The Effects On Inhibiting Skp2 Expression In Human Laryngeal Carcinoma Cell Line Hep-2 By RNA Interference

Research Background and ObjectiveLaryngeal carcinoma is a kind of malignant tumor which does great harm to human health and life. And it accounts for about 10-35% in all ENT malignant tumors. Among them, laryngeal squamous cell carcinoma (LSCC) is the most frequent one, and it accounts for 90%. The morbidity of LSCC in northeast is highest in China, and increases year by year. Laryngectomy is the main method for LSCC treatment. Nevertheless, satisfactory treatment results have not been achieved even though by laryngectomy accompanied with radiotherapy and/or chemotherapy, because of poorer quality of life after treatments and their limitation to deal with some patients in advanced stage and/or worse general body state. Therefore, to research the pathological mechanism of LSCC, and to further investigate gene therapy method are focal points in present LSCC research.As a recently discovered gene, Skp2 is related to proliferation and differentiation. It has been discovered in previous laryngeal carcinoma researches that Skp2 expression was positive correlated to clinical stage and lymph node metastasis. And it indicated that increased expression of Skp2 gene was probably related to invasion and metastasis of LSCC. Moreover, significant negative correlation is discovered between the expression of Skp2 gene and p27 antioncogene. And it indicated that increased expression of Skp2 maybe lead to decreased expression of p27 which as a negative regulatory factor in cell cycle through ubiquitin-proteasomes pathway in LSCC development. Therefore, the anticancer ability of p27 gene was inhibited, and cancer cells got better invasion and metastasis ability. To sum up, it is suspected that Skp2 probably is the target for the gene therapy of laryngeal malignant tumor.RNA interference is a recently developed method to block gene expression, which provides better expression inhibition of target gene than antisense oligonucleotides ribozyme technique. It is confirmed that chemosynthesized small interfering RNA (siRNA) was able to efficiently mediate RNA interference effect, but its active duration was short. Method applied siRNA expression vector especial lentivirus vector could prolong active duration effectively.We aimed to observe the influence of inhibition of Skp2 expression with RNA interference technique by lentivirus on proliferation and apoptosis of human laryngeal carcinoma cell line Hep-2 and p27 expression in cell experiments in vivo and animal experiments. And we suppose to provide experimental foundation for further research and treatment of laryngeal cancer.Methods1. RNAi vector construction for human Skp2 geneFour siRNA sequences of Skp2 gene cds region and a negative sequence were designed with computer-aided software. The sequences were directed cloned in eukaryotic expressive pSIHl-Hl-copGFP shRNA Vector. And five recombinant plasmids such as pSIHl-negative, pSIHl-siRNAl, pSIHl-siRNA2, pSIHl-siRNA3, pSIH1-siRNA4 were got.2. Inhabitation of Skp2 expression of human laryngeal carcinoma cell line Hep-2 in vivo(1) To select effective interference consequence targeted to Skp2 gene in Hep-2 cell:Recombinant plasmids were transfected into Hep-2 cell with liposome. Skp2 mRNA before and after transfection was detected with fluorescent realtime quantitated PCR, and the best inhibited interference consequence was selected for following experiments.(2) To construct the stable cell line interfered Skp2 gene with lentivirus system: Hep-2 cells were infected with lentivirus stock solution of tri-vehicle package contained selected pSIH-siRNA3 and pSIH-negative plasmids. The transfected effects were observed, and two stable Hep2-siRNA, Hep2-neg transfected cell lines were got.(3) To detect mRNA expression of Skp2 and p27 in stable transfected cell lines with fluorescent realtime quantitated PCR.(4) To detect protein expression of Skp2 and p27 in Hep2, Hep2-siRNA, Hep2-neg cells with Western blot. (5) To detect the proliferation of Hep2, Hep2-siRNA, Hep2-neg cells with MTT.(6) To detect the apoptosis of Hep2, Hep2-siRNA, Hep2-neg cells with flow cytometer.3. The effect of Skp2 expressive inhibition on human laryngeal carcinoma cell line Hep-2 in vitro(1) Athymic mice were divided into three groups. And cells of three groups were subcutaneous inoculated into each mouse back in dosage of 1.0×107个/ml.