腺病毒介导的双基因（anti-VEGF发夹状核酶+IL-24）治疗结肠癌的实验研究
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摘要
背景及目的:结肠癌的发病率不断上升,但结肠癌的治疗效果仍不理想,特别是复发和转移的患者。VEGF在肿瘤血管形成方面起着关键作用,VEGF在大多数结肠癌组织中高表达,并且其表达水平与结肠癌患者的复发和预后高度相关。发夹状核酶(hairpin ribozyme)是一种有催化活性的RNA,它能特异切割、降解含有能被其识别序列的靶mRNA,从而抑制该基因的表达。白细胞介素-24即黑素瘤分化相关基因-7(IL—24/Mda-7),是细胞因子白细胞介素-10基因家族成员。高表达白细胞介素-24在许多肿瘤中能引起肿瘤细胞凋亡和肿瘤生长抑制,但其对正常上皮细胞未产生明显的有害影响。本研究旨在构建带有双基因(抗VEGF-发夹状核酶+白细胞介素-24)的复制缺陷型腺病毒载体,并在细胞水平及移植瘤水平观察带有抗VEGF-发夹状核酶和白细胞介素-24的腺病毒对结肠癌VEGF表达的抑制作用以及对结肠癌生长的抑制作用。
方法:根据Genebank公布的VEGF165基因序列设计并合成编码抗VEGF165的发夹状核酶的双链DNA,所合成的双链DNA两端分别带有SalⅠ、BglⅡ酶切位点。将该双链DNA插入包含IRES(克隆于SalⅠ和NotⅠ之间)和人IL-24基因(克隆于XhoⅠ和XbaⅠ之间)的转移质粒pAdTrack-CMV-IRES-IL-24的相应多克隆位点上(pAdTrack-CMV-IRES-IL-24苏州大学细胞与分子生物学教研室构建并测序保存)。转移质粒pAdTrack-CMV-Rz/IL-24经PmeI酶切后与腺病毒骨架质粒pAdEasy-1在BJ5183大肠杆菌中同源重组,得到的重组质粒pAdTrack-CMV-Rz/IL-24-AdEasy-1经PacI酶切后用脂质体转染QBI-293A细胞,在293细胞中包装成完整病毒(命名为Ad-Rz/IL24)并复制扩增,获得重组腺病毒Ad-Rz/IL24。同时构建仅带有抗VEGF发夹状核酶的腺病毒(Ad-Rz)作为对照,带有IL-24的腺病毒(Ad-IL24)和空病毒载体(Ad-GFP)由苏州大学细胞与分子生物学教研室构建。RT-PCR及免疫荧光法检测病毒感染后抗-VEGF发夹状核酶及白细胞介素-24在HT-29细胞中的表达,Real-time PCR及ELISA检测其对VEGF mRNA及蛋白表达的抑制作用,MTT法和流式细胞仪检测Ad-Rz/IL24对HT-29细胞生长抑制和凋亡诱导作用。在Balb/c裸鼠皮下建立HT-29细胞动物移植瘤模型,观察Ad-Rz/IL24基因治疗对结肠癌移植瘤生长的抑制作用,免疫组化法检测白细胞介素-24、生长抑制和DNA损伤基因(GADD)及VEGF在移植瘤组织中的表达,CD34标记检测移植瘤组织微血管密度(MVD)。
结果:成功将抗-VEGF发夹状核酶和白细胞介素-24克隆至同一复制缺陷型腺病毒载体上。实验所构建复制缺陷型腺病毒Ad-Rz/IL24可以有效感染结肠癌HT-29细胞,Ad-Rz/IL24感染HT-29细胞后抗-VEGF发夹状核酶和白细胞介素-24可在HT-29细胞中高效表达。Ad-Rz/IL24能显著抑制HT-29细胞VEGF的表达,Ad-Rz/IL24、Ad-Rz、Ad-IL24感染48小时后HT-29细胞中VEGFmRNA的相对表达量分别为PBS组的(39.0±1.0)%,(45.0±1.0)%和(91.0±1.0)%,与PBS比较P＜0.05;Ad-GFP组(空病毒载体)中VEGF的相对表达量为PBS组的(122.0±2.0)%;在蛋白水平,Ad-Rz/IL24、Ad-Rz、Ad-IL24、Ad-GFP及PBS组细胞上清液中VEGF蛋白含量(OD.值)分别为0.37±0.28/million cells,0.46±0.35/million cells,0.56±0.30/million cells,0.74±0.13/million cells和0.81±0.16/million cells(P＜0.05)。MTT示Ad-Rz/IL24、Ad-IL-24可显著抑制人结肠癌HT-29细胞的生长(P＜0.05),Ad-Rz对人结肠癌HT-29细胞的生长有一定的抑制作用,其差异没有统计学意义(P＞0.05)。