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. 2021 Jul 1;16(7):e0253522. doi: 10.1371/journal.pone.0253522

MicroRNA-210-3p is transcriptionally upregulated by hypoxia induction and thus promoting EMT and chemoresistance in glioma cells

Hong Liu 1, Changjin Chen 1, Jinhao Zeng 1, Ziyi Zhao 1,*,#, Qiongying Hu 2,*,#
Editor: Michael Klymkowsky3
PMCID: PMC8248614  PMID: 34197482

Abstract

Background

Glioma is the most common and lethal form of brain cancer. It is highly malignant and is often characterized by chemoresistance and radioresistance, which are thought to mainly result from hypoxic microenvironments. Various tumour-promoting and tumour-suppressing microRNAs (miRNAs) have been identified in gliomas; however, it is still largely unknown how miRNAs are modified by hypoxia and subsequently affect glioma. In this study, we examined the expression of miR-210-3p, a well-characterized miRNA that responds to hypoxia in glioma cell lines.

Methods

The expressions of miR-9 and miR-210-3p were analysed by using qPCR. Cell viability was measured by performing CCK-8 after eechinomycin treatment or introduction of miR-210 for 24 or 48 h. The correlation of HIF-1α expression with TGF-β were analysed using the REMBRANDT database. The biomarkers of EMT, including E-cadherin, N-cadherin and Vimentin, were detected by western blot. Apoptotic cell death was measured by performing Annexin V-FITC/PI double staining followed by flow cytometry.

Results

We found that miR-210-3p was induced by a mechanism dependent on the hypoxia-induced transcriptional activity of HIF-1α. Then we established a positive association between the HIF-1α and TGF-β expression levels, and miR-210-3p upregulation induced TGF-β expression, indicating that hypoxia-induced HIF-1α activity upregulated TGF-β via miR-210-3p upregulation. Hypoxia-induced miR-210-3p activity was found to promote EMT by upregulating TGF-β, which subsequently enhanced the invasive ability in U87-MG cells. We further confirmed that miR-210-3p induced chemoresistance to TMZ in U87-MG cells via TGF-β upregulation under hypoxic conditions.

Conclusion

These results help to reveal the potential regulatory mechanisms of hypoxia-induced miR-210-3p expression that affect malignant behaviors and chemoresistance via TGF-β upregulation in glioma cells.

Introduction

Glioma is the most common and lethal brain tumor; it is highly malignant and is often characterized by chemoresistance and radioresistance [1]. Glioma is characterized by high mortality and recurrence rates [2], lethal invasiveness [3], and strong angiogenesis in hypoxic microenvironments [4]. A common therapeutic strategy consists of surgical removal, radiotherapy and chemotherapy [5]; however, glioma patients often have poor prognoses mainly due to malignant biological behaviors [6], indicating that glioma cells can migrate to and invade sites away from the primary tumor mass. Epithelial-to-mesenchymal transition (EMT), which is characterized by loss of the epithelial marker (E-cadherin) along with overexpression of mesenchymal markers (N-cadherin and Vimentin) [7], is a well-researched physiological process that results in more robust invasive or metastatic phenotypes [8]; furthermore, EMT is thought to limit total surgical resection and to contribute to therapeutic resistance, which can lead to tumor recurrence in several cancers [9, 10], including in glioma [11]. Many studies have focused on the mechanism of EMT-induced metastasis in glioma; however, the effects of EMT on glioma remain largely unknown.

Hypoxia is well-accepted as a promoting factor in cancer progression, and it contributes to malignant behaviors and metastasis [12]. Poor vasculature leads to a limitation in the delivery of oxygen and nutrients, frequently inducing necrosis in the interior regions of solid tumors. Hypoxia has also been reported to regulate miRNAs, small, single-stranded non-coding RNAs that have multiple critical roles in physiological processes in tumor cells under hypoxic conditions [13]. In gliomas, microRNAs have been identified and characterized as critical regulators in the malignant progression [14]. As the most responsive and influential miRNA, miR-210-3p is positively regulated by hypoxia-induced factor 1α (HIF-1α) [15–17], and it post-transcriptionally regulates several genes. In this manner, miR-210-3p upregulation mediated by HIF-1α exerts critical effects on cell cycle control, apoptosis, and aberrant regulation of cell morphology, polarization and malignant behaviors [18, 19]. However, the potential effects of miR-210-3p and its responsiveness to hypoxia remain largely unknown in glioma.

HIF-1α, which has been postulated to be a hallmark of hypoxia treatment, tightly regulates transforming growth factor-β (TGF-β) and its downstream signaling, resulting in multiple biological effects, including tumorigenesis, progression and metastasis [20–22]. TGF-β has been shown to be a master regulator of the initiation and maintenance of EMT phenotypes in many kinds of cancers [23], and persistent hypoxia exposure induces acquisition of the EMT phenotype via multiple mechanisms, one of which is transcriptional activation of TGF-β via HIF-1α. It is also believed that miRNAs that respond to hypoxic conditions may be involved in these interactions and exert biological effects.

Based on these observations, we hypothesized that hypoxia-responding miRNAs may be involved in regulating a network of physiological processes in glioma cells. We demonstrate here that hypoxia-induced miR-210-3p induction in glioma cells promotes EMT via TGF-β upregulation and induces chemoresistance, which indicates its tumor promoting roles in glioma.

Material and methods

Cell culture and treatment

Human glioma cell lines, U87-MG (from unknown origin), A172 and HS683 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and stored in liquid nitrogen in our laboratory. For each cell line, they were stored in liquid nitrogen before 3–5 passages. Cells were maintained in Dulbecco’s modified Eagle’s medium high glucose (DMEM-H) containing 10% Fetal bovine serum (FBS, Life Technologies, Grand Island, NY, USA), 100 U/mL penicillin and 100 μg/mL streptomycin, and kept in a 5% CO2 incubator at 37°C. Medium were replaced and cells were passaged every two or three days.

