Establishment Of Eukaryotic Expression Vector PEGFP-C3-JNK3 And SHSY5Y Cell Line Stably Expressing JNK3

Apoptosis is the cellular autonomously and orderly death regulating by gene. In the activity of organism, apoptosis and anti-apoptosis present the unified dynamic equilibrium. Between signal transduction pathways and signal molecules, there may exist complicated relationship to regulate the living and death of cells. Many research indicated that disequilibrium between apoptosis and anti-apoptosis may relate to occurrence and development of tumor. The c-Jun N-terminal kinases (JNKs) are members of the mitogen activated protein kinases (MAPKs) superfamily. Studies have verificated that MAPK signaling pathway can transduce and regulate cellular apoptosis, and JNKs may play an important role in the process.In vertebrate, JNKs are incoded by gene jnk1, jnk2 and jnk3. And the expressed protein including JNK1, JNK2 and JNK3 are Ser/Thr protein kinases, which are mostly found in cytoplasm. There are 10 JNK isoforms expressed in mammalian cells, which are derived from the alternative splicing of three genes. JNK1 and JNK2 isoforms are ubiquitous while JNK3 isoforms are expressed only in brain and testis and, to a lesser extent, heart. Unlike JNK1 and JNK2, JNK3 is unique in having an extended N-terminus of 38 amino acids. In recent years, studies have found that JNK3 is a mediator of nerve cell apoptosis and is consistently associated with nerve cell apoptosis induced by ischemic, hypoxic and poisonous chemical materials. However, researches on the relationships between JNK3 and tumors are not enough at present.In this research, we used DNA recombination technique to construct a new eukaryotic expression vector of human JNK3. Then, we transfected SHSY5Y cell with the vector and established the stably-transfectnd SHSY5Y cell line. Finally we analysed the expression of JNK3, and investigated its relevance to cellular apoptosis to evaluate the physiological function of JNK3.1. Construction and identification of eukaryotic expression vector of human JNK3. Methods: The full-length JNK3 cDNA fragment was amplified by PCR from eukaryotic expression vector pDBLeu-JNK3. The cDNA fragment was oriently inserted into another eukaryotic expression vector——pEGFP-C3. The positive clone was obtained after transformation and antibiotic screening,and it was identified by restriction endonuclease digestion, PCR, and DNA sequencing. Results: The JNK3 cDNA fragment was obtained and cloned into the downstream of CMV promoter of pEGFP-C3. The recombinant plasmid was identified by restriction PCR and endonuclease digestion. DNA sequencing confirmed that the sequence of the inserted element was correct. Conclusion: The eukaryotic expression vector pEGFP-C3-JNK3 was constructed successfully.2. Establishment of stably-transfected SHSY5Y cell line and research on the relationship between JNK3 and apoptosis. Methods: the recombinant eukaryotic expression vector pEGFP-C3-JNK3 was transfected into SHSY5Y cells by lipofectamineTM 2000. After selected by G418, stably-transfected SHSY5Y cell line was establish. The expression of JNK3 was observed by fluorescence microscope and identified by RT-PCR and Western Blot. Then we discovered that high express JNK3 could restrain proliferation and promote apoptosis induced by hydrogen dioxide(H₂O₂). It was verificated by MTT and flow cytometer(FCM). Results: SHSY5Y cell line stably transfected with pEGFP-C3-JNK3 vector was established after liposome transfection and G418 selection. JNK3 gene was efficiently transcribed and translated. And high expression of JNK3 could significantly restrain proliferation and promote apoptosis induced by H₂O₂. Conclusion: Stably-transfected SHSY5Y cell line was established successfully. High expression of JNK3 could obviously restrain proliferation and promote apoptosis induced by H₂O₂. The stably-transfected cell line provides a solid experimental foundation for further studies on the function of JNK3.