Mcl-1特异性shRNA联合凋亡素治疗肝细胞癌的实验研究
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摘要
原发性肝癌是全球第5大恶性肿瘤，在肿瘤相关死亡中排在第3位。手术切除和肝移植仍是当前可能治愈肝癌的主要方法，但绝大多数患者被确诊时已是晚期，失去手术时机。此外，现有的化疗药物并不能提高肝癌患者的生存率，因此，探求新的更有效的肝癌治疗方法是当前迫切需要解决的问题。
髓细胞白血病基因-1(myeloid cell leukemia-1 mcl-1)是bcl-2家族成员，其主要作用是促进细胞生存，抑制细胞凋亡。Mcl-1在肝癌组织中明显呈高表达状态，被认为与肝癌的发生密切相关。凋亡素(apoptin)是鸡贫血病毒VP3基因编码的蛋白，能选择性地诱导人转化细胞和肿瘤细胞凋亡，但对正常细胞无诱导凋亡作用。
RNA干扰(RN Ain terference，RNAi)技术是一种能够有效抑制目的基因表达的基因治疗工具，已被广泛应用于目的基因功能研究和肿瘤的基因治疗中。本研究针对mcl-1mRNA设计、构建并筛选出表达小发卡状RNA(short hairpin RNA，shRNA)的重组表达质粒，观察mcl-1特异性shRNA对肝癌细胞凋亡的影响，以及与凋亡素联合对肝癌增殖和凋亡的作用，为肝癌的治疗提供新的策略。
第一部分靶向mcl-1基因的shRNA真核表达质粒的构建与鉴定
目的设计并构建靶向mcl-1基因并含有EGFP基因的shRNA表达质粒。
方法根据shRNA的设计原则、载体pGenesil-1.1的酶切位点以及Gengank中mcl-1mRNA序列，设计并合成3对编码shRNA的寡核苷酸链，将寡核苷酸链退火形成互补双链，再克隆至载体pGenesil-1中，构建重组质粒mcl-1.1～mcl-1.3，将质粒转化DH5α菌株，提取质粒，行酶切鉴定和DNA测序鉴定。
结果所构建的重组质粒mcl-1.1～mcl-1.3经酶切鉴定及DNA测序证实符合设计要求，证实质粒mcl-1.1～mcl-1.3构建成功。
结论成功构建了靶向人mcl-1基因shRNA表达质粒，为一步利用shRNA表达质粒进行肝细胞癌治疗研究奠定了基础。
第二部分Mcl-1特异性shRNA的筛选及其对肝癌细胞凋亡的影响
目的从所构建的3个shRNA质粒中筛选出对mcl-1基因抑制作用最强的质粒，并观察该质粒对肝癌细胞HepG2凋亡的影响。
方法经Lipofectamine 2000介导将所构建的重组质粒mcl-1.1～mcl-1.3及阴性对照质粒HK转染HepG2。转染后48 h于荧光显微下观察并计算质粒的转染率，并分别利用RT-PCR和western blot检测细胞内mcl-1 mRNA和蛋白，确定3种质粒对mcl-1基因的抑制效率，筛选出对mcl-1基因抑制作用最强的质粒。采用Hoechst染色观察所选质粒转染HepG2细胞48 h后的凋亡形态学改变，及PI单染行流式细胞仪检测HepG2的凋亡率。
结果重组质粒mcl-1.1～mcl-1.3和阴性对照质粒HK对HepG2的转染率分别为64.00％±3.77％、64.20％±2.10％、63.70％±3.34％和62.5％±2.42％，各组间转染率无明显差异(P=0.59)。质粒mel-1.1～mcl-1.3组mcl-1 mRNA的相对含量分别为0.61±0.02、0.56±0.02和0.46±0.01，mcl-1蛋白的相对含量分别为0.54±0.01、0.48±0.03、0.36±0.01，与HK(mRNA：0.95±0.00，蛋白：0.88±0.01)和空白对照组(mRNA：0.97±0.01，蛋白：0.90±0.03)相比均存在显著性差异(均P=0.00)。质粒Mcl-1.3对mcl-1mRNA和蛋白的抑制率分别为52.27±0.00％和58.98±0.01％，均高于mcl-1.1(mRNA：36.26±0.12％，蛋白：38.80±0.02％)和mcl-1.2(mRNA：41.47±0.13％，蛋白：45.70±0.02％) (均P=0.00)。转染mcl-1.3的细胞经Hoechst染色出现明显凋亡核形态学表现。经流式检测，mcl-1.3转染组HepG2的凋亡率明显高于HK组(7.74％±0.97％VS 2.81％±0.46％P=0.00)
结论成功筛选出靶向mcl-1基因的shRNA真核表达质粒，其对肝癌细胞HepG2内的mcl-1基因表达具有明显抑制作用，并可直接导致肝癌细胞凋亡。利用shRNA沉默Mcl-1基因可能是肝癌基因治疗的有效途径之一。
第三部分Mcl-1特异性shRNA联合凋亡素对肝癌细胞增殖和凋亡的影响
