肝脏特异性表达人遗传印记基因PEG10转基因小鼠肝癌模型的建立
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摘要
目的原发性肝细胞癌(Hepatocellular carcinoma，HCC)是一种全球性高度恶性肿瘤，发病率、死亡率居高不下，严重影响人类健康。早期发现和切除肿瘤是提高肝癌患者长期生存率的重要因素。肝癌的发生是多病因、多基因、多阶段的复杂过程。导致发病的危险因素主要有：病毒感染、肝硬化、黄曲霉素、化学致癌物及寄生虫感染，过量摄入酒精等。目前认为，肝癌的发病机制与癌基因的激活，抑癌基因和DNA修复基因的失活有关。其中肿瘤相关基因启动子区域CpG岛的甲基化、印记缺失等表观遗传学改变在癌变中发挥重要作用。但是导致肝癌发病和进展的具体分子机制尚不清楚，影响了肝癌的早期临床诊断。大多数肝癌患者在发现症状时，已届病程的中晚期阶段，伴有不同程度的肝内、肝外转移，错过了手术治疗的最佳时机。寻找肝癌发病相关的特异性分子靶是早期诊断肝癌，特别是甲胎蛋白水平正常的肝癌和小肝癌迫切需要解决的问题。PEG10(paternally expressed gene 10)是一个父方表达／母方印记的遗传印记基因(genetic imprinting gene)。有研究表明：PEG10基因在大多数肝癌组织、血清甲胎蛋白水平正常的肝癌和小肝癌中有高表达；处于G2／M期的小鼠再生肝脏组织中也检测到PEG10的表达。这些结果提示我们：PEG10在肝脏再生和人肝细胞肿瘤发生中起着重要作用，阐明PEG10异常表达的分子基础有助于了解肝脏疾病的发病机制。白蛋白是肝脏表达的血清蛋白，由分化成熟的肝细胞合成，其他组织细胞中的白蛋白基因处于关闭状态。白蛋白基因(albumin gene，ALB)启动子序列(ALBpromoter，AGP)因高度保守性和包含的肝脏特异性表达元件，使组织特异性表达成为可能。自1989年Izban克隆了小鼠AGP序列，其在转基因动物及基因治疗中得到了广泛的应用，实现了基因转移的器官靶向性。本课题借助分子生物学方法构建包含有AGP和PEG10基因片段的转基因载体，制备肝脏特异性表达PEG10的转基因小鼠。采用酒精灌胃的方法处理各组小鼠，观察PEG10基因过表达是否会诱发其产生肝癌，增强罹患肿瘤的易感性。进一步探讨PEG10基因的生物学功能以及其作为肝癌基因治疗新靶点的可能性。方法提取人肝癌细胞HepG2总RNA，用RT-PCR方法获得PEG10基因全长DNA片段，与Simple T载体连接。经过酶切、测序鉴定后将PEG10插入到含有CMV启动子的真核表达载体pEGFP-N1中，构建重组质粒pEGFP-PEG10。pEGFP-PEG10转染L02细胞，经G418抗性筛选，挑选阳性克隆并扩大培养，阳性细胞命名为L02／PEG10，未转染任何质粒的正常L02细胞和转染空载体pEGFP-N1的L02细胞(L02／vector)为阴性对照组细胞，以表达内源性PEG10基因的HepG2细胞为阳性对照细胞。RT-PCR法检测各组细胞中PEG10 mRNA的表达水平，Western Blot及免疫细胞化学法检测PEG10蛋白的表达和亚细胞定位。用50-400mM H_2O_2分别处理L02／PEG10、正常L02细胞和L02／vector 6h、1 2h、24h后，进行hoechst33342染色。荧光显微镜观察细胞核的变化并抽提DNA进行琼脂糖凝胶电泳。提取小鼠外周血基因组DNA，用PCR方法获得AGP片段，与T载体连接。经过酶切、测序鉴定后将AGP片段插入到无启动子的真核表达载体pEGFP-1中，构建重组质粒pAGP-EGFP。pAGP-EGFP分别瞬时转染L02细胞、人宫颈癌细胞Hela、人结肠癌细胞SW480和人胰腺癌细胞Bxpc-3。pEGFP-1和pEGFP-N1分别为阴性对照和阳性对照质粒，转染上述四种细胞。瞬时转染24h、48h、72h、96h后，倒置荧光显微镜观察各组细胞中AGP启动EGFP表达的能力；瞬时转染72h后，流式细胞仪定量分析各组细胞EGFP的相对荧光强度。同时筛选出稳定转染pAGP-EGFP和pEGFP-N1的L02细胞株，流式定量分析AGP转录活性与CMV转录活性的差别。最后将PEG10基因片段连接到AGP的下游，构建转基因载体pAGP-PEG10-EGFP，将pAGP-EGFP作为转基因对照质粒。pAGP-PEG10-EGFP经BamH I、Age I双酶切，pAGP-EGFP经Xba I单酶切后，分别胶回收具有表达功能的线性化元件，经纯化和定量后，采用显微注射法导入小鼠受精卵雄原核，移植入假孕母鼠输卵管内，待其产仔。提取G0代小鼠鼠尾组织DNA，PCR检测目的基因在转基因小鼠中的整合阳性率。观察转基因阳性鼠的皮毛、饮食、精神、活动度等一般情况。若在饲养过程中出现小鼠死亡，则提取其心、肺、肾、胃、小肠、肝脏、脾脏、子宫、卵巢组织总RNA和总蛋白，RT-PCR和Western Blot检测PEG10mRNA和蛋白在上述组织中的表达。HE染色观察肝脏有无病理改变。分别取PEG10转基因阳性鼠、转基因对照鼠和野生型小鼠，给予小剂量酒精(0．3g／kg)灌胃，每天两次。期间观察各组小鼠的一般情况。6个月后，各组动物恢复至正常饲养条件。两个月后处死小鼠，取肝脏组织称重，进行HE染色，检测PEG10转基因小鼠是否产生肝癌。