Ad-Rz/IL24诱导的HT-29细胞凋亡率为(11.00±0.80)%,大约为Ad-IL-24所诱导凋亡的两倍(Ad-IL-24凋亡率:(5.60±0.27)%,P＜0.05)。Ad-Rz所诱导得HT-29细胞凋亡率约(1.83±0.18)%,与PBS组和Ad-GFP组比较差异有统计学意义(P＜0.05)。Ad-Rz/IL24可在一定程度上抑制HT-29细胞移植瘤的生长,与PBS比较统计学意义处于临界状态间(P=0.0619,秩和检验),而Ad-Rz、Ad-IL24对移植瘤生长的抑制作用无统计学意义(P＞0.05)。使用免疫组化方法在Ad-Rz/IL24及Ad-IL-24处理组移植瘤中均检测到了白细胞介素-24及GADD的表达,Ad-Rz/IL-24可以显著抑制移植瘤组织中VEGF的表达及微血管密度(MVD),各组MVD分别为:PBS 12.8±11.1,Ad-GFP 11.5±1.0,Ad-IL-24 6.0±1.0,Ad-Rz 4.3±0.5,Ad-Rz/IL24 3.5±0.6。(P＜0.05)。
结论腺病毒载体介导的双基因anti-VEGF核酶和IL-24(Ad-Rz/IL24)可显著降低结肠癌HT-29细胞中VEGF的表达和抑制HT-29细胞的生长,可显著减少移植瘤组织的血管生成,并可在一定程度上抑制移植瘤的生长。
Background and Objective: The morbidity of colon cancer is increasing, but the current treatment for colon cancer is still unsatisfactory, especially for relapsed and metastatic patients. VEGF plays a key role in tumor angiogenesis. In general, VEGF is commonly upregulated in colon cancer, and increased VEGF expression and tumor vascularization are correlated with tumor progression and a poor clinical prognosis in colon cancer. The hairpin ribozyme is a small RNA molecule with endoribonuclease activity that exhibits catalytic sequence-specific cleavage of the target RNA and down-regulates the expression of target gene.Mda-7/IL-24 (melanoma differentiation associated gene-7) is a member of the IL-10 subfamily .When expressed at high levels, Mda-7/IL-24 suppresses growth and induces programmed cell death (apoptosis) in a broad spectrum of human cancers, including colon cancer. In contrast, Mda-7/IL-24 does not induce apoptosis in, and has a negligible effect on the growth of, normal cells. In this study, we constructed an adenovirus that simultaneously expresses anti-VEGF hairpin ribozyme and human IL-24 and examined its VEGF expression inhibitory effect and anti-tumor efficacy against human colon cancer HT-29 cells in vitro and in vivo.
