The Effect Of PLAC8 On Proliferation、Cycle Distribution And Migration Of Lung Cancer-derived Cells And Molecular Mechanism

Placenta specific protein 8(PLAC8)is a highly conserved and cysteine-rich protein with an unique structure and tissue-specially expressed.PLAC8 plays an important role in cell physiological processes such as cell proliferation,differentiation,apoptosis,migration and invasion.Recent studies on PLAC8 have been mainly focused on tumorigenesis.However,the study of PLAC8 in lung cancer is still limited.Statistic analysis showed that the mRNA expression level of PLAC8 in lung cancer tissue was lower than that in normal lung tissue.Furthermore,the results of lung disease tissue chip showed that the protein expression level of PLAC8 in lung cancer tissue was lower than the adjacent normal lung tissue.Taken together,our previous study suggested that the expression of PLAC8 might be associated with lung cancer.miR-185-5p is an important microRNA with tumor suppressor gene properties.miR-185-5p could bind to the 3’UTR of target genes,leading to mRNA expression suppression or degradation,thereby affecting cell physiological activity.Bioinformatic analysis showed that there was a potential binding site for miR-185-5p in the 3’-UTR of PLAC8 mRNA,which is worthy of our further study.We detected the expression of PLAC8 in 11 lung cancer-derived cell lines.We selected H1299 in which PLAC8 was highly expressed to construct PLAC8 knockout cell line via CRISPR/Cas9 technology.Meanwhile,A549 with low expression of PLAC8 was employed to construct PLAC8 overexpression cell line.Further results of cellular experiment showed that knockout of PLAC8 in H1299 altered the expression levels of cell cyclins.The expression levels of P21waf/cip1,P27Kip1 and p-weel were increased,and the expression levels of G1 phase-related kinases such as CDK2,cyclinD1 and cyclinD3 were down-regulated,which induced G1 arrested and inhibited cell proliferation.Further migration experiments showed that knockout of PLAC8 inhibited cell migration.In A549,the overexpression of PLAC8 downregulated the expressions of kinase inhibitors P27Kip1 and P-weel,while upregulated the expressions of the key factor cdc2(CDK1)/cyclinB 1 complex.The overexpression of cdc2/cyclinB 1 reduced the percentage of G2/M cells,which promoted cell proliferation.Further migration experiments showed that the overexpression of PLAC8 promoted cell migration.Dual luciferase activity assay in 16HBE showed that miR-185-5p could inhibit the expression of PLAC8 protein by binding to the 3’UTR of PLAC8.Meanwhile,the expression level of the S-phase regulatory protein complex CDK2-cyclinA2 was decreased,which induced S phase arrest,and inhibited cell proliferation.Taken together,PLAC8 may regulate a series of physiological processes including cell proliferation,cycle and migration in A549,H1299 and 16HBE by altering the expression of a series of cell cyclins and migration proteins.Moreover,miR-185-5p might be an important regulated factor participated in the upstream signaling pathway.The current research provides a theoretical basis for further study on the molecular mechanism of PLAC8 and also provided a potential gene target for the treatment of lung cancer.