结果琼脂糖凝胶电泳、酶切及测序结果表明PEG10基因片段扩增无误，并按正确的顺序插入到Simple T载体中。RT-PCR和Western Blot结果显示：PEG10基因在L02／PEG10中顺利实现表达。免疫细胞化学显示：PEG10蛋白主要分布在胞浆中，部分在核膜中表达。经400mM H_2O_2作用24h后，正常L02细胞和L02／vector可见凋亡特有的改变，如胞浆和核固缩、染色质断裂、DNA电泳呈梯状条带，而L02／PEG10未发现上述凋亡特征。酶切与测序鉴定显示：AGP片段完整正确地插入到T载体中。在pEGFP-N1瞬时转染各组细胞的不同时段，荧光显微镜下都能观察到EGFP的高表达：pAGP-EGFP瞬时转染L02细胞72h后，荧光显微镜下观察到EGFP的表达，流式分析显示：AGP启动EGFP表达的相对荧光强度为CMV的1／4，其它转染细胞观察不到EGFP的表达。转染pAGP-EGFP的L02经G418稳定筛选2w后，AGP的转录活性达到与CMV相当的水平。胶回收AGP-PEG10转基因片段后，将其显微注射导入1656枚受精卵中，产仔162只，PCR检测阳性鼠1 3只，转基因整合阳性率为8％。转基因对照小鼠的制备共注射受精卵1782枚，产仔151只，有12只小鼠经PCR检测证实为EGFP基因整合阳性鼠，整合阳性率为7．9％。在饲养过程中，有一只PEG10转基因小鼠死亡，RT-PCR和Western Blot检测到其肝脏内PEG10 mRNA和蛋白的表达，HE染色提示该小鼠的肝脏呈肝细胞癌病理学表现，出现大量异形核细胞，核大深染。其他存活转基因阳性鼠生长状态良好。酒精灌胃结束后，有两只PEG10转基因小鼠死亡，肝脏组织HE染色显示呈肝细胞癌病理表现。剩余10只PEG10转基因小鼠逐渐出现精神欠佳、进食进水量减少、活动减少、行动迟缓、皮毛失去光泽、身体消瘦、疲乏无力的表现。对照组小鼠一般情况正常。处死小鼠后，转基因对照组小鼠和野生型小鼠的肝脏重量分别为(3．98±0．94)g和(3．54±0．72)g，均显著低于PEG10转基因小鼠(8．29±0．76)g，P＜0．05。HE染色结果提示对照组小鼠的肝细胞排列完整致密，细胞间质成分正常。PEG10转基因小鼠中有6只小鼠的肝脏细胞核深染呈蓝色，大小不等，异形性明显，呈低分化癌，其他四只PEG10小鼠的肝细胞周围和小叶中央下静脉周围可见纤维化。结论PEG10基因可抑制H_2O_2诱导的细胞凋亡，低浓度酒精灌胃处理小鼠6个月后，PEG10的过表达可增强试验动物罹患肝癌的易感性，诱发其产生肝癌。本研究一定程度上为PEG10作为肝癌基因诊断和治疗一个新的分子靶点提供了实验依据。
Objective Hepatocellular carcinoma (HCC) is a highly malignant disease worldwide that isassociated with a high morbidity and mortality.It has been the second cancer killer in Chinasince 1990s.Early detection and resection have been generally recognized as the mostimportant factors to achieve long-term survival for HCC patients.HCC is a polygenic and amultiple-step disease among which chronic infection with hepatitis B virus or hepatitis Cvirus,cirrhosis,intake of food contaminated with chemical carcinogens and aflatoxin B,parasitic infections,excessive consumption of alcoholic beverages are considered to be themajor risk factors for HCC development.Present molecular analyses have demonstrated thecausal relationship between HCC and various groups of genes including the activation ofoncogene,inactivation of tumor suppressor gene and DNA repair genes.Among these,methylation of CpG islands in the promoter of tumor-associated gene and loss of imprintingseem to play important roles in the mutation of gene.However,the detailed molecularmechanisms leading to the development and progression of HCC remain unclear.The poorclinical