To achieve hypoxia, cells were maintained in a modular incubator chamber (Billups Rothenberg Inc., Del Mar, CA) containing 5% CO2, 95% N2 for 24 to 48 h. To achieve HIF-1α stabilization, 100 μM CoCl2 for 24 to 48 h to mimic hypoxia.

To block transcriptional activity of HIF-1α, cells were pretreated with 1 ng/ml Echinomycin (EC), a transcriptional activity inhibitor of HIF-1α, for 4 h before following treatment.

To mimic TGF-β induced EMT, cells were treated with 5 ng/ml TGF-β for 24 to 48 h before following treatment.

CCK-8 assay

To evaluate the cell viability, a CCK-8 Cell Counting Kit (CCK8; Dojindo, Kunmamoto, Japan) was employed according to the manufacturer’ instruction. Briefly, cells were plated at a density of 2×104 cells per well in 96-well plates and allowed to attach overnight. After desired treatment, 10 μL of CCK-8 detecting reagent was added into each well for another 4-hour incubation at 37°C. Then, absorbance at 450 nm was recorded in an EnSpire® Multimode Plate Readers (PerkinElmer, China). The experiments were done in pentaplicate and repeated three times.

Reverse-transcriptional quantitative PCR (RT-qPCR)

Total RNA was extracted using TRIZol (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions.

To detect TGF-β and β-actin mRNA levels, the reverse-transcriptional Kit (Life Technologies, Grand Island, NY, USA) was employed followed by the manufacturer’s instruction. Then the cDNA was used as template under the following conditions: 35 cycles of 95°C for 10 seconds (s); 60°C for 1 minute (min) on an ABI7500 (Applied Biosystems, Foster City, CA, USA). The primers used for qPCR were as follows: β-actin forward, 5’-CATGTACGTTGCTATCCAGGC-3’ and reverse, 5’-CTCCTTAATGTCACGCACGAT-3’; TGF-β forward, CTGACGGCCACGAACTTCC; and reverse, 5’- GCACTGACATTTGTCCCTTGA-3. To detect miR-210-3p, miR-9 and U6, All-in-OneTM miRNA qRT-PCR Detection Kit (GeneCopoeia, Guangzhou, China) was used followed by manufacturer’s instruction. The approved primers for miR-210-3p (Cat. No.: HmiRQP0317), miR-9 (Cat. No.: HmiRQP0825) and HsnRNA U6 (Cat. No.: HmiRQP9001) were all bought from GeneCopoeia. The qPCR results were analyzed and expressed relative to the CT (threshold cycle) values and then converted to fold changes; 2.0-fold change was considered significant.

Western blot

Mouse anti-human HIF-1α antibody (Cat.: ab1), rabbit anti-human E-cadherin antibody (Cat.: ab1548), mouse anti-human N-cadherin antibody (Cat.: ab98952), mouse anti-human Vimentin antibody (Cat.: ab8069), rabbit anti-human TGF-β1 antibody (ab92486) and rabbit anti-human β-actin antibody (Cat.: ab179467) are primary antibodies were bought from Abcam (Cambridge, England) and diluted at 1:2000.

Total protein was prepared using RIPA buffer (Thermo Scientific, Waltham, MA, USA) followed the manufacturer’s instruction. Protein concentration was measured by performing BCA staining (Sigma–Aldrich, St. Louis, MO, USA) and 20 μg total protein of each sample was fractionated using 8–16% SDS-PAGE gel, and transferred to PVDF (Millipore, Billerica, MA, U.S.A) membranes. After transferring, blotted membranes were blocked with 5% milk/TBS buffer for 30 min on a shaker, and this was followed by an incubation with primary antibodies at 4°C overnight. Followed by three washes with PBS-T (containing 0.1% Tween-20), HRP-conjugated secondary antibodies were incubated with PVDF membranes for 1 hour at room temperature. Goat anti-mouse IgG H&L (HRP) (Cat.: ab97040) and goat anti-rabbit IgG H&L (HRP) (Cat.: ab7090) secondary antibodies were employed for specific primary antibody. Blots were developed using PierceTM ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

Transwell assay

To evaluate the invasive ability, transwell migration assay was employed using a 24-well transwell chemotaxis chamber technique (Millipore, Billerica, MA, USA). Briefly, DMEM (500 μL) with 10% FBS was added in the lower chamber. 1× 104 cells suspended in 200 μL medium were placed into the upper chamber (pore size, 8 μm) coated with 100 μl Matrigel (Millipore, Billerica, MA, USA). The plate was then incubated for 24 h at 37°C in a humidified atmosphere with 5% CO2. The Matrigel was removed and its upper surface was wiped away with a cotton swab to remove the unmigrated cells and fixed with 4% paraformaldehyde for 15 min followed by staining with 0.1% crystal violet for 10 min. After 3 washes with ice-cold PBS, the number of cells per view was counted in randomly under a light microscope (BL-AC10DS, Olympus, Tokyo, Japan). Each assay was performed in triplicate wells.

Transfection of miR-210-3p mimics, inhibitor or siRNA target to TGF-β mRNA

SiRNA was employed for knocking down the expression of TGF-β. Introduction of miR-210-3p mimics or inhibitor was performed to regulate miR-210-3p expression.

SiTGF-β (ON‐TARGETplus Human TGFBI siRNA SMART pool; L‐019370‐00‐0005, J‐019370‐06‐0002 and J‐019370‐08‐0002) and control nonspecific siRNA (ON‐TARGETplus Non‐targeting Control Pool; D‐001810‐10‐05) were purchased from Dharmacon (Lafayette, CO, USA).

MiR-210-3p mimics and miR-210-3p inhibitor were obtained from Ambion (Austin, TX, USA).

The transfection was performed using Lipofectamine 2000 (Life Technologies, Grand Island, NY, USA) according to the suggested concentration of manufacturer (Ribobio Co. Ltd, Guangzhou, China).

