| Background and ObjectionHepatocellular carcinoma(HCC) is one of the most common and the worst prognosis cancer in our country. According to the statistics, there are about six hundred thousand new HCC patients every year globally which make it rank fifth in occurrence of cancer and rank third in death of malignancies around the world. Nowadays there are various treatments for HCC. Among these treatments, surgical removal is the most and mainly used, but the high recurrence rate and high cancer spread rate after surgery make the whole-scale foreseen result bad. HCC is a highly vascular and typical cancer and show close connection with the richness of blood vessels in cancer. It can easily go through cell membrane and blood vessel to spread locally or remotely.Despite of the many study of proliferation and control for HCC, there is still no clear vision for the molecular mechanism and cancer spreading via angiogenesis. Therefore, it’s urgent to have a further study of how HCC angiogenesis making cancer spread to find out new treatments for HCC.URG4 (Up-regulated gene-4, also known as URGCP) which is a recently discovered can be raised by liver virus’ protein (hepatitis B Virus x, HBx) is a new genie, it locates in chromosome 7p13. It is firstly found and explained by Tufan and others. Experiments showed that, when hepatitis B virus antigen is positive, the level of URG4/URGCP rise, it prompt the growth and living of liver cell which will grow into HCC cell. When the level of URG4/URGCP rise in HCC and stomach cancer patient, it prompt the cell proliferation and canceration. Based on these foundings, there was always been a point view that URG4/URGCP is the original cancer key in multi-cancer illness. However, in HCC cell the effect of URG4/URGCP has not been made clear and there are few reports show URGCP/URG4’s boost for angiogenesis make caner from bad to worse.In recent years, medical expert at home and abroad found that cancer blood vessel is the foundation for cancer cell to proliferate and spread. Angiogenesis is essential for cancer to grow. High level blood proliferation and poor prognosis is closely connected to the high invasion of HCC. Poor proliferation of blood vessel in cancer cause the death and withering of cancer cell which make it hard for cancer to grow. Inhabiting the proliferation blood vessel is an effective way to inhabit the growth and spread of cancer cell. But only through suppressing the growth factor activity of blood vessel is not sufficient to hold the proliferation of blood vessel, it also need to boost the negative growth factor activity and work with growth inhibitor of blood vessel to make an effective effort. Nowadays the most wildly used blood vessel inhibitor is tube inhibition, endothelial inhibition, interferon, platelet factor and others. But these inhibitors only target the endothelial cell growth and can’t inhibit or eliminate the growth of cancer cell. Also, long time using of these inhibitors will cause thrombosis, high or low blood pressure and other problems. Therefore, it’s of top urgency to find out a targeted method to control the angiogenesis.It’s widely believed that X protein(HBx) on HBV virus plays a crucial role in virus reproduction and occurrence of HCC. Among the HCC related to HBV, virus’ HBx now is confirmed as an key factor to boost cell reproduction and living. HBx as a transactivator in hepatitis B Virus polluted liver cells has been found that it help transcript and regulate some downstream genie such as URG4,7,11,12 and 19.It showed in prophase URGCP/URG4 boost the growth of HCC cell HepG2 and it also showed high level of URGCP/URG4 caused poor clinical prognosis. Both evidences showed the close connection between URGCP/URG4 and occurrence of HCC. Also, the over-expression of URGCP/URG4 in osteosarcoma is also related with cancer re-occurrence, spreading and cancer cell proliferation activity, it shows URGCP/ URG4 genie is the prognostic marker of some cancer. Xie C and others have found the high level of URG4/URGCP genie expression in HCC tissue and clinical tissue, and it also has positive relation with Ki67 genie expression, the cell tissue having a higher level of URG4/URGCP expression of show stronger proliferation and spreading competence. Also, high level expression of URG4/URGCP genie expression contributes to phosphorylation of FOXO3a protein, which inactivates the protein and activate AKT/GSK3βpathway. Consequently, it leads to the high level expression of cell-cycle related protein cyclin D1 and low level expression of p21 and p27, which boost the proliferation and spreading of cell.Many cancers’prognosis is poor, one of the important factor is cancer can release cell factor to boost the growth of blood growth, making it possible for cancer cell to infiltrated and spread. Ji