Effects And Mechanisms Of Adenosine A_2A Receptor In Hepatocellular Carcinoma

Background and AimsHepatocellular carcinoma（HCC）is one of the most common malignant tumors in the world,ranking the third highest in cancer-related mortality.More than half of the world’s hepatocellular carcinoma deaths occur in China.At present,the treatment of hepatocellular carcinoma.Include surgical resection,Liver transplantation,interventional therapy,chemoradiotherapy and targeted therapy.Still,surgical resection is the main treatment for hepatocellular carcinoma.However,the recurrence rate is high after radical resection,and the high metastasis rate is still the main factor that affects the prognosis of HCC patients.Therefore,it is imperative and meaningful to explore the factors affecting the growth,invasion and metastasis of HCC and to develop new treatment measures.Adenosine is a precursor to the synthesis of adenosine triphosphate（ATP）,and,as an intermediate product of adenine nucleotide metabolism,adenosine is produced in almost all cells.Adenosine acts on various human systems through four adenosine receptors,A₁,A_2A,A_2B and A₃,and participates in the regulation of various physiological and pathological processes.Adenosine plays an important role in regulating many diseases in human body.Current studies have found that adenosine participates in many pathophysiological processes through adenosine A_2A receptor.It also plays a very important role in the regulation of tumor microenvironment.High concentration of adenosine inhibits the cytotoxicity of immune cells to tumor through adenosine A_2AA receptor.A large number of studies have reported that selective adenosine A_2A receptor antagonists can reactivate the immune function of immune cells in tumor tissues.The selective adenosine A_2A receptor antagonist is expected to be used in the adjuvant treatment of tumor.The role of promoting invasion and metastasis in thymoma,melanoma,non-small cell lung cancer,human head and neck squamous cell carcinoma through adenosine A_2A receptor has been reported.At present,there are no reports on the expression and role of adenosine A_2A receptor in hepatocytes.This study is intended to observe the expression of adenosine A_2AA receptor in hepatocellular carcinoma tissues and liver cancer cells,and to investigate the adenosine A_2A receptor’s roles of proliferation,angiogenesis and apoptosis in hepatocellular carcinoma.The relationship between the expression of adenosine A_2AA receptor and the prognosis of patients was analyzed.And also,the downstream signal pathway of adenosine A_2A receptor were preliminarily explored,which provided a theoretical basis for opening up a new therapeutic target for hepatocellular carcinoma.Methods1.Expression of adenosine A_2A receptor in hepatocellular carcinoma and paracancerous tissues（1）Collection of liver cancer tissues and paracancerous tissues:all specimens were collected from the second hepatic surgical department of the Eastern Hepatobiliary surgery Hospital（approved by the Ethics Committee of the Eastern Hepatobiliary surgery Hospital,with the knowledge and consent of the patient and his family.）（2）The expression of adenosine A_2A receptor in hepatocellular carcinoma and paracancerous tissues:the expression of adenosine A_2A receptor was investigated by Western Blot immunoblotting in each group.2.To detect the expression of adenosine A_2A receptor in hepatocellular carcinoma cell line and normal hepatocyte cell line in vitro.The expression of adenosine A_2A receptor in human hepatoma cell line HepG2 and normal liver cell line L02 was detected by real-time quantitative PCR.3.The construction of adenosine A_2A receptor interference lentivirus vector and the packaging of lentivirusThe oligol of adenosine A_2A receptor gene was designed and ligated to pds019-pl6.3-SHRNA-BSD vector,so the plasmid CL810PDS19-shADORA_2A was obtained.The result of sequencing was verified by restriction endonuclease digestion.The constructed adenosine A_2A interfering lentivirus vector CL810_PDS19-ADORA2A and packaging plasmid were co-transfected into 293T cells to package lentivirus.The titer of adenosine A_2A interference lentivirus LV478 was obtained.4.The establishment of a stable low expression cell line of adenosine A_2A receptorAdenosineA_2A packaged lentivirus infected HepG2 cells,after 72h infection was observed in proportion reached more than 90%,while not observed phenomenon of large area of cell death,the expression levels in the two groups for the detection of adenosine A_2A receptor qRT-PCR results showed that the experimental group of the adenosine A_2AA receptor RNA level significantly lower than the control group,the knockdown efficiency of the experimental group is more than 70%.so it can be used in the function and mechanism of adenosine A_2A receptor in the following experiment.5.The effect of adenosine A2A receptor on the biological behavior of hepatoma cell line was detected in vitro.In order to detect the effect of adenosine A_2A receptor on the biological behavior of liver cancer cell line,we set up the liver cancer cell line which expressed adenosine A_2AA receptor knocked down by plasmid transfection,and at the same time,we set up the hepatoma cell line transfected with disordered sequence plasmid which as a negative control group,there were also hepatoma cells with no sequences transfected,which is set as blank control group（KB group）.Our preliminary experiment results showed that,Adenosine at the concentration of 100μM had the strongest effects on the proliferation,invasion and anti-apoptosis of hepatoma cells.Therefore,in order to study the effect of adenosine on the biological behavior of hepatoma cells,we added three groups of cells into 100μM adenosine at the same time.The changes of various biological behaviors of hepatoma cells were observed in 100100μM adenosine environment.Therefore,shA_2A+adenosine group,NC+adenosine group and KB+adenosine group were set up at the same time.