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MiR-135a Targets SMAD2 to Promote Osteosarcoma Proliferation and Migration.

ABSTRACT: Osteosarcoma (OS) is an aggressive malignant neoplasm that commonly occurs in adults and adolescents. The objectives of this work were to verify the role of microRNA- (miR-) 135a in OS and determine whether it can regulate the growth and cellular migration of OS by targeting mothers against decapentaplegic homolog 2 (SMAD2). miR-135a and SMAD2 mRNA expression levels were measured using reverse transcription-quantitative PCR (RT-qPCR). Proliferation and migration of cells were studied using the Cell Counting Kit-8, EdU staining, and transwell invasion experiment. Additionally, a dual-luciferase reporter experiment was used to investigate the possible relationship between miR-135a and SMAD2's 3'-UTR. Immunohistochemistry was utilized to examine the expressions of SMAD2 and Ki67 in mouse tumor tissues to determine the influence of miR-135a on cancer progression in vivo. miR-135a was shown to be elevated in OS tissue samples as well as five cell lines. High expression levels of miR-135a were correlated with poor prognosis of OS patients. Cellular proliferation and migration were promoted by the upregulation of miR-135a with miR mimics; however, this effect was inhibited by SMAD2 overexpression. miR-135a was also shown to directly target the 3'-UTR of SMAD2. Animal experiments also demonstrated that miR-135a downregulation had an inhibitory effect on tumor growth in vivo. High expression levels of miR-135a promoted transplanted tumor development in vivo and the proliferation and migration of OS cells by targeting SMAD2. In summary, miR-135a may be a prospective therapeutic target for OS in the future.

SUBMITTER: Chen Y

PROVIDER: S-EPMC9020941 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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miR-135a Targets SMAD2 to Promote Osteosarcoma Proliferation and Migration.

Chen Yuanyuan Y Cai Bin B Lian Xiaofeng X Xu Jianguang J Zhang Tao T

Journal of oncology 20220413

Osteosarcoma (OS) is an aggressive malignant neoplasm that commonly occurs in adults and adolescents. The objectives of this work were to verify the role of microRNA- (miR-) 135a in OS and determine whether it can regulate the growth and cellular migration of OS by targeting mothers against decapentaplegic homolog 2 (SMAD2). miR-135a and SMAD2 mRNA expression levels were measured using reverse transcription-quantitative PCR (RT-qPCR). Proliferation and migration of cells were studied using the C ...[more]

PMID: 35466322

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Project description:BackgroundGlioblastoma multiformae (GBM) is the most aggressive type of malignant brain tumor with complex molecular profile. Overexpression of Na+/H+ Exchanger isoform 9 (NHE9) promotes tumor progression and correlates positively with insensitivity to radiochemotherapy and poor prognosis. However, molecular mechanisms responsible for increase in NHE9 levels beyond a critical threshold have not been identified.MethodsBioinformatics analysis, luciferase reporter assays, real-time PCR and western blotting were conducted to examine the expression profiles and identify microRNAs (miRNA) that target NHE9. Cell proliferation and migration assays were conducted in U87 glioblastoma cells to determine the consequence of miRNA mediated targeting of NHE9. Endosomal pH measurements, immunofluorescence microscopy and surface biotinylation experiments were conducted to characterize the mechanistic basis of regulation.ResultsWe show that microRNA 135a (miR-135a) targets NHE9 to downregulate its expression in U87 cells. MiR-135a levels are significantly lower in glioblastoma cells compared to normal brain tissue. Downregulation of NHE9 expression by miR-135a affects proliferative and migratory capacity of U87 cells. Selectively increasing NHE9 expression in these cells restored their ability to proliferate and migrate. We demonstrate that miR-135a takes a two-pronged approach affecting epidermal growth factor receptors (EGFRs) to suppress tumor cell growth and migration. EGFR activity is a potent stimulator of oncogenic signaling. While miR-135a targets EGFR transcripts to decrease the total number of receptors made, by targeting NHE9 it routes the few EGFRs made away from the plasma membrane to dampen oncogenic signaling. NHE9 is localized to sorting endosomes in glioblastoma cells where it alkalinizes the endosome lumen by leaking protons. Downregulation of NHE9 expression by miR-135a acidifies sorting endosomes limiting EGFR trafficking to the glioblastoma cell membrane.ConclusionsWe propose downregulation of miR-135a as a potential mechanism underlying the high NHE9 expression observed in subset of glioblastomas. Future studies should explore miR-135a as a potential therapeutic for glioblastomas with NHE9 overexpression.

Dataset Information

MiR-135a Targets SMAD2 to Promote Osteosarcoma Proliferation and Migration.

Publications

miR-135a Targets SMAD2 to Promote Osteosarcoma Proliferation and Migration.

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