目的观察mcl-1特异性shRNA与凋亡素联合作用对肝癌细胞增殖和凋亡的影响。
方法利用质粒mcl-1.3和pGensil-1.2构建靶向mcl-1基因但不含有EGFP基因的shRNA表达质粒pmcl-1，然后将质粒pmcl-1和pcDNA3.0-vp3转入肝癌细胞HepG2内，利用RT-PCR和western blot检测细胞内mcl-1和vp3的mRNA与蛋白含量及细胞色素C含量，采用MTT法测定细胞增殖情况，Annexin V-FITC／PI双染检测细胞的凋亡率。
结果所构建的重组质粒pmcl-1经酶切及DNA测序证实符合设计要求，证实构建成功。经RT-PCR和western blot检测显示，pmcl-1+pcDNA3.0-vp3转染组和pcDNA3.0-vp3转染组vp3阳性，pmcl-1转染组及阴性对照组和空白对照组vp3均为阴性。pmcl-1+pcDNA3.0-vp3组的vp3 mRNA含量为0.83±0.02，蛋白含量为1.48±0.06，与pcDNA3.0-vp3组相比无明显差异(mRNA：0.78±0.37，P=0.20；蛋白：1.40±0.08，P=0.81)。质粒pmcl-1、pcDNA3.0-vp3及pmcl-1+pcDNA3.0-vp3均可明显下调细胞内mcl-lmRNA和蛋白水平，促进线粒体细胞色素C的释放。其中pmcl-1+pcDNA3.0-vp3组的mcl-1 mRNA含量为0.34±0.05，蛋白含量为0.38±0.02，与pmcl-1组(mRNA：0.46±0.03，蛋白0.44±0.03)和pcDNA3.0-vp3组(mRNA：0.43±0.01，蛋白：0.43±0.01)相比，差异均有显著性(均P＜0.05)。pmcl-1+pcDNA3.0-vp3组的细胞色素C含量为1.21±0.09，明显高于pmcl-1组(0.96±0.03，P=0.01)和pcDNA3.0-vp3组(0.98±0.02，P=0.01)。与阴性对照及空白对照相比，质粒pmcl-1、pcDNA3.0-vp3和pmcl-1+pcDNA3.0-vp3处理组的HepG2细胞在转染后1～5天的生长均明显受到抑制，其中质粒pcDNA3.0-vp3对HepG2的生长抑制作用在各时间点均强于pmcl-1(均P=0.00)，pmcl-1联合pcDNA3.0-vp3对HepG2生长的抑制作用较两者单独作用时均强(均P＜0.05)。质粒pmcl-1、pcDNA3.0-vp3及pmcl-1+pcDNA3.0-vp3均可诱导HepG2的凋亡，其中质粒pcDNA3.0-vp3转染24h、48h和72h的凋亡率分别为12.00±1.57％、22.1±2.82％和41.80±3.37％，均高于相应时间pmcl-1组的凋亡率(分别为8.60±0.78％，P=0.03；11.07±0.95％，P=0.03；15.00±1.44％，P=0.00)，而pmcl-1联合pcDNA3.0-vp3转染HepG2 24h、48h和72h的凋亡率分别为17.10±1.04％、29.87±4.59％和52.40±1.73％，均较两者单独作用时均高(均P＜0.05)。
结论Mcl-1特异性shRNA与凋亡素有协同作用，两者联合可增强对肝癌细胞生长的抑制，增加肝癌细胞的凋亡，是治疗肝癌的有效方法之一。凋亡素诱导的肝癌细胞凋亡与其抑制mcl-1基因，促进线粒体细胞色素C的释放有关。
Hepatocellular carcinoma (HCC) is the 5th most common cancer and the 3~(rd) mostcommon cause of cancer-related mortality world wide. The tumour might be curable byresection or liver transplantation.Most patients with HCC show dvanced-stage tumor at thetime of diagnosis, and surgical treatment can only be achieved in a minority of patients.Inaddation, systemic chemotherapy has never been shown to improve survival in patientswith HCC. Therefore, new treatment regimens for patients with HCC are needed badly.
