摘要
目的:
构建ING4和IL-24双基因共表达腺病毒载体(Ad-ING4-IL-24),研究其对肝癌的抑瘤增效和化疗增敏效应及分子机制。
方法:
分别以pGEZ-Term、pAdTrack-CMV-IL-24和pAdTrack-CMV-ING4质粒为模板,PCR扩增IRES(SalI、NotI)、IL-24(XhoI、XbaI)和ING4(BglII、SalI)目的片段,依次亚克隆至pAdTrack-CMV转移载体构建pAdTrack-CMV- ING4-IRES-IL-24双基因共表达重组转移载体。将构建正确的pAdTrack-CMV-ING4-IRES-IL-24重组转移载体经PmeI酶切后与pAdEasy-1腺病毒骨架质粒在BJ5183大肠杆菌中同源重组,得到的同源重组质粒pAdEasy-1-pAdTrack-CMV-ING4-IRES-IL-24(简称为pAd-ING4-IRES-IL-24)经PacI酶切后再用脂质体转染QBI-293A细胞,经多轮感染和扩增后获得Ad-ING4-IRES-IL-24双基因共表达重组腺病毒载体。polyA+promoter介导的ING4和IL-24双基因共表达腺病毒载体(Ad-ING4-polyA+promoter-IL-24)的构建方法基本同上,唯一不同的是polyA+promoter片段的获得是:以pORF-mbcl-2a质粒为模板,PCR分别扩增polyA和promoter片段;再以此为模板,SOE-PCR扩增polyA+promoter融合片段。将Ad-ING4-IRES-IL-24(简称为Ad-ING4-IL-24)体外感染SMMC-7721、HepG2肝癌细胞及联合顺铂(CDDP)化疗药物作用,MTT法和FCM检测其体外抑制肝癌细胞生长和诱导凋亡的协同增效和化疗增敏效应,并建立SMMC-7721裸鼠人肝癌移植瘤模型,观察Ad-ING4-IL-24体内对裸鼠人肝癌移植瘤的抑瘤增效作用。侵袭小室检测Ad-ING4-IL-24体外对SMMC-7721肝癌细胞侵袭能力的影响。Western blot和免疫组化检测细胞周期和凋亡相关蛋白及肿瘤血管形成相关因子P53、P21、P27、Cox-2、Fas、Bcl-2、Bax、Caspase-3、VEGF、CD34的表达。ELISA和半定量RT-PCR法检测Ad-ING4-IL-24对SMMC-7721肝癌细胞促血管形成因子VEGF、IL-8和肿瘤转移相关因子MMP-2、MMP-9表达的影响。
结果:
成功构建了IRES和双启动子(polyA+promoter)介导的两种ING4和IL-24双基因共表达腺病毒载体(Ad-ING4-IRES-IL-24和Ad-ING4- polyA+ promoter- IL- 24)。腺病毒介导的ING4和IL-24双基因共表达(Ad-ING4-IL-24)体外能明显抑制SMMC-7721、HepG2肝癌细胞的生长和诱导凋亡及抑制SMMC-7721细胞的体外侵袭能力,体内同样能明显抑制SMMC-7721裸鼠人肝癌移植瘤的生长,且均具协同抑瘤增效作用;而且Ad-ING4-IL-24能提高CDDP化疗药物体外抑制SMMC-7721、HepG2肝癌细胞的生长和诱导凋亡的作用,具有显著的化疗增敏效应。分子机制检测结果表明:Ad-ING4-IL-24能明显上调SMMC-7721肝癌细胞P53、P21、P27、Fas、Bax、Caspase-3和下调Cox-2、Bcl-2等细胞周期和凋亡相关蛋白的表达;能明显下调肿瘤血管形成因子VEGF、IL-8、CD34的表达;还能明显下调肿瘤转移相关因子MMP-2、MMP-9的表达,且其效应均较Ad-ING4、Ad-IL-24更为显著。
结论:
1、国内外首次成功构建了两种ING4和IL-24双基因共表达腺病毒载体Ad-ING4-IRES-IL-24和Ad-ING4-polyA+promoter-IL-24。
2、Ad-ING4-IL-24双基因表达及联合CDDP化疗药物体外抑制SMMC-7721、HepG2肝癌细胞生长和诱导凋亡呈现明显的协同抑瘤增效作用和化疗增敏效应。
3、Ad-ING4-IL-24双基因表达体内抑制SMMC-7721裸鼠人肝癌移植瘤同样具有协同抑瘤增效作用。
4、Ad-ING4-IL-24双基因表达更具显著上调P53、P21、P27、Fas、Bax、Caspase-3和下调Cox-2、Bcl-2等细胞周期和凋亡相关蛋白表达的效应,这可能是其双基因表达对肝癌细胞体内外生长抑制具有协同抑瘤增效作用的重要机制之一。
5、Ad-ING4-IL-24双基因表达更具显著下调肿瘤血管形成因子VEGF、IL-8、CD34表达的效应,这可能是其双基因表达对SMMC-7721裸鼠人肝癌移植瘤体内生长抑制具有协同抑瘤增效作用的另一重要机制。
6、Ad-ING4-IL-24双基因表达更具显著抑制SMMC-7721肝癌细胞体外侵袭的效应,这与下调肿瘤转移相关因子MMP-2和MMP-9的表达可能密切相关。
Objective:
To construct a recombinant adenoviral vector co-expressing inhibitor of growth 4 (ING4) and interleukin-24 (IL-24) and explore its enhanced anti-tumor and chemosensitivity to anticancer drug cisplatin (CDDP) effects on hepatocellular carcinoma and its mechanism.
