A Study On G Protein-coupled Receptor Kinase4 (GRK4)-induced Cell Senescence And Its Anti-tumor Effects In CNE2 Cells

Objective:To investigate the mechanism of senescence in human nasopharyngeal carcinoma CNE2 cells induced by GRK4 and its anti-tumor effect.Methods:1.CNE2 cells were seeded onto?10cm plates and transfected with ectogenic pEGFP-GRK4 plasmid at 80%-90%confluence.After twenty-four hours transfection,cells were sorted by FACS and CNE2/GRK4（-）、CNE2/GRK4（+）cell population were gained.Immunofluorence staining and Western blot were used to verify the expression of GRK4 of sorted cells.MTT assay was used to evaluate the proliferative potential of sorted cells and flow cytometer was used to test cell cycle distribution.Then SA-β-gal staining was used to verify the cell senescence induced by GRK4.qRT-PCR was used to analyse expression levels of 77 senescence-related diferential genes come from America QIAGEN compony designed by Invitrogen,and further verified by Western blot.RNA-Seq.2.Tet-On 3G inducible expression system was used to establish GRK4-Tet-On 3G/CNE2 cell line that can inductively express GRK4.Cells were dealed with different concentration and flow cytometer was used to test cell cycle distribution.SPSS 21.0 was used for statistical interpretation.Results:1.The sorted GRK4（-）and GRK4（+）cells were successfully tested by IFS and Western blot.Overexpression of GRK4 inhibits CNE2 cell proliferation and induces cell cycle arrest in G₀/G₁,accompanied by positive result of SA-β-gal staning.There were 12 differential expressed genes of 77senescence-related genes tested by qRT-PCR in CNE2/GRK4（+）.The mRNA levels of CDK 6、CITED2、GADD45A、p15、p21、p57、RBL 1、TERT、THBS1 and TP63 were upregulated（p<0.05）,while PLAU、RBL were down-regulated（p<0.05）.The expression of diferential protein level was tested by Western blot,accompanied with upregulated p21、CDK6 and down-regulated SIRT1、cyclin D2、cyclin D3.Undetected p53 and phosphorylated p38 MAPK show that there were no relationship between GRK4and p53、p38.There were 1894 differentially expressed genes in the mRNA level,and GO analysis and KEGG pathway enrichment analysis were performed.2.GRK4-Tet-On 3G/CNE2 stable cell line was established.Cells,with/without GRK4 expression induced by Dox,was treated with DDP,and GRK4（+）cells showed S phase arrest,acoompanied by a large number of apoptotic cells（Pre-G₁ phase）.Conclusions:This work verifies that GRK4 is capable of inhibiting tumor cells proliferation,inducing cell cycle arrest and cell senescence in a p53、p38-independent pathway which involves a interact cellular signaling network and confirms that overexpression of GRK4 can improve the anti-tumor effct of DDP.With the establishment of GRK4-Tet-On 3G/CNE2stable cell line and completement of RNA-Seq,this study and provides valuble information to futher explore tumor cells senescence induced by GRK4 and related potential signaling pathway.