| Pancreatic carcinoma (Pca) is one of the malignant tumors that threaten human health and one of the most lethal human cancers. In recent years, the incidence of pancreatic carcinoma has been on the rise both at home and abroad, with a 3~7-fold increase, reportedly, within the past decade. Despite efforts and advances in medical science in the past 50 years, conventional treatment approaches, such as surgery, radiation, chemotherapy, or combinations of these, may allow for marginal increases in survival and remain under investigation, with current 5-year survival rates of only 4%. Surgical radical resection remains the only hope for cure, although only 5% to 20% of patients are candidates at the time of presentation due to the biologically aggressive nature of these tumors. Even in this select group of patients, only 25% can hope to survive for 5 years. Clearly, novel diagnostic and therapeutic strategies are needed. With the cohesive understanding of the genetic regulatory pathways involved in pancreatic carcinoma and the rapid development in genetic therapy, exploration in genetic diagnosis and therapy of pancreatic carcinoma has critical significance..In 1988, Amoguero C discovered that 95% (21/22) of human cases had K-ras mutation at the 12th codon. In research followed, people have realized that among all the genes contributing to the occurrence and development of pancreatic carcinoma, K-ras gene is the most involved one. About 80% of pancreatic ductal cancers have an activating point mutation in the K-ras gene,above 98% is the codon 12 mutation and the most common amino acid substitution is aspartic acid ( GAT ) for glycine (46%). The protein expressed by K-ras is called p21 protein. p21 protein, under the catalyzation of GTPase, binds to GTP, and breaks it down to GDP and phosphate. It is a modulator in receptor-mediated signal transduction from outside to the inside of the cell. In normal condition, the 12th codon of K-ras has a sequence of GGT, encoding glycin. When there is a mutation at this codon, changes occurs in the expressed p21 protein so that signal transduction is impacted. Accompanied with other factors, this process leads to carcinogenesis. It has been proved that inhibition of mutant K-ras in pancreatic carcinoma cell can make them unable to form tumor in nude mice,which gives us a promise to cure pancreatic carcimoma by genetic therapy. However,it is essential to effectively inhibit the activity of oncogenic K-ras, but not that of the wild-type K-ras protein in normal tissues,because normal wild-type K-ras plays a very important role in cellular activities, including cell proliferation, differentiation,and survival.Another problerm on transgenic technology is that exogenous gene provokes a strong interferon response in most mammalian cells. Both antisense oligonucleotides and ribozyme can not resolve them and are unsuccessful in vivo.RNA interference (RNAi),a newly rising technology recently, can resolve these problems. It is reported by Fire A in 1998 at first. RNAi is the sequence-specific gene silencing induced by double-stranded RNA (dsRNA). This phenomenon is conserved in a variety of organisms: Caenorhabditis elegans, Drosophila, plants, and mammals. RNAi is mediated by short interfering RNAs (siRNAs) that are produced from long dsRNAs of exogenous or endogenous origin by an endonuclease of the ribonuclease-III type, called Dicer. The resulting siRNAs are about 21–23 nucleotides (nt) long and are then incorporated into a nuclease complex, the RNA-inducing silencing complex, which then targets and cleaves mRNA containing a sequence identical to that of the siRNA. But the mechanisms of RNAi are not fully understood.Several persons inhibited the expression of mutant K-ras in pancreatic carcinoma cell by RNAi and made them unable to form tumor in nude mice.But in their results,the changes of proliferative ability and apoptosis are different.Our study is composed of 4 parts. 1 Diagnosis of K-ras mutation at the codon 12 of Exon one by enrichment of products of mutationObjective:To investigate the feasibility of the method,enrichment of products of mutation,in revealling the characteristics of K-ras at the 12th codon in two difference pancreatic carcinoma cell lines.Methods:After mutant-enriched PCR, the mutation characteristics in the codon 12 of BxPC-3 (wild type), AsPC-1 (mutation type) cell lines were analyzed by agar-gel electrophoresis. In the mean time, K-ras mutations in both cell lines were sequenced and the characteristics of the mutations were revealed.Results:By using two steps nest PCR, mutations in the mutation type cell lines were successfully diagnosed. Moreover, even after a 1/1,000 dilution of 2μg of total DNA from the cell lines, characteristics of mutation were still able to be diagnosed by mutant-enriched PCR; the bands from electrophoresis were clear. PCR products of the two cell lines were subjected into DNA sequencing, and the characteristics of the mutations in both cell lines was revealed as: BxPC-3, no mutation, with the sequence of the 12th codon of GGT, encoding glycin; AsPC-1, mutated from GGT to GAT at the 12th codon, encoding aspartic acid.Conclusions:Mutation-enriched PCR could be used as a sensitive method in detecting K-ras mutation in pamcreatic cancer cell lines.2 Construction of the plasmid expressing shRNA specific to K-rasAsn12 Objective:To construct a plasmid expressing shRNA specific to K-rasAsn12 by using pSilenCircle.Method:Two single-strand DNA sequences encoding mutant-specific shRNA specific to K-rasAsn12 were synthesized and then inserted into pSilenCircle. The recombinant plasmid was named as pSC-K-rasAsn12. By using the same method, pSC-GFP encoding shRNA with GFP was synthesized. Both plasmids were sequenced.Results:The OD260/280 data was 1.8 (pSC-K-rasAsn12) and 1.81 (pSC-GFP), the sequence of plasmids were correct by using sequenator. Conclusion:Construction of the plasmid expressing shRNA specific to K-rasAsn12 by using pSilenCircle is feasible.3 Inhibition of K-rasAsn12 expression by shRNA plasmid in human pancreatic cancer cell linesObjective:To evaluate the possibility of inhibition of K-rasAsn12 expression by using pSC-K-rasAsn12.Method:Both recombinant plasmids were transfected into human pacreatic cancer cell lines, AsPC-1 and BxPC-3, and the expression level of K-rasAsn12 was detected respectively by using the methods of semi-quantitative RT-PCR and Western Blot.Results:The results of RT-PCR indicated that the cells treated with pSC-K-rasAsn12 had a lower transcription of K-ras as compared with the controls (P<0.01). Western blot analysis demonstrated a significantely decreased expression of p21 protein in the cells transfected with pSC-K-rasAsn12 as compared with the controls (P<0.01).Conclusion:PSC-K-rasAsn12 can effectively inhibit the expression of K-rasAsn12, but not wild-type K-ras.4 The inhibition of tumor formation, prolideration and apoptosis of panxreatic cancer cell lines in nude mice by transfection of pSC-K-rasAsn12 Objective:To evaluate the effectiveness of the plasmid, pSC-K-rasAsn12, in inhibiting the proliferative and apoptosis of pancreatic cell lines, and tumor foemation in nude mice.Method:After the transfection of pSC-K-rasAsn12 and pSC-GFP, proliferative ability pancreatic cell lines was estimated by the cell proliferation assay by using the kit of CCK-8; apoptosis was detected by Flow Cytometry (FCM), and the ability of tumor formation was evaluated by injection of pancreatic cancer cells transfected with pSC-K-rasAsn12 to nude mice.Results:After the transfection of pSC-K-rasAsn12 into AsPC-1 cell line, cell proliferation revealed by cell growth curve was found to be decresed (P<0.01), while the apoptosis estimated by FCM was increased (P<0.01), and the capability of tumor formation in nude mice was almost lost, when compared with other cell lines transfected with pSC-K-rasAsn12 or pSC-GFPConclusion:Decreased proliferation and increased apoptosis of AsPC-1, as well as the loss of capability of tumor formation of the cells in nude mice following the transfected with pSC-K-rasAsn12 were evidenced in present study.
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