prognosis is largely attributed to most symptomatic HCC patients being diagnosedat the advanced inoperable stage,often accompanied by intra-or/and extra-hepaticmetastasis,which precluding their chance for surgical intervention.It is necessary toidentify new serologic HCC biomarkers that have a sufficient sensitivity and specificity for the diagnosis of HCC,especially in AFP-negative and/or smaller HCC cases.PEG10 is agenetic imprinting gene with an active paternal allele/silent maternal allele.It wasindentified as having an elevated level of expression in majority of the HCC patients,including those with negative serum AFP and small tumor size,and was also induced toexpress during G2/M phase in regenerating mouse liver.This result indicates that theinduction of PEG10 may play an important role in liver regeneration or carcinogenesis ofhuman hepatocyte.Understanding the molecular basis of the abnormal imprinting ofPEG10 will shed new light on the process that leads to liver cancer.Albumin is an abundantprotein in the adult serum.The promoter of albumin was activated in the terminallydifferentiated mature hepatocytes.The high conservation and strong liver-specificregulatory machinery of albumin gene promoter (AGP) cluster makes it possible a suitablepromoter for hepatic specific expression.AGP has been extensively used in transgenic miceto direct foreign gene expression in adult liver,since it was cloned by Izban in 1989.Thegoal of this research was to determine if over-expression of the PEG10 gene in the liveralone is sufficient to increase susceptibility to carcinogenesis.For this purpose,wegenerated lines of transgenic mice expressing PEG10 in the liver under the regulatorycontrol of the albumin promoter.The expression of PEG10 in the liver was quantified byRT-PCR and Western Blot analysis.Body weight and diet,activity were analyzed incontrol and transgenic mice.Control and transgenic mice were given a single intragastricinjection (0.3 g/kg) of ethanol for 6 month.Mice were euthanized at 2 month after ethanolinjection and the total liver weights were recorded.Liver tissues were fixed in 10%formalin.Section were obtained from paraffin-embedded tissue sample,stained withhematoxylin-eosin,and examined under a microscope and photographed.