Annexin V-FITC/PI double staining

To evaluate apoptotic cell death, the annexin V-FITC/PI double staining was performed according to the manual of Annexin V-FITC/PI apoptosis detection kit (Life Technologies, Grand Island, NY, US). For each sample, 1×106 cells were collected and stained with 5 μl Annexin V-FITC and 10 μl PI staining solution for 30 min in the dark at room temperature, and then the binding buffer was added to 500 μl of total volume and analyzed by performing flow cytometry on 3 laser Navios flow cytometers (Beckman Coulter, Brea, CA, USA). For each sample, 1×104 events were acquired.

Statistical analysis

All data are expressed as the mean±SD. One-way ANOVA followed by Bonferroni’s multiple comparison test were used for comparisons among experiments groups. The correlations were determined by a Pearson’s coefficient of correlation. All data analysis was performed using GraphPad Prism 5 software (GraphPad Inc., La Jolla, CA). A p-value<0.05 was considered statistically significant.

Results

MiR-210-3p is induced under hypoxic conditions via HIF-1α transcriptional activity

To evaluate the effects of hypoxia on the miR-210-3p expression level in glioma cell lines, including U87-MG, A172 and HS683, cells exposed to hypoxia for 24 and 48 h were used for quantitative PCR, and miR-9, which does not respond to hypoxic conditions, was used as a negative control [24, 25]. As shown in Fig 1A, hypoxia exposure significantly upregulated the miR-210-3p expression levels at 24 and 48 h in all three glioma cell lines; however, it did not affect miR-9 expression. To confirm whether the transcriptional activity of HIF-1α is involved in hypoxia-induced miR-210 upregulation, we cotreated cells with 1 ng/ml EC, and the miR-210 expression was measured 48 h after hypoxia exposure. As shown in Fig 1B, cotreatment with echinomycin abolished miR-210 upregulation after hypoxia exposure or CoCl2 treatment, demonstrating that HIF-1α transcriptional activity is critical for miR-210 upregulation. By considering that hypoxia exposure affected miR-210 similarly, we focused on the effect of hypoxia on U87 proliferation. Because HIF-1α plays critical roles under ischemia/hypoxia conditions [26], we further analyzed the effects of hypoxia and CoCl2 and the subsequent stimulated HIF-1α transcriptional activity on cell viability. Both hypoxia and CoCl2 exposure significantly decreased cell viability after 24 and 48 h. Addition of echinomycin significantly increased cell viability after 48-hour treatment (Fig 1C).

Fig 1. Hypoxia exposure upregulated miR-210-3p via transcriptional activity of HIF-1α.

Fig 1

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A. After hypoxia or CoCl2 exposure for 24 and 48 h, the expressing levels of miR-9 and miR-210-3p were evaluated by RT-qPCR. *P<0.05, vs. 24h normoxia group; #P<0.05, vs. 48h normoxia group. Square represents miR-210-3p/CoCl2 group; Triangle represents miR-9/hypoxia group; reverse triangle represents miR-9/CoCl2 group; circle represents miR-210-3p/hypoxia group. B. After co-treatment with 1 ng/ml echinomycin, the expressing levels of miR-9 and miR-210-3p in glioma cells were detected. *P<0.05, vs. hypoxia group; #P<0.05, vs. CoCl2 group. C. After echinomycin treatment or introduction of miR-210 for 24 or 48 h, cell viability was measured by performing CCK-8. *P<0.05, vs. hypoxia+echinomycin group; #P<0.05, vs. CoCl2+echinomycin group.

To further confirm the effect of HIF-1α induced by hypoxia on proliferation, we performed high content screening (HSC)-based cell viability assay. As it is shown in Fig 2A, consistently, Hypoxia exposure decreased cell viability, which was reversed by inhibition of HIF-1α transcriptional activity (Fig 2A). Cell cycle distribution was further analyzed by PI staining followed by flow cytometric assay, and expectedly, HIF-1α critically blocked entry of cell cycle (Fig 2B).

Fig 2. The effect of transcriptional activity of HIF-1α on proliferation.

Fig 2

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A. Cell viability was measured by performing high content screening (HSC)-based assay. B. Cell cycle distribution was analyzed by PI staining followed by flow cytometric assay. *P<0.05, vs. hypoxia group; #P<0.05, vs. CoCl2 group.

Hypoxia and CoCl2-induced HIF-1α upregulated TGF-β

By considering that HIF-1α and TGF-β is tightly associated, and HIF-1α is tightly regulated by TGF-β [27, 28], we searched for correlation between HIF-1α and TGF-β in Repository for Molecular Brain Neoplasia Data (REMBRANDT, E-GEOD-68848, http://www.ebi.ac.uk/arrayexpress/) database, and found that TGF-β protein was mildly positive associated with HIF-1α expression (Fig 3A). To confirm the effects of hypoxia on TGF-β, glioma cell lines were exposed to hypoxia or CoCl2 for 48 h and employed for TGF-β mRNA analysis. As it is shown in Fig 3B, hypoxia-induced upregulation of TGF-β was reversed by echinomycin cotreatment in all these three glioma cell lines. Expectedly, addition of 5 ng/ml TGF-β obviously induced HIF-1α, which indicates the positive feed-back loop between HIF-1α and TGF-β, that is induced by hypoxia or CoCl2 (Fig 3C and 3D, left panel). In normoxia condition, introduction of miR-210-3p mimics significantly upregulated TGF-β mRNA level, without being disturbed by echinomycin supplement (Fig 3D, right panel), demonstrated the positive association between miR-210-3p and TGF-β. We also introduced miR-210-3p into normoxia- or hypoxia-exposed cells, it was shown that (Fig 3E), the effect of hypoxia on TGF-β mRNA is similar with that of introduction of miR-210-3p mimics, indicated that hypoxia induced upregulation of TGF-β mRNA is mainly via upregulating miR-210-3p.

Fig 3. HIF-1α transcriptionally upregulated TGF-β in glioma cells.

