| Background and ObjectivePrimary hepatocellular carcinoma(HCC)is one of the most common human malignancy.HCC is the fifth most common malignancy worldwide and the third of china,there are nearly 600000 new cases of primary hepatocellular carcinoma(HCC)patients.HCC is a highly malignant,which making it the third leading cause of cancer-related death.In the past twenty years,the overall survival rate of primary HCC patients has not been improved mostly.There are two ways to treatment the HCC including surgical resection and liver transplantation,but most of these treatment is usually applicable to the tumor in the early stages.The invasion and metastasis rate of HCC are very high,Therefore,most patients are identifed at later stages of the disease,or the tumor is unresectable makes it unfavourable prognosis.The traditional systemic chemotherapy for these patients would cause serious adverse reaction,and so far,we did not find the effective treatments for these patients have lost the operation chance or the post-operation relapse,which make theose patients with a lower three-year survival rate(30-40%).Thus,the key to treatment for HCC is inhibitting the ability of invasion and metastasis of tumor cells,and further research about the liver cancer cell invasion and metastasis of molecular regulation mechanism has important clinical and scientific value.At present,the invasive mechanism of HCC are still under studying,the available mechanism of tumorigenesis,for instance gene mutations,including the proto-oncogene activation,losing,or variating,the reactivation of telomerase activity and epigenetic abnormalities,can not perfectly explain the development,invasion and metastasis of HCC of related molecular mechanism.The existing results show that there are distinct cell heterogeneity in hepatocellular carcinoma cells.In hepatocellular carcinoma cells,many cells show significant differences in molecular biology or genes,can led to the the differences in growth,invasion and metastasis ability of these tumor cells,and their sensitivity to drugs.Tumor cell morphological heterogeneity,functional heterogeneity,antigens and immunogenicity are several components of tumor heterogeneity.Tumor heterogeneity is one of the characteristics of a malignant tumor,in view of the characteristics of the liver cancer cells,study the different types of liver cancer cells may be a way to exploring different clinical biological behaviour of hepatocellular carcinoma.Researches show that the invasion and metastasis of tumor cells is not the characteristic of all tumor cells,but the characteristics of only few cells.So we suppose that some cells with the characteristics of invasion and metastasis whether showed abnormal transcription and expression of some regulatory proteins,and through regulating the expression of related proteins,making these cells get stronger invasion and metastasis ability or the ability of resistance to chemotherapy drugs,and make it to the invasion and metastasis tumor cells.About 30 years ago,Hedgehog(Hh)was initially discovered as a ‘‘segmentpolarity’’ gene that controls Drosophila embryonic cuticle pattern.Since the discovery of its vertebrate counterparts in the early 1990 s,enormous progress has been made in revealing the role of Hh signaling in development as well as the molecular underpinning of the Hh signaling cascade.It has been discovered Hh signaling contributes to embryonic development,and monitoring cell functions,such as cell differentiation and proliferation,and maintaining the steady state of the organismorgan.In mammalians,there are three Hedgehog homology genes including Sonic hedgehog(Shh),Indian hedgehog(Ihh)and Desert hedgehog(Dhh),encoding SHH,IHH,and DHH,respectively.The membrane receptor of Patched(Ptc)and Smoothened(Smo)control the Hh signaling.The Gli family including three(Gli-1,Gli-2and Gli-3)transcription factors,which plays an important role in the development of tumor.In the unactivated Hh signaling pathway,the three members of the gli family are not allowed to enter the nucleus,thus unable to exercise their transcriptional control function.It has been shown that Gli1 can regulate the expression of downstream target genes,including invasion and metastatic target genes.Increasing evidence has demonstrated that the Hh signaling pathway plays an important role in multiple tumor types,for example,basal cell carcinoma,retinoblastoma,esophageal cancers,gastric cancer,pancreatic cancer.There are also studies reported that the abnormal activated Hh signaling pathway is involved in the development of hepatocellular carcinoma and invasion and metastasis.However,the molecular mechanism underling the invasion and metastasis of liver cancer cells is still unclear.Transformation of epithelial mesenchymal(EMT)is epithelial convert to mesenchymal cells,it gives the cell invasion and metastasis ability,including stem cell properties reduce apoptosis and