The Effect Of Two-Way Gene Regulation Of Integrin α5β1 Expression On Human Aortic Vascular Smooth Muscle Cell Proliferation And Migration

Autogenous vein graft and transluminal angioplasty are the main effective treatment for vascular occlusive disease, but early restenosis after vascular treatment is a major problem troubling the effection. Vascular smooth muscle cells (VSMC) abnormal proliferation and migration, is the main reason leading to early vascular restenosis. Integrins are a family of glycoprotein receptor on cell surface and participate in cell adhesion with extracellular matrix (ECM), playing an important role in regulating cell growth, differentiation and proliferation. Integrinα5β1 is the main receptor of fibronectin (FN), linking the cell cytoskeleton by binding with ligand, which can cause the movement of VSMC, and also involves in vascular smooth muscle cell migration, proliferation and vascular injury repairing process. But the specific relationship between integrinα5β1 with proliferation and migration of VSMC, is still not clear. When integrinα5β1 is overexpressed by transgenic or induced gene silencing in VSMC, there has not been reported about whether it can induce VSMC proliferation and migration or not.ObjectiveTo explore the two-way control effects on proliferation and migration of VSMC, and investigate the changes of signaling pathway focal adhesion kinase (FAK) and integrin linked kinase (ILK), we constructed the lentiviral expression vector of integrinα5β1 and lentiviral vector of RNA silence interference (siRNA) of integrinα5β1, and induced integrinα5β1 overexpression and gene silencing in VSMC respectively. It would help us to understand the mechanisms of Integrinα5β1 regulating VSMC proliferation and migration, and provide experimental and theoretical evidence for precaution method for vascular early restenosis, as well as guide the clinical work.Methods1. Constructing the lentiviral expression vector of integrinα5,β1 and lentiviral vector of RNA silence interference(siRNA) of integrinα5,β11) Subcloning recombinant vector:The entire integrinα5 andβ1 cDNA were amplified by PCR from cDNA pCMV-SPORT6-ITGα5 and cDNA of pCMV-SPORT6-ITGβ1, which the upstream and downstream primers have KpnI and MluI. endonuclease sites, and then ligated with pGEM-T vector. The ligation products were transformed into the E.Coli DH5a cells. The positive recombinant clones pGEM-T-ITGα5, pGEM-T-ITGβ1 were selected and identified byα-complementation, PCR, restriction endonuclease digestion and DNA sequencing. The cloning vector and the Lentivirus were cut by KpnI and MluI. Then they were ligated and transformed. The enzyme analysis and gene sequencing analysis were used to verify the accuracy of recombinant vector pLent-ITGα5 and pLent-ITGβ1.2) Recombinant vector:According to the nucleotide sequence of the integrinα5 and integrinβ1 gene in Genbank and the principles of siRNA design, each 2 segment sequences was chosen:735-753nt,970-988nt and 600-618nt,1283-1301nt. The effective sequence of siRNA targeting integrinα5 andβ1 were designed. Both ends of hairpin target sequences with BamHI and XhoI endonuclease sites. The complementary DNA containing both sense and antisense of the targeting sequence was designed synthesized and cloned into the pRNAT-U6.2/Lenti vector which contained H1 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing integrinα5 orβ1 shRNA 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 to confirm the recombinant vector. PCR and gene sequencing analysis was used to verify the accuracy of recombinant vector.3) Lentiviruls packaging:Lentivirus packaging plasmids mixtures with Lentivirus-ITGα5 or Lentivirus-ITGβ1 or pRNAT-U6.2/Lenti-siITGα5-1 or pRNAT-U6.2/Lenti-siITGa5-2 or pRNAT-U6.2/Lenti-siITGβ1-1 or pRNAT-U6.2/ Lenti-silTGβ1-2 cotransfected 293FT cells. All virus stocks were produced by transfection Reagent Lipofectmaine 2000.The titer of virus was tested according to the expression level of GFP.2. Transfecting VSMC cells:Recombinate Lentivirus transfected into VSMC, ITGa5 and ITGβ1 gene up-regulated line and down-regulated line were established:Lentivirus-ITGa5,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 and pLentiGFP empty vector,pRNAT-U6.2/Leni empty vector were transfected into different VSMC, G418 screening method was used to obtain the stable transfection VSMC cells. They were named ITGα5 up-regulated line (EX-ITGα5), ITGβ1 up-regulated line (EX-ITGβ1); ITGα5 