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SP1/AKT/FOXO3 Signaling Is Involved in miR-362-3p-Mediated Inhibition of Cell-Cycle Pathway and EMT Progression in Renal Cell Carcinoma.

ABSTRACT: Emerging evidence has indicated that dysregulation of miR-362-3p is involved in the initiation and progression of several types of human cancers. However, the molecular mechanism of miR-362-3p in renal cell carcinoma (RCC) is still not completely clear. In this study, we found that miR-362-3p was frequently down-regulated in human RCC tissues. Overexpression of miR-362-3p in RCC cells significantly suppressed the proliferation, cell cycle and motility in vitro and in vivo via regulating AKT/FOXO3 signaling. We further confirmed that SP1 was a direct target of miR-362-3p. Knockdown of SP1 expression by a small interfering RNA (siRNA) phenocopied the effect of miR-362-3p overexpression in RCC cells. In conclusion, the current results provide evidence for the role of miR-362-3p in the pathogenesis of RCC and thus miR-362-3p may serve as an attractive candidate for RCC therapy.

SUBMITTER: Zhu H

PROVIDER: S-EPMC7214730 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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SP1/AKT/FOXO3 Signaling Is Involved in miR-362-3p-Mediated Inhibition of Cell-Cycle Pathway and EMT Progression in Renal Cell Carcinoma.

Zhu Hejia H Wang Song S Shen Haixiang H Zheng Xiangyi X Xu Xin X

Frontiers in cell and developmental biology 20200505

Emerging evidence has indicated that dysregulation of miR-362-3p is involved in the initiation and progression of several types of human cancers. However, the molecular mechanism of miR-362-3p in renal cell carcinoma (RCC) is still not completely clear. In this study, we found that miR-362-3p was frequently down-regulated in human RCC tissues. Overexpression of miR-362-3p in RCC cells significantly suppressed the proliferation, cell cycle and motility <i>in vitro</i> and <i>in vivo</i> via regul ...[more]

PMID: 32432112

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Project description:BackgroundHedgehog (Hh) signaling pathway, which is essential for cell proliferation and differentiation, is noted to be aberrantly activated in tumor from increasing studies in recent years. MicroRNAs (miRNAs) as an important non-coding RNA in cells have been proven to possess a regulatory role specific to the Hh signaling pathway. Here, in vitro and in vivo cellular/molecular experiments were adopted to clarify the regulatory mechanism linking miR-636 to the Hh signaling pathway in ovarian cancer (OVC).MethodsProtein-protein interaction analysis was performed to identify the hub gene in the Hh pathway. TargetScan database was used to predict the potential upstream regulators for Gli2. qRT-PCR was performed to test the expression of miR-636, while Western blot was conducted to detect the expression of proteins related to the Hh pathway and epithelial-mesenchymal transition (EMT). For cell functional experiments, HO-8910PM OVC cell line was used. MTT assay and wound healing assay were used to measure the effect of miR-636 on cell proliferation and migration. Flow cytometry was carried out to examine the effect of miR-636 on cell cycle, and Western blot was used to identify the change in expression of Hh and EMT-related proteins. Dual-luciferase reporter gene assay was implemented to detect the targeting relationship between miR-636 and Gli2. Xenotransplantation models were established for in vivo examination.ResultsGli2 was identified as the hub gene of the Hh pathway and it was validated to be regulated by miR-636 based on the data from TargetScan and GEO databases. In vitro experiments discovered that miR-636 was significantly lowly expressed in OVC cell lines, and overexpressing miR-636 significantly inhibited HO-8910PM cell proliferation, migration and induced cell cycle arrest in G0/G1 phase, while the inhibition of miR-636 caused opposite results. Dual-luciferase reporter gene assay revealed that Gli2 was the target gene of miR-636 in OVC. Besides, overexpressed miR-636 decreased protein expression of Gli2, and affected the expression of proteins related to the Hh signaling pathway and EMT. Rescue experiments verified that overexpression of Gli2 reversed the inhibitory effect of miR-636 on HO-8910PM cell proliferation and migration, and attenuated the blocking effect of miR-636 on cell cycle. The xenotransplantation experiment suggested that miR-636 inhibited cell growth of OVC by decreasing Gli2 expression. Besides, overexpressing Gli2 potentiated the EMT process of OVC cells via decreasing E-cadherin protein expression and increasing Vimentin protein expression, and it reversed the inhibitory effect of miR-636 on OVC cell proliferation in vivo.ConclusionmiR-636 mediates the activation of the Hh pathway via binding to Gli2, thus inhibiting EMT, suppressing cell proliferation and migration of OVC.Trial registrationThe experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of The Second Affiliated hospital of Zhejiang University School of Medicine (IR2019001235). Written informed consent was obtained from individual or guardian participants.

Dataset Information

SP1/AKT/FOXO3 Signaling Is Involved in miR-362-3p-Mediated Inhibition of Cell-Cycle Pathway and EMT Progression in Renal Cell Carcinoma.

Publications

SP1/AKT/FOXO3 Signaling Is Involved in miR-362-3p-Mediated Inhibition of Cell-Cycle Pathway and EMT Progression in Renal Cell Carcinoma.

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