| Contest & ObjectivesAcross the world, uterine cervical neoplasm is the most usual cancer of gynecologic for female patient and it’s a serious health risk to women. Epidemiological studies show that the changing tendency of attack age of cervical neoplasms varies by different period. High peak age of the cervical carcinoma in situ patients was from 30 to 35, invasive for 50 to 55 years old. The early stage of uterine cervix cancer usually does not have symptoms and signs, while unusually bleeding of lower abdomen, pelvic pain and sexual pain may occur in the end-stage cancer. Strong evidences exist to support occurrence of this disease is bound up with many risk, including HPV invasion, sexual behavior, vaginal delivery history, smoking, using oestrogen or progestin pills,impaired immunity, high-risk HPV invasion occupy major place. Nearly 4 decades, pathological changes of cervix could early discovery and treatment while the control about mortality and morbidity of cervical neoplasms have been remarkable as a result of thinprep cytologic test(TCT) widely used. Rough calculations verbalized that 500,000 instances of cervical neoplasms in clinical practice occur worldwide annually, developing cities in predominant. Still, ours is in it. Along with overcrowding, underdeveloped, the less health knowledge among our nation, the increasing sense of openness sex and HPV infection, the number of cervical neoplasms patients showed an upward trend and age of onset was younger trend in past 20 years. Every year, there is 130,000 new cases appear and about 50,000 person pass away. It’s urgently need that how to prevent cervical neoplasms and improve the recovery number of patients, because the etiology and pathogenic mechanism of uterine cervix cancer is still unclear. The traditional protocol is to combine surgery with chemoradiotherapy, but this way does more harm to the patient and significantly reduces living standard after treatment, yet the therapeutic effect is far from satisfying. Looking for a new therapeutic approach will be the key to future therapy of cervical neoplasms.Recently, a growing number of scholars have positioned the lever of molecular and gene as the way of prevention and treatment the malignant tumor. Various research reports that much abnormal animate of pathways exist in the course of this disease. People increasingly focused on JAK-STAT. It has achieved a crucial position to monitor viability, spread and dividing; meanwhile, it rules gene utterance by transferring many cytokines and growth factors into the nucleus at the gene level. Abnormal expression and activation of this pathway will promote the development and progression of tumors, so it is one effective molecular target for human tumors. The path accurately transmit cytokines, growth factors and other active substances by negative feedback methods so that to maintain homeostasis. In recent years, research indicates that more than 3 kinds of tumor-suppressing protein related to negative feedback regulation of JAK-STAT signal transduction, such as SOCS family; PTP, for instance SHP-1 and SHP-2; PIAS family. SOCS family have eight members such as CIS, SOCS 1-7, of which SOCS3 is most widely and deeply research. SOCS3 as an important molecule of SOCS family usually does not express or low expression under physiological condition, but the JAK / STAT signal transduction overactive can induce the expression. As SH2 domain could binds with phosphotyrosine to cut down the reaction of STATs and JAKs kinase, which form the negative feedback loop so as to maintain the balance of jak-stat pathways. Studies have shown that SOCS3 can play an active inhibition in a variety of tumors, and therefore it’s highly valued in gynecological tumors. It is believed that SOCS3 will as biomolecular targets and treatment of the molecular target in clinic diagnosis and prognosis of uterine cervix cancer with the development of molecular techniques and the deep research of SOCS3. In Cervical, whether by transforming SOCS3 to regulating the abnormal reaction of JAK-STAT channel, thereby changing outcome of Cervical, even cure malignancies, these problems will wait for further exploring in the later. RNA interference (RNAi) is to induce degradation of homologous messenger ma (mRNA) by use a short sequence specific double-stranded RNA (dsRNA), then generating PTGS phenomenon of specific gene. Small interfering RNA is a necessary factor in rnai and play central role in silencing pathways. It’s the instructional element in mRNA degradation. Lentiviral vector of human gene SOCS3 has been constructed by use siRNA as the tool and pLKO.1 as vector. To study connection between socs3 and Cervical by use virus as media to silence SOCS3 in human cancer cell lines. This will lay the foundation for the research of tumor prevention and treatment.METHODAccording to gene cDNA sequences and features of SOCS3 in Genbank, combined with siRNA design principles and features of lentiviral vector pLKO.1, we have designed upstream and downstream primers and synthesized by the company, after that it become shRNA Oligos by thermal treatment. Amplification pLKO.1 plasmid, handing plasmid by endonuclease, then recovering DNA plasmid from sepharose with extraction kit, finally purification plasmid DNA by DNA purification kit. Connection shRNA Oligos with empty vector, this recombinant was imported to competent cells of DH 5a, painted glass panels, choose monoclonal germ to test PCR in order to distinguish active recombinant that contain insertions. Amplification the active monoclonal and extract recombinant plasmid PLKo.l-SOCS3-siRNA in the light of manual. Bacteria contain clones pLKo.l-SOCS3-siRNA is sequenced in sequencing company after preserve E. coli DH 5 a.RESULT1. Obtain high concentrations plasmid pLKO.1 according to the Plasmid Minipreparation Kit and PAGE gel no obvious other bands.2. The plasmid of PLKO.1 was detected 7000bp and 1900bp fragments with gel ionophoresis after deaingl with AGE 1 and EcoRl, then recycle 7000bp fragment.3. Design siRNA Oligo according to gene cDNA sequences and features of SOCS3 in Genbank, it was annealed and connection the Oligos with restricted DNA products of pLKO.1, the recombinant was imported to E. coli DH 5a competent cells and select monoclonal colony PCR.4. Monoclonal colony PCR is sequenced in Takara company, the sequencing results is consistent with the gene sequence on GenBank. SOCS3 siRNA interference vector PIKO.1-SOCS3 was building successfully.ConclusionThe successful build of lentiviral vector to target SOCS3, which settle ground in farther investigation of SOCS3 about therapy and preventive of uterine cervical neoplasms, even in tumors. |
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