| Partâ… Objective Construct a lentiviral vector of RNA interference for LEFTY-A gene and identify its knockout effectiveness in human renal proximal tubular epithelial cells.Methods Two RNAi sequences are designed for LEFTY-A gene (NM003240). We design and synthesize their DNA Oligo of siRNA. After annealing, double-strand DNA is formed. We connect DNA ligases of T4 bacteriophage and pGCSIL-GFP which is cut out by the enzymes of Age I and EcoRI together, and then was transferred into competent cells. Choose positive bacteria which are detected by PCR amplification for sequencing analysis. After analysis, we extract pGCSIL-GFP which is named as PgcSIL1, PgcSIL2 respectively from the right clone with right sequence, and extract pHelper 1.0, pHelper 2.0 simultaneously. These three plasmids are co-transferred into Cell 293T by Lipofectamine 2000 and are packed by lentivirus. The concentrated virus solution is measured of viral biology titer. HK-2 cells, which are grouped of KD1 and KD2 respectively, are infected by four kinds of LEFTY-A RNAi lentiviral vector. The cells in negative control group (NC group) are infected by lentivirus. Group CON means that the cells are not infected. With the help of RT-PCR, we test the knockout effectiveness of these two kinds of LEFTY-A RNAi lentiviral vectors under the condition of different MOI (5 or 10). At last, we choose the group with highest knockout effectiveness and packed with numerous lentivirus.Results These two kinds of lentiviral vectors with the final concentration of 1×108TU/ml and 1×108 TU/ml, Pgcsill and Pgcsil2, are composed after the package of Cell 293T. The results of RT-PCR show that the 2-△△Ct value of Group CON, NC, KD1, KD2 with the MOI of 5 is 0.929±0.063,1.011±0.143,0.011±0.001 and 0.035±0.009 respectively. Through T-test, we compare the Group KD1, KD2 with NC and CON. The results of T-test are:tKD1-NC= 12.16 (p＜0.01),tKD2-NC=11.83 (p<0.01);tKD1-CON= 25.25 (p<0.01),tKD2-CON= 24.33 (p＜0.01). While the comparison between Group CON and NC with the MOI of 5 has no statistical significance. The results show that the 2-△△Ct value of Group CON, NC, KD1, and KD2 with the MOI of 10 is 0.965±0.104,1.016±0.178,0.009±0.002 and 0.008±0.001 respectively. Through T-test, we compare the Group KD1, KD2 with NC and CON. The results of T-test are:tKD1-NC= 9.83 (p＜0.01),tKD2-NC= 9.83 (p<0.01); tKD1-CON= 15.99 (p<0.01),tKD2-CON= 16.02 (p＜0.01). While the comparison between Group CON and NC with the MOI of 10 has no statistical significance. The knockout effectiveness of Pgcsill is larger than 95%, while the knockout effectiveness of Pgcsil2 is larger than 90%. According to the results, we choose the siRNA of Pgcsil 1 to pack the lentivirus with the final concentration of 1×109TU/ml.Conclusion We successfully constructed the lentiviral vector, Pscsill, of RNA interference for LEFTY-A gene. Pscsill are able to express green fluorescent protein (GFP) under the drive of CMV promoter. The expression will contribute to the judgment of knockout effectiveness, set up cell strain with the stable LEFTY-A gene knockout effectiveness. So that, lentiviral vectors of LEFTY-A RNAi gene can knockout the expression of LEFTY-A effectively.Part IIObjective After the infection of lentiviral vectors of LEFTY-A RNAi gene into HK-2 cell, the expression of LEFTY-A RNAi gene in HK-2 is restrained. We discuss the EMT effect when the cells face the stimulation of TGF-β1.Methods The K.D-2 cells are grouped into 4 groups randomly:TGF-β1 intervention group (lOng/ml, T), lentivirus infection group with TGF-β1+Pgcsill (lOng/ml, T+P) lentivirus infection group with Pgcsill (Pgcsill transfection, P), and normal HK-2 cell line group. At the same time, we test the knockout effectiveness of LEFTY-A gene. All cells in the infective groups with Pgcsill are cultured in the DMEM-F12 with two-antibodies after 36 hours infection. And then the cells in the infective groups are intervened with TGF-β1 (T+P group), or stroke-physiological saline solution (P group). Collect the cells after 12,24,48 hours intervention Extract cell total protein and part of