Negative Regulation of Serine Threonine Kinase 11 (STK11) through miR-100 in Head and Neck Cancer

Background: Serine Threonine Kinase 11 (STK11), also known as LKB1, is a tumor suppressor gene that regulates several biological processes such as apoptosis, energetic metabolism, proliferation, invasion, and migration. During malignant progression, different types of cancer inhibit STK11 function by mutation or epigenetic inactivation. In Head and Neck Cancer, it is unclear what mechanism is involved in decreasing STK11 levels. Thus, the present work aims to determine whether STK11 expression might be regulated through epigenetic or post-translational mechanisms. Methods: Expression levels and methylation status for STK11 were analyzed in 59 cases of head and neck cancer and 10 healthy tissue counterparts. Afterward, we sought to identify candidate miRNAs exerting post-transcriptional regulation of STK11. Then, we assessed a luciferase gene reporter assay to know if miRNAs directly target STK11 mRNA. The expression levels of the clinical significance of mir-100-3p, -5p, and STK11 in 495 HNC specimens obtained from the TCGA database were further analyzed. Finally, the Kaplan–Meier method was used to estimate the prognostic significance of the miRNAs for Overall Survival, and survival curves were compared through the log-rank test. Results: STK11 was under-expressed, and its promoter region was demethylated or partially methylated. miR-17-5p, miR-106a-5p, miR-100-3p, and miR-100-5p could be negative regulators of STK11. Our experimental data suggested evidence that miR-100-3p and -5p were over-expressed in analyzed tumor patient samples. Luciferase gene reporter assay experiments showed that miR-100-3p targets and down-regulates STK11 mRNA directly. With respect to overall survival, STK11 expression level was significant for predicting clinical outcomes. Conclusion: This is, to our knowledge, the first report of miR-100-3p targeting STK11 in HNC. Together, these findings may support the importance of regulation of STK11 through post-transcriptional regulation in HNC and the possible contribution to the carcinogenesis process in this neoplasia.

17β-estradiol binding to ERα promotes the progression of prolactinoma through estrogen-response element-induced CaBP-9k up-regulation

17β-estradiol (E2) is considered to be an important instigator of prolactinoma, and can positively regulate the expression of calbindin-D9k (CaBP-9k) which contains an estrogen responsive element (ERE) via estrogen receptors (ERs). However, the detailed mechanism of E2 in promoting CaBP-9k expression and their roles in prolactinoma progression remain unclear. Here, we aimed to characterize it. The luciferase gene reporter assay with luc-ERE transfection showed that E2 treatment significantly enhanced the transcriptional level of CaBP-9k, whereas CaBP-9k activity was reduced when GH3 and MMQ cells were treated with AZD9496, an antagonist of ERα. E2 treatment increased the protein expressions of CaBP-9k and ERα but not ERβ, whereas this effect was also abolished when cells were treated with AZD9496. Besides, immunoprecipitation (IP) and immunofluorescence assays demonstrated that CaBP-9k could directly interact with ERα not ERβ, and Chromatin IP (ChIP) assay showed that ERα could bind to ERE of the CaBP-9k promoter. Moreover, cell counting kit-8 (CCK-8) and flow cytometry assays showed that E2 treatment significantly enhanced cell viability and inhibited cell apoptosis, but these effects were all abolished when ERα was down-regulated by short hairpin RNA (shRNA) or inhibited by AZD9496, as well as CaBP-9K suppression in both GH3 and MMQ cell lines. Taken together, these findings indicated that E2 stimulation promoted prolactin cell proliferation and inhibited cell apoptosis through ERα-induced CaBP-9k up-regulation, which then accelerated the advanced progression of prolactinoma.

miR-150 Affects Wnt/β-Catenin Pathway Activity and Regulates MSC Osteogenic Differentiation

The Wnt/β-catenin pathway promotes osteoblast differentiation. Bioinformatics analysis showed a targeting relationship between miR-150 and β-catenin 3′-UTR. This study investigated miR-150's role in β-catenin expression and osteoblast differentiation of Adipose-Derived Mesenchymal Stem Cells (ADMSCs). Differentiation of ADMSCs into osteoblasts was induced and the expression of miR-150, β-catenin, RUNX2 and OPN on the 0th, 9th and 18th day during differentiation was detected. The dual luciferase gene reporter assay validated the relationship between miR-150 and β-catenin. ADMSCs were divided into angomir-NC group and angomir-150 group, and then induced for differentiation for 18 days followed by measuring the level of miR-150, β-catenin, RUNX2 and OPN and the degree of osteoblast differentiation by alizarin red staining. Compared with Day 0, miR-150 expression was gradually decreased on 9th and 18th day of osteoblast differentiation, and the expression of β-catenin, RUNX2 and OPN was gradually increased. There was a targeted regulation relationship between miR-150 and β-catenin. Compared with angomir-NC group, miR-150 expression was significantly increased in angomir-150 transfection group with decreased expression of β-catenin, RUNX2 and OPN protein and reduced degree of alizarin red staining. miR-150 expression is decreased and β-catenin expression is increased during osteoblast differentiation of ADMSCs. Up-regulation of miR-150 expression attenuates the ability of ADMSCs to differentiate into osteoblasts by inhibiting β-catenin expression and Wnt/β-catenin pathway.

Piper cubebaL. Methanol Extract Has Anti-Inflammatory Activity Targeting Src/Syk via NF-κB Inhibition

Piper cubebaL. is a plant in the Piperaceae family that is generally found in tropical countries and acts as an antioxidant and anti-inflammatory agent. Unfortunately, the molecular mechanism of the anti-inflammatory activity has not been fully investigated. In this study, we elucidated the anti-inflammatory mechanism by focusing on NF-κB signaling, which is considered a prototypical inflammatory signaling pathway in both innate and adaptive immune functions. Cellular activity and the molecular target of Pc-ME were identified in macrophage RAW264.7 cells and HEK293T cells by assessing NO production, cytokine expression by RT-PCR, luciferase gene reporter assay, and protein regulation in cytoplasm by Western blot upon NF-κB activation. Pc-ME reduced NO production without any cell toxicity; inhibited expression of proinflammatory cytokines such as iNOS and IL-6; downregulated NF-κB activation mediated by both MyD88 and TRIF; and diminished the phosphorylation of IκBα, IKKα/β, Akt, p85, Src, and Syk. Pc-ME inhibited Syk and Src autophosphorylation during overexpression in HEK cells, which confirmed our hypothesis that Syk and Src were signaling targets of Pc-ME. These findings indicate thatPiper cubebaL. has anti-inflammatory activity by targeting Src/Syk in the NF-κB pathway.