Methods: Designing and synthesizing the double strands DNA which encodes the anti-VEGF hairpin ribozyme according to declared VEGF165 gene sequence from GenBank. These oligonucleotide sequences, which include Bgl II and Sal I linker sites at each end, were inserted into the multiple cloning site (MCS) of the shuttle plasmid pTrack-CMV which harbored an IRES between Sal I and Not I digestion sites and human IL-24 cDNA between Xho I and Xba I sites (previously constructed by the Cell and Molecular Biology Institute of Soochow University). The resultant shuttle vector (pAdTrack-CMV-Rz/IL-24) was linearized by the restriction endonuclease PmeI and subsequently cotransformed into BJ5183 bacterial cells with the adenoviral backbone plasmid (pAdEasy-1). After PacI digestion, the recombinant pAd-Rz/IL-24 plasmid was transfected into human embryonic kidney 293 (QBI-293A) cells with lipofectamine (Sigma; CA) to package the viruses. The constructed adenovirus, Ad-Rz/IL24 ,were amplified in QBI-293A cells. The expression of anti-VEGF hairpin ribozyme and IL-24 mRNA in HT-29 cells were detected by RT-PCR and the inhibitory effect of VEGF expression were tested by Real-time PCR and Elisa. The effect of growth inhibition and apoptosis-inducing of Ad-Rz/IL24 on HT-29 cells were detected by MTT and FCAS. The effect and mechanism of Ad-Rz/IL24 treatment on colon carcinoma in vivo were observed and studied through the HT-29 human colon cancer subcutaneous model in nu/nu mice. The growth inhibition effect of Ad-Rz/IL24 on xenografts was teseted and IL-24, GADD, and VEGF expression in xenograft tumor tissue were detected by Immunohistochemical analysis. The microvessle density (MVD) of the xenograft tumor was determined by CD34 staining.
Results: The anti-VEGF ribozyme and IL-24 were successfully inserted into the adenoviral vector. The adenovirus of Ad-Rz/IL24 can effectively infect the colon cancer HT-29 cells. Anti-VEGF ribozyme and IL-24 could effectively express in HT-29 cells after treated by Ad-Rz/IL24. Ad-Rz/IL24 could inhibit the expression of VEGF mRNA in HT-29 cells .The relative expression of VEGF mRNA in HT-29 cell treated with Ad-Rz/IL24, Ad-Rz (adenovirue carrying anti-VEGF ribozyme ) or Ad-IL-24 (adenovirue carrying IL-24 )decreased to about (39.0±1.0)%, (45.0±1.0)%, or (91.0±1.0)% of that of PBS control group in HT-29 cells respectively(P<0.05) and the VEGF expression in cells treated with Ad-GFP (vacant vector) was about (122.0±2.0)%. The amount of VEGF protein in the supernatant of Ad-Rz/IL24, Ad-Rz ,Ad-IL-24 ,Ad-GFP or PBS treated cells were 0.37±0.28/million cells, 0.46±0.35/ million cells, 0.56±0.30/ million cells ,0.74±0.13/ million cells ,and 0.81±0.16/ million cells, respectively (P<0.05). MTT assay showed significant growth inhibition for colon cancer cell lines by Ad-Rz/IL-24 or Ad-IL-24 (p<0.05, compared to PBS), while cells treated with Ad-Rz did not show significant growth inhibition (p > 0.05). Ad-Rz/IL-24 treatment induced apoptosis in about (11.0±0.8) % of the cells, which was about twice as much as Ad-IL-24 treatment (The apoptosis induced by Ad-IL-24 is about (5.6±0.3) %). Ad-Rz treatment induced apoptosis in only about (1.8±0.2) % of the cells, but it was still significantly different from both the control and Ad-GFP-treated cells (p <0.05). The growth of xenograft tumors were inhibited to some extent by Ad-Rz/IL24 treatment, but the significance was under borderline (P=0.0619, CMH test, compared to PBS). IL-24 and GADD protein was detected only in the Ad-Rz/IL-24 and Ad-IL-24 treated groups. Ad-Rz/IL24 can significantly inhibit the VEGF expression in xenograft tumor tissue and the MVD of xenograft tumors treated with Ad-Rz/IL24, Ad-Rz ,Ad-IL-24 ,Ad-GFP or PBS were 3.5±0.6, 4.3±0.5, 6.0±1.0, 11.5±1.0 and 12.8±11.1, respectively(P<0.05).
