Generation Of Transgenic Mouse Model For Hepatocellular Carcinoma With Liver-specific Expression Of Human Genetic Imprinting Gene PEG10

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 Salâ… and Ageâ… restriction 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^TM2000.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 H₂O₂.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^TM2000,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 H₂O₂ 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 H₂O₂ 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.