Methods Firstly,PEG10 was amplified by reverse transcription polymerase chain reaction(RT-PCR) using cDNA synthesized from hepatoma cell line HepG2 total RNA.A pair ofprimers was designed according to the PEG10 cDNA sequence deposited in Genbank,which would produce 1027 bp fiagment with SalⅠand AgeⅠrestriction enzyme sites inserted at 5'and 3'ends.The amplified PEG10 gene fragments were inserted into pMD-18Simple T vectors by T-A cloning.Recombinant plasmid was identified with double enzymedigestion and confirmed by DNA sequencing.Then the eukaryotic expression vector ofPEG10 gene (pPEG10-EGFP) was constructed.Correct recombinant plasmidpPEG10-EGFP was transfected into liver cell line L02 by lipofectamine~(TM)2000.Positivecell clone was screened by culturing in the selective culture medium with G418 and definedas L02/PEG10,while the cell transfected with empty expression vector (pEGFP-N1) wasdefined as L02/vector.L02/vector and parental L02 cells were served as negative control,HepG2,with endogenous expression of PEG 10,as a positive control.RT-PCR and WesternBlot were carried out to detect the expression of target genes.PEG10 protein and itssubcellular localization were detected by immunocytochemistry.The apoptosis of L02 cellswas induced by 50-400 mM H_2O_2.Morphological changes of apoptotic cells weredetermined by fluorescence microscopy using hoechst33342 nuclei staining.DNA fragmentwas observed by agarose gel electrophoresis.Total DNA was extracted from mouse bloodfor PCR amplification of AGP.The amplified fragments were inserted into the multiplecloning sites of pMD-18 T vector and identified using restriction enzymes and sequencing,then cloned into the expression vector pEGFP-1.pAGP-EGFP was transfected into cervicalcarcinoma cell line Hela,L02,colorectal cancer cell line SW480 and pancreaticcarcinoma cell lines Bxpc-3 cells by lipofectamine~(TM)2000,pEGFP-1 and pEGFP-N1 wereserved as a negative and positive control,respectively.The expressions of EGFP weredetected by the fluorescence microscope as well as flow cytometer.Finally,PEG10 genefragments were inserted into the downstream of AGP to construct the liver-specifictransgenic vector (named pAGP-PEG10-EGFP).pAGP-EGFP was served as a negativecontrol.A 1716 bp linearized expression element of pAGP-PEG10-EGFP,which containedAGP and structural gene of PEG10,was purified from agarose gel and adjusted to a finalconcentration of 2μg/ml in TE.KM female mice were hormonally super-ovulated andmated with KM male mice.The fertilized eggs were collected from the oviduct 24h later. Transgenic fragment in TE was microinjected into the pronuclei of fertilized eggs.Then,the injected fertilized eggs were transplanted into the oviduct of pseudo-pregnant KM mice.Control transgenic mouse were established with the same methods by microinjecting 1.5Kbfragments contained AGP-EGFP into the pronuclei of fertilized eggs.All the newborn micewere screened and identified by PCR detecting genomic DNA from tail tissue.The activityand diet of positive transgenic mice were investigated.If the transgenic mouse were dead inthe raising process,the expression of PEG10 gene in the tissues of heart,lung,kidney.uterus,ovary,liver,spleen,stomach,small intestine were examined by RT-PCR andWestern Blot,respectively,In addition to that,liver tissues were fixed in formaldehyde andsectioned.The sections were stained with hematoxylin/eosin and examined undermicroscope to found whether it had tumor-like lesions.Mice were euthanized at 2 monthafter intragastric injection with low concentration alcohol (0.3g/kg) twice a day for sixmonth.the total liver weights were recorded.Liver tissues were fixed in 10% formalin.Section were obtained from paraffin-embedded tissue sample,stained withhematoxylin-eosin,and examined under a microscope and photographed.
