Abstract:

Objective To discuss the effect of interleukin-24 （IL24） on the biological behaviors of cervical cancer HeLa cells through regulation of V-Rel reticuloendotheliosis viral oncogene homolog B （RelB）， and to clarify the mechanism. Methods The HeLa cells were divided into control group ［pcDNA3.1（+） group， transfected with pcDNA3.1（+） plasmid］ and IL-24 overexpression group ［pcDNA3.1（+）-IL-24 group，transfected with pcDNA3.1（+）-IL-24 plasmid］. Real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods were used to detect the transfection efficiencies of the cells in two groups； cell counting kit-8 （CCK-8） method was used to detect the proliferation activities of the cells in two groups； flow cytometry was used to detect the apoptotic rates of the cells in two groups； Transwell chamber assay was used to detect the migration rates and invasion rates of the cells in two groups； transcriptome sequencing （RNA-seq） method and bioinformatics were used to explore the differentially expressed genes in two groups of IL-24； RT-qPCR and immunofluorescence staining methods were used to detect the expression level of RelB mRNA and protein in HeLa cells after IL-24 overexpression. In validation experiment， the HeLa cells were divided into pcDNA3.1（+） group， pcDNA3.1（+）-IL-24 group， HPV18 E6 overexpression group ［pcDNA3.1（+）- IL-24+E6 group， co-transfected with pcDNA3.1（+）-IL-24 plasmid and pcDNA3.1（+）-HPV18 E6 plasmid］， HPV18 E7 overexpression group ［pcDNA3.1（+）-IL-24+E7 group， co-transfected with pcDNA3.1（+）-IL-24 plasmid and pcDNA3.1（+）-HPV18 E7 plasmid］， and HPV18 E6/E7 overexpression group ［pcDNA3.1（+）-IL-24+E6+E7 group， co-transfected with pcDNA3.1（+）-IL-24 plasmid， pcDNA3.1（+）- HPV18 E6 plasmid， and pcDNA3.1（+）-HPV18 E7 plasmid］.RT-qPCR method was used to detect the expression levels of human papillomavirus（HPV18） E6 and HPV18 E7 mRNA and the expression level of RelB mRNA in the HeLa cells after transfection； Western blotting method was used to detect the expression level of RelB protein in the HeLa cells. Results Compared with pcDNA3.1（+） group， the expression levels of IL-24 mRNA and protein in the HeLa cells in pcDNA3.1（+）-IL-24 group were significantly increased （P<0.01）， the proliferation activity of the HeLa cells was decreased （P<0.001）， the migration rate of cells was decreased （P<0.05）， the invasion rate of cells was decreased （P<0.01）， and the apoptotic rate was increased （P<0.01）. The RNA-seq results showed that a total of 952 differentially expressed genes were identified， which were involved in multiple signaling pathways related to tumor occurrence and development， including nuclear factor-kappa B （NF-κB）， cyclic adenosine monophosphate （cAMP）， and phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt）. The protein-protein interaction （PPI） network analysis results showed that RelB was located in core regulatory region of the network. Compared with pcDNA3.1（+） group， the expression levels of HPV18 E6， HPV18 E7 and RelB mRNA in the HeLa cells in pcDNA3.1（+）-IL-24 group were significantly decreased（P<0.01）. Furthermore， after overexpression of HPV18 E6 and E7 in the HeLa cells in pcDNA3.1（+）-IL-24 group， the expression level of RelB mRNA and protein was significantly increased （P<0.05）. Conclusion Overexpression of IL-24 can inhibit the proliferation， migration and invasion of the HeLa cells and promote the cell apoptosis. The mechanism may be related to the inhibition of HPV18 E6 and E7 expressions and the down-regulation of RelB protein expression level.

Key words: Cervical neoplasm, Interleukin-24, Transcriptome sequencing, V-Rel reticuloendotheliosis viral oncogene homolog B, Human papillomaviru