Myeloid cell leukemia-1 (mcl-1) is a member of bcl-2 family. The main function ofmcl-1 is to promot cell survival and protect cells from apoptosis. Mcl-1 is overexpressed intissues of HCC and contributes to the developmen of HCC.Apoptin, a protein encoded by thechicken anemia virus (CAV) VP3 gene, has the ability to selectively induce apoptosis intransformed and malignant cells but not in normal cells.
RNA interference(RNAi) is a potent molecular biological tool in degrading expressionof aimed genes and has been applied extensively in studies of anti- tumor and gene function.In our researches,we constructed three recombination plasmids encoding shorthairpinRNA(shRNA) targeting mcl-1 gene and selected one that supresses the expressing ofmcl-1 gene most efficiently.The apoptosis of HepG2 cells induced by the plasmid wasinvestigated.Then the effcet of combination of the plasmid with apoptin on the proliferationand apoptosis of HepG2 cells was studied. Therefore, an effective therapeutic strategy forhepatocellular carcinoma will be initiated.
PartⅠConstruction and identification of eukaryotic expression plasmidsencoding the short hairpin RNA targeting Mcl-1 gene
Objective To design and construct short hairpin RNA(shRNA) eukaryotic expressionplasmids containing EGFP gene and targeting mcl-1 gene. Methods According to theprinciple of shRNA design and the restriction enzyme cutting site of pGenesil-1.1 vector,3pairs of shRNA oligonucleotide fragments were designed and synthesized based on thesequence of mcl-1mRNA in GenBank. Double strands were formed after annealing andinserted into pGenesil-1.1 vetor. The recombinant were named as mcl-1.1～mcl-1.3. Therecombinant plasmid were transformed into DH5α,The plasmids were extracted and identificated by restrictive enzyme digestion andsequencing analysis.Results The restriction enzyme digestion demonstrated that shRNAwas inserted into vector correctly, and sequencing analysis demonstrated that theirsequences were the same as the design. Conclusion The shRNA eukaryotic expressionplasmids targeting mcl-1 gene are constructed successfully. This lays a solid foundation forthe further research on the therapy of hepatocellular carcinoma with shRNA expressionplasmids targeting mcl-1 gene.
PartⅡThe screening of shRNA targeting mcl-1 gene and it's effect onapoptosis of hepatoeellular carcinima
Objective To select the plasmid that inhibits the expressing of mcl-1 gene mostefficiently,and to investigate the effect of the plasmid on the apoptosis of HepG2 cells.Methods Plasmids mcl-1.1～mcl-1.3 and negative control plasmid HK weretransfected into HepG2 cells via Lipofectamine 2000. Fourty-eight hours after transfection,the transfection rate of the plasmids was determined by using fluorescence microscopy.Theexpression levels of mcl-1 mRNA and protein were assayed by reverse transcriptase-polymerasechain reaction and Western blotting.The plasmid that silence mcl-1 gene mostefficiently was selected according to the levels of mcl-1 mRNA and protein. Fourty-eighthours after the selected plasmid transfected into HepG2, karyomorphological diversify ofapoptotic cells was detected by Hoechst staining. The apoptosis condition of the cells wasanalyzed by flow cytometry with PI single staining. Results The transfection rate of theplasmids mcl-1.1～mcl-1.3 and HK in HepG2 cells was 64.00%±3.77%, 64.20%±2.10%,63.70%±3.34% and62.5%±2.42% respectively (P=0.59).The mcl-1 mRNA and proteinlevels of mcl-1.1～mcl-1.3 group (mRNA: 0.61±0.02, 0.56±0.02 and 0.46±0.01, protein:0.54±0.01、0.48±0.03、0.36±0.01, respectively) were significantly lower than that of theblank control (mRNA: 0.97±0.01;protein: 0.90±0.03, all P=0.00) and negative control(mRNA: 0.95±0.00, protein: 0.88±0.01, all P=0.00). Compared with mcl-1.1 and mcl-1.2,mcl-1.3 had the strongest inhibitory effect on mcl-1 mRNA (52.27±0.00% vs 36.26±0.12%,41.47±0.13%,both P=0.00) and protein (58.98±0.01% vs 38.80±0.02%, 45.70±0.02 %, bothP=0.00). Karyomorphological diversify of apoptotic cells were obviously observed inmcl-1.3 group by hoechst33258 staining. The apoptosis rate of mcl-1.3 group was higherthan that of negative control group (7.74%±0.97% vs 2.81%±0.46%,P=0.00).ConclusionThe shRNA eukaryotic expression plasmid targeting at mcl-1 gene is selected successfully.The mcl-1 gene expression is suppressed significantly by the plasmid. The apoptosis ofHepG2 is promoted by the plasmid through silencing the expressing of mcl-1 gene.Silencing mcl-1 gene by shRNA maybe a potential approach for HCC gene therapy.
