RNA干扰沉默STAT5A基因诱导肝癌细胞HepG2凋亡
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摘要
目的：探讨针对STAT5A基因的siRNA表达载体对人肝癌细胞系HepG2细胞中STAT5A基因表达的特异性抑制作用及其对肝癌细胞凋亡的影响,为干扰STAT5基因作为肿瘤基因治疗新策略的应用提供实验依据。方法：设计2条针对STAT5A基因的RNA干扰靶序列GCTCTGAATTAGTC CTTGCTT和GCGCTTTAGTGACTCAGAAAT,合成具有该靶序列短发夹结构的寡核苷酸,退火形成双链DNA。将pYr-2.1表达载体经BsaI酶切后电泳分离,纯化回收大片段。将退火形成的双链DNA通过T4 DNA连接酶连接到线性化pYr-2.1质粒U6启动子下游,构建重组表达质粒pYr-2.1-hU6-STAT5A-shRNA-1和pYr-2.1-hU6-STAT5A-shRNA-2。将重组质粒转化感受态细胞DH5α,经Kanar抗性和CCDB致死基因筛选,挑选阳性克隆菌落进行摇菌扩增,并抽提纯化质粒,然后将抽提的重组质粒进行Xhol单酶切和琼脂糖电泳分析,同时做测序鉴定以确定插入的干扰片段准确无误,即针对STAT5A基因的siRNA表达载体构建成功。同法构建不针对任何基因的shRNA阴性对照pYr-2.1-HK。培养HepG2细胞,待细胞生长状态良好并达到指数生长期时,于转染前一日接种于6孔板中,并设置空白细胞对照组、HK阴性对照组、STAT5A-1干扰组、STAT5A-2干扰组。用表达绿色荧光蛋白(EGFP)的pYr-2.1-EGFP以检测肝癌细胞HepG2的转染效率,以每孔脂质体7.5μl、重组质粒3μg配成转染复合物,加入各孔HepG2细胞中进行转染(空白组不加任何试剂),转染后48h质粒pYr-2.1-EGPF上带有的EGFP基因得以表达,荧光显微镜下观察绿色荧光细胞所占比例,以此来判断转染效率。接着用TRIzol试剂分别提取各组细胞总RNA,并应用核酸定量分析仪及琼脂糖凝胶电泳检测所提取RNA样本的浓度、纯度以及完整性。以各组总RNA为模板,GAPDH(甘油醛-3-磷酸脱氢酶)为内对照进行两步法逆转录聚合酶链反应(RT-PCR),琼脂糖凝胶电泳分析,并用灰度半定量检测各组STAT5A mRNA的相对表达量；另在转染后72h进行FITC/PI双染每组细胞后流式细胞仪检测细胞凋亡情况。结果：①重组质粒pYr-2.1-hU6-STAT5A-shRNA-1和pYr-2.1-hU6-STAT5A-shRNA-2经XhoI酶切鉴定和测序鉴定证实目的序列已准确插入到预计位点,重组质粒构建成功。②以LipofectamineTM2000转染HepG2细胞后48h荧光显微镜下观察,转染效率约为65%以上③RT-PCR扩增产物凝胶电泳分析显示：各组泳道中内对照GAPDH扩增产物条带(410bp)的亮度相似,而STAT5A-1干扰组、STAT5A-2干扰组扩增产物条带(452bp)的亮度均明显弱于其它二组。灰度扫描半定量结果显示,空白细胞对照组、HK对照组、STAT5A-1干扰组、STAT5A-2干扰组HepG2细胞中STAT5A mRNA的相对表达量分别为1.038±0.013、1.053±0.016、0.353±0.014、0.450±0.012。经q检验方差分析,STAT5A-1干扰组、STAT5A-2干扰组STAT5 mRNA相对表达量分别与空白组、阴性对照组相比,分别下降66.0%和56.64%左右,差异有统计学意义(P<0.01),而空白细胞对照组与HK组之间相比,无显著性差异(P>0.05)。④通过流式细胞仪检测细胞凋亡率百分显示：STAT5A-1干扰组、STAT5A-2干扰组细胞凋亡显著,凋亡率分别为37.33%和33.93%；空白细胞对照组与HK组细胞凋亡无显著性差异。结论：①设计合成的shRNA干扰序列准确无误地克隆到pYr-2.1表达载体中,针对STAT5A基因的siRNA表达载体构、建成功。②针对STAT5A基因序列构建的RNAi体内表达载体在mRNA水平特异性地抑制HepG2细胞中STAT5A的表达。③运用RNA干扰沉默肝癌细胞中STAT5A基因表达,能诱导人肝癌细胞凋亡,可成为肿瘤基因治疗的有效方法。
Objectives:To construct two RNAi expression vector of STAT5A gene and explore the specific inhibitory effect of this vector on STAT5 A mRNA expression and the effect of induce apoptosis of HepG2 cells, and to lay the experimental foundation for the advanced research. Methods: Designed RNA interference target sequence targeted at STAT5A gene GCTCTGAATTAGTCCTTGCTT and GCGCTTTAGTGACTCAGAAAT, then synthesize two strands of oligonucleotides including a hairpin structure. The pYr-2.1 vector was digested with Bsal, then purified and retrieved the fragment by agarose electrophoresis separation. The double-strand DNA was inserted downstream from the U6 promoter of the linear pYr-2.1 vector by T4 ligase and the recombination expression plasmid pYr-2.1-hU6-STAT5A-1 and pYr-2.1-hU6-STAT5A-2 was constructed. The next step was transfecting the recombinant into the susceptible cell E.coli DH5a which was prepared by CaCl2 method, and choosing the positive colony which was screened by kanamycin resistance and CCDB to amplify by shaking, then extracting the plasmid. To make sure the interference fragment was accurate, that is to say, the siRNA expression vector targeting STAT5A had been constructed successfully, the extracted recombinant was digested by Xhol respectively, and to analyze the result by agarose electrophoresis and DNA sequencing. At the same time, we constructed the negative control recombinant pYr-2.1-HK that did not aim at any gene by the same method.HepG2 cells that were in good condition were plated in 6-well cell culture plates 24 hours prior to