Research On Relationship Between Glypican-3and Notch1in Hepatocellular Carcinoma

BACKGROUND&OBJECTIVEHepatocellular carcinoma (HCC) is a tumor of high malignant degree in clinic,which is one of the major diseases that affect human health. China is among the high incidence area of HCC, with an incidence of20.37per100thousand,which is the third highest incidence in common tumors. Every year there are about650thousand cases of new onset of liver cancer global, more than half of them happened in our country.In Asia, we represent more than80%of the cases of new onset yearly. Though many methods including surgical resection, liver transplantation, vascular intervention, ablation technology, radiotherapy, chemotherapy, can be performed in HCC in clinic practice,more than one half of HCC patients had the micrometastasis failing to dectect before radical surgery,which may be the direct reason of metastasis or recurrence of HCC after surgery. Metastasis and recurrence have become the crux of further improvement on survival rate of HCC patients. Therefore, to seek and predicate HCC’s metastasis and recurrence tendency, judge prognosis of HCC patients is the key and focus of HCC’s research, as well as its emphasis and difficulty.Glypican-3is a member of HSPG(heparan sulfate proteoglycan) family,which is a complex sugar compound and made up of protein, lipid and sugar through covalent bond. Glypican-3exists in the cell surface widely,which is anchored in the cell membrane through GPI(glycosyl phosphatidyl inositol). Glypican-3is also called GPC3, MXR7, OCI-5, GTXR2-2,the gene fragment length greater than900KB, which is one of the biggest genes in the human genome.Now,the HS chain of GPC3interacts with a large number of biological effec molecule,such as growth factor and its receptors,extracellular matrix proteins and adhesion molecules,to regulate cell proliferation, differentiation, adhesion and migration, etc.,that also may be involved in inhibiting or regulating the growth of most mesodermal tissues and organs.The studies found that GPC3participates in the control of signal transduction pathway which closely related to tumor development such as Wnt,Hedgehog (Hh),FGF,IGF,BMP,SMAD,TGF-β and so on. Researches also show that GPC3is highly expressed in HCC, colorectal carcinoma,Wilm’s tumor, neuroblastoma and hepatoblastoma, while loses its expression in lung adenocarcinoma, Ovarian cancer, breast cancer, mesothelioma, which suggests that GPC3play an important role in the development of tumor and may play a different role in different tumors. According to research, GPC3expresses significantly in human liver cancer tissue and it has closely relationship with the development of liver cancer.Notch signal transduction pathway consists of a set of cell membrane ligands, receptor and downstream molecules that is highly conserved in evolution.There are4kinds of Notch receptors(Notchl-4) and5kinds of ligands in mammals. Notch signal transduction process mainly occurs in cell-to-cell, so the Notch receptor is a single transmembrane molecule composed of heterodimer,and its ligand is a transmembrane molecule for the same.The main differences of the four receptors lie in EGF repeat sequences. Notch receptors as receptors,and as well as transcription factors play a huge role.The interaction between receptors and ligands in cell-to-cell can activate Notch signal transduction process, thereby to regulate gene transcription directly,making the cell gene expression is regulated by the neighboring cells. Notch signaling in the processes of cell differentiation, embryonic development, tissue self-renewal play an important role,and it is also involved in some pathological processes(including the tumor). Notch signaling as a cancer gene to promote tumor growth, but in some organizations it can also induce cell differentiation, inhibit proliferation of tumor.The change of Notch signaling in cancer stem cells may have played a key role. Studies have observed that Notch1signaling may lead to the cycle stagnation of liver cancer cell and apoptosis, it main plays the role of tumor suppressor genes in liver cancer, and it is related with the development of liver cancer closely.The purpose of our research is through the detection of GPC3and Notchl expression in hepatocellular carcinoma cell line,and through changing the expression level of GPC3to observe the change of Notchl expression level,and checking the expressions of GPC3and Notchl in hepatocellular carcinoma (HCC) tissues,thus to explore the relationship of GPC3and Notchl in hepatocellular carcinoma.This lay a theoretical basis for further to research molecular therapic target of hepatocellular carcinoma.METHODS1. Detection of expression of GPC3and Notchl in hepatocellular carcinoma cell linesQuantitive real-time PCR was used to detect the expression of GPC3mRNA and Notchl mRNA in hepatocellular carcinoma cell lines(MHCC97-H,SMMC-7721, HepG2,QGY-7701and Bel-7402).Next, Western blotting was used to detect the protein expression level of GPC3and Notchl in this five hepatocellular carcinoma cell lines.2.Construction of recombinant plasmid DNA of interference GPC3expression According to the information of GPC3gene, such as sequence and serial number,we designed four shRNA sequences(GPC3-809-shRNA,GPC3-715-shRNA, GPC3-1246-shRNA,GPC3-1720-shRNA).Next, DNA synthesis of complementary short fragments on the basis of the four shRNA sequences.DNA fragments were diluted, annealed and connected to pGPU6/GFP/Neo carrier of linear changing.After that, transformation, coated flat,overnight train and screened positive colonies to sequence.3. After transient transfection of MHCC97-H and SMMC-7721, GPC3was interfered,to observe the change