Cloning Of Human GPC3 CDNA And Its Expression In Hepatoma Cell MHCC97-L

Background and Objection:Primary hepatocellular carcinoma (HCC) accounts for 6% of the global cancer incidence, with age-adjusted annual incidence between 5.5 and 14.9 people per 100,000 population,more than half of which occurred in China. As a difficult curable malignant, HCC is at this moment the third cause of death for cancer worldwide,resulting in approximately 600,000 to 1,000,000 deaths annually. With the development of image analysis techniques and pathology detections including ultrasound, enhanced spiral CT and dynamic MRI, diagnosis and treatment of HCC made progresses,but the clinical outcome of patients haven't got corresponding improvements. The carcinogenisis and development of HCC are processes caused by uncontrolled cell proliferation which involves multiple factors and steps,but the specific molecular mechanisms are still unclear. Futher research in pathogenesis of HCC, especially the changes of common genes/moleculars in multiple signaling pathway in HCC development process,could help to define the sophisticated occurrence and development of HCC and find effective therapeutic targets, as well as providing novel specific biological markers for the diagnosis of HCC. The pathogenesis of HCC has been known to involve p53,β-catenin,TGF-βand the retinoblastomagene.p53 mutation occurs in one-third of HCC;β-catenin mutation is found in 13.1% of HCC and the activation of canonical Wnt-signaling pathway happens in 18% of HCC. In addition, we know very little about the key genes involved in the occurrence and development process of HCC.Thus the exploration of the effects of relevant genes on the pathogenesis of HCC will lay foundation for establishing new means for the diagnosis and treatment of HCC and improving clinical outcomes of patients with HCC.Hey-Chi Hsu et al. successfully cloned a fragment of cDNA specifically expressed in primary and recurrent HCC using mRNA DD,which were highly homology of OCI-5 cDNA and exactly the same as those isolated by Pilla with STSS-PCR(sequence tagged sites) from SGBS(Simpson-Golabi-Behmel Syndrome)patients,named Glypican-3(GPC3).Glypicans are family members of heparin sulfate proteoglycans(HSPGs) that regulate the activities of various growth factors in forms of co-receptors.The changes of expression level and regulating pattern are stage-and tissue-specific,suggesting that glypicans are involved in the regulation of morphogenesis and tumorgenesis. As interacting with core protein other than HS chain,GPC3 could regulates different types of growth factors according to different stage and tissue,for example,regulates cell proliferation and tissue development by affecting IGF-Ⅱ,Hh,BMP2,BMP7 and FGF-7 signaling transduction.Besides,secreted form of GPC3 may involved in a different mechanism of action as membrane form.GPC3 expression is down-regulated in several types of malignancies including ovarian cancer, mesothelioma, breast cancer,gastric cancer and lung cancer,whereas upregulated in HCC, malignant melanoma and Wilm's tumor.Act as a oncofetal protein,GPC3 expressed highly in embryonic liver and silenced in normal adult liver.Gradually upregulating expression during occurrence and progression of rat hepatocellular carcinoma indicates a role of GPC3 in the pathogenesis and development of HCC.A serial of researches have elucidated that GPC3 regulates signaling pathway of HCC cell proliferation and metastasis by affecting IGF-Ⅱ,Wnt,Hh,FGF,BMP and TGF-β,suggesting that GPC3 maybe an important prognosis factor and novel therapeutic target for HCC.This study constructed an eukaryotic expression vector with GPC3 gene and transfected into hepatoma cell line MHCC97-L,compared changes of biological behaviour before and after transfection,investigated the