Gz and other found compared to the tissue besides the HCC tissue, HCC tissue’s MVD is in positive relation with high level expression of TGF-β1、Smad7 genie and higher level MVD, activation of NF-κB is also in positive relation with the expression of TGF-β1 genie. TGF-β’s pathway make receptor complex activated by type Ⅰ and Ⅱ serine-threonine kinase and serine threonine kinase which cross the cell membrane. It activates type Ⅰ receptor phosphorylation receptor regulatory proteins Smad2 and Smad3 to activate the pathway for TGF-β/Smad. But in many cancer cells TGF-βcan also activate NF-κB.NF-κB is a key transcript factor to express proinflammatory genes, it can make cell secrete IL-6, IL-1,TNF-α and other molecules. In Lai HS and other’s study they found IL-6 can activate angiotensin to secrete JAK/STAT3 and JAK/p38/NF-κB to boost the growth of blood growth. NF-κB as a transcript factor, it plays an important role in cancer proliferation, spreading and blood vessel growth.In HCC cell, higher level expression of NF-κB is found, it boost the expression of MMP9 and VEGF by ERK/NF-κB. It’s still unclear of the control mechanism between the activation of NF-κB and signal TGF-β/Smad7、JAK/STAT3、AKT/NFKB,NFKB/STAT3. Some study shows URG4/URGCP can prompt the high level expression of NF-κB, but it need further studied for the activation of signal pathway of URG4/URGCP in HCC cells and how it boost the growth of liver caner blood vessel.Based on the above background, the primary endpoints for the study include:(1) To explore whether URG4/URGCP can boost HCC cell angiogenesis via activating NF-κB signal pathway and the corresponding mechanisms.(2) To investigate whether URG4/URGCP can be potentially used as a target molecule for the diagnose and treatment of HCC.Methods and materialsBy the experiments of real-time fluorescent quantitative PCR and immune protein imprinting, we check the level of URG4/URGCP expression in HCC cells and normal liver epithelial cells. Through the studying of formation of blood vessels using umbilical vein endothelial cells (HUVEC) and migration experiments, we check the cells which bearing the over-expression of URG4/URGCP and remove blood vessel growth ability.With the method of luciferase reporter gene, we check the activity of NF-κB transcription. We inhibit NF-κB by using over-expression resistance to degradation of (IκB)-α mutants and use real-time fluorescent quantitative PCR to detect the expression level of VEGFC, TNF-a, IL-6, IL-8 and MYC and use ELISA method to detect the expression level of VEGFC protein.1. Vectors, retrovirus infection and transfection. We use PCR to expand human’s full length subcloning URG4/URGCP sequence and clone it to the carrier of pMSCV-retro-puro to form the expression carrier of URG4/URGCP.As to remove URG4/URGCP genie, we used siRNA which counter to human URG4/URGCP genie sequence and clone it to pSuper-retro-puro plasmid to form URG4-Ri. For retroviruses generation and infection pls. Refer the the reported methods of Hahn and others (Mol Cell Biol.2002). NF-κB inhibitor expression plasmid pBabe-Puro-IκBα-mut [it can form anti-degradation κB inhibitor(IκB)-α mutant protein (IκBα-mut)] using liposome embedding method(Liposome 2000 instant transfection) to record HCC cells.2. Quantitative real-time RT-PCR.We use TRIzol reagent to extra all cell RNA and choose 2 μg RNA to make cDNA by using random primers. Real-time fluorescent quantitative PCR is done on ABI 7500. Steps are as follows:pre-denaturation 95℃, 10min, then finish 28 PCR circulation which include 95℃ 60s,58℃ 30s,72℃ 30 s; final step is 72℃ 5 min.The calculation of level of targeted genie expression is done by internal GAPDH comparison method, the formula is 2-[(Ct of Genes)-(Ct of GAPDH)].3. Western blotting.we extra all protein in cell and heat it for 5 mins 100℃.20μg protein sample is handled by SDS-PAGE electrophoresis, PVDF membrane electrophoresis, skimmed milk closing and then use rabbit polyclonal anti-URG4 antibody, anti IKK antibody, anti-IκBa antibody or anti-p-IκBα antibody to hybridize. After depriving, we use a-Tubulin antibody to re-hybridize, it’s used as a comparison to original sample.4. HUVEC tubule formation assay.In orifice palate, we add 200 ul to each of 24 orifice and aggregate for 30 mins 37℃. Draw the supernate of HCC cells nutrient solution and mix with HUVECs to make cell suspension. Draw 200μl and add it to each hole,37℃,5%CO2nesting for 24h. Take photo in optical microscope magnified 100 times, the growth for blood vessel is judged from the measured of blood capillary in each photo.5. The chicken chorioallantoic membrane (CAM) assay.Use 8 day-old fertilized egg in CAM. Make a hole on each egg surface and Dia is 1 cm. Remove shell to expose CAM. Place a Dia.0.5 cm filter paper at CAM position and eject 