| Part Ⅰ Clinical significance of amplification and expression of DcR3gene in human pancreatic carcinomaObjective To investigate the clinical significance of amplication and expression ofDcR3in hunman pancreatic carcinoma.Methods Real time-polymerase chain reaction （RT-PCR） was performed to detectthe amplification of DcR3DNA and the expression of DcR3mRNA in50pairs ofpancreatic tumor tissues and adjacent nontumorous tissues. DcR3protein expression andapoptotic cells were respectively detected by Immunohistochemistry （IHC） and terminaldUTP nick end-labeling （TUNEL） staining, then the correlations between them and therelationship between DcR3expression and clinical pathological factors were analyzed. Theserum levels of DcR3were examined by enzyme linked immunosorbent assay （ELISA）,and corrections between DcR3expression and prognosis were analyzed.Results21cases of pancreatic carcinoma displayed more than2times of DcR3amplification （accounting for42%）, while no such amplification was found innontumorous tissues. DcR3mRNA was overexpressed in tumor tissues compared withnontumous tissues（P<0.01）.21cases with DcR3amplification expressed higher levels ofDcR3mRNA than those of29cases without gene amplification （0.82±0.59vs0.34±0.11,P<0.05）. In addition,71.4%cases with DcR3gene amplification showed DcR3proteinoverexpression, and34.5%DcR3-positive without gene amplification （P<0.01）. Itindicated that DcR3overpression may be dependent on DcR3amplification. DcR3positivecells staining were located in cytoplasm, and the frequency of positive cells in tumortissues was higher than corresponding nontumous tissues （P<0.01）. Positive expression ofDcR3was correlated with tumor size and TNM stage. The level of DcR3in serum of pancreatic carcinoma patients was higher than that of pancreatic benign tumor beforeoperation （P<0.01）, and the level decreased notably afer tumor resection（P<0.01）. Wefollowed up this group of pantients, among which the median survival time was18.5months. Patients with higher levels of DcR3had significantly shorter survival time thanpatients with lower levels of DcR3（P<0.01）. Cox analysis showed that tumor size, TNMstage, lymph node metastasis, and levels of DcR3were factors associated with survivaltime.Conclusions DcR3amplification and overexpression exist in pancreatic tumortissues, and amplification may be one of the factors of DcR3overexpression. Theexpression of DcR3was correlated with cell apoptosis and clinic pathological parameters（tumor size and TNM stage）. The level of DcR3in serum was overexpressed in pancreaticcarcinoma, which was related to survival, and may be a parmeter for prognosis.Part Ⅱ construction and identification of miRNA expression vectorstargeting human DcR3geneObjective To design and construct miRNA RNAi expression vector targetinghuman DcR3gene and detect the suppression efficinecy of constructed vectors.Methods According to the encoding sequence of DcR3（NM003823） inGeneBank, the four specific pre-miRNA single strand DNA oligos were designed andsynthesized, then via annealing and ligating with PP-GFP-miR in order, four kind ofmiRNA RNAi expression vectors were constructed. After determination and sequencing,constructed vectors were thansfected into293T cells with vector which DcR3wasoverexpressed. The expression of DcR3was confirmed by Western blot.Results The recombinant plasmids targeting human DcR3gene were successfullyconstructed after indentified by digestion with restriction and DNA sequencing, and theinhibition effect of constructed vector with DcR3-miRNA-4was higher than the others.Conclusions The effective miRNA RNAi vector targeting human DcR3gene hasbeen successfully constructed, and it will be a useful tool to investigate the function ofDcR3. Part Ⅲ Construction and identification of DcR3targeted miRNAinterfering lentivirus vector in Panc-1cellsObjective To construct lentivirus interfering vector targeting DcR3gene anddetect the suppression efficiency of the constructed vector infected in Panc-1cells.Methods The recombined vector carrying DcR3-miRNA was co-transfected withpackaging plasmid and envelope plasmid into293T cells to produce virus. The virus titerwas measured. The DcR3expression and interferon effect were evaluated by RT-PCR andWestern blot after Panc-1cells infected with the recombinant virus.Results DcR3targeted lentivirus interfering vector was generated and the viruswere produced successfully. The virus titer was approximately7×109TU/ml. Flowcytometry showed that the infection eddiciency was above90%after the recombinedvector carrying DcR3-miRNA infected into Panc-1cells. RT-PCR and Western blotanalysis confirmed that the recombined lentivirus inhibited the expression of DcR3effectively （P<0.05；P<0.05, vs negative control, respectively）, and no inteferon effect（P>0.05vs negative control）.Conclusions The vector of LV-DcR3-miRNA was constructed and infected withPanc-1cells successfully, and it could inhibited the expression of DcR3effectively.Part IV Effect of cell proliferation and apoptosis by Silencing DcR3onhuman pancreatic carcinoma cells in vitroObjective To investigate the effect and the possible mechanism of cell growth,proliferation and apoptosis by