慢病毒介导的Lefty-A RNA干扰对TGF-β诱导HK-2细胞发生EMT转化的影响
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摘要
第一部分人Lefty-A基因RNAi漫病毒载体的构建及验证
目的构建人LEFTY-A基因RNA干扰(RNAi)序列的慢病毒载体并在工具细胞(人肾小管上皮细胞HK-2)中验证其对LEFTY-A基因的敲减效率。
方法针对人LEFTY-A基因(NM 003240)设计两条RNAi序列,设计并合成其siRNA的DNA Oligo,经退火形成双链DNA后,运用T4噬菌体DNA连接酶与经Age I和EcoRI双酶切后的pGCSIL-GFP连接,转化感受态细胞。使用载体多克隆位点两端的引物进行PCR扩增检测阳性菌落,挑选阳性克隆菌液进行测序分析。选择测序鉴定正确的克隆,抽提pGCSIL-GFP质粒(分别命名为PgcSIL1、PgcSIL2),同时抽提pHelper 1.0和pHelper 2.0质粒。用Lipofectamine 2000等三种质粒共转染293T细胞进行慢病毒包装,获得的病毒浓缩液采用逐孔稀释滴度测定法进行病毒生物学滴度测定。用构建的四种LEFTY-A RNAi慢病毒载体感染HK-2细胞分别设为KD1组和KD2组,阴性对照慢病毒感染的HK-2细胞设为NC组,CON为未感染病毒的细胞组。使用RT-PCR方法检验在不同的感染复数(MOI为5或10)下两种LEFTY-A RNAi慢病毒载体的干扰效率。选择敲减效率最高的siRNA序列进行慢病毒大包装。
结果经293T细胞包装后获得两种LEFTY-A RNAi的慢病毒载体,分别命名为Pgcsill和Pgcsil2,其各自的终浓度分别是1×108TU/ml和1×108 TU/ml。RT-PCR检测结果显示,低MOI值时(MOI=5), CON组、NC组、KD1组和KD2组的2-△△Ct值分别是0.929±0.063、1.011±0.143、0.011±0.001和0.035±0.009。各KD组和NC组比较,tKD1-NC＝12.16,tKD2-NC= 11.83,p值均小于0.01；各KD组和CON组比较,tKD1-CON= 25.25,tKD2-CON=24.33,p值也都小于0.01；而CON组与NC组比较,tcon-NC= 0.906,p值大于0.05,差异没有显著性。高MOI值时(MOI=10),CON组、NC组、KD1组和KD2组的2-△△Ct值分别是0.965±0.104、1.016±0.178、0.009±0.002和0.008±0.001。各KD组和NC组比较,tKD1-NC = 9.83, tKD2-NC=9.83,p值均小于0.01；各KD组和CON组比较,tKD1-CON=15.99, tKD2-CON=16.02,p值也都小于0.01；而CON组与NC组比较,tcon-NC=0.43,p值大于0.05,差异没有显著性。因此两个构建的慢病毒载体中Pgcsill在HK-2细胞上对LEFTY-A基因的表达都有非常显著的敲减效果,敲减效率均达到95%以上；Pgcsil2的敲减效率也达到90%以上。故选择Pgcsill的siRNA序列进行慢病毒大包装,包装后病毒的终浓度为1×109TU/ml。
结论本实验成功构建了LEFTY-A RNAi慢病毒载体Pscsill,能表达由CMV启动子驱动的荧光蛋白GFP,有助于帮助判断感染效率；可用来建立稳定表达LEFTY-A基因敲减的细胞株；在HK-2细胞中对LEFTY-A基因的表达有很高的敲减效率。因此该LEFTY-A RNAi慢病毒载体能够有效沉默细胞内LEFTY-A基因的表达,为进一步研究LEFTY-A基因对器官纤维化的作用奠定了基础。
第二部分LeftyA-RNAi增强TGF-β1的EMT诱导作用
目的利用LEFTY-A-RNAi慢病毒载体感染人肾小管上皮(HK-2)细胞,并探讨LEFTY-A基因表达受抑制后对其对TGF-β1刺激而发生EMT效应。