100 μl CM which is extracted from HCC cell culture solution or add CM collected from control cells’empty plasmid. Fertilized eggs are cultured 48hrs 37℃ wet 80-90% and then seal the opening with sterilized bondage. Then use fixative (Methanol 1:1 acetone) to fix for 15 mins and deprive CAM to take photos. By comparing with the control group to calculate relative value for level Ⅱ and level Ⅲ blood vessel number in experiment.6. HUVEC transwell migration assay. Mix HUVECs with 5%FBS’s CM, and put it on the top of polycarbonate transwell filter. Add 500μl 15% FBS culture solution in down room.Culture for 20hrs 37℃ then migrant to lower membrane surface cell and fix with 4% paraformaldehyde and hematoxylin stain for 15 mins. Counting from 10 random chosen filter paper under magnified 200 times and compare with control group to calculate the value.7.Luciferase reporter assay of NF-κB transcriptional activity. Use pNF-KB luciferase reporter and compared plasmid to detect NF-κB transcription activity. Add 3 set 1.4x104 HCC cells into 24 holes to grow. Use lipidosome 2000 to transfect 100 ng luciferase reporter plasmid or compared plasmid Ing pRL-TK Renilla. After 48hrs transfection for luciferase and Renilla signal, use dual luciferase reporter kit to detect.8. Enzyme-linked immunosorbent assay.VEGFC ELISA is done as instruction manual. In standard solution, make 3 complex holes in sample and negative control group, culturing 90 mins 36 ℃. Then wash palate, add specific anti-VEGFC antibody and culture 1 hrs 36℃, then wash palate and add second antibody to culture 1 hr. Read the optical density under OD450.9.Statistical analysis.All test data come from 3 independent repeated experiments, Through the group design two samples t test to compare the difference between the experiment group and control group, each index of difference. When the two groups variance not neat, the use of t’test. All the quantitative indicators are the mean+standard deviation said, with alpha=0.05 was the level of test, using two-sided test. Data establishment and data processing of database using SPSS 13.0 statistical analysis software. Result1.In HCC cell lines, URG4/URGCP protein and mRNA expression are upregulated.In URG4/URGCP protein expression in the liver cell lines of Western blotting experiments, compared with Normal liver epidermal cell line L02, URG4/URGCP protein expression in all seven HCC cell lines(Hep3B、MHCC-97H、 HepG2、SMMC-7721、QGY-7703、Huh7、Bel-7402) were increased obviously. there was significant difference between them. In HCC cell lines and normal liver cells URG4/URGCP mRNA expression of real-time quantitative PCR experiments, compared with normal liver epidermal cell line L02, URG4/URGCP protein and mRNA expression in all seven HCC cell lines were increased obviously. there was significant difference between them.2.Over-expression of URG4/URGCP promotes the angiogenic capacity and expression of VEGFC in HCC cells.In URG4/URGCP protein expression of Western blotting experiments, there was significant difference between them. In HUVEC tubule formation assay, human umbilical vein endothelial cells tubular structure generated number of the experimental group (URG4/QGY7703 URGCP hepatocellular carcinoma cell line and Hep3B) were greater than the control group (Vector QGY7703 hepatocellular carcinoma cell line and Hep3B), there was significant difference between them.In HUVEC transwell migration assay, the migrated quantity of HUVEC of the experimental group (QGY7703 and Hep3B of URG4/QGY7703) was higher than that of the control group (QGY7703 and Hep3B of Vector), there was significant difference between them.In CAM assay, newly generated number of blood vessels of the experimental group (QGY7703 and Hep3B of URG4/QGY7703) were higher than that of the control group (QGY7703 and Hep3B of Vector), there was significant difference between them.In RT-PCR experiments, mRNA expression of vascular endothelial growth factor C content of the experimental group (QGY7703 and Hep3B of URG4/QGY7703) were higher than that of the control group (QGY7703 and Hep3B of Vector), there was significant difference between them.In enzyme-linked immunosorbent experiments, the amount of vascular endothelial growth factor C protein expression of the experimental group (QGY7703 and Hep3B of URG4/QGY7703) were higher than that of the control group (QGY7703 and Hep3B of Vector), there was significant difference between them.3.Silencing URG4/URGCP reduces the angiogenic capacity and expression of VEGFC in HCC cells.In URG4/URGCP protein immunoblot experiments, URG4/ URGCP protein expression’s amount of the experimental group (QGY7703 and Hep3B of URG4/QGY7703) is lower than that of the control group (QGY7703 and Hep3B of RNAi vector), there was significant difference between them. In HUVEC tubule