Results Agarose gel electrophoresis,restriction enzyme digestion and DNA sequencingshowed that eukaryotic expression pPEG10-T vector harboring PEG10 gene wassuccessfully constructed.PEG10 gene mRNA and protein were expressed in L02/PEG10cells and PEG10 protein was mainly distributed in the cytoplasm and associated with thenuclear membrane.After treatment with 400 mM H_2O_2 for 24 h,distinct morphologiccharacteristic of cell apoptosis such as karyopyknosis and conglomeration were notobserved in L02/PEG10.Ladder-like DNA fragmentation was observed in adose-dependent manner in both L02 and L02/vector cell lines,but not in L02/PEG10.Agarose gel electrophoresis,restriction enzyme digestion and DNA sequencing showedthat AGP was successfully cloned into pEGFP-1.After transiently transfected withpEGFP-N1,the expressions of EGFP in tour different kinds of celllines were detected byfluorescence microscopy.The expression of EGFP was observed in L02 cell by fluorescence microscopy,at 72h post transfection with pAGP-EGFP.Flow cytometerresults showed that the mean fluorescence intensity of EGFP driven by AGP is a quarter ofthat driven by CMV promoter.AGP could not drive the expression of EGFP in other threecelllines.After two weeks screened by G418,the level of EGFP expression in L02transfected with pAGP-EGFP reached to a considerable level as compared with that of L02transfected with pEGFP-N1.AGP-PEG10 fragment was microinjected into 1656 KMmouse fertilized ova and later produced 162 newborns.The expression of PEG10 genecould be detected in 13 mice,positive rate of transgenic integration was 8%.AGP-EGFPfragment was microinjected into 1782 KM mouse fertilized ova and later produced 151newborns.The expression of EGFP gene could be detected in 12 mice,positive rate oftransgenic integration was 7.9%.Positive transgenic mice were examined every day.Of the13 PEG10 transgenic mice,one mouse was died within 4 weeks of age and PEG10 wassuccessfully expressed in the liver of this transgenic mouse.Other transgenic micedeveloped to maturity.All experimental mice were kept alive till corresponding time spotsexcept two PEG10 transgenic were dead.At autopsy,three mice that died of“natural”causes and six PEG10 transgenic mice had hepatomegaly and had grossly visible lesions inthe liver,suggesting the presence of liver cancer.On histologic examination,the hepatocytehyperplasia was characterized by the mussy framework of hepatic lobules,the hepatic cellnuclear anachromasis with nuclear atypia,which means liver-specific expression of PEG10gene was sufficient to aggravate the hepatocarcinogenesis induced by alcohol.The moreobvious fibrosis in the periphery of hepatic lobules and more infiltration of inflammatorycells in portal area were found in the other four survived PEG10 transgenic mice.Theaverage weight of liver between the PEG10 transgenic mice and wild type mice weresignificantly different (P＜0.05).
Conclusions Our results demonstrate that PEG10 over-expression markedly inhibits cellapoptosis induced by H_2O_2 on human liver cell line L02.AGP presented a strongtranscriptional activity in L02 cell among the tested cell lines.It will serve as a suitable tool for transgenic mouse in the future.PEG10 gene could be expressed in the liver of thetransgenic mice and increased susceptibility to carcinogenesis in liver tissues,which isuseful for studying the function of PEG10 in vivo and discuss its significance in thehepatocarcinogenesis.These findings indicate that PEG10 gene may be the key eventsduring the hepatocarcinogenesis and progression of HCC and a promise target gene forgene therapy of HCC in the future.
