MicroRNA-374a Governs Aggressive Cell Behaviors of Glioma by Targeting Prokineticin 2.

MicroRNA-374a has been abnormally expressed in several cancer types; however, its role in glioma remains unclear. Therefore, we aimed to investigate whether microR-374a participated in the progression of glioma. Expression of microR-374a in glioma cell lines and normal cell line was measured by quantitative real-time polymerase chain reaction. Luciferase reporter assay and Western blot were used to detect the targets of microR-374a. In vitro functional experiments were conducted to investigate the biological role of microR-374a. Low expression of microR-374a was found in glioma cell lines. Prokineticin 2 was identified as a direct target of microR-374a in glioma. Investigations on the mechanisms related to glioma progression showed that microR-374a inhibited glioma cell proliferation, cell cycle progression, and cell invasion through targeting Prokineticin 2. Taken together, these results revealed that microR-374a functions as tumor suppressor by targeting Prokineticin 2, suggesting it might be a novel therapeutic target for glioma.

Introduction

Glioma is the most frequently diagnosed and lethal type of brain tumor.[1] Over the past decades, numerous tumor suppressor genes and oncogenes were identified and reported to be associated with the pathogenesis of glioma, which have greatly improved our understanding of the progression glioma.[2,3] Hence, it is essential to deeply investigate the underlying mechanisms to advance the finding of novel therapeutic targets for glioma.[4]

MicroRNAs (miRNAs) are endogenous noncoding RNAs that are capable to negatively modulate gene expression mainly through binding to 3′-untranslated region (3′-UTR) of target mRNAs.[5] Importantly, miRNAs were reported to play pivotal roles in regulating cell functions, including cell proliferation, migration, invasion, and apoptosis.[6] Moreover, miRNAs have been demonstrated to have dual function in the pathogenesis of human cancers, namely, oncogenic and tumor suppressive role.[7-9] Recent studies demonstrated that miRNAs including miR-128 and miR-200a were downregulated, while the expression of miRNAs including miR-93 and miR-1908 were upregulated in glioma.[10-13] Hence, the characterization of novel miRNAs in the pathogenesis of glioma will advance our insight into the development of glioma.

The miR-374a has been reported to function as either tumor suppressor or oncogene.[14-16] The miR-374a was reported to promote osteosarcoma cell proliferation via downregulating axis inhibition protein 2 expression.[14] In addition, upregulation of miR-374a promoted gastric cancer growth both in vitro and in vivo through targeting SRC kinase signaling inhibitor 1.[15] Very recently, reduced expression miR-374a of was found in bladder carcinoma.[16] It was also found that miR-374a downregulation was associated with poor prognosis of bladder carcinoma, which suggested that miR-374a might be an important target for cancer treatment.[16] However, the expression status of miR-374a in glioma and the associated molecular mechanisms through which miR-374a modulates glioma cell behaviors remains largely unknown.

Here, we aimed to investigate the significance of miR-374a expression in glioma. We reported miR-374a expression was downregulated in both glioma tissues and cell lines. Prokineticin 2 (Prok2), with a binding site in its 3′-UTR for miR-374a, was identified as a direct target of miR-374a. Moreover, the effects of miR-374a and Prok2 on glioma cells were explored. It was shown that miR-374a inhibits glioma cell proliferation and invasion through modulating Prok2.

Materials and Methods

Cell Culture

Glioma cell lines T98, U87, H4, and A172 and normal human astrocytes (NHAs) were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were incubated in Dulbecco modified Eagle medium (DMEM; Thermo Fisher Scientific, Inc, Waltham, Massachusetts) plus 10% fetal bovine serum (FBS; Invitrogen, Thermo Fisher Scientific, Inc) and 1% penicillin/streptomycin mixture (Beyotime, Haimen, Jiangsu, China) at 37°C in a humidified incubator containing 5% CO2 and 95% air.

Cell Transfection

The open reading frame for Prok2 cloned into pcDNA3.1 was built by GenScript (Nanjing, China). The miR-374a mimic (5′-UUAUAAUACAACCUGAUAAGUG-3′), miR-374a inhibitor (5′-CACUUAUCAGGUUGUAUUAUAA-3′), and negative control miRNA (miR-NC; 5′-CAGUACUUUUGUGUAGUACAA-3′) were designed and synthesized by GenePharm (Shanghai, China). Transfection was conducted using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Inc) according to the manufacturer’s protocols. Quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot were conducted to measure the transfection efficiency.