transfection, and set up four experimental groups, they were blank control group, negative control group and STAT5A-1 group、STAT5A-2 group. We used pYr-2.1-EGFP that for transfection rate, and added-7.5μl LipofectamineTM 2000+3μg plasmid/well, and then transfected the compound to the HepG2 cells(do not add any reagent to blank control group). At 48 hours post transfection, the EGFP (enhancement green fluorescent protein) gene of pYr-2.1-EGFP recombinant could express, so transfection rate could be judged by observing the ratio of green fluorescence cells under the fluorescence microscope. The total RNA from each well of cells was isolated by TRIzol reagent at the same time. And the concentration, purity and integrality of total RNA were detected by GeneQuant pro RNA/DNA and agarose gel electrophoresis. The semi-quantitative two-step RT-PCR which took the total RNA as template and took GAPDH as the control was used to determine the interference efficiency. Agarose gel electrophoresis and semi-quantitative gray scale scan were used to detect the relative expression of STAT5A mRNA in every group. And the cell apoptosis was observed by means of flow cytometry FCM by FITC/PI double staining after gathered every group cells at 72h after transfection. Results:①The recombinant plasmids pYr-2.1-hU6-STAT5A-1 and pYr-2.1-hU6-STAT5A-2 were digested with Xhol to agarose gel electrophoresis and sequence analysis, showed that the target sequence had inserted into the predicted site precisely and the recombining plasmids were successfully constructed.②Observe the HepG2 cells under the fluorescence microscope 48h after transfection, and judge the transfection rate that LipofectamineTM 2000 to HepG2 cells was about 65% according to the ratio of the cells shinning green fluorescence.③The result of electrophoretic analysis of the amplification product of RT-PCR showed that the lightness of the strips of GAPDH (410bp) in the four channels were similar, while the luminosity of STAT5A-1 group、STAT5A-2 group strip (452bp) were obviously weaker compared with the other two groups. And the result of the semi-quantitative gray scale scan showed that the relative expression quantities of STAT5A mRNA in blank control group, negative control group and STAT5A-1group、STAT5A-2 group were 1.038±0.013、1.053±0.016、0.353±0.014、0.450±0.012. These data were analyzed by q test of ANOVA that the relative expression of STAT5A mRNA of STAT5A-1group、STAT5A-2 group was about 66.0% and 56.64% lower than the others groups, and the difference had statistical significance (P<0.01).But the comparation among the other two groups had no significant difference (P>0.05).④The result of analysis of flow cytometry:After transfection the STAT5A-1 group、STAT5A-2 group apoptosis rate began to increase, the rate of apoptosis is 37.33% and 33.93%. And the difference among negative control group and blank group were no statistically significant. Conclusions:①The shRNA interference sequence that had designed and synthesized had cloned to pYr-2.1 expression vector accurately, and the siRNA expression vector that aimed at STAT5A constructed successfully.②The RNAi expression vector in vivo that aimed at this target sequence efficiently and specifically inhibited the expression of STAT5A mRNA in HepG2 cells.③RNA interference STAT5A gene in HepG2 cells can induce apoptosis of human hepatocelluar cancer cell.
