| Objectives To explore the formation of mutant p53/mi R-223-3p feedback loop in lung squamous cell carcinoma cells,and to explore the influence and potential mechanism of mutant p53/mi R-223-3p feedback loop on lung squamous cell proliferation and migration through vivo and in vitro experiments.Methods(1)Based on the previous research results of the research group on lung squamous cell carcinoma,the expression of mi R-223-3p in the tumor tissue of the tumor was significantly reduced compared with that in the tumor group of lung squamous cell carcinoma.Compared with tissue adjacent to carcinoma,lung squamous carcinoma tissue in mi R-223-3p expression reduced),through the CCK-8 experiment,plate cloning experiment,flow cytometry and nude mice tumor growth further validation of mi R-223-3p effects on pulmonary squamous cancer cell proliferation;The effect of mi R-223-3p on the migration of lung squamous cell carcinoma was observed by Transwell chamber experiment and scratch-mark experiment.(2)The downstream target p53 and p27kip1 of mi R-223-3p were predicted by software,and the regulation of mi R-223-3p was observed by using luciferase reporter gene experiment,qrt-pcr experiment and Western blot experiment.(3)The p53 gene in lung squamous cell carcinoma cells was sequenced by Sanger sequencing and the regulation of mi R-223-3p was observed by qrt-pcr.(4)The effects of p53 and p27kip1 on proliferation and cell migration of lung squamous cell carcinoma were observed by CCK-8 and Transwell cell experiments.(5)The effects of p53 expression on m RNA and protein levels of p27Kip1 were observed by q RT-PCR experiment and Western blot experiment,as well as the effects of p53 and p27Kip1 on apoptosis of lung squamous cell carcinoma cells.The effects of p53 and p27kip1 on cell cycle were observed by flow cytometry.By immunofluorescence assay,the localization of p27Kip1 in lung squamous cell carcinoma cells and the change of p53 expression were observed in p27Kip1 localization.Results(1)In the lung squamous cell carcinoma(SK-MES-1,NCI-H520),overexpression of mi R-223-3p can significantly inhibit tumor cell proliferation,clone formation and migration,while down-regulation of mir-223-3p results in the opposite effect.In vivo experiment,the volume,weight and expression of Ki67 were significantly inhibited by up-regulated mi R-223-3p.(2)Mi R-223-3p inhibits the expression of both proteins in lung squamous cell carcinoma cells by combining p53 and p27Kip1 3 ’UTR.(3)Sanger sequencing results showed that the p53 gene in lung squamous cell carcinoma was mutated,and the expression of mi R-223-3p was significantly increased after the reduction of p53.(4)After the reduction of p53 or p27kip1 in the lung squamous cell carcinoma cells,the proliferation of tumor cells significantly decreased,and the number of cells passing through the Transwell cell decreased significantly compared with the control group.(5)In lung squamous cell carcinoma cells,p27Kip1 m RNA and protein content were both increased after the reduction of p53.After the reduction of p53,the number of cleaved PARP protein and cell cycle S cells increased significantly,and after the reduction of p27kip1,cleaved PARP protein significantly increased but the cell cycle did not change significantly.Immunofluorescence experiments showed that p27Kip1 was mainly located in cytoplasm.After the reduction of p53,p27kip1 was mainly located in the nucleus.In A549 cells,H1299 cells and BEAS-2B cells,p27kip1 was mainly located in cytoplasm.Conclusions In the lung squamous cell carcinoma,p53 has a missense mutation,and the mutant p53 interacts with mi R-223-3p in tumor cells to form a negative feedback loop.The mutant p53/ mi RNA-223-3p feedback loop was applied to p27kip1 in the cell,so that the content of p27kip1 was decreased in cytoplasm and the nucleus increased(that is,the reduction of the nucleotide ratio),thereby inhibiting the proliferation and migration of pulmonary squamous cell carcinoma.But p27kip1’s localization in cells is not dependent on p53 status. |
PDF Full Text Request