Study On EEF1D Gene RNAi By Lentivirus Mediated The Growth And Apoptosis Of SKOV3/DDP Xenografts In Nude Mice

Objective To explore the effect of lentiviral vector-mediated RNAi eukaryotic translation elongation factor 1?(EEF1D)gene silencing on the proliferation and apoptosis of human ovarian cancer xenografts in nude mice.Methods According to human EEF1 D gene design and screen the best si RNA which can down regulate the gene expression and construct the sh RNA expression vector p LJM1-sh RNA.The SKOV3 and SKOV3/DDP cell lines stably transfected with p LJM1-sh RNA-EEF1 D were constructed by lentiviral expression vector.RT-PCR and Western blot were used to detect the expression of EEF1 D gene in stable transfected cell line.Human ovarian cancer cell line SKOV3/DDP was injected into the right scapular region of BABL/c nude mice to establish a human ovarian cancer subcutaneous tumor model,The tumor diameter of 0.3~0.4cm mice were randomly divided into 4 groups,including control group,DDP+saline group,DDP+sh Scrambled group and DDP+sh EEF1 D group were given corresponding drug concentration.Sacrificed after 4 weeks,tumor volume was measured to calculate tumor inhibition rate and to observe the local infiltration and metastasis,The morphological changes of the cells were observed by HE staining.TUNEL assay was used to detect apoptosis.Western blot was used to detect the expression of tumor related proteins.Results 1.The results showed that the recombinant lectiviral vector p LJM1-sh RNA-EEF1 D was successfully constructed.The measured lentivirus titer was 3.08 × 107 TU/m L.In vitro lentiviral infection experiments showed that SKOV3 sh EEF1 D and SKOV3//DDP sh EEF1 D knocked down the expression of EEF1 D m RNAcompared with SKOV3 sh Scrambled and SKOV3/DDP sh Scrambled(F = 95.94,P <0.05),at the protein level,EEF1 D expression decreased in SKOV3 sh EEF1 D and SKOV3/DDP sh EEF1 D groups(F = 133.96,P <0.05).2.In vivo experiments showed that the growth of subcutaneous tumor in nude mice in DDP + sh EEF1 D group was significantly inhibited;the inhibition rate was 56.57%.The results of HE staining showed that the nuclei of DDP + sh EEF1 D group were highly concentrated,and no cytoplasm surrounding the cells formed empty halo,and the demarcation between cells was unclear.3.The results of TUNEL showed that the apoptotic index in DDP + sh EEF1 D group was 61.71 ± 9.26%,and in control group and DDP + sh Scrambled group were 4.13 ± 1.14% and 9.48 ± 2.38%.4.Western blot results showed that DDP + sh EEF1 D group induced increase of Cleaved caspase-3 protein expression,promoted Bax expression and inhibited Bcl-2 expression,Compared with control group and DDP + sh Scrambled group,the differences were statistically significant(P <0.05).Conclusion Inhibition of EEF1 D gene expression can inhibit the growth of subcutaneous xenografts in human ovarian cancer cell SKOV3/DDP cells and promote its apoptosis in BABL/c mice.The possible mechanism is to regulate the expression of Bax/Bcl-2 gene by activating the PIK3/AKT signaling pathway.