The thermocycling conditions were as follows: 95°C for 10 min followed by 95°C for 10 sec and 60°C 60 sec for 40 cycles. Gene expression was quantified by 2^−ΔΔCT method (15). Relative mRNA levels for each gene were normalized, and values are expressed as the ratio of target gene mRNA to GAPDH expression.

Values are expressed as the mean ± standard deviation. Statistical analyses of differences between the groups were performed using Student's t-test and analysis of variance. SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. P<0.05 was considered to indicate a statistically significant difference.

The results of PCR, enzyme analysis and DNA sequencing analysis confirmed that the ITGα5 and ITGβ1 genes had been successfully cloned (full length, 3,150+176 bp and 2,397+176 bp, respectively), and the pGEM-T-ITGα5 and pGEM-T-ITGβ1 recombinant clones were confirmed by gene sequencing analysis. Enzyme analysis and gene sequencing indicated that the lentiviral expression vectors Lentivirus-ITGα5 and -ITGβ1 had been successfully constructed. PCR and DNA sequencing demonstrated that the lentivirus RNAi vectors psiRNA-ITGα5 and -ITGβ1 were also successfully constructed (Fig. 1). The resulting lentiviral vectors were named pRNAT-U6.2/Lenti-siITGα5 and pRNAT-U6.2/Lenti-siITGβ1. The viral titers were all >7.7×10⁵ IU/ml, and were determined by detecting the expression levels of GFP.

As shown in Fig. 2A, the mRNA expression levels of ITGα5 and ITGβ1 were measured by RT-qPCR. The expression levels in the control groups were as follows: Con-si, ITGα5 mRNA (0.252±0.026) and ITGβ1 mRNA (0.516±0.056); Con-Ex, ITGα5 mRNA (0.251±0.021) and ITGβ1 mRNA (0.505±0.062); and Con, ITGα5 (0.238±0.021) and ITGβ1 (0.471±0.051). There were no significant differences between the expression levels in the control groups (P>0.05). The mRNA expression levels of ITGα5 in the EX-ITGα5 and D-EX cells were significantly increased (P<0.05), and were significantly decreased in the si-ITGα5-1 and D-si cells (P<0.05), as compared with those in the control groups. In addition, the mRNA expression levels of ITGβ1 in the EX-ITGβ1 and D-EX cells were significantly increased (P<0.05), while being significantly decreased in the si-ITGβ1-1 and D-si cells (P<0.05). The same trend was observed with regards to the protein expression levels of ITGα5 and ITGβ1, as detected by western blot analysis (Fig. 2B). As observed under an inverted microscope, the EX-ITGα5, EX-ITGβ1 and D-EX cells grew faster, as compared with the control cells, which exhibited better cell morphology and were spindle-shaped. The cells of the si-ITGα5, si-ITGβ1 and D-si groups had a similar morphology to that of the control cells; however, their growth rate was slightly slower as compared with that of the control cells (Fig. 3).

Fig. 4 shows the proliferative activity of the cells. There were no significant differences between the control groups, and on the second day there were no significant differences in cell proliferation between any of the groups. From the third day, the absorbance values of MTT were significantly increased in the EX-ITGβ1 and D-EX cells (P<0.05), and were significantly decreased in the si-ITGβ1 and D-Si cells (P<0.05), as compared with those in the control group; however, there was no significant difference between the EX-ITGα5 and si-ITGα5 cells (P>0.05). These results suggested that upregulation of ITGβ1 expression is able to induce cell proliferation, and downregulation of ITGβ1 expression may hinder cell proliferation. By contrast, ITGα5 expression had no effect on proliferation of the cells.

A Transwell chamber migration assay was used to measure the number of cells that passed through an artificial basement membrane (Fig. 5). The numbers of migrated cells in the control groups were as follows: Con-Ex, 32.9±4.4; Con-si, 36.6±4.1; and Con, 35.2±4.7, and there was no statistical difference between them. There was no difference between the number of migrated cells in the EX-ITGα5 (41.5±5.6) and si-ITGα5 (29.3±3.9) groups, as compared with that in the control groups (P>0.05); however, the number of migrated cells in the EX-ITGβ1 (62.3±6.8) and D-EX (65.7±7.2) groups was markedly increased (P<0.05). In addition, the number of migrated cells in the si-ITGβ1 (16.2±2.1) and D-si (14.8±1.7) groups was significantly decreased (P<0.05). These results indicated that ITGβ1 may affect the migratory ability of VSMCs. However, ITGα5 had no effect on VSMC cell migration.

The results of the flow cytometric cell cycle analysis showed that, among the three control groups, the proportion of cells in G₀/G₁, G₂/M and S phase were similar (P>0.05; Fig. 6). There was a higher proportion of cells in G₀/G₁ phase, as compared with that in S phase and G₂/M phase. In the EX-ITGα5, EX-ITGβ1 and D-EX groups, the number of cells in G₀/G₁ phase was lower, whereas the number of cells in S phase was increased compared with that in the control groups. In addition, there was a higher proportion of cells in G₀/G₁ phase in the si-ITGα5, si-ITGβ1 and D-si groups, and a lower proportion of cells in S and G₂/M phase as compared with that in the control groups. These results suggested that upregulation of ITGβ1 expression may be able to induce cell division.

The expression levels of ILK in the control groups were as follows: Con-Ex (0.142±0.011), Con-si (0.129±0.012) and Con (0.137±0.012), and there were no differences detected between them (P>0.05). The mRNA expression levels of FAK in the EX-ITGβ1 (0.357±0.0194) and D-EX cells (0.419±0.033) were significantly increased as compared with those in the control groups (P<0.01; Fig. 7). The mRNA expression levels of FAK in the si-ITGβ1 (0.054±0.008) and D-si (0.034±0.004) cells were decreased compared with those in the control (P<0.01). There were no marked changes in ILK mRNA expression levels between the groups. These results indicated that ITGβ-1 may be a major regulator of FAK mRNA expression.