mechanism of action involving the pathogenesis of HCC and built basis for GPC3 becoming effective targets of diagnosis,prognosis and treatment of HCC. Methods:l.Acquistion of target gene GPC3 Designed Primers and conduct PCR amplification according to the GPC3 sequences contained in pDNR-LIB vector, identified the purity and relative molecular weight of PCR product with 1% agarose gel electrophoresis,recycle GPC3 DNA fragments with DNA gel extraction kit.2.Construction of recombinant vector pEGFR-c3-GPC3 Mix the recycling GPC3 DNA with pEGFR-c3 digested by XhoI and EcoRI for ligation reaction.3.Cloning and identification of recombinant vector pEGFR-c3-GPC3 Transform competent E.coli DH5αwith ligating product,filter positive colonies with kanamycin resistance plate,selected randomly positive clone,cultured with medium containing kanamycin and then extacted pEGFP-c3-GPC3 which identified with 1% agarose gel electrophoresis and ultraviolet spectrophotometer.Digested by XhoI and EcoRI for evaluation,the new colony was named pEGFR-c3-GPC3 and sequenced by Sanying biotech co.,wuhan.1.Transfection of MHCC97-L with recombinant vector pEGFR-c3-GPC3 Transfected hepatoma cell line MHCC97-L with Lipofectamine2000 and pEGFR-c3-GPC3,obtained MHCC97-L/GPC3 stably expressing GPC3 with G418 selection(200μg/ml).The control group was MHCC97-L transfected with pEGFR-c3.Isolated colonies were planted in 96 well plates and cultured with medium containing less G418(100μg/ml).Transferred cells to 24 well plates after covering the 96 well plates,expanding culturing with complete medium containing G418(100μg/ml) for further identification and biological research.2.Detection of GPC3 expression and observation of the biological behavious of stable transfected cell line Detected the expression level of mRNA and protein with fluorescence quantitative PCR and western blot,respectively.Evaluated the in vitro effect of GPC3 on MHCC97-L with MTT method.Results:1.GPC3 gene in pDNR-LIB was digested by EcoR I and Xho I, agarose gel electrophoresis showed 1.7Kb and 4.1Kb straps conforming with the length of pDNA-LIB linear plasmid and GPC3 gene.2.Agarose gel electrophoresis with PCR amplification products showed 1.7Kb strap.3. Agarose gel electrophoresis with digesting products of pEGFR-c3-GPC3 showed 1.7Kb and 4.7Kb straps.The ligating products matched GPC3 sequences in GenBank with sequencing.1.MHCC97-L stably transfected with pEGFR-c3-GPC3 and pEGFR-c3 could grow in selective DMEM medium with 200μg/ml G418 for 2 weeks. MHCC97-L/GPC3 stably expressing GPC3 and control cell line MHCC97-L/C3 were established.2.Results of fluorescent quantitative PCR Comparing with MHCC97-L/C3 and MHCC97-L (2-△△Ct=1),the expression of mRNA in MHCC97-L/GPC3 was upregulated (95%CI (1.715,1.791);t=-44.714,P<0.001)3.Results of western blot The expression of protein in MHCC97-L/GPC3 was upregulated comparing MHCC97-L/C3 and MHCC97-L (P<0.001;P<0.001)4.Evaluation of 3 groups of cells with MTT method showed that MHCC97-L/GPC3 has a significant enhanced proliferation ability (P<0.001), compared with MHCC97-L/C3 and MHCC97-L.Conclusion:1. We successfully constructed eukaryotic expression vector pEGFR-c3-GPC3, restriction enzyme digestion of DNA sequence analysis confirmed that the sequence of inserting gene coincident with GPC3 gene sequences in GenBank. 2.GPC3 stably expressing cell line MHCC97-L/GPC3 was established with pEGFR-c3-GPC3 transfection and G418 selection.Fluorescent quantitative PCR and western blot detection showed a significant elevation of mRNA and protein levels of GPC3 in MHCC97-L/GPC3.Growth curve showed that the proliferation of MHCC97-L/GPC3 was significantly accelerated,comparing with MHCC97-L and MHCC97-L/C3,which suggesting upregulation of GPC3 could stimulate the growth of hepatoma cell line MHCC97-L.