PartⅢThe effect of combination of the shRNA targeting mcl-1 gene withapoptin on proliferation and apoptosis of hepatocellular carcinoma
Objective To investigate the effect of combination of the shRNA targeting mcl-1 genewith apoptin on proliferation and apoptosis of hepatocellular carcinoma. Methods Plasmidpmcl-1 targeting mcl-1 gene, without EGFP gene, was constructed based on the plasmidsmcl-1.3 and pGensil-1.2. The plasmids pmcl-1 and pcDNA3.0-vp3 were transfected intoHepG2 cells.The mRNA and protein levels of mcl-1 and vp3 were analyzed by RT-PCRand western blot respectively. Cytochrome C was also assayed by western blot. Theproliferation of cells was measured by methyl thiazolyl tetrazolium(MTT), the apoptosiscondition of the cells was analyzed by flow cytometry with Annexin V V-FITC/PI doublestaining. Results The restriction enzyme digestion and DNA sequencing analysisdemonstrated that the recombinant plasmid pmcl-1 was constructed successfully. Analyzedby RT-PCR and western blot, The mRNA and protein of vp3 were positive in the group ofpmcl-1+pcDNA3.0-vp3 and pcDNA3.0-vp3,while negative in the group of pmcl-1,negative control and blank control. The vp3 mRNA and protein levels ofpmcl-1+pcDNA3.0-vp3 group was no difference with that of pcDNA3.0-vp3 group(mRNA:0.83±0.02 vs 0.78±0.37,P=0.20; protein:1.48±0.06 vs 1.40±0.08, P=0.81).The plasmidspmcl-1,pcDNA3.0-vp3 and pmcl-1+ pcDNA3.0-vp3 all had the potential to down-regulatemRNA and protein levels of mcl-1.They also promoted cytochrome C release frommitochondrion. The mcl-1 mRNA and protein level of pmcl-1+ pcDNA3.0-vp3 group(mRNA: 0.34±0.05,protein: 0.38±0.02) were significantly lower than that of pmcl-1 group(mRNA:0.46±0.03,protein:0.44±0.03) and pcDNA3.0-vp3 group (mRNA:0.43±0.01,protein: 0.43±0.01)(all P＜0.05).The cytochrome C level of pmcl-1+ pcDNA3.0-vp3group(1.21±0.09) was higher than that of pmcl-1 group (0.96±0.03,P=0.01) and pcDNA3.0-vp3 group(0.98±0.02, P=0.01). Compaired with negative and blank control, theproliferation of HepG2 at 1～5 d after transfection was significantly supressed by pmcl-1,vp3 and pmcl-1+vp3 (P＜0.05). Viable cells at 1～5d after treated with pcDNA3.0-vp3 wereless than that treated with pmcl-1 and more than that treated with pmcl-1+pcDNA3.0-vp3.Compaired with negative and blank control, the apoptosis of HepG2 cells was promoted byplasmid pmcl-1, pcDNA3.0-vp3 and pmcl-1+ pcDNA3.0-vp3(P＜0.05). The apoptosis at24h,48h and 72h after transfected with pcDNA3.0-vp3 (12.00±1.57%, 22.1±2.82% and41.80±3.37% respectively) was higher than that transfected with pmc1-1(8.60±0.78%,P=0.03; 11.07±0.95%, P=0.03;15.00±1.44%, P=0.00 respectively).While the apoptosis at24h,48h and 72h of pmcl-1+ pcDNA3.0-vp3 group (17.10±1.04%, 29.87±4.59% and52.40±1.73% respectively) was higher than that of pmcl-1 group andpcDNA3.0-vp3(P＜0.05). Conclusion The proliferation of HepG2 is suppressed and theapoptosis of HepG2 is enhanced by the shRNA targeting mcl-1 gene combined withapoptin. The shRNA targeting mcl-1 gene combined with apoptin is a potential approachfor HCC therapy.The apoptosis induced by apoptin is correlate with the inhibition of mcl-1gene expressing and the releasing of cytochrome C from mitochondrion.