(2) Carcinoma development was observed. Athymic mice were executed after 30 days, and carcinoma tissues were separated subcutaneously. The size and weight of carcinoma were compared.(3) Regular pathologic examinations were performed with histological section of carcinoma. The protein expression of Skp2 and p27 were detected in athymic mice carcinoma of three groups with immunohistochemistry.Results1. pSIHl-negative, pSIHl-siRNAl, pSIHl-siRNA2, pSIHl-siRNA3 and pSIHl-siRNA4 recombinant plasmids targeted to Skp2 specific siRNA were successfully constructed with PCR and DNA sequencing.2. Inhibition ratios of Skp2 mRNA in Hep-2 cells after transfected by five recombinant plasmids are 12%,42%,14%,46% and 29%. And the transfective efficiency is merely 50%. Among them, the inhibitory effect of pSIHl-siRNA3 group on Skp2 gene was biggest in Hep-2 cells.3. The transfective efficiency increased to about 90%, and transfected cell lines were able to passage stably after Hep-2 cells were infected by virus stock solution contained packaged pSIHl-negative and pSIH1-siRNA3 plasmids with lentivirus system.4. The Skp2 and p27 mRNA expression in Hep2, Hep2-siRNA and Hep2-neg groups were detected with fluorescent realtime quantitated PCR. The Skp2 mRNA expression in Hep2-siRNA cells decreased about 74%, and p27 mRNA expression increased about 38% contrasted to Hep2 cells. But apparent change of gene expression has not been found in Hep2-neg group. 5. Skp2 and p27 protein expressions in Hep2, Hep2-siRNA and Hep2-neg groups were detected with Western blot. Skp2 protein expression in Hep2-siRNA cells decreased about 76%, and p27 protein expression increased about 89% contrasted to Hep2 cells. Apparent change of protein expression has not been found in Hep2-neg group either.6. Cell proliferation in Hep2-siRNA group apparently slowed down, and apparent change of cell proliferation had not been found between Hep2-neg and Hep2 group with MTT.7. The apoptosis ratios were (2.86±0.21)%, (3.58±0.16)% and (16.64±0.17)% in Hep2, Hep2-neg and Hep2-siRNA group with flow cytometer. The apoptosis ratio in Hep2-siRNA group was apparently bigger than Hep2 and Hep2-neg group. But significant difference of cell apoptosis had not been found between Hep2-neg and Hep2 group.8. Tumorigenesis activity apparently decreased, tumor growth slowed down and tumor volume obviously appeared less in athymic mice carcinoma cell transfected with positive plasmid. But no significant difference was found in oncogenicity and growth activity between negative plasmid group and controls. Weight of tumors was apparently lightened, and oncoinhibition ratio was 79.55% in positive plasmid group on the 30th day after tansfection. No significant difference was found in tumor weight between negative plasmid group and controls.9. Necrosis, active cellular proliferation and obvious cancer nest were observed in tumor center of bearing cancer mice in negative plasmid group and controls by HE dyeing. Most of tumor was composed of parenchyma, and mesenchymal was rare. Less necrosis and more mesenchymal were observed in tumor center in positive plasmid group. There was no significant difference on pathological character between negative plasmid group and controls.10. Increased expression of Skp2 protein and decreased expression of p27 protein were observed in tumor of bearing cancer mice in negative plasmid group and controls by immunohistochemistry. But decreased expression of Skp2 protein and increased expression of p27 protein were found in tumor of bearing cancer mice in positive plasmid group. Conclusions1. The mRNA and protein expressions of oncogene Skp2 in human laryngeal carcinoma cell line Hep-2 were specifically and efficiently inhibited by lentivirus-mediated siRNA expression vectors which were designed focused on Skp2 gene, but the expression of anti-oncogene p27 was increased. Meanwhile, the proliferation of carcinoma cell was inhibited, and apoptosis rate increased.2. The expression of Skp2 and p27 was obviously negative correlated in laryngeal carcinoma generation and development. The inhabitation effects of siRNA targeted to Skp2 on Hep-2 cell were probably derived from the increased expression of p27 gene.3. It is supposed that inhabitation of Skp2 expression with RNA interference technique maybe worked in gene therapy for laryngeal carcinoma.