结果琼脂糖凝胶电泳、酶切及测序结果表明PEG10基因片段扩增无误，并按正确的顺序插入到Simple T载体中。RT-PCR和Western Blot结果显示：PEG10基因在L02／PEG10中顺利实现表达。免疫细胞化学显示：PEG10蛋白主要分布在胞浆中，部分在核膜中表达。经400mM H_2O_2作用24h后，正常L02细胞和L02／vector可见凋亡特有的改变，如胞浆和核固缩、染色质断裂、DNA电泳呈梯状条带，而L02／PEG10未发现上述凋亡特征。酶切与测序鉴定显示：AGP片段完整正确地插入到T载体中。在pEGFP-N1瞬时转染各组细胞的不同时段，荧光显微镜下都能观察到EGFP的高表达：pAGP-EGFP瞬时转染L02细胞72h后，荧光显微镜下观察到EGFP的表达，流式分析显示：AGP启动EGFP表达的相对荧光强度为CMV的1／4，其它转染细胞观察不到EGFP的表达。转染pAGP-EGFP的L02经G418稳定筛选2w后，AGP的转录活性达到与CMV相当的水平。胶回收AGP-PEG10转基因片段后，将其显微注射导入1656枚受精卵中，产仔162只，PCR检测阳性鼠1 3只，转基因整合阳性率为8％。转基因对照小鼠的制备共注射受精卵1782枚，产仔151只，有12只小鼠经PCR检测证实为EGFP基因整合阳性鼠，整合阳性率为7．9％。在饲养过程中，有一只PEG10转基因小鼠死亡，RT-PCR和Western Blot检测到其肝脏内PEG10 mRNA和蛋白的表达，HE染色提示该小鼠的肝脏呈肝细胞癌病理学表现，出现大量异形核细胞，核大深染。其他存活转基因阳性鼠生长状态良好。酒精灌胃结束后，有两只PEG10转基因小鼠死亡，肝脏组织HE染色显示呈肝细胞癌病理表现。剩余10只PEG10转基因小鼠逐渐出现精神欠佳、进食进水量减少、活动减少、行动迟缓、皮毛失去光泽、身体消瘦、疲乏无力的表现。对照组小鼠一般情况正常。处死小鼠后，转基因对照组小鼠和野生型小鼠的肝脏重量分别为(3．98±0．94)g和(3．54±0．72)g，均显著低于PEG10转基因小鼠(8．29±0．76)g，P＜0．05。HE染色结果提示对照组小鼠的肝细胞排列完整致密，细胞间质成分正常。PEG10转基因小鼠中有6只小鼠的肝脏细胞核深染呈蓝色，大小不等，异形性明显，呈低分化癌，其他四只PEG10小鼠的肝细胞周围和小叶中央下静脉周围可见纤维化。结论PEG10基因可抑制H_2O_2诱导的细胞凋亡，低浓度酒精灌胃处理小鼠6个月后，PEG10的过表达可增强试验动物罹患肝癌的易感性，诱发其产生肝癌。本研究一定程度上为PEG10作为肝癌基因诊断和治疗一个新的分子靶点提供了实验依据。
Objective Hepatocellular carcinoma (HCC) is a highly malignant disease worldwide that isassociated with a high morbidity and mortality.It has been the second cancer killer in Chinasince 1990s.Early detection and resection have been generally recognized as the mostimportant factors to achieve long-term survival for HCC patients.HCC is a polygenic and amultiple-step disease among which chronic infection with hepatitis B virus or hepatitis Cvirus,cirrhosis,intake of food contaminated with chemical carcinogens and aflatoxin B,parasitic infections,excessive consumption of alcoholic beverages are considered to be themajor risk factors for HCC development.Present molecular analyses have demonstrated thecausal relationship between HCC and various groups of genes including the activation ofoncogene,inactivation of tumor suppressor gene and DNA repair genes.Among these,methylation of CpG islands in the promoter of tumor-associated gene and loss of imprintingseem to play important roles in the mutation of gene.However,the detailed molecularmechanisms leading to the development and progression of HCC remain unclear.The poorclinical prognosis is largely attributed to most symptomatic HCC patients being diagnosedat the advanced inoperable stage,often accompanied by intra-or/and extra-hepaticmetastasis,which precluding their chance for surgical intervention.It is necessary toidentify new serologic HCC biomarkers that have a sufficient sensitivity and specificity for the diagnosis of HCC,especially in AFP-negative and/or smaller HCC cases.PEG10 is agenetic imprinting gene with an active paternal allele/silent maternal allele.It wasindentified as having an elevated level of expression in majority of the HCC patients,including those with negative serum AFP and small tumor size,and was also induced toexpress during G2/M phase in regenerating mouse liver.This result indicates that theinduction of PEG10 may play an important role in liver regeneration or carcinogenesis ofhuman hepatocyte.Understanding the molecular basis of the abnormal imprinting ofPEG10 will shed new light on the process that leads to liver cancer.Albumin is an abundantprotein in the adult serum.The promoter of albumin was activated in the terminallydifferentiated mature hepatocytes.The high conservation and strong liver-specificregulatory machinery of albumin gene promoter (AGP) cluster makes it possible a suitablepromoter for hepatic specific expression.AGP has been extensively used in transgenic miceto direct foreign gene expression in adult liver,since it was cloned by Izban in 1989.Thegoal of this research was to determine if over-expression of the PEG10 gene in the liveralone is sufficient to increase susceptibility to carcinogenesis.For this purpose,wegenerated lines of transgenic mice expressing PEG10 in the liver under the regulatorycontrol of the albumin promoter.The expression of PEG10 in the liver was quantified byRT-PCR and Western Blot analysis.Body weight and diet,activity were analyzed incontrol and transgenic mice.Control and transgenic mice were given a single intragastricinjection (0.3 g/kg) of ethanol for 6 month.Mice were euthanized at 2 month after ethanolinjection and the total liver weights were recorded.Liver tissues were fixed in 10%formalin.Section were obtained from paraffin-embedded tissue sample,stained withhematoxylin-eosin,and examined under a microscope and photographed.