Fig 3

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A. The correlation of HIF-1α expression with TGF-β. Data were analyzed using the REMBRANDT database. Increased HIF-1α expression levels were associated with increased expression levels of TGF-β. B. The expressing levels of TGF-β is detected after hypoxia was detected by RT-qPCR. *P<0.05, vs. hypoxia group; #P<0.05, vs. CoCl2 group. C. HIF-1α was semi-quantitatively measured by western blot after TGF-β exposure. *P<0.05, vs. normoxia group; #P<0.05, vs. hypoxia group. D. After introduction of miR-210-3p mimics or inhibitor, the mRNA levels of TGF-β are detected by RT-qPCR. *P<0.05, vs. NC mimics group. E. After hypoxia exposure with or without miR-210-3p mimics introduction, the mRNA levels of TGF-β are detected by RT-qPCR. *P<0.05, vs. NC mimics group.

Introduced miR-210 mimics upregulated TGF-β and promoted malignant behaviors independent on transcriptional activity of HIF-1α

To confirm the regulation of miR-210 on TGF-β1 mRNA, miR-210 mimics was transfected, while miR-9 mimics was also transfected as a negative control. Efficient introduction of miR-210 or miR-9 was detected which was not affected by addition of echinomycin (Fig 4A). Expectedly, introduction of miR-210 mimics, but not miR-9 mimics, significantly upregulated TGF-β1 mRNA level, which was not affected by inhibition of HIF-1α transcriptional activity (Fig 4B). Then we focused on the effects of miR-210 mimics on malignant behaviors, including cell cycle distribution, invasion and colony formation. As shown in Fig 4C, miR-210 mimics decreased proportion of G1/G0 phase independent on the presence of HIF-1α transcriptional activity. The promoting effects of miR-210 mimics on invasion and colony formation were also observed (Fig 4D and 4E). Taken together, it is demonstrated that the effects of miR-210 on transcriptional level of TGF-β1 and malignant behaviors of glioma cells, is independent on the HIF-1α transcriptional activity induced by hypoxia.

Fig 4. miR-210 mimics upregulated TGF-β1 transcriptionally and promoted proliferation, invasion and colony formation abilities.

Fig 4

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A. After miR-210 mimics, or miR-9 mimics were transfected, RT-qPCR was performed to detect the amount of overexpressed miR-210 or miR-9. B. The TGF-β1 mRNA level was measured by performing RT-qPCR. C. Cell cycle was measured by performing PI staining followed by flow cytometric assay. *P<0.05, NC mimics group. D. Invasion was measured by performing transwell assay. *P<0.05, NC mimics group. E. Colony formation was performed to detect the tumor formation in vitro.

Hypoxia-induced miR-210-3p promoted EMT potentially via upregulating TGF-β1

We then aim to figure out whether miR-210-3p induced by hypoxia exposure affects EMT, which is promoted by TGF-β expression [29]. hallmarkers of EMT, including E-cadherin, N-cadherin and Vimentin were detected by western blot. As it is shown in Fig 5A (Left panel), Hypoxia exposure obviously promoted EMT, which was reversed by echinomycin co-treatment, indicated that transcriptional activity of HIF-1α induced by hypoxia is critical for EMT promotion. Meanwhile, the similar effect on EMT was also observed in miR-210-3p inhibitor introducing group, demonstrated that miR-210-3p transcriptionally induced by HIF-1α potentially plays critical role in promoting EMT after hypoxia exposure. To evaluate the effect of miR-210-3p on EMT, hallmarkers of EMT were detected under normoxia condition after miR-210-3p mimics introduction. As it is presented in Fig 5A (right panel), introduction of miR-210-3p mimics obviously promoted EMT, which is similar to the effect of addition of 5 ng/ml of TGF-β. Hypoxia exposure converted a cobblestone-like or a short spindle-shaped epithelial profile to a stick-like or long spindle shaped mesenchymal morphology in glioma cells, which was reversed by addition of echinomycin or inhibitor of miR-210 (Fig 5B). These results suggested that miR-210-3p plays critical roles in EMT promotion, which is potentially via regulating TGF-β expression. Subsequently, invasive ability of cells was evaluated by performing Transwell assay, and consistent with the profiling changes of hallmarkers of EMT, invasive ability presented same tendency expectedly, which further confirmed the effects of miR-210-3p on EMT (Fig 5C).

Fig 5. miR-210-3p promoted EMT potentially via regulating TGF-β.

Fig 5

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A. Western blot was performed to detect hallmarkers of EMT, including E-cadherin, N-cadherin and Vimentin. B. Morphological presentation of glioma U87MG cells after hypoxia exposure. C. Transwell assay was performed to detect the effects of miR-210-3p on invasive ability. For upper panel, *P<0.05, vs. Normoxia group; #P<0.05, vs. Hypoxia group. For lower panel, *P<0.05, vs. NC mimics group.

MiR-210-3p induced chemoresistance in U87-MG potentially via upregulating TGF-β

It is reported that hypoxia-induced TGF-β is positively associated with chemoresistance in cancer cells [26, 27], this promoted us to evaluate the effect of miR-210 and subsequent stimulated TGF-β on chemoresistance in U87-MG cells. After introduction of miR-210-3p mimics, cells were treated with a range concentration of TMZ from 7.5 to 480 μM for 24 h and analyzed by performing CCK-8 assay. As it is shown in Fig 6A (left panel), miR-210-3p introduction obviously desensitized U87-MG to TMZ, which is similar to that of treatment of 5 ng/ml of TGF-β (right panel). This demonstrated that both miR-210-3p introduction and TGF-β treatment promoted chemoresistance in U87-MG. To further confirm whether miR-210-3p promotes chemoresistance by upregulating TGF-β, TGF-β was knockdown in miR-210-3p-introduced cells, and the results presented that TGF-β knockdown obviously sensitized U87-MG to TMZ (Fig 6B). Then, the apoptotic death rate after 100 μM TMZ treatment was performed, and expectedly, miR-210-3p significantly decreased apoptotic cell death, which was reversed by TGF-β knockdown (Fig 6C).

