Construction And Identification Of Mirna Interference Vector Targeting Alpha -1,3 Fucosyltransferaseⅳ Gene In Hcc

BackgroundsIn worldwide, the mortality rate of Hepatocellular carcinoma ( HCC ) ranks third of all cancer mortality rate, and it is the second leading cause of death in China. The survival rate is effectively improved by comprehensive therapy including surgical treatment, chemotherapy and other treatments. Even if radical resection, the 1-year and 3-year recurrence rate were 20%-64% and 57%-81%. Due to metastasis and recurrence of HCC, it will be difficult to improve the survival rate of HCC significantly. Overcoming the metastasis and recurrence is a keypoint in the treatment of HCC. There are three pathways of the HCC metastasis. The cancer cells transfer to other sites by blood stream, lymphnode and invading into adjacent organs. Previous studies have demonstrated that HCC is prone to intrahepatic metastasis by portal system. The key step for intrahepatic metastasis is adhesion of HCC cells to the endothelial cells of portal vein.Theα1,3-fucosyltransferase ( FUT ) protein is a kind of enzyme for synthesis of SleX and Lewis X in epithelial cells. The fucosylation step in SLeX synthesis was catalyzed by FUT, a terminal glycosyltransferase. They have nine subtypes. SLeX antigens are synthesized by FUT4. Cancer cells and embryonic tissue that express SleX antigens on their cell surfaces have also been known to adhere to endothelial E-selectin and are associated with malignancy and metastasis. SleX antigen is the smallest ligand with three kinds of selectin protein( E, L, P- selectin ). SleX antigen is an adhesion molecule, and expression of this antigen in gastrointestinal cancer cells, colonic cancer and HCC. It is reportedly correlated with tumor progression, distant metastasis and postoperative recurrence. It is confirmed by pathological examination that 64.10% of HCC can express SleX antigen in primary foci. Higher positive rate of SleX antigen are found in pathological examinations of the patients'who have portal vein tumor thrombus, extrahepatic metastasis, satellite focus and recurrence in three months after surgery. Due to these characters, metastasis of tumor cells can be blocked by monoclonal antibodies preventing SleX antigen adhesion to endothelial cell, which have been proved to function well. Some of the methods for simulating the polymer to suppress the adhesion of cancer cells to endothelia cells have achieved to inhibit the cancer metastasis. But all methods used in the studies have some disadvantages and limitations when applied in practice. Therefore, we plan to apply micro-RNA to interference the enmyze of the HepG2 cell according previous found and studies. Reduced expression of SleX antigen on the HepG2 surface is achieved by miRNA silencing FUT4. Thus the adhesion of SleX antigen to E-selectin was influenced by the interference. The organism can remove these tumor cells because tumor cells are difficult to stay in the tissues and organs.The key of our experiment is designing and synthesising miRNA sequence of FUT4 gene. We firstly design four optimal interfercence sequences targeting FUT4-mRNA based on principles of miRNA designing. Every kind of sequences annealed with vector to form miRNA interference vector, then plasmid amplificating and transfecting cells. We analyzed the effect of interference vector on cells by a series of experiments. We then selected the best carrier to perform the experiment.Objective:1. To explore the design and synthesis miRNA sequence of FUT4 gene.2. To construct target gene vectors ( miRNApcDNA?6.2- GW/EmGFPmi ) expressing green fluorescnet protein were constructed and identified.3. To evaluate the efficiency of miRNA interference on the silence of FUT4 gene .Methods: According to the sequence of FUT4 in GeneBank, we designed FUT4-miRNA interference fragments corresponding with FUT4 using miRNA design software. Interference fragments were synthesized by Invitrogen company, and then were connected with pcDNA?6.2-GW/EmGFPmi miRNA expression vectors which target to FUT4. Plasmids were amplified by transforming into competence E.coli TOP10 and the sequence were identified by Invitrogen company. We also observed the expression of FUT4 under fluorescence microscope ,which is also demonstrated by Fluorescence quantitative PCR and western-blot.Results: Sequencing results confirm that vectors contain the fragments of miRNA and connect correctly. The purity of Plasmids were very high and do not include other DNA impurities by analysis of Agarose Gel Electrophoresis. Results of fluorescence quantitative PCR indicated that the FUT4 miRNA level is lesser in experimental group compared to that in the control group (P<0.05), but there was no significant difference between negative control group and blank control group (P>0.05). Western-blot results showed that the expression of FUT4 protein was significantly reduced in the experimental group than those in control group (P>0.05).Conclusions:1. There is no homology between four groups of miRNA sequence fragments and human proto-oncogene that testing by BLAST. There is no homology between negative control sequence fragment and the target gene.2. Each fragments connect to the vector properly which were complete.3. Plasmid successfully transfected human hepatoma HepG2 cells. The efficiency of transfection is 40%. There is no endotoxin and lower nucleic acid.4. Human hepatoma HepG2 cells exist transcription and translation by plasmid.5. Interference fragment in plasmid of 059-1 group has the best efficient which is 44%-53%.