Methods:
The internal ribosome entry site (IRES), IL-24, and ING4 fragments were amplified by PCR using pGEZ-Term, pAdTrack-CMV-IL-24, and pAdTrack-CMV-ING4 plasmids as templates, respectively. The IRES (SalI、NotI), IL-24 (XhoI、XbaI), and ING4 (BglII、SalI) fragments were subcloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-ING4-IRES-IL-24 and identified by PCR, double endonuclease digestion, and DNA sequencing. The pAdTrack-CMV-ING4-IRES-IL-24 transfer vector linearized with PmeI digestion and pAdEasy-1 backbone vector were further cotransformed into the bacteria BJ5183 competent cells for homologous recombination. The resultant pAdEasy-1-pAdTrack-CMV-ING4-IRES-IL-24 (pAd-ING4-IRES-IL-24) homologous recombinant plasmids purified from the above BJ5183 cells were transformed into the bacteria DH5αcompetent cells to abundantly amplify pAd-ING4-IRES-IL-24 plasmids. Then they were linearized with PacI digestion and transfected into the human embryonic kidney 293 (QBI-293A) cells by Lipofectamine2000, leading to formation of the recombinant adenoviruses Ad-ING4-IRES-IL-24 co-expressing ING4 and IL-24. The other kind of recombinant adenoviruses Ad-ING4-polyA+promoter-IL-24 was similarly prepared as described above, except that the polyA+promoter fusion fragments were obtained by SOE-PCR using pORF-mbcl-2a plasmids as templates. The in vitro enhanced growth-suppressing, apoptosis-inducing, and chemosensitivity to anticancer drug cisplatin (CDDP) effect of Ad-ING4-IL-24 co-expressing ING4 and IL-24 on SMMC-7721 and HepG2 human hepatocellular carcinoma cells were assessed by MTT assay and FCM, and its in vivo enhanced anti-tumor effect on hepatocellular carcinoma was further observed using SMMC-7721 human hepatocellular carcinoma transplanted tumor in athymic nude mouse model. The in vitro effect of Ad-ING4-IL-24 on SMMC-7721 cells’invasive ability was addressed using a Transwell Chamber system. The expression of cell cycle and apoptosis- and angiogenesis-related proteins (P53, P21, P27, Cox-2, Fas, Bcl-2, Bax, Caspase-3, VEGF, IL-8, and CD34) in SMMC-7721 hepatocellular carcinoma cells and SMMC-7721 transplanted tumor was determined by Western blot, ELISA, semi-quantitative RT-PCR, and immunohistochemistry analysis, respectively. The effect of Ad-ING4-IL-24 on the expression of metastasis-related factors (MMP-2 and MMP-9) in SMMC-7721 hepatocellular carcinoma cells was also examined by semi-quantitative RT-PCR.
Results:
Two kinds of adenoviral vector co-expressing ING4 and IL-24, Ad-ING4-IRES-IL-24 and Ad-ING4-polyA+promoter-IL-24, were successfully constructed. Adenovirus-mediated ING4 and IL-24 co-expression significantly inhibited SMMC-7721 and HepG2 hepatocellular carcinoma cell growth, induced cell apoptosis, suppressed SMMC-7721 cell invasiveness, and retarded SMMC-7721 human hepatocellular carcinoma transplanted tumor growth in athymic nude mouse, exhibiting synergetic anti-tumor effect. Furthermore, Ad-ING4-IL-24 significantly enhanced the chemosensitivity to anticancer drug CDDP in SMMC-7721 and HepG2 cells in vitro. Ad-ING4-IL-24 had more marked effect in up-regulating the expression of P53, P21, P27, Fas, Bax, and Caspase-3 and down-regulating the expression of Cox-2, Bcl-2, VEGF, IL-8, CD34, MMP-2, and MMP-9 compared with Ad-ING4 or Ad-IL-24.
Conclusions:
1. Two kinds of adenoviral vector co-expressing two suppressor gene of ING4 and IL-24 (Ad-ING4-IRES-IL-24 and Ad-ING4-polyA+promoter-IL-24), for the first time, were successfully constructed at home and abroad.
2. Ad-ING4-IL-24 co-expressing ING4 and IL-24 had significant synergetic and enhanced chemosensitivity to CDDP effect in suppressing SMMC-7721 and HepG2 human hepatocellular carcinoma cell growth and inducing cell apoptosis in vitro.
3. Ad-ING4-IL-24 co-expressing ING4 and IL-24 had significant synergetic effect in retarding SMMC-7721 human hepatocellular carcinoma transplanted tumor growth in athymic nude mouse.
4. Ad-ING4-IL-24 co-expressing ING4 and IL-24 had more potent effect in up-regulating the expression of P53, P21, P27, Fas, Bax, and Caspase-3 and down-regulating the expression of Cox-2 and Bcl-2, which may be responsible for its synergetic anti-tumor effect on SMMC-7721 human hepatocellular carcinoma cells in vitro and in vivo in athymic nude mouse model.
5. Ad-ING4-IL-24 co-expressing ING4 and IL-24 had more potent effect in down-regulating the expression of angiogenesis-related factors VEGF, IL-8, and CD34, which may be another important mechanism involved in its synergetic effect in suppressing SMMC-7721 human hepatocellular carcinoma transplanted tumor growth in vivo in athymic nude mouse model.
6. Ad-ING4-IL-24 had more potent effect in suppressing in vitro SMMC-7721 human hepatocellular carcinoma cell’s invasiveness, which may be associated with down-regulating the expression of metastasis-related factors MMP-2 and MMP-9.
引文
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