Effects Of CD55 Gene Silence In Biological Behaviour Of Pancreatic Cancer

Objective:To study the expression of CD55 in human pancreatic cancer and clilic Significance:Methods:The expressions of CD55 protein and mRNA in 12 pairs of pancreatic cancer and adjacent non-tumorous tissues were detected by immunohistochemical staining, Western blot and quantitative Real-time PCR. The expressions of CD55 protein of additonal unpaired 31 pancreatic cancer tissues were also detected by immunohistochemical staining.The expressions of CD55 protein in six human pancreatic cancer cell lines were also detected by Western blot;We detected the expression of CD55 protein and mRNA in seven human pancreatic carcinoma to search for cell lines as objects for study.Results:In 12 pairs of pancreatic cancer and adjacent non-tumorous tissues RT-qPCR showed that the expression of CD55 mRNA of pancreatic carcinoma tissues(the median: 4.77895) was significantly higher than that (the median:2.45131) of matched adjacent non-tumorous tissues (P<0.05),Immunohistochemical staining demonstrated that the positive expression rate and numbers of positive staining cells of pancreatic carcinoma tissues (100% strong positive) were notablely higher than those of matched adjacent non-tumorous tissues (only 25% weak posive) (P<0.05), Western blot proved that the expression of CD55 protein of 91.7% pancreatic carcinoma tissues (the median:5.57578) was higher than that (the median:1.8713) of matched adjacent non-tumorous tissues(P<0.05); by immunohistochemical staining we found that 38 of 43 pancreatic carcinoma express CD55 protein and the expression of CD55 protein was positively related to the degree of differentiation,lymphatic metastasis and clinical stage,but not related to age and gender.The relative expression levels of CD55 protein in BxPC-3(the relative value: 3.159)and PANC-1 (the relative value:1.726) cell lines were higher than other cell lines.Conclusions:CD55 is abnormally activated in pancreatic carcinoma and is related positively to the degree of differentiation,lymphatic metastasis and clinical stage.It might play an important role in the carcinogenesis and progress of pancreatic carcinoma and is expected to become a new therapeutic target for pancreatic carcinoma.The BxPC-3 and PANC-1 cell lines might be chosed for experiments of RNA interference. Objective:To construct and identify the human CD55-shRNA lentiviral vector.The CD55 gene silencing effect was detected after transfecting BxPC-3 cell lines with it.Methods:I searched in pubmed for RNA interference sequence targeting CD55. The lentiviral vector of shRNA targeted against CD55 was constructed by pGCSIL-GFP.We demonstrated the above recombinant through PCR and sequence analysis of positive clones and we named it as CD55-RNAi.Then we packaged the CD55-RNAi by co-transfecting 293T with three packaging plasmids into lentiviral particles (CD55-RNAi-LV).At last,we detected the titre of the lentiviral particles by titration dilution.Afeter culturing stably tansfected cell lines by limiting dilution analysis,We detected the expressions of CD55 protein and mRNA by RT-qPCR and Western blot to confirm CD55 gene silencing effect.Results:PCR identification and sequence analysis of positive clones suggested the inserted sequences were correct. We packaged the CD55-RNAi successfully into CD55-RNAi-LV and the titre of the lentiviral particles is 6.00×108Tu/ml. The infection efficacy of BxPC-3 cell line was above 60%.We found that the inhibitory effect of the CD55 gene and protein levels were respectively 75.92% and 88.64% in CD55-RNAi-LV stably tansfected cell lines cultured by limiting dilution analysis.Conclusions:The human CD55-shRNA lentiviral vectors were constructed successfully. The CD55 gene silencing effect in CD55-RNAi-LV stably tansfected BxPC-3 cell line is effective. Objective:To study the specific silencing effect of lentiviral vector-mediated RNA interference targeting CD55 (CD55-RNAi-LV) on cell migration,cell aggression and the growth in human pancreatic cancer cell line BxPC-3.Methods:The CD55-RNAi-LV and NC-GFP-LV(the control virus,direct gift) was transfected into BxPC-3 cells and we established stably tansfected cell lines by limiting dilution analysis.The BxPC-3 cells stably transfected with the CD55-RNAi-LV(experimental groups) and those stably transfected with the NC-GFP-LV (control groups) and without any treatment (blank groups) were compared. Cell migration and cell aggression was examined by Transwell assay. And the expressions of E-cadherin, MMP-2 and MMP-9 were detected by RT-qPCR and Western blot. And We used flow cytometry to detect biological behavior of target cells,such as cycle, proliferation and cell apoptosis. Moreever,cells were injected into nude mices subcutaneously with cell concentration of 1.0×107/200μl respectively. The growth of tumor was examined for 7 weeks.Results:The cell migration in experimental group (average of the number of migratory cells:79.83±7.62) was significantly suppressed compared with that in control (103.5±10.17) or in blank groups(105.9±9.53) by Transwell assay (P<0.05). The cell aggression in experimental group (average of the number of aggressive cells:25.41±4.96) was obviously suppressed compared with that in control (54.95±6.58) or in blank groups(57.62±7.63)(P<0.05). Western blot and RT-qPCR both proved that the expression of E-cadherin mRNA and protein in experimental group was higher than that in control or in blank groups; and showed that the expression of MMP-2 protein and mRNA and the expression of active MMP-9 protein in experimental group cells were significantly less than those in control or in blank groups (P<0.01). The cells in experimental group(the mean of the apoptosis rate: (33.84±1.09)%)were found to exhibit significantly higher apoptosis than that in control (the mean of the apoptosis rate:(3.32+0.19)%) or in blank groups (the mean of the apoptosis rate:(15.45+0.47)%) by flow cytometry (P<0.05). The cells in experimental group (the mean of the proliferation rate:(30.06±0.97)%) were detected to show less proliferation than that in control (the mean of the proliferation rate:(42.28±2.31)%) or in blank groups (the mean of the proliferation rate:(40.41±1.29)%) by BrdU flow cytometry (P<0.05).The target cells stagnated in phase Gl after transfection(P<0.05). The subcutaneous tumors in the experimental group occurred later and smaller than those in control or blank groups(P<0.05).Conclusion:Silencing CD55 gene might lower cell migration and aggression, also suppress the growth of human pancreatic cancer cell line BxPC-3 in vitro and in vivo and may be a promising therapeutic target for pancreatic carcinoma.