Conclusion: Dual-gene, anti-VEGF hairpin ribozyme and interleukin-24, mediated by adenovirus(Ad-Rz/IL24) can significantly down-regulate the expression of VEGF in HT-29 cells and inhibit tumor growth of colon cancer HT-29 cells. Forthermore, Ad-Rz/IL24 can significantly inhibit the tumor angiogenesis and inhibit the xenograft tumor growth to some extent in vivo.
方法:根据Genebank公布的VEGF165基因序列设计并合成编码抗VEGF165的发夹状核酶的双链DNA,所合成的双链DNA两端分别带有SalⅠ、BglⅡ酶切位点。将该双链DNA插入包含IRES(克隆于SalⅠ和NotⅠ之间)和人IL-24基因(克隆于XhoⅠ和XbaⅠ之间)的转移质粒pAdTrack-CMV-IRES-IL-24的相应多克隆位点上(pAdTrack-CMV-IRES-IL-24苏州大学细胞与分子生物学教研室构建并测序保存)。转移质粒pAdTrack-CMV-Rz/IL-24经PmeI酶切后与腺病毒骨架质粒pAdEasy-1在BJ5183大肠杆菌中同源重组,得到的重组质粒pAdTrack-CMV-Rz/IL-24-AdEasy-1经PacI酶切后用脂质体转染QBI-293A细胞,在293细胞中包装成完整病毒(命名为Ad-Rz/IL24)并复制扩增,获得重组腺病毒Ad-Rz/IL24。同时构建仅带有抗VEGF发夹状核酶的腺病毒(Ad-Rz)作为对照,带有IL-24的腺病毒(Ad-IL24)和空病毒载体(Ad-GFP)由苏州大学细胞与分子生物学教研室构建。RT-PCR及免疫荧光法检测病毒感染后抗-VEGF发夹状核酶及白细胞介素-24在HT-29细胞中的表达,Real-time PCR及ELISA检测其对VEGF mRNA及蛋白表达的抑制作用,MTT法和流式细胞仪检测Ad-Rz/IL24对HT-29细胞生长抑制和凋亡诱导作用。在Balb/c裸鼠皮下建立HT-29细胞动物移植瘤模型,观察Ad-Rz/IL24基因治疗对结肠癌移植瘤生长的抑制作用,免疫组化法检测白细胞介素-24、生长抑制和DNA损伤基因(GADD)及VEGF在移植瘤组织中的表达,CD34标记检测移植瘤组织微血管密度(MVD)。
结果:成功将抗-VEGF发夹状核酶和白细胞介素-24克隆至同一复制缺陷型腺病毒载体上。实验所构建复制缺陷型腺病毒Ad-Rz/IL24可以有效感染结肠癌HT-29细胞,Ad-Rz/IL24感染HT-29细胞后抗-VEGF发夹状核酶和白细胞介素-24可在HT-29细胞中高效表达。Ad-Rz/IL24能显著抑制HT-29细胞VEGF的表达,Ad-Rz/IL24、Ad-Rz、Ad-IL24感染48小时后HT-29细胞中VEGFmRNA的相对表达量分别为PBS组的(39.0±1.0)%,(45.0±1.0)%和(91.0±1.0)%,与PBS比较P＜0.05;Ad-GFP组(空病毒载体)中VEGF的相对表达量为PBS组的(122.0±2.0)%;在蛋白水平,Ad-Rz/IL24、Ad-Rz、Ad-IL24、Ad-GFP及PBS组细胞上清液中VEGF蛋白含量(OD.值)分别为0.37±0.28/million cells,0.46±0.35/million cells,0.56±0.30/million cells,0.74±0.13/million cells和0.81±0.16/million cells(P＜0.05)。MTT示Ad-Rz/IL24、Ad-IL-24可显著抑制人结肠癌HT-29细胞的生长(P＜0.05),Ad-Rz对人结肠癌HT-29细胞的生长有一定的抑制作用,其差异没有统计学意义(P＞0.05)。Ad-Rz/IL24诱导的HT-29细胞凋亡率为(11.00±0.80)%,大约为Ad-IL-24所诱导凋亡的两倍(Ad-IL-24凋亡率:(5.60±0.27)%,P＜0.05)。Ad-Rz所诱导得HT-29细胞凋亡率约(1.83±0.18)%,与PBS组和Ad-GFP组比较差异有统计学意义(P＜0.05)。Ad-Rz/IL24可在一定程度上抑制HT-29细胞移植瘤的生长,与PBS比较统计学意义处于临界状态间(P=0.0619,秩和检验),而Ad-Rz、Ad-IL24对移植瘤生长的抑制作用无统计学意义(P＞0.05)。使用免疫组化方法在Ad-Rz/IL24及Ad-IL-24处理组移植瘤中均检测到了白细胞介素-24及GADD的表达,Ad-Rz/IL-24可以显著抑制移植瘤组织中VEGF的表达及微血管密度(MVD),各组MVD分别为:PBS 12.8±11.1,Ad-GFP 11.5±1.0,Ad-IL-24 6.0±1.0,Ad-Rz 4.3±0.5,Ad-Rz/IL24 3.5±0.6。(P＜0.05)。
结论腺病毒载体介导的双基因anti-VEGF核酶和IL-24(Ad-Rz/IL24)可显著降低结肠癌HT-29细胞中VEGF的表达和抑制HT-29细胞的生长,可显著减少移植瘤组织的血管生成,并可在一定程度上抑制移植瘤的生长。
