The Reasearch Of Effects And Mechanism Of Silencing Plce1 Gene Mediated By Lentiviral Vector On Esohageal Cancer Eca109cell Xenograft Growth In Nude Mice

ObjectiveTo investigate the effects and mechanisms of the recombinant lentiviral vector for RNA interference(RNAi)of PLCE1 gene on the growth of subcutaneous tumor of esophageal cancer Eca109 cells in nude mice.MethodConstruct one shRNA-PLCE1 plasmid and a negative control plasmid and transfected into Eca109 cells.The mRNA and protein levels of PLCE1 in group of Eca109-negative control,Eca109-shRNA-PLCE1 were determined by real-time fluorescence quantitative PCR and western blotting and confirm the plasmid of Eca109-shRNA-PLCE1 obvious silencing effect for lentivirus packaging.BALB/C nude mice were inoculated subcutaneously with Eca109 cells to establish the subcutaneous tumor model of esophageal squamous carcinoma.The experimental group was injected with 3 × 107 Eca109 cells that had been stably transfected with PLCE1 siRNA lentivirus;and the control group received 3 × 107 Eca109 GEF control cells at the left axillary subcutaneous tissue.The tumor volume and weight of the nude mice were observed upon tumor formation,and the growth of the tumor was observed every three days.After 18 days of inoculation,the mice were then sacrificed,and the stripping surgery of the tumor was weighed and photographed.The tumor volume was measured before and after treatment,and the growth curve of tumor in the nude mice was drawn.The expression of PLCE1,Ki67,Bcl-2 and Beclin-1 protein was detected by immumohistochemical(IHC)and immunofluorescence.TUNEL stain was applied to detect apoptosis in tumor tissue.ResultsThe expression level of PLCE1 mRNA and protein was detected by qRT PCR and Western bolt show the Eca109-shRNA-PLCE1 group were obviously decreased(P<0.05).The mean rate of tumor growth in the Eca109-sh RNA-PLCE1 group was much slower than in the negative control group(P<0.05).The volume of dissected tumors in the experimental group was(0.88±0.30)mm3,,which is decreased than that in negative controls(61.65±6.44)mm3,the difference was significantly(P=0.0020).The dissected tumors in the experimental group and the negative control group had weights of(4.29±1.39)mg and(410±80.97)mg,the difference was significantly(P<0.0001).These data strongly support that PLCE1 promotes tumorigenicity in vivo.The immunohistochemical staining results shows that the expression of PLCE1,Ki-67 and Bcl-2 was decreased in PLCE1 interference group compared to negative controls(P all< 0.05).The immunofluorescence result shows that fluorescence intensity of PLCE1,Ki67 was reduced in Eca109-shRNA-PLCE1 group than negative controls,but the fluorescence intensity of Beclin-1 was increased in Eca109-shRNA-PLCE1 group.The number of apoptotic cells in the Eca109-shRNA-PLCE1 group was(19.00±2.64)%,significantly higher than in the negative control group(10.67±2.08)% transfected with shRNA-GEF(P =0.0064).ConclusionSilencing PLCE1 may inhibit growth of subcutaneous tumor of esophageal squamous carcinoma by promoting autophagy and apoptosis in nude mice.Targeting PLCE1 can suppress tumor growth,which would be helpful in developing a new therapy approach for ESCC.