Objectives:To construct two RNAi expression vector of STAT5A gene and explore the specific inhibitory effect of this vector on STAT5 A mRNA expression and the effect of induce apoptosis of HepG2 cells, and to lay the experimental foundation for the advanced research. Methods: Designed RNA interference target sequence targeted at STAT5A gene GCTCTGAATTAGTCCTTGCTT and GCGCTTTAGTGACTCAGAAAT, then synthesize two strands of oligonucleotides including a hairpin structure. The pYr-2.1 vector was digested with Bsal, then purified and retrieved the fragment by agarose electrophoresis separation. The double-strand DNA was inserted downstream from the U6 promoter of the linear pYr-2.1 vector by T4 ligase and the recombination expression plasmid pYr-2.1-hU6-STAT5A-1 and pYr-2.1-hU6-STAT5A-2 was constructed. The next step was transfecting the recombinant into the susceptible cell E.coli DH5a which was prepared by CaCl2 method, and choosing the positive colony which was screened by kanamycin resistance and CCDB to amplify by shaking, then extracting the plasmid. To make sure the interference fragment was accurate, that is to say, the siRNA expression vector targeting STAT5A had been constructed successfully, the extracted recombinant was digested by Xhol respectively, and to analyze the result by agarose electrophoresis and DNA sequencing. At the same time, we constructed the negative control recombinant pYr-2.1-HK that did not aim at any gene by the same method.HepG2 cells that were in good condition were plated in 6-well cell culture plates 24 hours prior to transfection, and set up four experimental groups, they were blank control group, negative control group and STAT5A-1 group、STAT5A-2 group. We used pYr-2.1-EGFP that for transfection rate, and added-7.5μl LipofectamineTM 2000+3μg plasmid/well, and then transfected the compound to the HepG2 cells(do not add any reagent to blank control group). At 48 hours post transfection, the EGFP (enhancement green fluorescent protein) gene of pYr-2.1-EGFP recombinant could express, so transfection rate could be judged by observing the ratio of green fluorescence cells under the fluorescence microscope. The total RNA from each well of cells was isolated by TRIzol reagent at the same time. And the concentration, purity and integrality of total RNA were detected by GeneQuant pro RNA/DNA and agarose gel electrophoresis. The semi-quantitative two-step RT-PCR which took the total RNA as template and took GAPDH as the control was used to determine the interference efficiency. Agarose gel electrophoresis and semi-quantitative gray scale scan were used to detect the relative expression of STAT5A mRNA in every group. And the cell apoptosis was observed by means of flow cytometry FCM by FITC/PI double staining after gathered every group cells at 72h after transfection. Results:①The recombinant plasmids pYr-2.1-hU6-STAT5A-1 and pYr-2.1-hU6-STAT5A-2 were digested with Xhol to agarose gel electrophoresis and sequence analysis, showed that the target sequence had inserted into the predicted site precisely and the recombining plasmids were successfully constructed.②Observe the HepG2 cells under the fluorescence microscope 48h after transfection, and judge the transfection rate that LipofectamineTM 2000 to HepG2 cells was about 65% according to the ratio of the cells shinning green fluorescence.③The result of electrophoretic analysis of the amplification product of RT-PCR showed that the lightness of the strips of GAPDH (410bp) in the four channels were similar, while the luminosity of STAT5A-1 group、STAT5A-2 group strip (452bp) were obviously weaker compared with the other two groups. And the result of the semi-quantitative gray scale scan showed that the relative expression quantities of STAT5A mRNA in blank control group, negative control group and STAT5A-1group、STAT5A-2 group were 1.038±0.013、1.053±0.016、0.353±0.014、0.450±0.012. These data were analyzed by q test of ANOVA that the relative expression of STAT5A mRNA of STAT5A-1group、STAT5A-2 group was about 66.0% and 56.64% lower than the others groups, and the difference had statistical significance (P<0.01).But the comparation among the other two groups had no significant difference (P>0.05).④The result of analysis of flow cytometry:After transfection the STAT5A-1 group、STAT5A-2 group apoptosis rate began to increase, the rate of apoptosis is 37.33% and 33.93%. And the difference among negative control group and blank group were no statistically significant. Conclusions:①The shRNA interference sequence that had designed and synthesized had cloned to pYr-2.1 expression vector accurately, and the siRNA expression vector that aimed at STAT5A constructed successfully.②The RNAi expression vector in vivo that aimed at this target sequence efficiently and specifically inhibited the expression of STAT5A mRNA in HepG2 cells.③RNA interference STAT5A gene in HepG2 cells can induce apoptosis of human hepatocelluar cancer cell.