方法将实验HK-2细胞随机分成4组,TGF-β1干预组(lOng/ml, T), TGF-β1+ Pgcsill慢病毒感染组(10ng/ml,T+P), Pgcsill慢病毒感染组(Pgcsill transfection, P),单纯HK-2细胞培养组(Normal HK-2 cell line, N)。检测构建的病毒载体对该细胞LEFTY-A基因表达的敲减效率。所有Pgcsill感染组细胞均于感染36h后,换用DMEM-F12(含双抗)培养,同时给予TGF-β1 (T+P组)或同体积的无菌生理盐水(P组)进行干预。所有组分别于干预后12、24、48h收集细胞,部分提取细胞总蛋白,部分提取总RNA,-70℃保存。Western-blot法检测HK-2细胞中Lefty-A、a-SMA、CTGF蛋白的表达水平。RT-PCR法检测培养HK-2细胞中Lefty-A、a-SMA、CTGF mRNA的表达。ELISA法检测细胞上清液中Col-I的表达。
结果重组Lefty-A RNAi慢病毒载体可显著降低HK-2细胞Lefty-A mRNA和蛋白的表达。两次RT-PCR结果均显示,KD组的mRNA表达只有NC组的15%左右,实验
与NC组(1.00±0.04)和CON组(0.99±0.10)比较,KD组(0.18±0.04)Lefty-A mRNA的表达下降显著(tKD/NC=28.07, P<0.01;tKD/CON= 12.70, P<0.01),而NC组与CON组相比较差异无统计学意义(tCON/NC= 0.17, P>0.05);实验二：与NC组(1.02±0.25)和CON组(0.91±0.14)比较,KD组(0.11±0.04) Lefty-A mRNA的表达下降显著6.19, P<0.05;tKD/CON=9.57, P<0.01);而NC组与CON组相比较差异无统计学意义tCON/NC=0.70, P>0.05)。在各组蛋白上样量相同的条件下,KD组(0.33±0.13)Lefty-A蛋白表达只有NC组(1.82±0.18)的18%(t=11.63,P<0.01)；KD组蛋白表达与CON组(1.79±0.15)比较也下降显著(t=1 2.72,P<0.01)；而CON组与NC组比较,差异无显著性(t=0.19,P>0.05)。经TGF-β1刺激12h后,正常HK-2细胞中,a-SMA蛋白几乎没有表达；T组可见胞浆中a-SMA和CTGF表达上调,但CTGF表达量的比较中P值>0.05；T+P组细胞中a-SMA (P< 0.05)、CTGF (P< 0.05)蛋白的表达均明显升高；P组细胞两蛋白的表达与正常比较未见变化。到24h时,T组胞浆中a-SMA (P<0.05)与CTGF (P<0.05)蛋白表达继续上调,而T+P组的上调幅度更大,而P组蛋白表达与正常比较仍无明显变化。到48h时,T+P组胞浆中a-SMA (P<0.01)和CTGF (P<0.01)的表达量已远远高于T组；与P组或N组比较差异仍具有显著性意义。RT-PCR结果显示,a-SMA、CTGF mRNA的表达基本与各自蛋白表达的变化趋势
一致。P组或N组中两基因的表达在三个时间点均无明显变化。而T组和T+P组中两基因的表达均高于P组和正常对照。并随时间的延长,T+P组中a-SMA、CTGF mRNA的表达水平与T组的差异逐渐增大。ELISA检测结果显示,P组或N组HK-2细胞培养上清液中Col-I的含量基本在三个观察时间点均无明显变化。在个时间点T+P组和T组的测量值均显著高于P组或N组。而主要差异变化体现在T+P组和T组之间,随RNA干扰时间的延长,T+P组上清液中Col-I的水平与T组的差异逐渐增大。
结论构建的介导LEFTY RNAi的慢病毒载体在HK-2细胞中具有较好的干扰效果。而LEFTY-A基因表达被干扰后,HK-2细胞对TGF-β1刺激的反应显著增强,细胞表达a-SMA蛋白而发生EMT的速度明显增快。反证了Lefty-A在纤维化产生过程中的关键作用。所以,通过慢病毒载体介导的RNAi可在体外有效降低HK-2细胞LEFTY-A基因的表达,这种LEFTY-A基因和蛋白的强制低表达增强了HK-2细胞在体外对于TGF-β1刺激的反应,加快了细胞发生EMT的进程,促进了纤维化的发生。
PartⅠ
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 (NM_003240). 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 II
Objective 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.