Fig 6. miR-210-3p induced chemoresistance potentially via regulating TGF-β.

Fig 6

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A. After introduction of miR-210-3p mimics, or treated with 5 ng/ml of TGF-β for 24 h, chemosensitivity to TMZ was evaluated by performing CCK-8 assay. B. After knockdown of TGF-β, chemosensitivity to TMZ was evaluated by performing CCK-8 assay. C. Apoptotic cell death was measured by performing Annexin V-FITC/PI double staining followed by flow cytometry after 24-hour treated with 100 μM TMZ. *P<0.05, vs. NC mimics group.

Hypoxia-induced miR-210-3p partially regulates U87-MG via activating NF-κB signalling pathway

To investigate the underlying mechanism of the regulatory role of miR-210-3p induced by hypoxia exposure in U87MG, we found that hypoxia exposure significantly increased, while addition of echinomycin or QNZ reduced NF-κB-dependent luciferase activity in U87MG cells, which indicated that hypoxia-activated NF-κB is, at least partially, depending on HIF-1α activity (Fig 7A). To confirm whether miR-210-3p was involved in activation of NF-κB pathway, we introduced miR-210 mimics with or without addition of QNZ. Expectedly, miR-210 mimics significantly activated NF-κB pathway, which was reversed by addition of QNZ. Moreover, cellular fractionation and western blot analysis revealed that hypoxia exposure enhanced, while addition of echinomycin or QNZ reduced nuclear accumulation of NF-κB/p65 (Fig 7C). By performing RT-qPCR analysis, it was further showed that hypoxia exposure increased the expression levels of multiple NF-κB signalling downstream metastasis-related target genes, including TWIST1, MMP13 and IL11 in U87MG cells (Fig 7D). We then analysed invasion ability and found that hypoxia-activated NF-κB promoted cell invasion in U87MG (Fig 7E).

Fig 7. Hypoxia-induced miR-210 activates NF-κB signaling pathway and promotes invasion.

Fig 7

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A. After hypoxia exposure with echinomycin or QNZ, the transcriptional activity of NF-κB was measured by performing luciferase reporter assay. *P<0.05, vs. Normoxia group; #P<0.05, vs. Hypoxia group. B. After introduction of miR-210 mimics, with or without QNZ, the transcriptional activity of NF-κB was measured by performing luciferase reporter assay. *P<0.05, vs. NC mimics/mock group; #P<0.05, vs. miR-210 mimics/mock group. C. Western blot was performed to detect nuclear NF-κB/p65 expression. The nuclear p84 and HDAC1 were used as the nuclear protein markers. D. RT-qPCR analysis of TWIST1, MMP13 and IL11 was performed after hypoxia exposure with addition of echinomycin or QNZ. *P<0.05, vs. Normoxia group; #P<0.05, vs. Hypoxia group. E. Invasion ability was performed to detect the effect of hypoxia on invasion via activating NF-κB signaling. *P<0.05, vs. Normoxia group; #P<0.05, vs. Hypoxia group.

Discussion

Our study revealed that hypoxia-dependent miR-210-3p induction is transcriptionally upregulated by HIF-1α and that it positively increased TGF-β expression of in glioma cells. Subsequently, this TGF-β upregulation potentially promotes EMT, invasive ability and chemoresistance to TMZ. Interestingly, TGF-β is responsible for upregulating HIF-1α, which indicates a feedback loop between HIF-1α and TGF-β. These results indicate that hypoxia-induced miR-210-3p expression may act as an oncogene to promote invasiveness by promoting EMT to protect in glioma cells from chemotherapy.

Accumulating evidence has revealed that a group of hypoxia-relevant microRNAs are tightly associated with the oxidative stress response and subsequent tumor progression in several kinds of cancer cells [30]. Li and colleagues reported that miR-137, a novel hypoxia-responsive microRNA, is tightly involved in the regulation of tumor progression and that it exerts protective effects in vivo by inhibiting mitophagy by targeting a key regulator of mitophagy [31]. It has also been reported that miR-101 is responsible for hypoxia exposure and that it promotes angiogenesis via the heme oxygenase-1/vascular endothelial growth factor axis by targeting cullin3, indicating that it has protective roles in human umbilical vein endothelial cells [32]. All of these data inspired us to dissect the exact roles of microRNAs in glioma cells.

Based on our results, it appears that HIF-1α induction via both hypoxia exposure and CoCl2 treatment transcriptionally upregulated miR-210-3p, revealing the specific regulatory mechanism of miR-210-3p under hypoxic conditions. To address our aim of revealing the effects of miR-210-3p on hypoxia-induced EMT and invasiveness in glioma cells, we treated hypoxia-exposed cells with echinomycin or introduced a miR-210-3p inhibitor (Fig 3B). We observed that both of these two treatments had similar effects on invasiveness, indicating that transcriptional induction of miR-210-3p may play critical roles in these processes. We also evaluated the effects of miR-210-3p on other malignant behaviors, including proliferation, colony formation and tumor formation; however, while echinomycin treatment clearly affected these phenomena, the miR-210-3p inhibitor had no effect, indicating that miR-210-3p is mainly involved in the regulation of invasiveness. Echinomycin, a potent small-molecule and cell-permeable inhibitor of hypoxia-inducible factor-1 (HIF-1) DNA-binding activity [33], was employed to inhibit HIF-1 transcriptional activity and thus to investigate the regulation of HIF-1α on miR-210-3p. HIF-1α functions as a transcriptional regulator and also interacts with proteins, or even long non-coding RNA and thus exerts multiple functions [34, 35]. By considering this, echinomycin was employed instead of siRNA targeting to HIF-1 mRNA to specifically inhibit HIF-1α transcriptional activity without disturbing other functions.