of Notchl expression levelTransfected GPC3-shRNA into hepatocellular carcinoma cell lines by lipofection technique.Next, selected one of the most effective shRNA sequences.After that,used this most effective shRNA sequences to transfect MHCC97-H and SMMC-7721. Quantitive real-time PCR and Western blotting to detect the expressions of GPC3and Notchl,thus to observe the change of Notchl expression level when GPC3was interfered.4. Identification of expression of GPC3and Notchl in hepatocellular carcinoma tissuesUsing immunohistochemical method and image analysis technology of computer,we detected the expressions of GPC3and Notchl in the tissues of30HCC.5. Statistical methodsSPSS13.0software was used for statistical analysis. Enumeration date used Nonparametric Test;measurement data used Independent-Samples T Test and One-Way ANOVA of Compare Means;correlation analysis used Bivariate. P values of<0.05were considered statistically significant.Inspection level of alpha=0.05, the test on both sides.RESULTS 1. Expression of GPC3and Notch1in hepatocellular carcinoma cell linesThe results of quantitive real-time PCR showed that the expression of GPC3in five cell lines of HCC was significantly different from each other(F=112.516, P=0.000),and the expression of Notchl in five cell lines of HCC was significantly different from each other too(F=123.793, P=0.000). Using2-delta delta Ct method, QGY-7701as the control group,the results found that the expression of GPC3in Bel-7402,QGY-7701,HepG2,SMMC-7721and MHCC97-H were in turn increased(P<0.05),but the expression of Notchl were in turn reduced(P<0.05).The results of Western blotting showed that the expression of GPC3in Bel-7402,QGY-7701,HepG2,SMMC-7721and MHCC97-H were in turn increased, but the expression of Notch1were in turn reduced.2. Screening of four shRNA sequencesWe used four recombinant plasmid DNA of interference GPC3expression and NC-shRNA to transfect MHCC97-H.After48hours, observed under fluorescence microscope and counted the transfection efficiency:GPC3-809-shRNA is82.5%, GPC3-1246-shRNA is55.7%,GPC3-1720-shRNA is47.3%,GPC3-715-shRNA is76.2%, NC-shRNA is79.6%.To this five cells after transfection, the results of quantitive real-time PCR showed that the expression of GPC3was significantly different from each othe(F=63.871, P=0.000),and the lowest is GPC3-809-shRNA(P <0.05).The results of Western blotting also indicated that the expression of GPC3in GPC3-809-shRNA is lowest. In conclusion that, GPC3-809-shRNA group has the highest transfection efficiency,and its interfered efficiency is highest also.3. Change of the expression level of Notchl after interfering GPC3expressionWe used GPC3-809-shRNA and NC-shRNA to transfect MHCC97-H and SMMC-7721respectively.After48hours, the results of quantitive real-time PCR showed that the expression of GPC3in MHCC97-H after GPC3-809-shRNA interference is0.281±0.081times as much as in MHCC97-H after NC-shRNA interference(t=6.221,P=0.003).The expression of Notchl in MHCC97-H after GPC3-809-shRNA interference is5.243±0.308times as much as in MHCC97-H after NC-shRNA interference(t=13.636,P=0.000). The expression of GPC3in SMMC-7721after GPC3-809-shRNA interference is0.427±0.110times as much as in SMMC-7721after NC-shRNA interference(t=5.645,P=0.005).The expression of Notchl in SMMC-7721after GPC3-809-shRNA interference is3.782±0.200times as much as in SMMC-7721after NC-shRNA interference(t=10.605,P=0.008).The results of Western blotting showed that the expressions of GPC3of GPC3-809-shRNA interference were less than NC-shRNA interference in MHCC97-H and SMMC-7721,while the expressions of Notchl of GPC3-809-shRNA interference were higher than NC-shRNA interference in MHCC97-H and SMMC-7721.4. The expression of GPC3and Notch1in hepatocellular carcinoma tissuesThe results of immunohistochemical semiquantitative and quantitative description show that in high,medially and poorly differentiated HCC tissues, the expression levels of GPC3in turn increase, with significant difference (P<0.05) or highly significant difference (P<0.01).The expression levels of Notchl in turn reduce, with significant difference (P<0.05) or highly significant difference (P<0.01).The Spearman rank correlation analysis of semiquantitative described results show that the expression between GPC3and Notchl is highly significant negative correlation (rp=0.607, P=0.607).The Pearson correlation analysis of quantitative described results show that the expression between GPC3and Notchl is highly significant negative correlation too(r=-0.692,P=0.000).CONCLUSION1. In five HCC cell lines (Bel-7402,QGY-7701,HepG2,SMMC-7721, MHCC97-H), the expression levels of GPC3are increased in turn,but the expression levels of Notchl are reduced in turn. The expression levels of GPC3and Notchl are inverse in this five HCC cell lines.2. Application of RNAi technology,GPC3-targeted shRNA can induce GPC3gene silencing to achieve the purpose of closure of GPC3gene. In the design of four shRNA sequences,the transfection efficiency of GPC3-809-shRNA is highest,and its interfered efficiency is highest to target GPC3.The results show that silencing the expression of GPC3gene, the expression level of Notchl gene has changed, its expression level is rised.This prompt that between GPC3and Notchl may exist the negative mutual regulated mechanism.3. The higher the degree of differentiation of HCC tissue was,the lower the GPC3expression,and the higher the Notchl expression; the degree of differentiation was lower,the higher the GPC3expression,and the lower the Notchl expression.The expressions of GPC3and Notch1show a negative correlation in HCC.From the level of organization verifies the relationship between GPC3and Notch1.