down-regulated linel (si-ITGα5-1), ITGα5 down-regulated line2 (si-ITGα5-2), ITGβ1 down-regulated linel (si-ITGβ1-1), ITGβ1 down-regulated line2 (si-ITGβ1-2), pLentiGFP empty vector line (Con-Ex), and pRNAT-U6.2/Leni empty vector (Con-si), accordingly. After stable transfection, Lentivirus-ITGα5 was transfected into EX-ITGβ1 line, got the ITGα5 and ITGβ1 gene all up-regulated line, named D-EX. pRNAT-U6.2/Lenti-siITGα5-1 was transfected into si-ITGβ1-2, got the ITGα5 and ITGβ1 gene all down-regulated line, named D-si; Real time PCR and Western blot were used to detect the changes of integrinα5 andβ1 gene and protein in all the stable transfection cells. Observe cell growth by microscope.3. Real time PCR was used to detect the changes of FAK and ILK gene in the stable transfection cells.4. The changes of invasion and migration abilities were measured by Transwell chamber invasion assay in the stable transfection cells.5. MTT assay was used to detect the proliferative activity in the stable transfection cells. 6. The changes of cell cycle were detected by flow cytometry assay in the stable transfection cells.Results1. Constructing vector:1).The results of PCR, enzyme analysis and DNA sequencing analysis have confirmed the right ITGα5 and ITGβ1 gene were cloned(full length 3150bp+176bp and 2397bp+176bp), recombinant clones pGEM-T-ITGα5,pGEM-T-ITGβ1 confirmed by gene sequencing analysis. Enzyme analysis and gene sequencing, lentiviral expression vector Lentivirus-ITGα5 and ITGβ1 were successfully constructed.2). PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of integrinα5 andβ1 producing psiRNA-integrinα5 andβ1 were constructed successfully. They were named pRNAT-U6.2/Lenti-siITGα5-1,pRNAT-U6.2/ Lenti-siITGα5-2,pRNAT-U6.2/Lenti-siITGβ1-1 and pRNAT-U6.2/Lenti-siITGβ1-2;3). The titer of all the virus were above 7.7×105IU/mL according to testing the expression level of GFP.2. The established cell lines:Integrinα5 andβ1 mRNA expression level were measured by Real-time fluorescence quantitative PCR, the level of the control groups were as follows: Con-si (ITG a 5 mRNA 0.252±0.026, ITGβ1 mRNA 0.516±0.056),Con-Ex (ITGα5 mRNA 0.251±0.021, ITGβ1 mRNA 0.505±0.062) and Con(ITGα5 0.238±0.021, ITGβ1 0.471±0.051), there were no differences between them (P>0.05);Compared with the above control groups, the ITGα5 mRNA of EX-ITGα5 (0.632±0.102,0.534±0.061) and D-EX (0.036±0.005,0.124±0.017) were significantly increased (P<0.05); and those of si-ITGα5-1 (0.033±0.004, 0.459±0.038) and D-si (0.036±0.005,0.124±0.017) were significantly lower (P <0.05);Compared with the control groups, the ITGβ1 mRNA of EX-ITGβ1 (0.271±0.031,1.172±0.11) and D-EX (0.036±0.005,0.124±0.017) were significantly increased (P<0.05), those of si-ITGβ1-1 (0.033±0.004,0.459±0.038), and D-si were significantly decreased (P<0.05);The same trend occurred in the changes of protein ITGα5 and ITGβ1 detected by Western blot.Observed under inverted microscope, cells of EX-ITGα5,EX-ITGβ1 and D-E growthed faster than the control groups cells,which having better cell morphology and spindle shape; cells of si-ITGα5,si-ITGβ1 and D-si were consistent with the control groups cells, but the growth rate was slightly slower than the control group cells, and spindle cell morphology was less than cells of EX-ITGα5, EX-ITGβ1 and D-EX, but the floating shrinking cells were more.3. FAK mRNA expression level was measured by Real-time fluorescence quantitative PCR, the level of the control groups were as follows:Con-Ex (0.142±0.011),Con-si (0.129±0.012) and Con (0.137±0.012),there were no difference between them (P>0.05). Compared with the control groups, the FAK mRNA expression level of EX-ITGα5 (0.165±0.014), those of EX-ITGβ1 (0.357±0.0194) and D-EX (0.419±0.033) were higher, the difference were significant (P<0.05); but those of EX-ITGβ1 (0.357±0.0194) and D-EX (0.419±0.033)were markedly higher (P<0.01). Compared with the control groups, the FAK mRNA expression level of si-ITGα5 (0.111±0.009),si-ITGβ1 (0.054±0.008) and D-si (0.034±0.004) were gone down (P<0.05), but si-ITGβ1 (0.054±0.008) and D-si (0.034±0.004) were significantly lower (P<0.01);ILK mRNA expression level measured by Real-time fluorescence quantitative PCR, the level of the control groups were as follows:Con-Ex (0.211±0.019),Con-si (0.194±0.017) and Con (0.203±0.016),there were no difference between them (P >0.05). Compared with the control groups, the ILK mRNA expression level of EX-ITGα5 (0.216±0.021) and