Methods Firstly,PEG10 was amplified by reverse transcription polymerase chain reaction(RT-PCR) using cDNA synthesized from hepatoma cell line HepG2 total RNA.A pair ofprimers was designed according to the PEG10 cDNA sequence deposited in Genbank,which would produce 1027 bp fiagment with SalⅠand AgeⅠrestriction enzyme sites inserted at 5'and 3'ends.The amplified PEG10 gene fragments were inserted into pMD-18Simple T vectors by T-A cloning.Recombinant plasmid was identified with double enzymedigestion and confirmed by DNA sequencing.Then the eukaryotic expression vector ofPEG10 gene (pPEG10-EGFP) was constructed.Correct recombinant plasmidpPEG10-EGFP was transfected into liver cell line L02 by lipofectamine~(TM)2000.Positivecell clone was screened by culturing in the selective culture medium with G418 and definedas L02/PEG10,while the cell transfected with empty expression vector (pEGFP-N1) wasdefined as L02/vector.L02/vector and parental L02 cells were served as negative control,HepG2,with endogenous expression of PEG 10,as a positive control.RT-PCR and WesternBlot were carried out to detect the expression of target genes.PEG10 protein and itssubcellular localization were detected by immunocytochemistry.The apoptosis of L02 cellswas induced by 50-400 mM H_2O_2.Morphological changes of apoptotic cells weredetermined by fluorescence microscopy using hoechst33342 nuclei staining.DNA fragmentwas observed by agarose gel electrophoresis.Total DNA was extracted from mouse bloodfor PCR amplification of AGP.The amplified fragments were inserted into the multiplecloning sites of pMD-18 T vector and identified using restriction enzymes and sequencing,then cloned into the expression vector pEGFP-1.pAGP-EGFP was transfected into cervicalcarcinoma cell line Hela,L02,colorectal cancer cell line SW480 and pancreaticcarcinoma cell lines Bxpc-3 cells by lipofectamine~(TM)2000,pEGFP-1 and pEGFP-N1 wereserved as a negative and positive control,respectively.The expressions of EGFP weredetected by the fluorescence microscope as well as flow cytometer.Finally,PEG10 genefragments were inserted into the downstream of AGP to construct the liver-specifictransgenic vector (named pAGP-PEG10-EGFP).pAGP-EGFP was served as a negativecontrol.A 1716 bp linearized expression element of pAGP-PEG10-EGFP,which containedAGP and structural gene of PEG10,was purified from agarose gel and adjusted to a finalconcentration of 2μg/ml in TE.KM female mice were hormonally super-ovulated andmated with KM male mice.The fertilized eggs were collected from the oviduct 24h later. Transgenic fragment in TE was microinjected into the pronuclei of fertilized eggs.Then,the injected fertilized eggs were transplanted into the oviduct of pseudo-pregnant KM mice.Control transgenic mouse were established with the same methods by microinjecting 1.5Kbfragments contained AGP-EGFP into the pronuclei of fertilized eggs.All the newborn micewere screened and identified by PCR detecting genomic DNA from tail tissue.The activityand diet of positive transgenic mice were investigated.If the transgenic mouse were dead inthe raising process,the expression of PEG10 gene in the tissues of heart,lung,kidney.uterus,ovary,liver,spleen,stomach,small intestine were examined by RT-PCR andWestern Blot,respectively,In addition to that,liver tissues were fixed in formaldehyde andsectioned.The sections were stained with hematoxylin/eosin and examined undermicroscope to found whether it had tumor-like lesions.Mice were euthanized at 2 monthafter intragastric injection with low concentration alcohol (0.3g/kg) twice a day for sixmonth.the total liver weights were recorded.Liver tissues were fixed in 10% formalin.Section were obtained from paraffin-embedded tissue sample,stained withhematoxylin-eosin,and examined under a microscope and photographed.