引文
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3. Buitenhuis M, Baltus B, Lammers JW, et al. Signal transducer and activator of transcription 5a(STAT5a)is required for eosinophil differentiation of human cord blood-derived CD34+ cells [J]. Blood,2003,101(1):134-142.
4. Darnell J E Jr. Transcription factors as targets for cancer therapy[J]. Nat Rev Cancer,2002,2(10):740-749.
5.赵振军,左连富,单保恩.香加皮杠柳苷抑制STAT5信号通路诱导SMMC-7721细胞凋亡的实验研究[J].临床检验杂志,2008,26(1)：46-48.
6. Olas B,Wachowicz B,Majsterek L, et al. Resveratrol may reduce oxidative stress induce by platinum compounds in human plasma,blood platelets and lymphocytes[J]. Anticancer Drugs,2005,16(6):659-665.
7. Lee EO, Kwon BM, Song GY, et al. Heyneanol a induces apoptosis via cytochrome C release and caspase activation in Human leukemic LJ937cells[J]. Life Sci,2004,74 (18):2313-2326.
8. Yoshikawa H, Matsubara K, Qian G S, et al. SOCS-1, a negative regulator of the JAK / STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth suppression activity[J]. Nat Genet,2001,28(1):29-35。
9.王禹,张颖超,韩喜春,癌基因STAT5在肝细胞肝癌中的表达和意义[J].免疫学杂志,2009,25(1)：75-76.
10. Wall NR, Shi Y. Small RNA:can RNA interference be exploited for therapy [J]? Lancet,2003,362(9393):1401-1403.
11. Caplen NJ. Gene therapy progress and prospects. Down regulating gene expression: the impact of RNA interference [J]. Gene Ther,2004,11(16): 1241-1248.
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15.马向涛,余力伟.STAT5反义寡核苷酸联合5-氟尿嘧啶对胃癌细胞增殖与凋亡的影响.世界华人消化杂志,2006,14(13)：1257—1261。
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2. Barash I. STAT5 in the mammary gland:controlling normal development and cancer [J]. J Cell Physiol,2006,209(2):305—313.
3. Buitenhuis M, Baltus B, Lammers JW, et al. Signal transducer and activator of transcription 5a(STAT5a)is required for eosinophil differentiation of human cord blood-derived CD34+ cells [J]. Blood,2003,101(1):134-142.
4. Darnell J E Jr. Transcription factors as targets for cancer therapy[J]. Nat Rev Cancer,2002,2(10):740-749.
5.赵振军,左连富,单保恩.香加皮杠柳苷抑制STAT5信号通路诱导SMMC-7721细胞凋亡的实验研究[J].临床检验杂志,2008,26(1)：46-48.
6. Olas B,Wachowicz B,Majsterek L, et al. Resveratrol may reduce oxidative stress induce by platinum compounds in human plasma,blood platelets and lymphocytes[J]. Anticancer Drugs,2005,16(6):659-665.
7. Lee EO, Kwon BM, Song GY, et al. Heyneanol a induces apoptosis via cytochrome C release and caspase activation in Human leukemic LJ937cells[J]. Life Sci,2004,74 (18):2313-2326.
8. Yoshikawa H, Matsubara K, Qian G S, et al. SOCS-1, a negative regulator of the JAK / STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth suppression activity[J]. Nat Genet,2001,28(1):29-35。
9.王禹,张颖超,韩喜春,癌基因STAT5在肝细胞肝癌中的表达和意义[J].免疫学杂志,2009,25(1)：75-76.
10. Wall NR, Shi Y. Small RNA:can RNA interference be exploited for therapy [J]? Lancet,2003,362(9393):1401-1403.
11. Caplen NJ. Gene therapy progress and prospects. Down regulating gene expression: the impact of RNA interference [J]. Gene Ther,2004,11(16): 1241-1248.
12. Kisseleva T, Bhattacharya S. Braunstein J, et al. Signaling through the JAK/STAT pathway, recent advances and future changes[J]. Gene,2002, 285(1-2):1-24.
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