（1）the knockdown effect of adenosine A_2A receptor on the proliferation of hepatoma cell lines:The relative optical density of six groups cells at 48h,72h and 96h were measured by using the method of CCK-8,and the relative optical density curves were plotted.（2）The knockdown effect of adenosine A_2A receptor on apoptosis:Cells in six groups were stained by Annexin-V and 7-AAD,and the proportion of apoptosis was detected by flow cytometry.（3）The knockdown effect of adenosine A_2A receptor on the expression of VEGFThe expression of VEGFmRNA in six group is detected using qRT-PCR（4）The knockdown effect of adenosine A_2A receptor on angiogenesis:AThe same amount of cells were co-cultured with HUVECs cells for 24 hours,the number of tubular structures produced by HUVECs on matrix glue was observed under inverted 200-fold microscope.Three visual fields were randomly selected to calculate the angiogenesis length.（5）The knockdown effect of adenosine A_2A receptor on the invasive power of hepatoma cells:the cells of the same amount of cells from the six groups were inoculated into the glial transwell chamber for 24 hours respectively,after 24 hours,the cells were stained with crystal violet.The number of cells in the lower chamber was counted.6.Relationship between overexpression of adenosine A_2A receptor and prognosis of patients with hepatocellular carcinomaSPSS 23.0 software was used for data analysis.The overall survival（OS）and Disease-free survival（DFS）curves were plotted by Kaplan-Meier method.Log-rank test was used to compare the difference signifience of survival curves between different groups.univariate and multivariate analysis of the influencing factors of OS and DFS were carried out using Cox proportional risk regression model.The best cutoff value of adenosine A_2A receptor expression level was determined by X-tile software,and the test level（α）were 0.05.7.Primary study on the downstream signal transduction pathway mediated by adenosine A_2A receptorWestern Blot was used to detect the expression level of AKT、p-AKT、ERK and p-ERK protein in six groups of cells.Results1 The result of qRT-PCR demonstrate that the expression of adenosineA_2A receptor was 2.015±0.078 in hepatocellular carcinoma tissues and 1.329±0.071 in homologous paracancerous tissue.The expression of adenosineA_2A receptor in hepatocellular carcinoma tissues was higher than that in homologous paracancerous tissues,and the difference was also significant in the result of Western-blot.In a word,The expression of adenosineA_2A receptor in hepatocellular carcinoma tissues was higher than that in homologous paracancerous tissues.2.The expression of adenosine A_2A receptor in HepG2 cell line was significantly higher than that in L02 cell line,and significant difference was observed between the two groups.3.The proliferation effect of adenosine A_2A receptor was detected by CCK-8 cell proliferation assay,and the effect of adenosine on the proliferation of hepatoma cell line was almost disappeared after the expression of adenosine A_2A receptor was knocked down.4.Flow cytometry showed that the knockdown of adenosine A_2A receptor inhibited the antiapoptotic effect of adenosine.5.The results of qRT-PCR showed that adenosine A_2A receptor silencing attenuated the effect of adenosine on VEGF expression.6.The results of HUVECs angiogenesis test showed that the knockdown of adenosine A_2A receptor inhabits the effect of adenosine on angiogenesis.7 Transwell cell invasion assay showed that the knockdown ofadenosine A_2AA receptor inhabits the ability of adenosine to promote the invasion of hepatocellular carcinoma.8.The results of multi-factor analysis showed that the high expression of adenosine A_2A was an independent risk factor affecting OS in patients with hepatocellular carcinoma.9.The preliminary results of Western Blot showed that adenosine could promote the expression of p-AKT and p-ERK,and the knockdown of adenosine A_2A receptor inhabits the expression of p-AKT and p-ERK.ConclusionOur study found that adenosine A_2A receptor is highly expressed in hepatocellular carcinoma tissues and hepatoma cells.Adenosine can promote the proliferation of liver cancer cells,inhibit the apoptosis of liver cancer cells,and promote angiogenesis as well.After the knockdown of adenosine A_2A receptor,the positive regulation of adenosine on hepatoma cells was obviously weakened.Overexpression of adenosine A_2A receptor is an independent risk factor for OS in patients with hepatocellular carcinoma.The preliminary study on the downstream signal pathway of adenosine A_2A receptor showed that Adenosine A_2A receptor may mediate PI3K/AKT and RAS/RAF/MEK/ERK signaling pathways.