enhancing immune suppression,etc.The process of not only affect the growth of cells,is also involved in tissue healing processes such as organ fibrosis and cancer development.EMT can be seen as the beginning of tumor invasion and metastasis.The following characteristic changes can occur when tumor cells occur EMT,including losing the expression of E-Cadherin,and raising the expression of N-Cadherin,vimentin,etc.E-cadherin has very important effect to maintain cell shape,cell movement and adhesive ability.When in the case of certain physiological or pathological stimulus,the loseing of E-cadherin in tumor cells,decreasing the adhesion ability of cell,thereby promoting tumor cells shed from the primary lesion,infringement of surrounding tissues or distant metastasis.It has been found that in the EMT process of tumor cells,the m RNA and protein levels of the key regulatory transcription factor Twist1 were significantly increased.Therefore,we hypothesized that Twist1 may be an important molecular basis for regulating the occurrence of EMT in hepatocellular carcinoma cells.This project intends to research the key transcription factor Gli1 in Hh signaling pathway in the regulation of invasion and metastasis of HCC,to explore the related molecular mechanism,and to provide the possible experimental basis to assess recurrence,metastasis and prognosis of liver cancer,and to provide a new theoretical basis for the treatment of liver cancer.Methods1.Western blotting was applied to selecte Gli1 protein high expression and Gli1 protein low expression cell lines in the Huh7,hep G2,PLC,SMMC-7721 and Bel-7404 human liver cancer cells.2.The GLI1 expression vector was constructed in plentis-pgk-gtp-sffv-gli1,and was prepared as a virus Lentiviral-GLI1.With Lentiviral-Gli1 infection will be prepared to screen out the high expression of Gli1 protein of human liver cells,the Lentiviral-Gli1 group and the blank control group(N group)do not do any processing,light control group(NC group)transfection only empty carrier.3.According to the Gen Bank database design and synthesis of three Gli1-si RNA through high expression plasmid transfection to screen out Gli1 protein of human liver cancer cells,respectively p GCsi-U6-GLi1 si RNA-1(si RNA-1 group),p GCsi-U6-GLi1 si RNA-2(si RNA-2 group),p GCsi-U6-GLi1 si RNA-3(si RNA-3 groups),and blank control group(N group)is not carrying any sequence and the negative control group transfection negative control sequence(NC group).4.Real-time fluorescence quantitative PCR(RT-q PCR)and Western blotting was applied to detect the expression levels of Gli1-m RNA and Gli1 protein,and the best si RNA sequences were screened.5.Cell scratch experiments was applied to observe Lentiviral-Gli1 groups and select the best si RNA sequences of cell and Western blotting was applied to detect the exprssion of Twist1,E Cadherin,N-Cadherin,Vimentin.Results:1.Western blotting results suggested that compared with Huh7,hep G2,PLC and smmc-7721 the expression of Gli1 protein level in Bel-7404 cells increased(P < 0.05),and compared with Hep G2,PLC,smmc-7721 and Bel-7404 the expression of Gli1 protein in Huh7 cells increased(P < 0.05).2.In Huh7 cells,which were selected for low expression of Gli1 protein,the expression levels of Gli1 protein and Gli1 m RNA were increased in Lentiviral-Gli1 group(P<0.05).In addition,the expression of Twist1 protein in Lentiviral-Gli1 group was up-regulated,and the expression of e-cadherin protein was down-regulated,and the expression of N-cadherin and Vimentin protein was up-regulated,and the cell migration ability was increased(P<0.05).3.In Bel-7404 cells with high expression of Gli1 protein,compared with si RNA-1,si RNA-3,N and NC the expression of Gli1 m RNA level in si RNA-2 are the lowest(P < 0.05).4.Compared with N group,the expression of Twist1 protein in the sirna-2 group was down-regulated,the expression of e-cadherin protein was up-regulated,and the expression of N-cadherin and Vimentin protein was down-regulated,and the cell migration ability decreased(P < 0.05).Conclusions:1.The expression of Gli1 protein in the human hepatoma carcinoma cell of Bel-7404 was increased Compared with in the Huh7,hep G2,PLC and smmc-7721,The expression of Gli1 protein in the human hepatoma carcinoma cell of Huh7 was declined compared with in the Bel-7404,hep G2,PLC and smmc-7721.2.Si RNA interference Gli1 can own-regulated the expression of Twist1 in human liver cancer Bel-7404 and inhibits epithelial-interstitial cell transformation and inhibits the capacity of cell migration.3.Used GLI1 expression vector p Lentis PGK-GTP-SFFV-GLI1 made by virus protein overexpression of Hedgehog signaling pathways of the key transcription GLI1 can raise human liver Twist1 Huh7 cells the expression of protein levels,and promoted the transformation of epithelial-mesenchymal cells and enhance the capacity of cell migration. |
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