髓细胞白血病基因-1(myeloid cell leukemia-1 mcl-1)是bcl-2家族成员，其主要作用是促进细胞生存，抑制细胞凋亡。Mcl-1在肝癌组织中明显呈高表达状态，被认为与肝癌的发生密切相关。凋亡素(apoptin)是鸡贫血病毒VP3基因编码的蛋白，能选择性地诱导人转化细胞和肿瘤细胞凋亡，但对正常细胞无诱导凋亡作用。
RNA干扰(RN Ain terference，RNAi)技术是一种能够有效抑制目的基因表达的基因治疗工具，已被广泛应用于目的基因功能研究和肿瘤的基因治疗中。本研究针对mcl-1mRNA设计、构建并筛选出表达小发卡状RNA(short hairpin RNA，shRNA)的重组表达质粒，观察mcl-1特异性shRNA对肝癌细胞凋亡的影响，以及与凋亡素联合对肝癌增殖和凋亡的作用，为肝癌的治疗提供新的策略。
第一部分靶向mcl-1基因的shRNA真核表达质粒的构建与鉴定
目的设计并构建靶向mcl-1基因并含有EGFP基因的shRNA表达质粒。
方法根据shRNA的设计原则、载体pGenesil-1.1的酶切位点以及Gengank中mcl-1mRNA序列，设计并合成3对编码shRNA的寡核苷酸链，将寡核苷酸链退火形成互补双链，再克隆至载体pGenesil-1中，构建重组质粒mcl-1.1～mcl-1.3，将质粒转化DH5α菌株，提取质粒，行酶切鉴定和DNA测序鉴定。
结果所构建的重组质粒mcl-1.1～mcl-1.3经酶切鉴定及DNA测序证实符合设计要求，证实质粒mcl-1.1～mcl-1.3构建成功。
结论成功构建了靶向人mcl-1基因shRNA表达质粒，为一步利用shRNA表达质粒进行肝细胞癌治疗研究奠定了基础。
第二部分Mcl-1特异性shRNA的筛选及其对肝癌细胞凋亡的影响
目的从所构建的3个shRNA质粒中筛选出对mcl-1基因抑制作用最强的质粒，并观察该质粒对肝癌细胞HepG2凋亡的影响。
方法经Lipofectamine 2000介导将所构建的重组质粒mcl-1.1～mcl-1.3及阴性对照质粒HK转染HepG2。转染后48 h于荧光显微下观察并计算质粒的转染率，并分别利用RT-PCR和western blot检测细胞内mcl-1 mRNA和蛋白，确定3种质粒对mcl-1基因的抑制效率，筛选出对mcl-1基因抑制作用最强的质粒。采用Hoechst染色观察所选质粒转染HepG2细胞48 h后的凋亡形态学改变，及PI单染行流式细胞仪检测HepG2的凋亡率。
结果重组质粒mcl-1.1～mcl-1.3和阴性对照质粒HK对HepG2的转染率分别为64.00％±3.77％、64.20％±2.10％、63.70％±3.34％和62.5％±2.42％，各组间转染率无明显差异(P=0.59)。质粒mel-1.1～mcl-1.3组mcl-1 mRNA的相对含量分别为0.61±0.02、0.56±0.02和0.46±0.01，mcl-1蛋白的相对含量分别为0.54±0.01、0.48±0.03、0.36±0.01，与HK(mRNA：0.95±0.00，蛋白：0.88±0.01)和空白对照组(mRNA：0.97±0.01，蛋白：0.90±0.03)相比均存在显著性差异(均P=0.00)。质粒Mcl-1.3对mcl-1mRNA和蛋白的抑制率分别为52.27±0.00％和58.98±0.01％，均高于mcl-1.1(mRNA：36.26±0.12％，蛋白：38.80±0.02％)和mcl-1.2(mRNA：41.47±0.13％，蛋白：45.70±0.02％) (均P=0.00)。转染mcl-1.3的细胞经Hoechst染色出现明显凋亡核形态学表现。经流式检测，mcl-1.3转染组HepG2的凋亡率明显高于HK组(7.74％±0.97％VS 2.81％±0.46％P=0.00)
结论成功筛选出靶向mcl-1基因的shRNA真核表达质粒，其对肝癌细胞HepG2内的mcl-1基因表达具有明显抑制作用，并可直接导致肝癌细胞凋亡。利用shRNA沉默Mcl-1基因可能是肝癌基因治疗的有效途径之一。
第三部分Mcl-1特异性shRNA联合凋亡素对肝癌细胞增殖和凋亡的影响
目的观察mcl-1特异性shRNA与凋亡素联合作用对肝癌细胞增殖和凋亡的影响。