目的构建人LEFTY-A基因RNA干扰(RNAi)序列的慢病毒载体并在工具细胞(人肾小管上皮细胞HK-2)中验证其对LEFTY-A基因的敲减效率。
方法针对人LEFTY-A基因(NM 003240)设计两条RNAi序列,设计并合成其siRNA的DNA Oligo,经退火形成双链DNA后,运用T4噬菌体DNA连接酶与经Age I和EcoRI双酶切后的pGCSIL-GFP连接,转化感受态细胞。使用载体多克隆位点两端的引物进行PCR扩增检测阳性菌落,挑选阳性克隆菌液进行测序分析。选择测序鉴定正确的克隆,抽提pGCSIL-GFP质粒(分别命名为PgcSIL1、PgcSIL2),同时抽提pHelper 1.0和pHelper 2.0质粒。用Lipofectamine 2000等三种质粒共转染293T细胞进行慢病毒包装,获得的病毒浓缩液采用逐孔稀释滴度测定法进行病毒生物学滴度测定。用构建的四种LEFTY-A RNAi慢病毒载体感染HK-2细胞分别设为KD1组和KD2组,阴性对照慢病毒感染的HK-2细胞设为NC组,CON为未感染病毒的细胞组。使用RT-PCR方法检验在不同的感染复数(MOI为5或10)下两种LEFTY-A RNAi慢病毒载体的干扰效率。选择敲减效率最高的siRNA序列进行慢病毒大包装。
结果经293T细胞包装后获得两种LEFTY-A RNAi的慢病毒载体,分别命名为Pgcsill和Pgcsil2,其各自的终浓度分别是1×108TU/ml和1×108 TU/ml。RT-PCR检测结果显示,低MOI值时(MOI=5), CON组、NC组、KD1组和KD2组的2-△△Ct值分别是0.929±0.063、1.011±0.143、0.011±0.001和0.035±0.009。各KD组和NC组比较,tKD1-NC＝12.16,tKD2-NC= 11.83,p值均小于0.01；各KD组和CON组比较,tKD1-CON= 25.25,tKD2-CON=24.33,p值也都小于0.01；而CON组与NC组比较,tcon-NC= 0.906,p值大于0.05,差异没有显著性。高MOI值时(MOI=10),CON组、NC组、KD1组和KD2组的2-△△Ct值分别是0.965±0.104、1.016±0.178、0.009±0.002和0.008±0.001。各KD组和NC组比较,tKD1-NC = 9.83, tKD2-NC=9.83,p值均小于0.01；各KD组和CON组比较,tKD1-CON=15.99, tKD2-CON=16.02,p值也都小于0.01；而CON组与NC组比较,tcon-NC=0.43,p值大于0.05,差异没有显著性。因此两个构建的慢病毒载体中Pgcsill在HK-2细胞上对LEFTY-A基因的表达都有非常显著的敲减效果,敲减效率均达到95%以上；Pgcsil2的敲减效率也达到90%以上。故选择Pgcsill的siRNA序列进行慢病毒大包装,包装后病毒的终浓度为1×109TU/ml。
结论本实验成功构建了LEFTY-A RNAi慢病毒载体Pscsill,能表达由CMV启动子驱动的荧光蛋白GFP,有助于帮助判断感染效率；可用来建立稳定表达LEFTY-A基因敲减的细胞株；在HK-2细胞中对LEFTY-A基因的表达有很高的敲减效率。因此该LEFTY-A RNAi慢病毒载体能够有效沉默细胞内LEFTY-A基因的表达,为进一步研究LEFTY-A基因对器官纤维化的作用奠定了基础。
第二部分LeftyA-RNAi增强TGF-β1的EMT诱导作用
目的利用LEFTY-A-RNAi慢病毒载体感染人肾小管上皮(HK-2)细胞,并探讨LEFTY-A基因表达受抑制后对其对TGF-β1刺激而发生EMT效应。
方法将实验HK-2细胞随机分成4组,TGF-β1干预组(lOng/ml, T), TGF-β1+ Pgcsill慢病毒感染组(10ng/ml,T+P), Pgcsill慢病毒感染组(Pgcsill transfection, P),单纯HK-2细胞培养组(Normal HK-2 cell line, N)。检测构建的病毒载体对该细胞LEFTY-A基因表达的敲减效率。所有Pgcsill感染组细胞均于感染36h后,换用DMEM-F12(含双抗)培养,同时给予TGF-β1 (T+P组)或同体积的无菌生理盐水(P组)进行干预。所有组分别于干预后12、24、48h收集细胞,部分提取细胞总蛋白,部分提取总RNA,-70℃保存。Western-blot法检测HK-2细胞中Lefty-A、a-SMA、CTGF蛋白的表达水平。RT-PCR法检测培养HK-2细胞中Lefty-A、a-SMA、CTGF mRNA的表达。ELISA法检测细胞上清液中Col-I的表达。
结果重组Lefty-A RNAi慢病毒载体可显著降低HK-2细胞Lefty-A mRNA和蛋白的表达。两次RT-PCR结果均显示,KD组的mRNA表达只有NC组的15%左右,实验
