Previous studies have shown that the expression of microRNA-4458 (miR-4458) is dysregulated in hepatocellular carcinoma and colon cancer. In this study, we investigated the direct target of miR-4458 and its biological functions in human lung cancer cells. By using the database TargetScan, we identified Lin28B, an oncogene, as a direct target gene of miR-4458. In dual-luciferase reporter assay, we found that miR-4458 mimics dose-dependently inhibited the luciferase activity of the wild-type 3'UTR of Lin28B in human lung cancer A549 and NCI-H1299 cell lines without affecting its mutant forms, whereas anti-miR-4458, an inhibitor of miR-4458, dose-dependently promoted the luciferase activity of the wild-type 3'UTR of Lin28B in A549 and NCI-H1299 cell lines without affecting its mutant forms. Overexpression of miR-4458 significantly decreased the protein levels of Lin28B in the cells, and inhibited the cell growth and colony formation. Conversely, knockdown of miR-4458 with anti-miR-4458 significantly increased the protein levels of Lin28B, and promoted the cell proliferation, which could be reverted by knockdown of Lin28B expression. In addition, we detected the expression of Lin28B using RT-PCR in 40 human lung cancer tissues and matched peritumoral tissues, and found that Lin28B was overexpressed in lung cancer tissues and negatively correlated with miR-4458 expression (r=-0.694, P<0.05). We conclude that miR-4458 is a tumor suppressor, and Lin28B is the direct target of miR-4458. These results suggest the modulation of miR-4458/Lin28B expression offers a potential therapeutic strategy for lung cancer.

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BackgroundPrevious investigations have revealed that miR‐563 is associated with a number of diseases including the ossification of posterior longitudinal ligament, Parkinson's disease or drug resistance to leukemia. Yet, the role of miR‐563 and its molecular mechanism in the initiation and progression of cancers has not been previously explored. In this study, we aimed to provide clues to the function of miR‐563 and its direct target in lung cancer.MethodsOnline informatics software was applied to predict the target genes of miR‐563. MiR‐563 targeting LIN28B was evaluated through the luciferase reporter gene analysis. The effect of miR‐563 on LIN28B at the level of RNA and protein was detected using RT‐PCR and immunoblotting. The ability of proliferation of human lung cancer A549 was examined by MTT assay. RNA interference targeting LIN28B was examined through immunoblotting. The level of miR‐563 and LIN28B and their correlation were analyzed in 27 cases of lung tumor tissues by real‐time PCR.ResultsOncogenic LIN28B was identified as one of the target genes of miR‐563 in lung cancer cells. MiR‐563 dose‐dependently decreased the LIN28B RNA level and subsequently its protein level in the cells. Cell proliferation was suppressed by ectopic miR‐563 expression and was accelerated after endogenous miR‐563 was knocked down by its inhibitor. However, silence in LIN28B reversed promotion of cell proliferation by the inhibition of miR‐563. In lung cancer tissues, miR‐563 was decreased and negative correlation of miR‐563 and LIN28B was shown.ConclusionMiR‐563 plays a tumor suppressive role in lung cancer progression via targeting oncogenic LIN28B.

The tumor microenvironment (TME) enhances regulatory T (T reg ) cell stability and immunosuppressive functions through up-regulation of lineage transcription factor Foxp3, a phenomenon known as T reg fitness or adaptation. Here, we characterize previously unknown TME-specific cellular and molecular mechanisms underlying T reg fitness. We demonstrate that TME-specific stressors including transforming growth factor–β (TGF-β), hypoxia, and nutrient deprivation selectively induce two Foxp3-specific deubiquitinases, ubiquitin-specific peptidase 22 ( Usp22 ) and Usp21 , by regulating TGF-β, HIF, and mTOR signaling, respectively, to maintain T reg fitness. Simultaneous deletion of both USPs in T reg cells largely diminishes TME-induced Foxp3 up-regulation, alters T reg metabolic signatures, impairs T reg -suppressive function, and alleviates T reg suppression on cytotoxic CD8 + T cells. Furthermore, we developed the first Usp22 -specific small-molecule inhibitor, which dramatically reduced intratumoral T reg Foxp3 expression and consequently enhanced antitumor immunity. Our findings unveil previously unappreciated mechanisms underlying T reg fitness and identify Usp22 as an antitumor therapeutic target that inhibits T reg adaptability in the TME.