Experimental Study Of Human Hepatic Cancer Cells Inhibition By PAMAM-eIF4E-shRNA Nanoparticles

Background and ObjectiveHepatocellular carcinoma (HCC) which has a bad prognosis and a low survival rate in five years,is the most common malignant tumor in our country.By now there isn't any effectiveness therapy,including radiotherapy, chemotherapy,or surgery.eukaryotic translation initiation factors(elFs) play an important part in the translation initiation phase of a eukaryotic cell.In this family,the factor named eIF4E (Eukaryotic translation initiation factor 4E)can specifically bind with the cap of mRNA 5'(m7GpppN) and adjust its translation and expression.The eIF4E has a high expression in many tumors such as the cancer of head and neck, larynx, lung, mammary gland, thyroid gland, esophagus, stomach, bile duct, colon, and so on.It's high level of expression is closely concerned with the generation, infiltration,and metastasis of the tumor.Small interferencing RNA (siRNA) can silence the target gene specifically and efficiently.It has a wide perspective in therapy of tumors.PAMAM-Dendrimer is a new kind vector of nanoparticles,which has a construction of arborization.It has many advantages compared to the nanoparticles vector used before. For example,it has a bigger capacity, is harmless to human body,etc.This research made a synthesis of siRNA targeting the gene of eIF4E,and used the PAMAM-Dendrimer to carry the siRNA into the cells of HCC,and observe Its effect to the tumor cells.This research may provide a new method to the therapy of HCC.Methods1.Detection of the expression level of eIF4E in HCC cells and tissuesWe used the method of Western Blot to detect the expression levels of protein eIF4E in the human normal mature hepatic cells, human hepatic cancer cell lines HepG2, Hep3B, and Huh7.We used the method of Immunohistochemical to detect the levels of eIF4E in 46 groups of people HCC tissues and its adjacent hepatic tissues.2.Building for eIF4E of PAMAM-D-shRNA vector complexUsing siRNA design software, from the human eIF4E mRNA coding region (GenBank NMBC035166) of the start codon (AUG) downstream to find two "AA" started two joint sequence length 21nts,we design and synthesis the two series, and design a non-specific sequence as control, and make the alignment with human genome BLAST and verify them both single genes. We made the synthesis of single-stranded DNA oligonucleotides above, and then annealed the formation of double-stranded shRNA.Under the room temperature, RPMI1640 medium without serum, respectively we added the shRNA and PAMAM-D vectors, shRNA and liposome,.After mixed reaction, we obtained the PAMAM-D-shRNA complex and liposome-shRNA complex.3.Hepatoma cells ransfection with PAMAM-D-shRNA vector complex, detection the effect caused by reducing the eIF4E expression on the biological characteristics(proliferation and apoptosis) of HCC cells(1)Cell cultureUnder the temperature of 37℃,5% CO2, we put the liposome-shRNA and PAMAM-D-shRNA with HCC cells together respectively.(2)Detection of the eIF4E mRN A expression by the method of Real Time PCRWe extracted the RNAs from cells, prepared cDNAs by reverse transcription reaction,.After PCR amplification,we made image analysis on the ABI Prism 7300 SDS Software which comes with the equipment.(3)Cell viability assay by thiazole blue (MTT)Inoculated cells, after the cells adherent to the test tube,we divided them into three groups, which were respectively added liposome-shRNA and PAMAM-D-shRNA, and the control group without medication. Per hole plus MTT 20ul, lay up for four hours. Made a MTT test respectively at the 1,2,3,4,5,6,7 days. Cells relative inhibitory rate (%)=(1-the experimental group OD/control OD)×100%.(4)Cell cycle and apoptosis rate detected by Flow cytometryUsing PI to detect cell cycle and apoptosis,with TUNEL method to detect the apoptosis rate by Flow cytometry.4.Statistical analysisStatistical analyses were performed with SPSS software(16.0 version),with a P value<0.05 as statistically significant and P<0.01 as very significant.Results1.eIF4E has higher expression levels in hepatic cancer cell lines and hepatocellular carcinoma tissues than in normal hepatic tissues(1)Western Blot Detection of human normal mature hepatic cells and human hepatic tumor cell lines with protein expression of eIF4E, showed that HepG2 cell line has the highest expression of eIF4E protein. (2)Used Immunohistochemical method to detect pathological wax blocks of 46 groups.There are 32 groups of people HCC eIF4E protein levels higher than in noncancerous tissue, no significant difference in 6 groups, in 8 groups human hepatic tissues'protein levels of eIF4E were lower than adjacent tissues.2.successful construction of PAMAM-D-shRNA vector complexBased on the shRNA design principles, we made the synthesis of specific DNA fragments. And then annealed,synthetic the DNA template transcription, purification. Put the shRNA and PAMAM-D vector to 1μg shRNA:0.65μg PAMAM-D proportion together,through electrostatic interaction,they can form the complex of PAMAM-D-shRNA vector.3.Reduced of eIF4E expression can inhibit proliferation of hepatic tumor cells(1)Detection of the eIF4E mRNA expression by the method of Reverse transcription-polymerase chain reaction (RT-PCR)Under the same condition, PAMAM-D-shRNA group performed a stronger effect on reducing eIF4E gene expression.It is visible that PAMAM-D as a carrier of shRNA has a higher transfection efficiency.(2) Cell viability assay by thiazole blue (MTT)The results of the 1,2,3,4,5,6,7 day MTT tests,all showed P<0.05,indicating statistically significant difference.PAMAM-D-shRNA group has higher inhibitory rate in hepatic cancer cells.(3)Using PI to detect cell cycleAt G0-G1 phase, liposome-shRNA group has higher rate of cell apoptosis; at S and G2-G1 phase, PAMAM-D-shRNA group has a higher rate of cell apoptosis.(4) detection of the apoptosis rate by Flow cytometry with TUNEL method With TUNEL method, P was 0.013, P<0.05 indicated significant difference. PAMAM-D-shRNA group has a higher cell apoptosis index than liposome group.Conclusions1.In HepG2 and other hepatoma cell lines and most human HCC tissues,eIF4E expression was increased.2.Using PAMAM-D as a carrier, can efficiently transfer shRNA into the cell.3.The shRNA targeting eIF4E infection of hepatic cells, can down-regulate the expression of eIF4E gene, and to promote tumor cell apoptosis. 4.eIF4E is expected to become a target for treatment of hepatic cancer in molecular biology. PAMAM-D is expected to become the more efficient and safer new carrier in the dehepaticy of shRNA.