与NC组(1.00±0.04)和CON组(0.99±0.10)比较,KD组(0.18±0.04)Lefty-A mRNA的表达下降显著(tKD/NC=28.07, P<0.01;tKD/CON= 12.70, P<0.01),而NC组与CON组相比较差异无统计学意义(tCON/NC= 0.17, P>0.05);实验二：与NC组(1.02±0.25)和CON组(0.91±0.14)比较,KD组(0.11±0.04) Lefty-A mRNA的表达下降显著6.19, P<0.05;tKD/CON=9.57, P<0.01);而NC组与CON组相比较差异无统计学意义tCON/NC=0.70, P>0.05)。在各组蛋白上样量相同的条件下,KD组(0.33±0.13)Lefty-A蛋白表达只有NC组(1.82±0.18)的18%(t=11.63,P<0.01)；KD组蛋白表达与CON组(1.79±0.15)比较也下降显著(t=1 2.72,P<0.01)；而CON组与NC组比较,差异无显著性(t=0.19,P>0.05)。经TGF-β1刺激12h后,正常HK-2细胞中,a-SMA蛋白几乎没有表达；T组可见胞浆中a-SMA和CTGF表达上调,但CTGF表达量的比较中P值>0.05；T+P组细胞中a-SMA (P< 0.05)、CTGF (P< 0.05)蛋白的表达均明显升高；P组细胞两蛋白的表达与正常比较未见变化。到24h时,T组胞浆中a-SMA (P<0.05)与CTGF (P<0.05)蛋白表达继续上调,而T+P组的上调幅度更大,而P组蛋白表达与正常比较仍无明显变化。到48h时,T+P组胞浆中a-SMA (P<0.01)和CTGF (P<0.01)的表达量已远远高于T组；与P组或N组比较差异仍具有显著性意义。RT-PCR结果显示,a-SMA、CTGF mRNA的表达基本与各自蛋白表达的变化趋势
一致。P组或N组中两基因的表达在三个时间点均无明显变化。而T组和T+P组中两基因的表达均高于P组和正常对照。并随时间的延长,T+P组中a-SMA、CTGF mRNA的表达水平与T组的差异逐渐增大。ELISA检测结果显示,P组或N组HK-2细胞培养上清液中Col-I的含量基本在三个观察时间点均无明显变化。在个时间点T+P组和T组的测量值均显著高于P组或N组。而主要差异变化体现在T+P组和T组之间,随RNA干扰时间的延长,T+P组上清液中Col-I的水平与T组的差异逐渐增大。
结论构建的介导LEFTY RNAi的慢病毒载体在HK-2细胞中具有较好的干扰效果。而LEFTY-A基因表达被干扰后,HK-2细胞对TGF-β1刺激的反应显著增强,细胞表达a-SMA蛋白而发生EMT的速度明显增快。反证了Lefty-A在纤维化产生过程中的关键作用。所以,通过慢病毒载体介导的RNAi可在体外有效降低HK-2细胞LEFTY-A基因的表达,这种LEFTY-A基因和蛋白的强制低表达增强了HK-2细胞在体外对于TGF-β1刺激的反应,加快了细胞发生EMT的进程,促进了纤维化的发生。
PartⅠ
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 (NM_003240). 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 II
Objective 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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14. Wada T, Furuichi K, Sakai N, Iwata Y, Yoshimoto K, Shimizu M, Takeda SI, Takasawa K, Yoshimura M, Kida H, Kobayashi KI, Mukaida N, Naito T, Matsushima K, Yokoyama H (2000) Upregulation of monocyte chemoattractant protein-1 in tubulointerstitial lesions of human diabetic nephropathy. Kidney Int58:1492-1499
15. Okada H, Moriwaki K, Konishi K, Kobayashi T, Sugahara S, Nakamoto H, Saruta T, Suzuki H (2000) Tubular osteopontin expression in human glomerulonephritis and renal vasculitis. Am J Kidney Dis 36:498-506
16. Okada H, Moriwaki K, Kalluri R, Imai H, Ban S, Takahama M, Suzuki H (2000) Inhibition of monocyte chemoattractant protein-1 expression in tubular epithelium attenuates tubulo interstitial alteration in rat Good pasture syndrome. Kidney Int 57: 927-936
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24. D'Agord Schaan B, Lacchini S, Bertoluci MC, et al. Increased renal GLUT1 abundance and urinary TGF-beta 1 in streptozotocin-induced diabetic rats: implications for the development of nephropathy complicating diabetes. Horm Metab Res,2001,33:664-9.
