Identification Of A Novel Tumor Marker For Hepatocellular Carcinoma And Its Primary Founction Studies

Hepatocellular carcinoma(HCC), one of the most frequent malignant tumors ,is the second common cause of cancer death in China, where its mortality rate is 20.37/105. There are a lot of methods to treat the HCC now, including Surgical resection, radiotherapy, chemotherapy and biotherapy . Surgical resection is the most effective method for curing this disease, but a large amont of cases are not adapted to surgery because of their intrahepatic or distant metastases at the time of diagnosis.Therefore, it is very important to detect HCC at earlier period. By reasons of convenience, inexpensiveness, and the satisfactory accuracy, tumor marker of HCC have been used as an effective method for detecting malignant tumors for a long time. These tumor marker can be divided into 4 categories:â‘ oncofetal antigens and glycoprotein antigens;â‘¡enzaymes and isoenzymes;â‘¢cytokines;â‘£relative genes.Although all of above tumor marker plays a very important role in detecting the HCC, but for their own limited reason , no one can be used lonely except mixing together to determine the presence of cancer cells in tissues. In order to find a novle and more specific tumor marker for HCC, we have done the experiment of 2-D electrophoresis and gene chip to find the differentially expressed genes between hepatocellular carcinoma and normal tissue. There are a lot of famous tumor marker for HCC( for example AFP, VEGF, AFU etc) have been creening out , but at the mean time, 6 unkown molecular related with HCC were discoved . LIN-28B is the most obviously changed genes between HCC and normal tissue.AIM Through detecting the differently expressed level of LIN-28B gene between hepatocellular carcinoma , its corresponding paracancerous tissues and non-tumor tissues to evaluate if LIN-28B gene can be used as a novle tumor marker for HCC. Then, by over-expressing or silencing the LIN-28B gene to investigate the function of this gene in the development and progression of hepatocellular carcinoma, which may be provide a new target for curing the hepatocellular carcinoma.METHODS Firstly, construct expressing vector containing human LIN-28B gene, then induce the expression of this gene and purify the fusion protein, and use target protein to make specific and high titer antibody.Secondly , real-time quantitative PCR and Western blot techniques were used to detect the expression of LIN-28B in mRNA level and protein level respectively between HCC and their corresponding paracancerous tissues and normal liver tissues. Finally , construct LIN-28B expressing vector which stable transfected into MCF-7 cell line and RNAi vector targeting the LIN-28B gene which stable transfected into HepG2 cell line, by using the method of MTT, clone formation and Flow cytometry to investigate the effection of cellular proliferation and cell cycle after overexpression or silenceing the LIN-28B gene .RESULTS Successfully constructed a LIN-28B vector which expressed in Escherichia coli. A specific and high titer anti- LIN-28B polyclonal serum in 1:64000 was gained.The Real-time quantitative PCR analysis showed that the level of LIN-28B mRNA had obviously different between poorly-differentiated tumor tissues(100%),moderately-differentiated tumor tissues( 71.4% ) and. well-differentiated tumor tissues( 20% ),whereas the gene can not detected or little expressed in non-tumor tissue and corresponding paracancerous tissues. The Western Blot analysis showed that the protein level of this gene was similar with the mRNA.Stable transfected LIN-28B gene into MCF-7 can promote its proliferation rate, on the contray silencing the expression of LIN-28B gene in HepG2 can decrease its proliferation rate and hold the cell cycle in G1 phase.CONCLUSIONS In summary ,on the one hand , The expression level of LIN-28B protein is correlated with the malignant level of HCC, and the more it expressed ,the higher malignant level of HCC. All these indicated that this gene can be a novle tumor marker for HCC and to predict the prognosis of HCC. On the other hand ,LIN-28B gene can promote the proliferation rate of tumor cell lines , which indicated that it can be a new target for the therapy of the HCC.