Construction Of ATGL Eukaryotic Plasmid And Its Role In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is a common malignant tumor,including hepatocellular carcinoma(liver cancer primary,PLC)and secondary liver cancer(liver cancer secondary,SLC).China is the country with the highest incidence and mortality of liver cancer,the number of people accounted for more than 50% of the world,the mortality rate accounted for 45%,ranking first in the world.At present,the first choice of treatment of primary liver cancer is surgery,but most of the patients lost the opportunity to surgery when disease found.The 5 year survival rate of HCC is only 15%.Primary liver cancer patients with chemotherapy,radiofrequency ablation therapy and traditional Chinese medicine comprehensive treatment can increase the survival rate of patients,improve the quality of life,but the effect is not satisfactory.Proliferation and metastasis are two of malignant characteristics of liver cancer,targeted therapy of liver cancer proliferation and metastasiscan inhibit the progression of liver cancer and improve the living standards of patients.However,there is no effective drugs significantly inhibited the proliferation and metastasis of hepatocellular carcinoma;to find new drugs target to inhibition of the proliferation and metastasis of hepatocellular carcinoma,we investigated the role of ATGL on hepatocellular carcinoma,and provide theoretical and experimental basis for the future treatment of hepatocellular carcinoma by ATGL.We first designed primersbaseing on the ATGL sequence in Pubmed,using liver cancer cDNA as the template,and amplifiedATGL gene by PCR followed by constructed to the pcDNA3.1+ vectorthrough the molecular cloning.Next step was sequencing to verify the success of its construction.ATGL plasmid was transfected into HCC cell lines,which was highly expressed,and its mRNA level was nearly 20 thousand times higher than that of the control group.The proliferation of hepatocellular carcinoma was detected by MTT assay and plate clone;and the metastasis of hepatocellular carcinoma was assessed by Wound healing assay and migration assay.Western blot was detectedexpression of p-AKT and p-ERK.First,analysis of proliferation of ATGL-transfected hepatocellular carcinoma cell lines HepG2 and Hep3 B byMTT assay,we found that ATGL significantly promoted liver cancer proliferation.Clone formation experiment also showed that overexpression of ATGL facilitated tumor growth.Second,Wound healing assay and migration assay strongly implied that overexpression of ATGL has no effect on metastasis of hepatocellular carcinoma.Third,the p-AKT level and p-ERK protein level in hepatocellular carcinoma cells treatment witn ATGL overexpression were detected by Western blotting,amd the result showed that ATGL can upregulate the phosphorylation of AKT,but no ERK phosphorylation.We successfully constructed ATGL eukaryotic expressing plasmid and translated in liver cancer cells.Furthermore,ATGL could significantly promote the proliferation of hepatocellular carcinoma cells,but no metastasis.Moreover,upregulation of p-AKT level by ATGL,rather than p-ERK,indicating that ATGL promoted the proliferation of hepatocellular carcinoma cells via p-AKT signaling pathway.