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26. McGlinchey PG, Spence MS, Patterson CC, et al. The matrix metalloproteinase-3 (MMP-3) 5A/6A promoter polymorphism is not associated with ischaemic heart disease:analysis employing a family based approach. Dis Markers,2004,20: 289-94.
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1. Razzaque MS, Taguchi T (1999) Localization of HSP47 incisplatn-treated rat kidney:possible role in tubulointerstitial damage. Clin Exp Nephrol 3:222-228
2. Cheng M, Razzaque MS, Nazneen A, Taguchi T (1998) Expression of the heat shock protein 47 in gentamicin-treated rat kidneys. Int J Exp Pathol 79:125-132
3. Razzaque MS, Shimokawa I, Nazneen A, Higami Y, Taguchi T(1998) Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heats hock protein 47. Cell Tissue Res 293:471-478
4. Razzaque MS, Nazneen A, Shinagawa T, Taguchi T (2000) Upregulation of heat shock protein 47 in the hypertensive kidney:possible role in nephrovascular sclerotic process (abstract). Am J Hypertens 13:282
5. Liu D, Razzaque MS, Cheng M, Taguchi T (2002) The renalexpression of heat shock protein 47 and collagens in acute and chronic experimental diabetes in rats. Histochem J 33:623-630
6. Eddy AA (2000) Molecular basis of renal fibrosis. Pediatr Nephrol 15:290-230
7. Razzaque MS, Harada T, Taguchi T (1996) Distribution and cellular origin of type Ⅲ, type Ⅳ and type Ⅵ collagens in tubulointerstitial damage in various renal diseases. Clin Nephrol,46:213-215
8. Razzaque MS, Harada T, Taguchi T (1996) Significance of increased accumulation of type Ⅵ collagen and transforming growth factor beta 1 in tubulointerstitial damage in hypertensive nephrosclerosis:an immunohistochemical study. J Int Med Res24:199-208
9. Razzaque MS, Cheng M, Horita Y, Nishihara M, Harada T, Taguchi T (1995) Immunohistochemical analysis of type Ⅲ and Ⅳ collagens in tubulointerstitial damage in human benign nephrosclerosis. J Int Med Res 23:480-486
10. Scheinman JI, Tanaka H, Haralson M, Wang SL, Brown O (1992) Specialized collagen mRNA and secreted collagens in human glomerular epithelial, mesangial, and tubular cells. J Am Soc Nephrol 2:1475-1483
11. He JS, Hayashi K, Horikoshi S, Funabiki K, Shirato I, Tomino Y(2001) Identification of cellular origin of type Ⅰ collagen in glomeruli of rats with crescentic glomerulonephritis induced by anti-glomerular basement membrane antibody. Nephrol Dial Transplant 16:704-711
12. Mampaso FM, Wilson CB (1983) Characterization of inflammatory cells in autoimmune tubulointerstitial nephritis in rats. Kidney Int 23:448-457
13. Matsumura R, Umemiya K, Nakazawa T, Ochiai K, Kagami M, Tomioka H, Tanabe E, Sugiyama T, Sueishi M (1998) Expression of cell adhesion molecules in tubulointerstitial nephritis associated with Sjogren's syndrome. Clin Nephrol 49:74-81
14. Wada T, Furuichi K, Sakai N, Iwata Y, Yoshimoto K, Shimizu M, Takeda SI, Takasawa K, Yoshimura M, Kida H, Kobayashi KI, Mukaida N, Naito T, Matsushima K, Yokoyama H (2000) Upregulation of monocyte chemoattractant protein-1 in tubulointerstitial lesions of human diabetic nephropathy. Kidney Int58:1492-1499
15. Okada H, Moriwaki K, Konishi K, Kobayashi T, Sugahara S, Nakamoto H, Saruta T, Suzuki H (2000) Tubular osteopontin expression in human glomerulonephritis and renal vasculitis. Am J Kidney Dis 36:498-506
16. Okada H, Moriwaki K, Kalluri R, Imai H, Ban S, Takahama M, Suzuki H (2000) Inhibition of monocyte chemoattractant protein-1 expression in tubular epithelium attenuates tubulo interstitial alteration in rat Good pasture syndrome. Kidney Int 57: 927-936
17. Steinmann-Nggli K, Ziswiler R, kung M, el al. J Am soc Nephrol,1998,9(3): 397-407
18. Suzuki D,Miyazaki M,Jinde K,el al. Kidney Int,1997; 52(1):111-119
19. Gonzalez-Avila G, Iturria C, Vadillo-Ortega F, Ovalle C, Montano M (1998) Changes in matrix metal loproteinases during the evolution of interstitial renal fibrosis in a rat experimental model. Pathobiology 66:196-204
20. Jones CL, Fecondo J, Kelynack K, Forbes J, Walker R, Becker G(1995) Tissue inhibitor of the metalloproteinases and renal extracellular matrix accumulation. Exp Nephrol 3:80-86
21. Tang WW, Feng L, Xia Y, Wilson CB (1994) Extracellular matrixaccumulation in immune-mediated tubulointerstitial injury. Kidney Int 45:1077-1084
22. Sugimoto H, Grahovac G, Zeisberg M, et al. Renal fibrosis and glomerulosclerosis in a new mouse model of diabetic nephropathy and its regression by bone morphogenic:protein-7 and advanced glycation end product inhibitors. Diabetes, 2007,56:1825-33.
