The Function Of The RNAi Lenti-virus Targeting PD-L1to Pancreatic Cancer Cell Patu8988

Objective To investigate pancreatic carcinoma and normal pancreatic tissuespecimen of PD-L1（programed de ath ligand-1） gene expression. To screen targetinghuman PD-L1mRNA siRNA effective sequence, and to construct RNA interferencelenti-virus targeting PD-L1. The reconstructed lenti-virus transfect human pancreaticcarcinoma cell line Patu8988, detecting the interfering efficiency. To lay the foundation forfurther investigation on the function and mechanism of PD-L1gene in pancreaticcarcinoma.Methods Experiment is divided into two parts1.Collecting22pancreatic carcinoma tissue samples from clinical resection, and at thesame time in22patients with normal pancreas tissue specimens. ImmunohistochemicalS-P method was used to detect PD-L1expression.2. Three plasmid expression vectors coding for shRNA targeting PD-L1genesequence were constructed．And then they wree transfected stably into293T cells whichPD-L1gene was over-expressed. The PD-L1gene silencing effect was measured byWestern blotting respectively. The most effective sequence of shRNA targeting PD-L1waschosen. Recombinant lentivirus carrying PD-L1-siRNA was produced by293T cells, alsothe virus titer was measured. Flow cytometry was used to measure the infection efficiencyafter the LV-PD-L1-siRNA transfection into Patu8988cell. PD-L1gene knockdown wereobserved by Real-time PCR and ELISA test.Results1.PD-L1in pancreatic carcinoma tissue of the significantly higher than normalexpression in the pancreas（χ~2=11.023，P<0.01）. 2.Transfection of293T cells with shRNA plasmids resulted in an inhibition of PD-L1protein expressions respectively. The most potent effect of silencing PD-L1geneexpression was conducted by PD-L1-shRNA-3. The shRNA fragment targeting againstPD-L1was successfully packaged into the lentivirus，and the titer was approximately5x109TU/ml. The infection efficiency was above90%when green fluorescent protein(GFP) stably expressed in Patu8988cell. The mRNA and protein inhibitory rates of PD-L1were very high.3.Conclusion1.Pancreatic carcinoma tissue PD-L1are excessively expressed, PD-L1may plays animportant role in the developments of pancreatic cancer.2.Successful screening the effective RNA interference sequence targeting PD-L1gene,and successfully constructed RNAi lenti-virus targeting PD-L1gene. Laying thefoundation for further investigation on the function and mechanism of PD-L1gene inpancreatic carcinoma.