si-ITGα5 (0.191±0.018) had no differences (P>0.05); the ILK mRNA expression level of EX-ITGβ1 (0.247±0.024) and D-EX (0.256±0.023) were higher, the difference were significant (P<0.05); Compared with the control groups, the ILK mRNA expression level of si-ITGβ1 (0.159±0.015),D-si (0.153±0.014) were lower, the difference were significant (P<0.05)From those data, we could know, up-regulation expression of ITG a 5 have only a small increase role on FAK, but no effect on ILK; Up-regulation expression of ITGβ-1 had stronger role on FAK and ILK than that of ITG a 5.4. Transwell chamber invasion assay:the number of passed through the artificial basement membrane of the control groups were as follows:Con-Ex (32.9±4.4),Con-si (36.6±4.1) and Con (35.2±4.7),there were no difference between them (P<0.05); Compared with the control groups,the number of EX-ITGα5 (41.5±5.6) and si-ITGα5 (29.3±3.9) had no difference between them (P>0.05); the number of EX-ITGβ1 (62.3±6.8) and D-EX (65.7±7.2) were higher (P<0.05), the number of si-ITGβ1 (16.2±2.1) and D-si (14.8±1.7) were lower (P<0.05).From those data, we could know, there was no effect on VSMC cell invasion and metastasis by regulating ITG a 5 expression only.5. MTT assay showed the proliferative activity of the cells, there were no differences between the control groups (P>0.05); and the speed of cell proliferation among all the groups had no significant difference on the second day (P> 0.05); from the third day, the absorbance values A of MTT were significantly higher among EX-ITGβ1 and D-EX (P<0.05); and si-ITGβ1 and D-EX were significantly lower (P<0.05); but there had no significant difference among EX-ITGα5 and si-ITGα5 (P> 0.05). From those data, we could know, up-regulation ITGβ1 expression could induce cell proliferation, in the other, down-regulation ITGβ1 expression could hinder cell proliferation, regulating ITGα5 gene expression had no effect on cells growth.6. Flow cytometry results showed that, among the three control groups, the distribution of the proportion of cells in G0～G1,G2～M,and S phase were basically similar(P>0.05). G0～G1 phase were more than S phase, which were the general trend among the three control groups; Among EX-ITGα5,EX-ITGβ1 and D-EX, cells in G0～G1 phase were much lower, and G2～M phase were much higher (P<0.05); EX-ITGβ1 and D-EX had more S phase(P>0.05); there were more G0～G1 phase in si- ITGα5±si-ITGβ1 and D-si, and less G2～M phase(P>0.05); among si-ITGβ1 and D-si, cells in S phase were much lower(P>0.05); from those data, we could know, up-regulation ITGβ1 expression could induce cell division, on the other hand, down-regulation ITGβ1 expression could hinder cell proliferation.Conclusion1. Lentiviral expression vector Lentivirus-ITGα5 and ITGβ1 were successfully constructed. Lentivirus RNAi vector of integrinα5 andβ1 producing psiRNA-integrinα5 andβ1 were constructed successfully, also. ITGα5 and ITGβ1 up-regulation expression and down-regulation expression VSMC cell lines were established (Including separate high and low expression of ITGα5, ITGβ1 cell lines,all high and low expression of ITGα5, ITGβ1 cell lines). Form this, different functions of the ITGα5 and ITGβ1 subunits of the foundation could be explored deeply further.2. During integrin-mediated proliferation and migration of VSMC, focal adhesion kinase (FAK) and integrin linked kinase (ILK) were involved in the signaling pathway. In the process of signal transduction, the change of FAK was stronger than that of ILK. FAK may be the main structural basis for the signal conduction of regulating VSMC proliferation and migration.3. Up-regulation expression of ITGβ1, VSMC could be induced to re-enter the cell division cycle, and the ability of invasion, proliferation, and migration could be induced stronger. On the other hand, down-regulation expression of ITGβ1, VSMC could be hindered to re-enter the cell division cycle, and the ability of growth, proliferation, and migration could be induced inferior. There were no effect on the ability of growth, proliferation, and migration by only regulating expression of VSMC gene ITGα5.4. This indicates that integrinβ1 was really involved in the progress of proliferation and migration in VSMC. Down-regulation expression of integrinβ1 gene in VSMC by RNAi, the abnormal proliferation and migration of VSMC could be inhibited. This might provide a possible therapeutic target of prevention and treatment for early vascular restenosis.