方法利用质粒mcl-1.3和pGensil-1.2构建靶向mcl-1基因但不含有EGFP基因的shRNA表达质粒pmcl-1，然后将质粒pmcl-1和pcDNA3.0-vp3转入肝癌细胞HepG2内，利用RT-PCR和western blot检测细胞内mcl-1和vp3的mRNA与蛋白含量及细胞色素C含量，采用MTT法测定细胞增殖情况，Annexin V-FITC／PI双染检测细胞的凋亡率。
结果所构建的重组质粒pmcl-1经酶切及DNA测序证实符合设计要求，证实构建成功。经RT-PCR和western blot检测显示，pmcl-1+pcDNA3.0-vp3转染组和pcDNA3.0-vp3转染组vp3阳性，pmcl-1转染组及阴性对照组和空白对照组vp3均为阴性。pmcl-1+pcDNA3.0-vp3组的vp3 mRNA含量为0.83±0.02，蛋白含量为1.48±0.06，与pcDNA3.0-vp3组相比无明显差异(mRNA：0.78±0.37，P=0.20；蛋白：1.40±0.08，P=0.81)。质粒pmcl-1、pcDNA3.0-vp3及pmcl-1+pcDNA3.0-vp3均可明显下调细胞内mcl-lmRNA和蛋白水平，促进线粒体细胞色素C的释放。其中pmcl-1+pcDNA3.0-vp3组的mcl-1 mRNA含量为0.34±0.05，蛋白含量为0.38±0.02，与pmcl-1组(mRNA：0.46±0.03，蛋白0.44±0.03)和pcDNA3.0-vp3组(mRNA：0.43±0.01，蛋白：0.43±0.01)相比，差异均有显著性(均P＜0.05)。pmcl-1+pcDNA3.0-vp3组的细胞色素C含量为1.21±0.09，明显高于pmcl-1组(0.96±0.03，P=0.01)和pcDNA3.0-vp3组(0.98±0.02，P=0.01)。与阴性对照及空白对照相比，质粒pmcl-1、pcDNA3.0-vp3和pmcl-1+pcDNA3.0-vp3处理组的HepG2细胞在转染后1～5天的生长均明显受到抑制，其中质粒pcDNA3.0-vp3对HepG2的生长抑制作用在各时间点均强于pmcl-1(均P=0.00)，pmcl-1联合pcDNA3.0-vp3对HepG2生长的抑制作用较两者单独作用时均强(均P＜0.05)。质粒pmcl-1、pcDNA3.0-vp3及pmcl-1+pcDNA3.0-vp3均可诱导HepG2的凋亡，其中质粒pcDNA3.0-vp3转染24h、48h和72h的凋亡率分别为12.00±1.57％、22.1±2.82％和41.80±3.37％，均高于相应时间pmcl-1组的凋亡率(分别为8.60±0.78％，P=0.03；11.07±0.95％，P=0.03；15.00±1.44％，P=0.00)，而pmcl-1联合pcDNA3.0-vp3转染HepG2 24h、48h和72h的凋亡率分别为17.10±1.04％、29.87±4.59％和52.40±1.73％，均较两者单独作用时均高(均P＜0.05)。
结论Mcl-1特异性shRNA与凋亡素有协同作用，两者联合可增强对肝癌细胞生长的抑制，增加肝癌细胞的凋亡，是治疗肝癌的有效方法之一。凋亡素诱导的肝癌细胞凋亡与其抑制mcl-1基因，促进线粒体细胞色素C的释放有关。
Hepatocellular carcinoma (HCC) is the 5th most common cancer and the 3~(rd) mostcommon cause of cancer-related mortality world wide. The tumour might be curable byresection or liver transplantation.Most patients with HCC show dvanced-stage tumor at thetime of diagnosis, and surgical treatment can only be achieved in a minority of patients.Inaddation, systemic chemotherapy has never been shown to improve survival in patientswith HCC. Therefore, new treatment regimens for patients with HCC are needed badly.
