miR-210 Expression Is Strongly Hypoxia-Induced in Anaplastic Thyroid Cancer Cell Lines and Is Associated with Extracellular Vesicles and Argonaute-2

Abstract

Hypoxia, or low oxygen tension, is frequently found in highly proliferative solid tumors such as anaplastic thyroid carcinoma (ATC) and is believed to promote resistance to chemotherapy and radiation. Identifying hypoxic cells for targeted therapy may thus be an effective approach to treating aggressive cancers. Here, we explore the potential of the well-known hypoxia-responsive microRNA (miRNA) miR-210-3p as a cellular and extracellular biological marker of hypoxia. We compare miRNA expression across several ATC and papillary thyroid cancer (PTC) cell lines. In the ATC cell line SW1736, miR-210-3p expression levels indicate hypoxia during exposure to low oxygen conditions (2% O₂). Furthermore, when released by SW1736 cells into the extracellular space, miR-210-3p is associated with RNA carriers such as extracellular vesicles (EVs) and Argonaute-2 (AGO2), making it a potential extracellular marker for hypoxia.

Keywords: HIF1-alpha; anaplastic thyroid cancer; extracellular vesicles; hypoxia; miR-210; miRNA; microRNA.

Figure 1

HIF1-α-induced miR-210-3p expression during hypoxic culture of SW1736 cells. (A). Cartoon illustration of hypoxic malignant tumor. In low oxygen conditions, miR-210 expression is regulated by HIF. (B). HIF1-α stabilization verified by Western blot (WB) and densitometry analysis after 24, 48, 72, 96 and 120 h of hypoxia (2% O₂) or normoxia (21% O₂). Actin was used as a loading control. (C). RT-qPCR analysis of mir-210-3p expression in hypoxia vs. normoxia at 24, 48, 72, 96 and 120 h of hypoxia relative to normoxia. Error bars represent the standard deviation of three biological repeats. (D). RT-qPCR analysis of HIF-α expression relative to normoxia (E). Images of SW1736 cells before and after 72 h of normoxia or hypoxia. (F). Wound healing assay of SW1736. (G). Percent viability of SW1736 cells after 72 h of normoxia or hypoxia. SW1736 cell counts after 72 h of normoxia or hypoxia. (H). SW1736 cell culture media glucose concentration after 72 h of normoxia or hypoxia.

Figure 2

miR-210 expression in ATC and PTC cell lines. (A). The RNA-seq analysis of differentially expressed pre-miRNAs between anaplastic thyroid cancer (ATC) cell lines SW1736 and C643 and papillary thyroid cancer (PTC) cell lines BCPAP and TPC-1. Log₂ fold change > ±1. q value < 0.05 n = 3. Bar included to highlight miR-210. (B). RT-qPCR of miR-210 basal expression and hypoxia (2%O₂)-induced expression in ATC vs. PTC cell lines. (C). The ATC vs. PTC qPCR analysis of miR-210-3p expression during hypoxia time course study at 0, 2, 4, 8, 24, 48 h hypoxia.

Figure 3

SW1736 cellular miRNA expression in hypoxia at 72 h. (A). The MD plot showing differential expression of miR-210-3p, pre-miR-210 as well as mRNA and lncRNA transcripts in hypoxia vs. normoxia datasets. Significantly deregulated genes (adj. p-value < 0.05) are shown in color. (B). Heatmap of the normalized read counts for all differentially expressed transcripts with −0.5 < LFC < 0.5 and adj. p-value < 0.05 (n = 66 in total). (C). Gene ontology, KEGG and pathway analysis of the deregulated genes in normal vs. hypoxic cells. (D). The NCI-Nature Pathway Interaction Database (PID) image visualizing HIF-1-alpha transcription network with the indicated protein-coding genes differentially expressed in datasets from hypoxic cells (in yellow).

Figure 4

Characterization of cellular and extracellular fractions and AGO2 detection in hypoxic SW1736 culture. (A). Workflow diagram schematic of EV and protein separation from SW1736 cell culture media (CCM) by differential centrifugation and size-exclusion chromatography (SEC). Three independent experiments were performed. (B). Western blot analysis of tetraspanin CD63 and CD81 expression in SW1736 cell lysates and enrichment in hypoxic and normoxic SEC fractions after 72 h. (C). SP-IRIS detection of tetraspanins CD81, CD63 and CD9 in hypoxic and normoxic EV-enriched SEC fractions after 72 h. (D). Transmission electron micrograph (EM) of hypoxic and normoxic EV and protein SEC fractions after 72 h. (E). The SEC EV fraction particle counts per cell from normoxia and hypoxia. Data points represent three independent experiments by nano-flow cytometry. (F). miR-210-3p SEC pooled EV, mixed and protein qPCR analysis after 72 h hypoxia or normoxia, derived from 100 mLs of culture media. Data points represent three independent experiments. (G). AGO2 detection in SEC mixed and protein fractions by immunoprecipitation followed by Western blot.