Lentiviral-mediated RNAi targeting DcR3on Panc-1cells.Methods Panc-1Cells were divided into three groups: LV-RNAi （infected withrecombinant lentivirus of DcR3RNAi）; LV-NC （infected with vacant virus, as negativecontrol）; Control （non-infected cells as the blank control）. Cell growth and proliferationwere assessed by CCK8（Cell Count Kit）, colony formation assay andEdU（5-ethynyl-2’-deoxyuridine） incorporation assay. Cell cycle distribution and cellapoptosis were detected by flow cytometry analysis. The genchip was used to study thegene change between LV-RNAi and LV-NC, and the result of genchip were confirmed through RT-PCR and Western blot.Results There was no significant effect on cell viability, cell cycle and apoptosiswith FasL or silencing DcR3alone. Whereas when LV-RNAi treated with sFasL for24h,LV-RNAi could inhibit cell viability compared with LV-NC by CCK8assay （P<0.05）.Analysis of clonogenicity indicated that LV-RNAi cells with FasL displayed much fewerand smaller colonies than LV-NC with FasL （P<0.05）. The relative colony number ofLV-RNAi was reduced by nearly36%. Similarly, we found that the relative cellproliferation rate was （69.8±5.93）%for control,（67.6±5.55）%for LV-NC and（39.2±3.83）%for LV-RNAi, showing a significant difference （P<0.05, LV-RNAi vs.LV-NC）. Treating with sFasL24h, the percentage of cells at G0/G1phases in LV-RNAiwas significantly higher than that of LV-NC （P<0.05） and percentage of G2in LV-RNAiwas lower than that of LV-NC （P<0.05）. Analysis of apoptosis by flow cytometry showedthat apoptotic rate was （5.7±0.86）%for control,（4.6±0.73）%for LV-NC and （12.8±1.62）%for LV-RNAi, showing a significant difference （P<0.05, LV-RNAi vs. LV-NC）. We got thesimilar results by flow cytometry analysis when three groups were cocultured with mFasL.Coculturing with mFasL24h, the percentage of cells at G0/G1phases in LV-RNAi wassignificantly higher than that of LV-NC （P<0.05） and percentage of G2in LV-RNAi waslower than that of LV-NC （P<0.05）. Analysis of apoptosis by flow cytometry showed thatapoptotic rate was （3.2±0.35）%for control,（2.8±0.26）%for LV-NC and （11.1±1.49）%forLV-RNAi, showing a significant difference （P<0.05, LV-RNAi vs. LV-NC）.Genechip detection and Gene Ontology analysis showed that silencing DcR3withsFasL（25ng/ml） induced cell apoptosis and G0/G1arrest by regulating signal transductionpathway of cell cycle and apoptosis. The relative mRNA levels of FADD, caspsae3andcaspase8in LV-RNAi were higher than LV-NC （P<0.01; P<0.01; P<0.01, respectively）,and the change fold in PCR was corresponded well with the result of genchip. There wasno significant difference of the relative expression of pERK when silencing DcR3alone（P>0.05）. Whereas when LV-RNAi was treated with sFasL, it could decrease pERKexpression compared with LV-NC by Western blot （P<0.05）.Conclusions It indicated that Panc-1cells were resistant to FasL/Fas-mediatedapoptosis. However, silencing DcR3expression could enhance the effect of FasF/Fas,inhibit tumor cells proliferation and induce cell apoptosis. FADD, Caspase3, Caspase8andpERK involved in apoptotic progress mediated by FasL when silencing DcR3. Part Ⅴ Treatment with LV-DcR3-miRNA inhibited the growth ofpancreatic carcinoma in vivoObjective To observe the inhibition of the growth of human pancreatic carcinomaxenografts in nude mice by LV-DcR3-miRNA.Methods After establishing subcutaneous tumor model and orthotopic tumor modelin nude mice, the mice were treated with LV-DcR3-miRNA, LV-NC or saline in each group,respectively. Efficiency of Silencing DcR3gene was verified by IHC. In subcutaneoustumor model, the growth of xenografts was observed and cell apoptosis was detected byTUNEL and transmission electron microscopy. In orthotopic tumor model, the invasionand metastasis of tumor were observed and survival time of the mice with orthotopicimplantation was analyzed.Results The DcR3expression of LV-DcR3-miRNA group was decreased comparedwith LV-NC group （P<0.05）. In subcutaneous tumor model, intratumor injection withLV-DcR3-miRNA could significantly inhibit the growth of tumor （P<0.05vs. LV-NC）, andthe tumor weight at sacrific in LV-DcR3-miRNA was lighter than that in LV-NC （P<0.05）.TUNEL analysis showed that the apoptotic cell increased in LV-DcR3-miRNA group thanin LV-NC group （P<0.05）. Cells in Control and LV-NC observed under the transmissionelectron microscope were plump with intact karyotheca. The presence of euchromatin andfine particle-like heterochromatin were clearly observed. The nucleolus had a net-likeappearance, indicating vigorous nuclear proliferation. The cancer cells in LV-RNAi groupshowed obvious apoptotic features including decreased cell volume, vanished inter-cellularconnection, increased and marginal distribution of heterochromatin in the cell nucleusagglomerated below the karyotheca. In orthotopic tumor model, the invasion andmetastasis of tumor were improved after silencing DcR3. Survival analysis indicated thatsurvival time of LV-DcR3-miRNA group was longer than LV-NC group （P<0.05）.Conclusions Silencing DcR3expression by gene therapy could induce tumor cellapoptosis and prolong the survival time of tumor-bearing mice. |
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