Myeloid cell leukemia-1 (mcl-1) is a member of bcl-2 family. The main function ofmcl-1 is to promot cell survival and protect cells from apoptosis. Mcl-1 is overexpressed intissues of HCC and contributes to the developmen of HCC.Apoptin, a protein encoded by thechicken anemia virus (CAV) VP3 gene, has the ability to selectively induce apoptosis intransformed and malignant cells but not in normal cells.
RNA interference(RNAi) is a potent molecular biological tool in degrading expressionof aimed genes and has been applied extensively in studies of anti- tumor and gene function.In our researches,we constructed three recombination plasmids encoding shorthairpinRNA(shRNA) targeting mcl-1 gene and selected one that supresses the expressing ofmcl-1 gene most efficiently.The apoptosis of HepG2 cells induced by the plasmid wasinvestigated.Then the effcet of combination of the plasmid with apoptin on the proliferationand apoptosis of HepG2 cells was studied. Therefore, an effective therapeutic strategy forhepatocellular carcinoma will be initiated.
PartⅠConstruction and identification of eukaryotic expression plasmidsencoding the short hairpin RNA targeting Mcl-1 gene
Objective To design and construct short hairpin RNA(shRNA) eukaryotic expressionplasmids containing EGFP gene and targeting mcl-1 gene. Methods According to theprinciple of shRNA design and the restriction enzyme cutting site of pGenesil-1.1 vector,3pairs of shRNA oligonucleotide fragments were designed and synthesized based on thesequence of mcl-1mRNA in GenBank. Double strands were formed after annealing andinserted into pGenesil-1.1 vetor. The recombinant were named as mcl-1.1～mcl-1.3. Therecombinant plasmid were transformed into DH5α,The plasmids were extracted and identificated by restrictive enzyme digestion andsequencing analysis.Results The restriction enzyme digestion demonstrated that shRNAwas inserted into vector correctly, and sequencing analysis demonstrated that theirsequences were the same as the design. Conclusion The shRNA eukaryotic expressionplasmids targeting mcl-1 gene are constructed successfully. This lays a solid foundation forthe further research on the therapy of hepatocellular carcinoma with shRNA expressionplasmids targeting mcl-1 gene.
PartⅡThe screening of shRNA targeting mcl-1 gene and it's effect onapoptosis of hepatoeellular carcinima
Objective To select the plasmid that inhibits the expressing of mcl-1 gene mostefficiently,and to investigate the effect of the plasmid on the apoptosis of HepG2 cells.Methods Plasmids mcl-1.1～mcl-1.3 and negative control plasmid HK weretransfected into HepG2 cells via Lipofectamine 2000. Fourty-eight hours after transfection,the transfection rate of the plasmids was determined by using fluorescence microscopy.Theexpression levels of mcl-1 mRNA and protein were assayed by reverse transcriptase-polymerasechain reaction and Western blotting.The plasmid that silence mcl-1 gene mostefficiently was selected according to the levels of mcl-1 mRNA and protein. Fourty-eighthours after the selected plasmid transfected into HepG2, karyomorphological diversify ofapoptotic cells was detected by Hoechst staining. The apoptosis condition of the cells wasanalyzed by flow cytometry with PI single staining. Results The transfection rate of theplasmids mcl-1.1～mcl-1.3 and HK in HepG2 cells was 64.00%±3.77%, 64.20%±2.10%,63.70%±3.34% and62.5%±2.42% respectively (P=0.59).The mcl-1 mRNA and proteinlevels of mcl-1.1～mcl-1.3 group (mRNA: 0.61±0.02, 0.56±0.02 and 0.46±0.01, protein:0.54±0.01、0.48±0.03、0.36±0.01, respectively) were significantly lower than that of theblank control (mRNA: 0.97±0.01;protein: 0.90±0.03, all P=0.00) and negative control(mRNA: 0.95±0.00, protein: 0.88±0.01, all P=0.00). Compared with mcl-1.1 and mcl-1.2,mcl-1.3 had the strongest inhibitory effect on mcl-1 mRNA (52.27±0.00% vs 36.26±0.12%,41.47±0.13%,both P=0.00) and protein (58.98±0.01% vs 38.80±0.02%, 45.70±0.02 %, bothP=0.00). Karyomorphological diversify of apoptotic cells were obviously observed inmcl-1.3 group by hoechst33258 staining. The apoptosis rate of mcl-1.3 group was higherthan that of negative control group (7.74%±0.97% vs 2.81%±0.46%,P=0.00).ConclusionThe shRNA eukaryotic expression plasmid targeting at mcl-1 gene is selected successfully.The mcl-1 gene expression is suppressed significantly by the plasmid. The apoptosis ofHepG2 is promoted by the plasmid through silencing the expressing of mcl-1 gene.Silencing mcl-1 gene by shRNA maybe a potential approach for HCC gene therapy.