We also observed that miR-210-3p is positively associated with the TGF-β mRNA level (Fig 2D); however, without knowing the status of TGF-β release into supernatant, the confirmation of our hypothesis was limited. To determine whether TGF-β is involved in miR-210-3p-induced malignancy, glioma cells were cultured with TGF-β supplementation under normoxic conditions, and we observed that miR-210-3p-promoted invasiveness was similar to the effects of TGF-β addition. By measuring apoptotic cell death under hypoxic conditions, we found that miR-210-3p protected the cells from TMZ-induced apoptosis, which is also similar to the effect of TGF-β supplementation, and this effect was reversed by TGF-β knockdown with a TGF-β-targeted siRNA. These data suggest that miR-210-3p exerts its malignancy-promoting effects in glioma cells via a mechanism mainly dependent on TGF-β expression, which is consistent with the previous report showing that miR-210-3p exerts as a promoter of malignancies in glioma [36]. However, we failed to further confirm the effects of miR-210-3p induced TGF-β on cellular behaviors by adding TGF-β neutralizing antibody, which is a limitation in this study and is worth doing in further study.

Previous work showed that hypoxia-responsive miR-210-3p modulates mitochondrial respiration in the placenta by targeting mitochondrial-related genes [37]. In particular, miR-210-3p modified the maintenance of mitochondrial membrane potential to improve mitochondrial function, which is a primary promoting factor of cancer progression. According to our results, despite of the roles of miR-210-3p on mitochondrial functions, HIF-1α induced miR-210-3p may also promote malignant behaviors, including invasiveness and EMT in glioma cells, which may also be involved in the regulation of mitochondrial function.

Supporting information

S1 File

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Acknowledgments

The authors would like to thank Mr. Tao Hong for language editing and suggestions for statistical analysis.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

Ziyi Zhao. 82074298 for Ziyi Zhao The General Program (Key Program, Major Research Plan) of National Natural Science Foundation of China http://isisn.nsfc.gov.cn/egrantweb/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Michael Klymkowsky

25 Jan 2021

PONE-D-21-00617

MicroRNA-210-3p is transcriptionally upregulated by hypoxia induction and thus promoting EMT and chemoresistance in glioma cells

PLOS ONE

Dear Dr. Zhao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Before I send the manuscript out for review, there are number of points that I think need to be clarified. 

  • In the methods, what exactly does cells "were stored immediately to avoid being passaged for too many times" mean - how many times were they passaged?. 

  • The levels of hypoxia under various culture conditions need to be specified.  Are the cultures actually anoxic?  How are final O2 levels measured?  How do they compare with O2 levels in situ, that is, within a tissue? This speaks to the physiological relevance of these studies. 

  • The use of CoCl2 treatment to mimic hypoxia seems problematic, not withstanding its convenience.  Do the authors have comparative RNA SEQ analyses (for example) to establish how closely CoC2 treatment and hypoxia conditions resemble each other?  This seems necessary given that "CoCl2 influences the transcription of distinct sets of genes that were not affected by low oxygen‐induced hypoxia, indicating that genes induced by both models of hypoxia do not overlap (Vengellur et al., 2005)." (from Muñoz‐Sánchez and Chánez‐Cárdenas, 2019). 

  • The authors should use the full name echinomycin rather than the non-standard abbreviation (EC)  
    • and should note that "echinomycin strongly inhibits the activity of HIF‐1 under hypoxic conditions, and also interferes with the activity of other transcription factors. These results demonstrate the lack of specificity of this molecule. Moreover, it is demonstrated that echinomycin induces an increase in HIF‐1 activity under normoxic conditions, parallel to an increase in the expression of HIF‐1 target genes. (Vlaminck et al., 2007)

  • The authors should present their studies in the context of related work on miRNAs and C6 glioma cells (He et  al., 2020. "MiR‑210‑3p Inhibits Proliferation and Migration of C6 Cells by Targeting Iscu". Neurochemical research.)

  • and probably should have cited: Malzkorn et al. 2010) Identification and functional characterization of microRNAs involved in the malignant progression of gliomas. Brain Pathol 20:539–550

Please submit your revised manuscript by Mar 06 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Michael Klymkowsky, Ph.D.

Academic Editor

PLOS ONE

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PLoS One. 2021 Jul 1;16(7):e0253522. doi: 10.1371/journal.pone.0253522.r002

Author response to Decision Letter 0

16 Mar 2021

1. Thank you for providing the following data availability statement:

"All relevant data are within the manuscript and its Supporting Information files."

However, we note you have mentioned the following in your manuscript:

The datasets used during the present study are available from the corresponding author upon

reasonable request."

* Can you please clarify which statement is correct?

Answer：dear Sir, the statement “All relevant data are within the manuscript and its Supporting Information files.” Is correct. We modified the statement in manuscript as “All data are fully available without restriction” in “Availability of data and materials” section

Attachment

Submitted filename: response to reviewers.docx

Click here for additional data file. (19.4KB, docx)

Decision Letter 1

Michael Klymkowsky

24 May 2021

PONE-D-21-00617R1

MicroRNA-210-3p is transcriptionally upregulated by hypoxia induction and thus promoting EMT and chemoresistance in glioma cells

PLOS ONE

Dear Dr. Zhao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please address review #1 comments in detail, you can indicate areas of future study and caution, but I do not think new experiments are necessary.  

Please submit your revised manuscript by Jul 08 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Michael Klymkowsky, Ph.D.

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Comments to the Author

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Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

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Reviewer #1: Partly

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Liu et al. is an interesting addition to the field. The authors report that hypoxia results in the up-regulation of miRNA-210-3p through the induction of HIF-1alpha and that miRNA-210-3p expression influences EMT and chemoresistance in glioma cells through a mechanism that involves the regulation of TGF-beta. While overall this is a good study, some points should be further addressed.

Major:

1. The authors do a nice job of demonstrating that miR-210-3p is up-regulated with hypoxia. While the data using EC would suggest this up-regulation is due to HIF-1a, some additional evidence, beyond the use of EC, is needed. What happens to miR-210 expression under hypoxic conditions when HIF-1a is knocked-down. Does the miR-210-3p promoter have a HIF-1a binding site(s)?