Background and Objective: The morbidity of colon cancer is increasing, but the current treatment for colon cancer is still unsatisfactory, especially for relapsed and metastatic patients. VEGF plays a key role in tumor angiogenesis. In general, VEGF is commonly upregulated in colon cancer, and increased VEGF expression and tumor vascularization are correlated with tumor progression and a poor clinical prognosis in colon cancer. The hairpin ribozyme is a small RNA molecule with endoribonuclease activity that exhibits catalytic sequence-specific cleavage of the target RNA and down-regulates the expression of target gene.Mda-7/IL-24 (melanoma differentiation associated gene-7) is a member of the IL-10 subfamily .When expressed at high levels, Mda-7/IL-24 suppresses growth and induces programmed cell death (apoptosis) in a broad spectrum of human cancers, including colon cancer. In contrast, Mda-7/IL-24 does not induce apoptosis in, and has a negligible effect on the growth of, normal cells. In this study, we constructed an adenovirus that simultaneously expresses anti-VEGF hairpin ribozyme and human IL-24 and examined its VEGF expression inhibitory effect and anti-tumor efficacy against human colon cancer HT-29 cells in vitro and in vivo.
Methods: Designing and synthesizing the double strands DNA which encodes the anti-VEGF hairpin ribozyme according to declared VEGF165 gene sequence from GenBank. These oligonucleotide sequences, which include Bgl II and Sal I linker sites at each end, were inserted into the multiple cloning site (MCS) of the shuttle plasmid pTrack-CMV which harbored an IRES between Sal I and Not I digestion sites and human IL-24 cDNA between Xho I and Xba I sites (previously constructed by the Cell and Molecular Biology Institute of Soochow University). The resultant shuttle vector (pAdTrack-CMV-Rz/IL-24) was linearized by the restriction endonuclease PmeI and subsequently cotransformed into BJ5183 bacterial cells with the adenoviral backbone plasmid (pAdEasy-1). After PacI digestion, the recombinant pAd-Rz/IL-24 plasmid was transfected into human embryonic kidney 293 (QBI-293A) cells with lipofectamine (Sigma; CA) to package the viruses. The constructed adenovirus, Ad-Rz/IL24 ,were amplified in QBI-293A cells. The expression of anti-VEGF hairpin ribozyme and IL-24 mRNA in HT-29 cells were detected by RT-PCR and the inhibitory effect of VEGF expression were tested by Real-time PCR and Elisa. The effect of growth inhibition and apoptosis-inducing of Ad-Rz/IL24 on HT-29 cells were detected by MTT and FCAS. The effect and mechanism of Ad-Rz/IL24 treatment on colon carcinoma in vivo were observed and studied through the HT-29 human colon cancer subcutaneous model in nu/nu mice. The growth inhibition effect of Ad-Rz/IL24 on xenografts was teseted and IL-24, GADD, and VEGF expression in xenograft tumor tissue were detected by Immunohistochemical analysis. The microvessle density (MVD) of the xenograft tumor was determined by CD34 staining.