PartⅢThe effect of combination of the shRNA targeting mcl-1 gene withapoptin on proliferation and apoptosis of hepatocellular carcinoma
Objective To investigate the effect of combination of the shRNA targeting mcl-1 genewith apoptin on proliferation and apoptosis of hepatocellular carcinoma. Methods Plasmidpmcl-1 targeting mcl-1 gene, without EGFP gene, was constructed based on the plasmidsmcl-1.3 and pGensil-1.2. The plasmids pmcl-1 and pcDNA3.0-vp3 were transfected intoHepG2 cells.The mRNA and protein levels of mcl-1 and vp3 were analyzed by RT-PCRand western blot respectively. Cytochrome C was also assayed by western blot. Theproliferation of cells was measured by methyl thiazolyl tetrazolium(MTT), the apoptosiscondition of the cells was analyzed by flow cytometry with Annexin V V-FITC/PI doublestaining. Results The restriction enzyme digestion and DNA sequencing analysisdemonstrated that the recombinant plasmid pmcl-1 was constructed successfully. Analyzedby RT-PCR and western blot, The mRNA and protein of vp3 were positive in the group ofpmcl-1+pcDNA3.0-vp3 and pcDNA3.0-vp3,while negative in the group of pmcl-1,negative control and blank control. The vp3 mRNA and protein levels ofpmcl-1+pcDNA3.0-vp3 group was no difference with that of pcDNA3.0-vp3 group(mRNA:0.83±0.02 vs 0.78±0.37,P=0.20; protein:1.48±0.06 vs 1.40±0.08, P=0.81).The plasmidspmcl-1,pcDNA3.0-vp3 and pmcl-1+ pcDNA3.0-vp3 all had the potential to down-regulatemRNA and protein levels of mcl-1.They also promoted cytochrome C release frommitochondrion. The mcl-1 mRNA and protein level of pmcl-1+ pcDNA3.0-vp3 group(mRNA: 0.34±0.05,protein: 0.38±0.02) were significantly lower than that of pmcl-1 group(mRNA:0.46±0.03,protein:0.44±0.03) and pcDNA3.0-vp3 group (mRNA:0.43±0.01,protein: 0.43±0.01)(all P＜0.05).The cytochrome C level of pmcl-1+ pcDNA3.0-vp3group(1.21±0.09) was higher than that of pmcl-1 group (0.96±0.03,P=0.01) and pcDNA3.0-vp3 group(0.98±0.02, P=0.01). Compaired with negative and blank control, theproliferation of HepG2 at 1～5 d after transfection was significantly supressed by pmcl-1,vp3 and pmcl-1+vp3 (P＜0.05). Viable cells at 1～5d after treated with pcDNA3.0-vp3 wereless than that treated with pmcl-1 and more than that treated with pmcl-1+pcDNA3.0-vp3.Compaired with negative and blank control, the apoptosis of HepG2 cells was promoted byplasmid pmcl-1, pcDNA3.0-vp3 and pmcl-1+ pcDNA3.0-vp3(P＜0.05). The apoptosis at24h,48h and 72h after transfected with pcDNA3.0-vp3 (12.00±1.57%, 22.1±2.82% and41.80±3.37% respectively) was higher than that transfected with pmc1-1(8.60±0.78%,P=0.03; 11.07±0.95%, P=0.03;15.00±1.44%, P=0.00 respectively).While the apoptosis at24h,48h and 72h of pmcl-1+ pcDNA3.0-vp3 group (17.10±1.04%, 29.87±4.59% and52.40±1.73% respectively) was higher than that of pmcl-1 group andpcDNA3.0-vp3(P＜0.05). Conclusion The proliferation of HepG2 is suppressed and theapoptosis of HepG2 is enhanced by the shRNA targeting mcl-1 gene combined withapoptin. The shRNA targeting mcl-1 gene combined with apoptin is a potential approachfor HCC therapy.The apoptosis induced by apoptin is correlate with the inhibition of mcl-1gene expressing and the releasing of cytochrome C from mitochondrion.
引文
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