2. As regards the regulation of TGF-beta, the authors clearly show an involvment of both TGF-beta and miR-210 in the hypoxic response, but no evidence is presented to directly link these molecules. How do the authors propose that miR-210 is regulating TGF-beta? If miR-210-3p is regulating TGF-beta, then addition of TGF-beta to the media of cells in which TGF-beta mRNA and miR-210-3p have been knock-down, should have the same or similar cellular effects as HIF-1alpha stimulation.

3. Does inhibition of TGF-beta and NF-kB block the observed cellular effects of HIF-1alpha? The authors state in the discussion, "that miR-210-3p is positively associated with the TGF-beta mRNA level; however, without knowing the status of TGF-beta release into supernatant, the confirmation of our hypothesis was limited". The authors should assay both the synthesis/stability of TGF-beta mRNA as well as the levels of TGF-beta in the media following hypoxia, in the presence or absence of miR-210-3p. Also use of neutralizing TGF-beta antibody could help determine how much of the effects contributed by miR-210-3p are mediated through TGF-beta.

4. Use of a second relevant chemotherapeutic agent is recommended to enhance the significance of the findings described in U87MG cells.

5. The X-axis of graphs in the same panel (Figures 1 and 4) should be on the same scale.

6. In Figue 3D, it would be helpful to show the effects of adding miR-210-3p to hypoxic cells on TGF-beta mRNA.

7. In Figure 4C, the addition of the miR-210 mimics results in a reduction of cells in G1/Go but no significant increase in S or G2 is visible. Is there an increase in the sub-G1/G0 population? Additionally, treatment with EC causes an increase in G2, but in the absence of an EC only control it is difficult to determine if miR-210-3p has any influence.

8. In Figure 5C, how do the authors explain the lack of an additive effect of adding miR210-3p mimics and TGF-beta.

Minor:

1. There are several grammatical errors that need to be addressed.

2. As presented by the Editorial Manager, many of the figures were out of order and not of good quality making them difficult to follow.

Reviewer #2: (No Response)

**********

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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PLoS One. 2021 Jul 1;16(7):e0253522. doi: 10.1371/journal.pone.0253522.r004

Author response to Decision Letter 1

28 May 2021

Reviewer #1: The manuscript by Liu et al. is an interesting addition to the field. The authors report that hypoxia results in the up-regulation of miRNA-210-3p through the induction of HIF-1alpha and that miRNA-210-3p expression influences EMT and chemoresistance in glioma cells through a mechanism that involves the regulation of TGF-beta. While overall this is a good study, some points should be further addressed.

Major:

1. The authors do a nice job of demonstrating that miR-210-3p is up-regulated with hypoxia. While the data using EC would suggest this up-regulation is due to HIF-1a, some additional evidence, beyond the use of EC, is needed. What happens to miR-210 expression under hypoxic conditions when HIF-1a is knocked-down. Does the miR-210-3p promoter have a HIF-1a binding site(s)?

Answer: Thanks for the suggestion. HIF-1a functions as a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions, activates the transcription of numerous genes. Meanwhile, HIF-1a protein interacts with redox regulatory protein APEX1 and thus activates NCOA1 and CREBBP. In the purpose of investigating the transcriptional role of HIF-1a on miR-210-3p, we employed EC co-treatment, instead of HIF-1a knockdown which will disturb the interaction between HIF-1a and APEX1.

It is still don’t known whether HIF-1a directly binds to miR-210-3p promoter region. In further investigation, it is worth performing ChIP-seq to screen HIF-1a binding site(s) in promoter region of HIF-1a.

2. As regards the regulation of TGF-beta, the authors clearly show an involvment of both TGF-beta and miR-210 in the hypoxic response, but no evidence is presented to directly link these molecules. How do the authors propose that miR-210 is regulating TGF-beta? If miR-210-3p is regulating TGF-beta, then addition of TGF-beta to the media of cells in which TGF-beta mRNA and miR-210-3p have been knock-down, should have the same or similar cellular effects as HIF-1alpha stimulation.

Answer: No evidence was shown to present the direct link between TGF-b and miR-210. In figure 3B, inhibition of HIF-1a activity by EC addition decreased TGF-b mRNA. This indicated that HIF-1a activity upregulate TGF-B mRNA. In figure 4B, it is also showed that introduction of miR-210 mimics upregulated TGF-b mRNA, which is not reversed by EC co-treatment. This indicates that HIF-1a upregulated TGF-b mRNA dependent on miR-210-3p. In figure 5c, addition of TGF-b present similar effect with miR-210 mimics on promoting invasion. These results indirectly indicated that HIF-1a regulates TGF-b via regulating miR-210-3p.

3. Does inhibition of TGF-beta and NF-kB block the observed cellular effects of HIF-1alpha? The authors state in the discussion, "that miR-210-3p is positively associated with the TGF-beta mRNA level; however, without knowing the status of TGF-beta release into supernatant, the confirmation of our hypothesis was limited". The authors should assay both the synthesis/stability of TGF-beta mRNA as well as the levels of TGF-beta in the media following hypoxia, in the presence or absence of miR-210-3p. Also use of neutralizing TGF-beta antibody could help determine how much of the effects contributed by miR-210-3p are mediated through TGF-beta.

Answer: In figure 6, figure 7, inhibition of TGF-beta and NF-kB block the observed cellular effects of HIF-1alpha. Thanks for the precious suggestion. In this study, we mainly focused on the regulatory effects of miR-210-3p on TGF-b mRNA, so more attention was put into the identification of regulatory effect of miR-210-3p on TGF-b mRNA. In further research, we’d like to quantitatively measure the contribution of miR-210-3p on cellular behavior through TGF-b.

We’ve also discussed this as a limitation in this study.

4. Use of a second relevant chemotherapeutic agent is recommended to enhance the significance of the findings described in U87MG cells.

Answer: Temozolomide (TMZ) is an alkylating agent currently used as first-line therapy in standard treatment of GBM. Temozolomide (TMZ) chemotherapy has been widely accepted as the new standard of care for patients with newly diagnosed GBM. That’s why we employed TMZ in this study.