total-RNA which are conserved under the temperature of-70℃. We use Western-blot to test the expression level of Lefty-A, a-SMA, CTGF protein in HK-2 cell, and also use RT-PCR to test the expression level of Lefty-A,α-SMA, CTGF's mRNA in HK-2 cell. Test the expression of Col-I in supernatant liquid by ELISA.Results Lentiviral vector of LEFTY-A RNAi can reduce the expression of LEFTY-A mRNA and protein in HK-2 cells effectively. The results of two times RT-PCR both shows that the expression of mRNA in KD group is only 15% of which in the NC group. Experiment I:Comparing with NC group (1.00±0.04) and CON group (0.99±0.10), the expression of LEFTY-A mRNA in KD group (0.18±0.04) reduce significantly (tKD/NC=28.07, P<0.01; tKD/CON= 12.70, P<0.01), while the difference between NC and CON group has no statistical significance (tCON/NC= 0.17, P>0.05). Experiment II:Comparing with NC group (1.02±0.25) and CON group (0.91±0.14), the expression of LEFTY-AmRNA in KD group (0.11±0.04) reduce significantly (tKD/NC=28.07, P<0.01;tKD/CON= 12.70, P＜0.01), while the difference between NC and CON group has no statistical significance (tCON/NC=0.70, P>0.05). With the condition of same quality of loading protein, the LEFTY-A protein expression of KD group (0.33±0.13) is only 18%(t=11.63, P<0.01) of the expression in NC group (1.82±0.18). The comparison of protein expression between KD and NC (1.79±0.15) group also has statistical significance (t=12.72, P<0.01). But the comparison between CON and NC group has no statistical significance (7=0.19, P>0.05). After 12 hours stimulation of TGF-β1, there is almost no expresses of a-SMA protein in the normal HK-2 cells. In T group, expression of a-SMA and CTG in cytoplasm is up regulated, while the comparison of CTGF in different groups has no statistical significance (P>0.05). In T+P group, the expression of a-SMA and CTG in cytoplasm is up regulated and with statistical significance when compared with others (P<0.05). In P group, both the expression of two proteins has no difference. After 24 hours stimulation of TGF-β1, expression of a-SMA and CTG in cytoplasm in T group is up regulated continuously, while the up regulated range in T+P group is even larger. In P group, the expression has no difference when compared with normal cells. After 48 hours stimulation of TGF-β1, the expression of a-SMA (P<0.01) and CTGF (P＜0.01) in T+P group is obviously higher than which in the T group. When the expression in T+P group compared with which in the P and N group, the results still have significantly difference. The results of RT-PCR shows:the expression of mRNA is coordinate with the expression of protein. In all the three point-in-time, the gene expression both in P and N group has no variation. In T+P and P group, the expression of both groups is higher than P and control group, and is higher along with the time. In T+P group, the difference of the expression level of a-SMA, CTGF mRNA with T group is increased gradually along with the time. The result of ELISA shows:the expression of Col-I in supernatant liquid in P and N group has no differences along with the time. At any time point, the expression in T+P and T group is higher than P or N group. The most significantly difference is expressed between the comparison of T+P and T group. Along with the time extension of RNAi, the difference between T+P and T group is larger and larger.Conclusion The lentiviral vector of LEFTY-A gene has very good knockout effectiveness in HK-2 cells. After the expression interference of LEFTY-A gene, the reaction of HK-2 cell when facing the stimulation of TGF-β1 increase significantly. The speed of the expression of a-SMA and then produce EMT increases significantly. These results prove the key importance, from another aspect, of LEFTY-A during fibrosis. Through RNAi, this kind of compellent low-expression of LEFTY-A gene and protein enhance the reaction of HK-2 cells when facing the stimulation of TGF-β1 in vitro. Totally, these accelerate the process of EMT in cells and thus promote the fibrosis.
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