| Objective Primary liver cancer(PLC) is one of common malignant tumors in China.There are about 600 to 700 thousand cases in China.With high invasive features,it is easy to lead both intrahepatic and extrahepatic metastasis rapidly.The main reason for the pathogenesis of hepatic cell carcinoma(HCC)is hepatitis B virus (HBV) infection in China.There are bout 120 million carriers of hepatitis B virus ,about 120 thousand people died of HCC each year .The research about HBV was mostly concentrated in the epidemiology, but the specific mechanism of inducing the liver cancer is still unclear. In recent years, many researches were concerned aboutHBX protein in HBV genome,it has a strong trans-activation in tumors, play an importent role in the development of HCC.However,the randomness of HBV integration and gene mutation are easily led to the complexity of HCC occurrence.Hypoxia is a very common phenomenon in most of solid tumors.HIF is one of important transcriptional regulators maintaining oxygen balance in tumor cells.HIF promote the transcription of target genes by binding to the hypoxia response element (HRE)of target genes Under hypoxic conditions.The protein products play a variety of biological functions, such as participating in the formation and development of tumor blood vessels,energy metabolism of tumor cells,the proliferation and survival of tumor cells. A number of studies show that hypoxia and oxidative stress are known as the induction mechanism of HIF.HIF pathway is a key signaling pathway activated under hypoxic conditions.Initiating a variety of diseases, HIF can lead the expression change of VEGF.VEGF is widely recognized as the most important factor of stimulating angiogenesis of tumor. VEGF is distributed in brain, kidney, liver, spleen, lungs,bones and other organizations both in human and animal.participating in normal embryonic development, follicular maturation and physiological regulation of kidney etl.It plays an important role and in inflammation,wound healing, especially in tumor microvessel formation, invasion and metastasis.In recent years, studies have shown that VEGF expression in PCC was significantly higher than that of normal liver tissues,played an important role in the growth, invasion and metastasis of HCC.If the HBV infection and hypoxia can synergistically induced VEGF expression in hepatocellular carcinoma cells is unclear.Methods In this study,we selected human hepatoma HepG2 cells and HepG2.2.15 cells,cultured HepG2 cells and HepG2.2.15 cells in normoxia and hypoxic conditions invitror espectively.The cell morphology was observed by the inverted light microscope.The change of VEGF mRNA and its protein was detected under different conditions within different time points(2h,6h,12h,24h,48h).The relative expression levels of VEGFmRNA and its protein in two cells were detected by reverse transcription polymerase chain reaction(RT-PCR) and Westen-Blot respectively.By analyzing the expression differences of VEGFmRNA and its protein before and after the HBV infection,in normoxic and hypoxic conditions,we can explore the role of HBV infection and hypoxia in HCC invasion and metastasis,in order to provide experimental basis for treatment of liver cancer from aspects of anti-angiogenic theory and etiology.This study include the following three aspects:Cultured HepG2 and HepG2.2.15 cells in hypoxic condition;Detecting the relative expression levels of VEGFmRNA in two cells by reverse transcription polymerase chain reaction (RT-PCR).Detecting the relative expression levels of VEGF protein in two cells by Western-Blot.Results VEGFmRNA and VEGF protein was expressed in two cells both before and after HBV infection.The expression levels of VEGFmRNA in HepG2.2.15 cells is higher than in HepG2 cells after transfection of HBV(p<0.05).In normoxic conditions, HepG2 and HepG2.2.15 cells expressed VEGFmRNA.After hypoxia treatment,the expression levels of VEGFmRNA of two cells is significantly higher than in the normoxia conditions. Compared with the normoxia groups ,the expression levels of VEGFmRNA in hypoxic groups were statistically significance(p <0.05).And the level of VEGFmRNA expression in hypoxia groups is gradually increased with the prolongation of time(r=0.482 and 0.975,respectively).Compared with the HepG2 cells,HepG2.2.15 cells,which is transfected by HBV,expressed higher levels of VEGF protein.The difference was statistically significant(p<0.05).In normoxic conditions,HepG2 and HepG2.2.15 cells expressed VEGF protein.After hypoxia treatment,the expression levels of VEGF protein of two cells is significantly higher than in the normoxia conditions.Compared with the normoxia groups ,the expression levels of VEGF protein in hypoxic groups were statistically significance(p <0.05).And the level of VEGF protein expression in hypoxia groups is gradually increased with the prolongation of time(r =0.823 and 0.822,respectively).Conclusionâ‘ When change the normoxic conditions to hypoxic conditions,the expression levels of VEGF in PLC cells increased.With the time increasing in hypoxic condition,the level of VEGF transcription and translation is increasing.Which indicate that the Hepatoma cells under the hypoxic emergency can express VEGF to adapt the hypoxic condition in the short term through a series of regulatory pathways within cells.â‘¡The Hepatoma cells infected by HBV is also have an increasing trend in VEGF expression.With the time increasing in hypoxic condition,the level of VEGF expression is increasing.Which indicated that HBV infection may be synergistic stimulation of hypoxia, affecting the level of VEGF expression at the gene level.The VEGF expression level is increased, which may be the important regulatory aspects on the adaptation to hypoxic microenvironment and to maintain their growth and invasion with the synergy of HBV infection.Then by regulating the expression of other target genes,the VEGF Promote the growth of hepatoma cells.Anti-VEGF therapy,in order to improve the hypoxic environment within HCC cells,make the technique of anti-VEGF treatment become a mature way.
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