5. The X-axis of graphs in the same panel (Figures 1 and 4) should be on the same scale.

Answer: It has been modified.

6. In Figue 3D, it would be helpful to show the effects of adding miR-210-3p to hypoxic cells on TGF-beta mRNA.

Answer: It has been added.

7. In Figure 4C, the addition of the miR-210 mimics results in a reduction of cells in G1/Go but no significant increase in S or G2 is visible. Is there an increase in the sub-G1/G0 population? Additionally, treatment with EC causes an increase in G2, but in the absence of an EC only control it is difficult to determine if miR-210-3p has any influence.

Answer: Sorry for the mistake in figure 4C (right panel). The sub G2/M was incorrectly calculated via flow cytometry results. It is re-calculated!

In figure 4c, miR-210/echinomycin group was compared with miR-210 group, the populations of G1/G0, S, or G2/M were not obviously different, this indicated that in cells transfected with miR-210 mimics was not affected by echinomycin addition.

8. In Figure 5C, how do the authors explain the lack of an additive effect of adding miR210-3p mimics and TGF-beta.

Answer: The addition of miR-210-3p mimics is sufficient to stimulate invasion. Added TGF-β is no longer enhance this cellular behavior.

Minor:

1. There are several grammatical errors that need to be addressed.

Answer: The grammatical errors have been checked carefully.

2. As presented by the Editorial Manager, many of the figures were out of order and not of good quality making them difficult to follow.

Answer: The figures were modified for a better understanding. Thanks for the suggestion.

Decision Letter 2

Michael Klymkowsky

3 Jun 2021

PONE-D-21-00617R2

MicroRNA-210-3p is transcriptionally upregulated by hypoxia induction and thus promoting EMT and chemoresistance in glioma cells

PLOS ONE

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Reviewer #1: All comments have been addressed

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Reviewer #1: Yes

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Reviewer #1: Most comments have been adequately addressed. Previous Comment 1 was directed at the specificity of EC. Many "specific inhibitors" are not so specific and if knock-down of the supposed target is not employed the authors should discuss briefly the limitations of EC (i.e., what is its specificity--IC50--for HIF and whether it is known to affect other enzymes; in order for the reader to have a full appreciation of the possible limitations and implications of using this methodology. As regards previous Comment 2, the authors should make sure that the train of thought and interpretation of the findings as presented in their response to the Reviewer's comment is clearly made to the reader in the manuscript.

All other points were sufficiently addressed.

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PLoS One. 2021 Jul 1;16(7):e0253522. doi: 10.1371/journal.pone.0253522.r006

Author response to Decision Letter 2

4 Jun 2021

"Please respond to the reviewer's specific comments and outline those responses for me.."

The reviewer comments, in section 6 are:

"Reviewer #1: Most comments have been adequately addressed. Previous Comment 1 was directed at the specificity of EC. Many "specific inhibitors" are not so specific and if knock-down of the supposed target is not employed the authors should discuss briefly the limitations of EC (i.e., what is its specificity--IC50--for HIF and whether it is known to affect other enzymes; in order for the reader to have a full appreciation of the possible limitations and implications of using this methodology. As regards previous Comment 2, the authors should make sure that the train of thought and interpretation of the findings as presented in their response to the Reviewer's comment is clearly made to the reader in the manuscript.

Answer: Echinomycin (Quinomycin A) is potent small-molecule and cell-permeable inhibitor of hypoxia-inducible factor-1 (HIF-1) DNA-binding activity. In discussion section, we discussed the reason why echinomycin was employed instead of siRNA targeting to HIF-1 mRNA as a limitation of this manuscript. The methodology of this choice was explained to make it more clear for reader to have a full appreciation of this limitation. The modified section was described as follows (3rd paragraph, discussion section):

Based on our results, it appears that HIF-1α induction via both hypoxia exposure and CoCl2 treatment transcriptionally upregulated miR-210-3p, revealing the specific regulatory mechanism of miR-210-3p under hypoxic conditions. To address our aim of revealing the effects of miR-210-3p on hypoxia-induced EMT and invasiveness in glioma cells, we treated hypoxia-exposed cells with echinomycin or introduced a miR-210-3p inhibitor (Figure 3B). We observed that both of these two treatments had similar effects on invasiveness, indicating that transcriptional induction of miR-210-3p may play critical roles in these processes. We also evaluated the effects of miR-210-3p on other malignant behaviors, including proliferation, colony formation and tumor formation; however, while echinomycin treatment clearly affected these phenomena, the miR-210-3p inhibitor had no effect, indicating that miR-210-3p is mainly involved in the regulation of invasiveness. Echinomycin, a potent small-molecule and cell-permeable inhibitor of hypoxia-inducible factor-1 (HIF-1) DNA-binding activity [33], was employed to inhibit HIF-1 transcriptional activity and thus to investigate the regulation of HIF-1α on miR-210-3p. HIF-1α functions as a transcriptional regulator and also interacts with proteins, or even long non-coding RNA and thus exerts multiple functions [34,35]. By considering this, echinomycin was employed instead of siRNA targeting to HIF-1 mRNA to specifically inhibit HIF-1α transcriptional activity without disturbing other functions.

All other points were sufficiently addressed."

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Decision Letter 3

Michael Klymkowsky

8 Jun 2021

MicroRNA-210-3p is transcriptionally upregulated by hypoxia induction and thus promoting EMT and chemoresistance in glioma cells

PONE-D-21-00617R3

Dear Dr. Zhao,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Michael Klymkowsky, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Michael Klymkowsky

18 Jun 2021

PONE-D-21-00617R3

MicroRNA-210-3p is transcriptionally upregulated by hypoxia induction and thus promoting EMT and chemoresistance in glioma cells

Dear Dr. Zhao:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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PLOS ONE Editorial Office Staff

on behalf of

Dr. Michael Klymkowsky

Academic Editor

PLOS ONE

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