Induction Of Apoptosis And Inhibition Of Proliferation In Hep-2 By Survivin Gene Targeting

Laryngeal cancer is one of the most common malignant neoplasm in head and neck.Although a number of the patients with laryngeal cancer were saved by surgery operation, radiotherapy, chemotherapy and Laser, there are about 34-34% patients with laryngeal cancer died of recurrence and metastasis of carcinoma. Advancing of molecular biological technique, gene therapy of carcinoma will bring hope to the patients with carcinoma .To investigate the ability of induction of apoptosis and inhibition of Hep-2 proliferation by down-regulation survivin expression using antisense survivin RNA, the identification of new molecular targets may lead to more effective treatment for laryngeal cancer.More and more studies show suppression of apoptosis plays a critical role in tumor initiation,progression.A candidate molecule for the interface between apoptosis and cell cycle was recently identified as survivin, a new member of the inhibitor of apoptosis protein (IAP) family. It is different from the other IAPs. Survivin is expressed during embryonal development and over-expressed in a variety of human tumors, but lacks expression in terminally differentiated adult tissues. Data suggested that survivin is aberrantly expressed in lung cancer, breast cancer, rectal cancer, gastric cancer, hepatic cancer, esophageal squamous cell carcinoma and head and neck squamous cell carcinoma, melanoma, dermopathic squamous cell carcinoma, prostate cancer, uterine and ovarian cancer, Leukemia, lymphoma and neuroblastoma. Because survivin, unlike other potential therapeutic targets, is expressed in few adult tissue, survivin-specific therapies are likely to produce few, if any, side effects. It may be a potential effective therapeutic target.Antisense RNA is the RNA that is complementary with the mRNA. The antisense RNA could combine with target mRNA specifically and inhibit the translation of target mRNA and suppress the function of the mRNA. In comparison of the antisense oligonucleotides technique, antisense RNA technique has the advantages of simple designing, strong specifity, easy operating, and being economic and time constanting.1.Immuohistochemistry: The expression of survivin was detected by immunohistochemistry in 42 cases laryngeal carcinoma , 20 cases their neighboring noncancerous tissues , 20 cases keratosis of larynx and 10 cases normal laryngeal tissue.The results show that the expression of survivin protein in laryngeal carcinoma (66.6%) was higher significantly than in neighboring noncancerous tissues (40%) and in keratosis of larynx(35% )(P≤0.001), the expression of survivin protein were not detected in normal laryngeal mucosa. The up-regulation expression of survivin in laryngeal carcinoma suggested that survivin may play a role in the pathway of carcinogensis.2.Construction of antisense survivin RNA plasimid:we designed two pairs of the oligonucleotid primer which comprised the EcoR Ⅰand Bgl Ⅱ endonuclease site respectively.( in terms of the sequence of human survivin from Gene Bank accession NM:-001168): ①Sense: 5'- GAATTCATGGGTGCCCCGACGTTGCC- 3'antisense: 5'- AGATCTTTCTTATTGTTGGTTTCC- 3'②Sense: 5'-GAATTCGTCCCTGGCTCCTCTACT-3' antisense: 5' -AGATCTCAAATCCATCATCTTACGC-3'Total RNA was extracted from Hep-2 cells with Trizol. We got the cDNA of survivin from Hep-2 by RT-PCR. They are 370bp and 480bp fragment respectively. At first, the specific cDNA fragment was cloned into pMD18-T vector. The recombinant plasmid was confirmed by DNA sequencing. Then, the eukaryon expression vector pEGFP-C1 and the recombinant plasmid were digested with EcoR Ⅰand Bgl Ⅱ.The released cDNA with the molecular size of 370 bp(480bp) was subcloned into lined pEGFP-C1. Two recombined antisense survivin RNA eukaryon expression vector were constructed and we named them pEGFP/survivin-1 and pEGFP/survivin-2 respectivetly.3. Cell culture and transfectionthe human laryngeal cancer cell lines Hep-2 were cultured in RPMI1640 (GIBCO,INC))supplemented with 10%FCS; lipofectamine 2000 (Invitrogen) was used...