Results Agarose gel electrophoresis,restriction enzyme digestion and DNA sequencingshowed that eukaryotic expression pPEG10-T vector harboring PEG10 gene wassuccessfully constructed.PEG10 gene mRNA and protein were expressed in L02/PEG10cells and PEG10 protein was mainly distributed in the cytoplasm and associated with thenuclear membrane.After treatment with 400 mM H_2O_2 for 24 h,distinct morphologiccharacteristic of cell apoptosis such as karyopyknosis and conglomeration were notobserved in L02/PEG10.Ladder-like DNA fragmentation was observed in adose-dependent manner in both L02 and L02/vector cell lines,but not in L02/PEG10.Agarose gel electrophoresis,restriction enzyme digestion and DNA sequencing showedthat AGP was successfully cloned into pEGFP-1.After transiently transfected withpEGFP-N1,the expressions of EGFP in tour different kinds of celllines were detected byfluorescence microscopy.The expression of EGFP was observed in L02 cell by fluorescence microscopy,at 72h post transfection with pAGP-EGFP.Flow cytometerresults showed that the mean fluorescence intensity of EGFP driven by AGP is a quarter ofthat driven by CMV promoter.AGP could not drive the expression of EGFP in other threecelllines.After two weeks screened by G418,the level of EGFP expression in L02transfected with pAGP-EGFP reached to a considerable level as compared with that of L02transfected with pEGFP-N1.AGP-PEG10 fragment was microinjected into 1656 KMmouse fertilized ova and later produced 162 newborns.The expression of PEG10 genecould be detected in 13 mice,positive rate of transgenic integration was 8%.AGP-EGFPfragment was microinjected into 1782 KM mouse fertilized ova and later produced 151newborns.The expression of EGFP gene could be detected in 12 mice,positive rate oftransgenic integration was 7.9%.Positive transgenic mice were examined every day.Of the13 PEG10 transgenic mice,one mouse was died within 4 weeks of age and PEG10 wassuccessfully expressed in the liver of this transgenic mouse.Other transgenic micedeveloped to maturity.All experimental mice were kept alive till corresponding time spotsexcept two PEG10 transgenic were dead.At autopsy,three mice that died of“natural”causes and six PEG10 transgenic mice had hepatomegaly and had grossly visible lesions inthe liver,suggesting the presence of liver cancer.On histologic examination,the hepatocytehyperplasia was characterized by the mussy framework of hepatic lobules,the hepatic cellnuclear anachromasis with nuclear atypia,which means liver-specific expression of PEG10gene was sufficient to aggravate the hepatocarcinogenesis induced by alcohol.The moreobvious fibrosis in the periphery of hepatic lobules and more infiltration of inflammatorycells in portal area were found in the other four survived PEG10 transgenic mice.Theaverage weight of liver between the PEG10 transgenic mice and wild type mice weresignificantly different (P＜0.05).
Conclusions Our results demonstrate that PEG10 over-expression markedly inhibits cellapoptosis induced by H_2O_2 on human liver cell line L02.AGP presented a strongtranscriptional activity in L02 cell among the tested cell lines.It will serve as a suitable tool for transgenic mouse in the future.PEG10 gene could be expressed in the liver of thetransgenic mice and increased susceptibility to carcinogenesis in liver tissues,which isuseful for studying the function of PEG10 in vivo and discuss its significance in thehepatocarcinogenesis.These findings indicate that PEG10 gene may be the key eventsduring the hepatocarcinogenesis and progression of HCC and a promise target gene forgene therapy of HCC in the future.
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