Results: The anti-VEGF ribozyme and IL-24 were successfully inserted into the adenoviral vector. The adenovirus of Ad-Rz/IL24 can effectively infect the colon cancer HT-29 cells. Anti-VEGF ribozyme and IL-24 could effectively express in HT-29 cells after treated by Ad-Rz/IL24. Ad-Rz/IL24 could inhibit the expression of VEGF mRNA in HT-29 cells .The relative expression of VEGF mRNA in HT-29 cell treated with Ad-Rz/IL24, Ad-Rz (adenovirue carrying anti-VEGF ribozyme ) or Ad-IL-24 (adenovirue carrying IL-24 )decreased to about (39.0±1.0)%, (45.0±1.0)%, or (91.0±1.0)% of that of PBS control group in HT-29 cells respectively(P<0.05) and the VEGF expression in cells treated with Ad-GFP (vacant vector) was about (122.0±2.0)%. The amount of VEGF protein in the supernatant of Ad-Rz/IL24, Ad-Rz ,Ad-IL-24 ,Ad-GFP or PBS treated cells were 0.37±0.28/million cells, 0.46±0.35/ million cells, 0.56±0.30/ million cells ,0.74±0.13/ million cells ,and 0.81±0.16/ million cells, respectively (P<0.05). MTT assay showed significant growth inhibition for colon cancer cell lines by Ad-Rz/IL-24 or Ad-IL-24 (p<0.05, compared to PBS), while cells treated with Ad-Rz did not show significant growth inhibition (p > 0.05). Ad-Rz/IL-24 treatment induced apoptosis in about (11.0±0.8) % of the cells, which was about twice as much as Ad-IL-24 treatment (The apoptosis induced by Ad-IL-24 is about (5.6±0.3) %). Ad-Rz treatment induced apoptosis in only about (1.8±0.2) % of the cells, but it was still significantly different from both the control and Ad-GFP-treated cells (p <0.05). The growth of xenograft tumors were inhibited to some extent by Ad-Rz/IL24 treatment, but the significance was under borderline (P=0.0619, CMH test, compared to PBS). IL-24 and GADD protein was detected only in the Ad-Rz/IL-24 and Ad-IL-24 treated groups. Ad-Rz/IL24 can significantly inhibit the VEGF expression in xenograft tumor tissue and the MVD of xenograft tumors treated with Ad-Rz/IL24, Ad-Rz ,Ad-IL-24 ,Ad-GFP or PBS were 3.5±0.6, 4.3±0.5, 6.0±1.0, 11.5±1.0 and 12.8±11.1, respectively(P<0.05).
Conclusion: Dual-gene, anti-VEGF hairpin ribozyme and interleukin-24, mediated by adenovirus(Ad-Rz/IL24) can significantly down-regulate the expression of VEGF in HT-29 cells and inhibit tumor growth of colon cancer HT-29 cells. Forthermore, Ad-Rz/IL24 can significantly inhibit the tumor angiogenesis and inhibit the xenograft tumor growth to some extent in vivo.
引文
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