23. Chow FY, Nikolic-Paterson DJ, Atkins RC, et al. Macrophages in streptozotocin-induced diabetic nephropathy:potential role in renal fibrosis. Nephrol Dial Transplant,2004,19:2987-96.
24. D'Agord Schaan B, Lacchini S, Bertoluci MC, et al. Increased renal GLUT1 abundance and urinary TGF-beta 1 in streptozotocin-induced diabetic rats: implications for the development of nephropathy complicating diabetes. Horm Metab Res,2001,33:664-9.
25. Asano T, Tada M, Cheng S, et al. Prognostic Values of Matrix Metalloproteinase Family Expression in Human Colorectal Carcinoma. J Surg Res,2007.
26. McGlinchey PG, Spence MS, Patterson CC, et al. The matrix metalloproteinase-3 (MMP-3) 5A/6A promoter polymorphism is not associated with ischaemic heart disease:analysis employing a family based approach. Dis Markers,2004,20: 289-94.
27. Fogo AB. Renal fibrosis:not just PAI-1 in the sky. J Clin Invest,2003,112:326-8.
28. Cochrane AL, Kett MM, Samuel CS, et al. Renal structural and functional repair in a mouse model of reversal of ureteral obstruction. J Am Soc Nephrol,2005,16: 3623-30.
29. Blobel CP (1997) Metalloprotease-disintegrins:links to cell adhesionand cleavage of TNFα and notch. Cell 90:589-592
30. Huovila AP, Almeida EA, White JM (1996) ADAMs and cell fusion. Curr Opin Cell Biol 8:692-699
31. Hurskainen TL, Hirohata S, Seldin MF, Apte SS (1999) ADAMTS5, ADAM-TS6, and ADAM-TS7, novel members of a newfamily of zinc metalloproteases. General features and genomicdistribution of the ADAM-TS family. J Biol Chem 274: 25555-255563
32. Massague J (2000) How cells read TGF-beta signals. Nat Rev Mol Cell Biol 1:69-78
33. Bottner M, Krieglstein K, Unsicker K (2000) The transforminggrowth factor-betas: structure, signaling, and roles in nervous system development and functions. J Neurochem 75:2227-2240
34. Mauviel A. Transforming growth factor-beta:a key mediator of fibrosis. Methods Mol Med,2005,117:69-80.
35. Lan HY. Tubular epithelial-myofibroblast transdifferentiation mechanisms in proximal tubule cells. Curr Opin Nephrol Hypertens,2003,12:25-9.
36.姚颐,张杰.肾间质纤维化相关信号转导通路的研究进展.国际泌尿系统杂志,2007,27：831-834.
37. Flanders KC. Smad3 as a mediator of the fibrotic response. Int J Exp Pathol,2004, 85:47-64.
38. Razzaque MS, Taguchi T. Cellular and molecular events leading to renal tubulointerstitial fibrosis. Med Electron Microsc,2002,35:68-80.
39. Wang W, Koka V, Lan HY. Transforming growth factor-beta and Smad signalling in kidney diseases. Nephrology (Carlton),2005,10:48-56.
40. Basile DP (2001) Transforming growth factor-beta as a target for treatment in diabetic nephropathy. Am J Kidney Dis 38:887-892
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