formation assay, human umbilical vein endothelial cells tubular structure generated number of the experimental group (QGY7703 and Hep3B of URG4/QGY7703) was lower than that of the control group (QGY7703 and Hep3B of RNAi vector), there was significant difference between them.In HUVEC transwell migration assay, the quantity of migrated HUVEC of the experimental group (QGY7703 and Hep3B of URG4/QGY7703) was lower than that of the control group (QGY7703 and Hep3B of RNAi vector), there was significant difference between them.In CAM assay, the number of new blood vessels of the experimental group (QGY7703 and Hep3B of URG4/QGY7703) was lower than the control group (QGY7703 and Hep3B of RNAi vector), there was significant difference between them.In RT-PCR experiments, mRNA expression of vascular endothelial growth factor amount of the experimental group (QGY7703 and Hep3B of URG4/QGY7703) was lower than the control group (QGY7703 and Hep3B of RNAi vector), there was significant difference between them.In enzyme-linked immunosorbent experiments, vascular endothelial growth factor C protein expression supernatant fluid’s amount of the experimental group (gene knockout URG4/URGCP QGY7703 hepatocellular carcinoma cell line and Hep3B) was lower than that of the control group (QGY7703 and Hep3B of RNAi vector), there was significant difference between them.4.Over-expression of URG4/URGCP promotes the activation of NF-κB signaling pathway.In the experiments about the luciferase report gene, when URG4/ URGCP expressed, liver cell line QGY-7703 and HEP3B nf-kappa B transcriptional activity was higher than the control group, difference was statistically significant; when URG4/URGCP kept silence, liver cell line QGY-7703 and the experimental group in HEP3B nf-kappa B transcription activity was lower than the control group, difference was statistically significant.In protein immunoblot experiments, when URG4/URGCP gene expressed, liver cell lines QGY7703 and Hep3B the amount of protein expression of the p-IKK and p-IkBa experimental group was higher than the control group, the difference was statistically significant, while the difference in both IKK and IkBa protein expression between the two groups had no statistical significance; when QGY7703 URG4/URGCP gene kept silence, liver cell lines and Hep3B the amount of protein expression of the p-IKK and p-IkBa experimental group was lower than the control group, difference was statistically significant, while the difference in both IKK and IkBa protein expression between the two groups had no statistical significance.In RT-PCR experiments, when URG4/URGCP expressed, gene expression amount in the downstream of the experimental nuclear factor groups TNF alpha, IL-6, IL-8 and MYC in hepatocellular carcinoma cell line QGY-7703 and in the Hep3B was higher than the control group (empty carrier), difference is statistically significant; when URG4/URGCP kept silence, gene expression amount in the downstream of the experimental nuclear factor groups TNF alpha, IL-6, IL-8 and MYC in hepatocellular carcinoma cell line QGY-7703 and in the Hep3B was lower than the control group, the difference was statistically significant.5.Inhibition of NF-κB signaling activity inhibits the ability of URG4/URGCP to enhance the angiogenic capacity of HCC cells.We further explored whether URG4/URGCP increased the angiogenic capacity of HCC cells by activating NF-κB signaling.In luciferase reporter assay of NF-κB transcriptional activity, the luciferase activity of experimental groups, QGY7703 andHep3B, were both lower than that of control groups (empty carriers). There was significant difference among them.In HUVEC tubule formation assay, the Quantity of generated HUVEC structures of experimental groups, QGY7703 and Hep3B, were both lower than that of control groups, There was significant difference among them.In HUVEC transwell migration assay, the Quantity of migrated HUVEC of experimental groups, IκBα mutant expression group, were both lower than that of control groups(empty carrier), There was significant difference among them.In chicken chorioallantoic membrane (CAM) assay, the Quantity of newly generated number blood vessels were lower than that of control groups(empty carrier). There was significant difference among them.ConclusionThis study show, in HCC cells, when URG4/URGCP rises, it activates NF-κB signal pathway to boost HCC angiogenesis. URG4/URGCP could be the potential targeted molecule in HCC inhibiting blood vessels. It provides a promising strategy for treatment of HCC, a new molecular target for future research and development of diagnosis and treatment of caner genie targeting therapy and a scientific foundation for future drug development and clinical diagnosis of HCC. |
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