The Initial Research On The Inhibition Effects Of PTTG1 Gene By RNA Interference In Laryngeal Carcinoma Hep-2 Cells

Tumor is a multi-gene, multi-factor disease, the majority of tumors are related to onco-genes'mutation and over-expression, and related for anti-oncogenes'mutation and inactivation. It has always been an important path to research new therapies of anti-tumor by inhibiting onco-genes'expression.The phenomenon of RNA interference(RNAi) was discovered in 1995. It is a mechanism of post-transcriptional gene silencing. In recent years, the RNAi has become the focus of gene therapy. RNAi technology is based on the principle of alkali repair principle, small interfering RNAs(siRNAs) guide RNA-induced silencing complex(RISC) to identify and combine to target mRNA specifically, then the degradation of mRNA was triggered and the generation of cancer-related proteins was inhibited leading to cancer cells apoptosis or dividing toward maturity, thereby the tumor's proliferation was inhibited.Pituitary tumor transforming gene(PTTG) plays an crucial role in proliferation, differentiation and apoptosis of normal cells. Studies have shown that PTTG1 gene's high expression are closely related to a variety of tumors'occurrence and development. It has a practical significance and potential clinical application to block PTTG1 gene's expression in order to prevent or delay the growth of human laryngeal carcinoma Hep-2 cells by RNAi technology.Based on the PTTG1 gene's mRNA sequence(NM₀04219.2) from GenBank, the online siRNA design software provided by Applied Biosystems company was used to find two different siRNA target sites, then two pairs of complementary single-stranded DNA were syntheticed. The two pairs of complementary single-stranded DNA were mixed after diluted respectively, and two different double-stranded DNA molecules were obtained by annealing, which are named PTTG1a and PTTG1b. The 5'end of both PTTG1a and PTTG1b is EcoRâ… recognition site,while 3'end is BamHâ…  recognition site and five consecutive thymine nucleotide(T) as the termination signal of hU6 promoter; five nucleotides constitute"Loop"in the middle of each sequence. Eukaryotic expression vector plk0.1-puro were digested by EcoRâ… and BamHâ… restriction enzymes, and the 6312bp fragments were recoveried, then, the fragments were recombined together with PTTG1a and PTTG1b respectively, to construct two recombinant plasmids which can express short hairpin RNA(shRNA). The results of double-enzyme digestion and sequencing showed that two recombinant plasmids was successfully constructed and were named as plk0.1-puro/PTTG1a and plk0.1-puro/PTTG1b respectively. The spectrophotometer was used to measur the concentration of plasmid solution, the result showed that the purity of both plasmid are high enough to meet the requirement of transfection experiments.At the same time, the puromycin solution was diluted to eight different concentrations, which were added into cultured human laryngeal cancer Hep-2 cell lines, and the effects to cells'activition of puromycin were observed.Recombinant plasmid plk0.1-puro/PTTG1a and plk0.1-puro/PTTG1b were diluted to different concentrations, and was transfected into Hep-2 cells mediated by liposome LipofectamineTM2000. Then puromycin were used to kill Hep-2 cells without plasmids, and survival cells were collected respectively twelve days later after transfection. The total RNA and protein were extracted from the cells,β-actin was used as internal-control, RT-PCR and Western blot techniques were used to detect PTTG1 gene's mRNA and protein relative expression levels. The results showed that, with the increase of plasmids'concentration, PTTG1 gene's mRNA and protein level decreased gradually compared with the blank control; when 1000ng or 1200ng plasmids were transfected in each hole while used 6-well plates, PTTG1 gene's mRNA and protein can be decreased at least 70% by plk0.1-puro/PTTG1a, while plk0.1-puro/PTTG1b's inhibition rate was less than 20%. Therefore, follow-up experiments were carried out by plk0.1-puro/PTTG1a. When Hep-2 cells were cultured in 6-well plates, the recombinant plasmid plk0.1-puro/PTTG1a and plk0.1-puro was transfected respectively into Hep-2 cells with the same concentrations 1000ng each well mediated by liposome LipofectamineTM2000, then puromycin were added to kill cells without plasmids. After transfection, all of the cells were collected after 7 days, 12 days and 18 days respectively, the total RNA and protein were extracted. RT-PCR and Western blot techniques were used to detect PTTG1 gene's mRNA and protein relative expression levels takingβ-actin as internal-control. The results showed that, with the extension of time, PTTG1 gene's mRNA and protein expression level decreased compared with the blank control; when the recombinant plasmid plk0.1-puro/PTTG1a were transfected into Hep-2 cells more than 12 days, PTTG1 gene's mRNA and protein expression level decreased at least 70%.In general, the siRNA sequences with inhibition efficiency more than 70% have good application prospects. In this study, 12 days after the recombinant plasmid plk0.1-puro/PTTG1a were transfected into human laryngeal cancer Hep-2 cells, PTTG1 gene's mRNA and protein relative expression levels decreased more than 70%, and the PTTG1 gene's expression were inhibited effectively, thus, the recombinant plasmid plk0.1-puro/PTTG1a has a good application value in studies of PTTG1 gene.