RNA Isolation and RT-qPCR

Total RNA of the cells was isolated using Trizol reagent (Invitrogen, Thermo Fisher Scientific, Inc) according to the manufacturer’s protocol. RNA sample was reverse transcribed to complementary DNA (cDNA) using first-strand cDNA synthesis kit (Beyotime). The RT-qPCR was conducted with SYBR Green qPCR Mix (Beyotime) at ABI 7500 PCR equipment (Applied Biosystems, Foster City, California). The following primer sequences were used: miR-374a forward primer: 5′-CGGCGGTTATAATACAACCTG-3′, reverse primer: 5′-GCCACCATGGAGGGCGGATT-3′; U6 small nuclear RNA (U6 snRNA) forward primer: 5′-GTGCTCGCTTCGGCAGCACATATAC-3′; and reverse primer: 5′-AAAAATATGGAACGCTCACGAATTTG-3′. Relative miR-374a expression level was normalized to U6 snRNA and calculated with the 2(−ΔΔCT) method.[17]

Protein Isolation and Western Blot

Total protein of the cells was extracted using RIPA lysis buffer (Beyotime). Fifty-microgram protein sample was heat denatured and then isolated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to NC membrane (Beyotime). The membranes were blocked with skim milk and incubated with specific antibodies: anti-Prok2 (ab87360; Abcam, Cambridge, Massachusetts) and anti-GAPDH (ab181602; Abcam). Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (ab205718; Abcam) at room temperature after washing 3 times with Tris Buffered Saline Tween (TBST). Protein bands were developed using an ECL kit (Beyotime) and analyzed with Image Lab Software (Bio-Rad, Kidlington, United Kingdom).

Cell Proliferation Assay

Cells to be investigated were seeded into 96-well plates at the density of 5 × 103 cells/well. At 0, 1, 2, and 3 days after incubation, rate of cell proliferation was analyzed by adding MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (Beyotime) into the plate. Remaining formazan crystals were dissolved with DMSO, and optical density was measured using spectrophotometer (Thermo Fisher Scientific, Inc).

Cell Cycle Assay

Cells to be investigated were harvested by trypsinization and fixed by 80% precold ethanol. Then, the cells were washed with cold PBS and then incubated with 50 mg/mL propidium iodide (Beyotime) and RNase (50 mg/mL) in dark for 20 minutes at room temperature. Cell cycle distribution was measured at flow cytometer (FACSCalibur; BD Biosciences, Bedford, Massachusetts) and analyzed using FlowJo software (Ashland, Oregon).

Cell Invasion Assay

Cells of 1 × 105 were seeded in the upper chamber in DMEM using a Matrigel-coated membrane (Corning, New York, New York). The lower chamber was filled with DMEM containing 10% FBS. After incubation for 24 hours, noninvasive cells on the top of the membrane were removed with cotton swab. Invasive cells were fixed with 100% methanol for 30 minutes and stained with Crystal violet (Beyotime). Five independent fields were randomly selected and counted to calculate the average invasive numbers.

Tube Formation Assay

Cells were seeded onto 24-well plates containing 200 μL of 10 mg/mL Matrigel (Corning). After incubation at 37°C, cells were imaged at ×40 magnification on a Nikon (Minato-ku, Tokyo, Japan) TE-2000U inverted microscope. The number of tubes was counted in 3 individual wells.

Luciferase Reporter Assay

The wild-type (wt) 3′-UTR of Prok2 was cloned from the genome and inserted into psiCHECK-2 vector (Promega, Madison, Wisconsin). The mutant (mut) 3′-UTR of Prok2 construct was built using site-directed mutagenesis kit (Takara, Dalian, China). Cells were cotransfected with Prok2-wt or Prok2-mut and miR-374a mimic or miR-NC. After 48 hours transfection, luciferase activity was measured using dual-luciferase reporter system (Promega) with firefly luciferase activity as internal control.

Statistical Analysis

Data were presented as the mean (standard derivation). Multiple comparisons were performed using 1-way analysis of variance followed by Tukey multiple comparison test. Other comparisons were analyzed using 2-tailed Student t test. P < .05 was regarded as statistically significant.

Results

MicroR-374a Was Significantly Downregulated in Glioma Cell Lines

The RT-qPCR was conducted to measure the expression levels of miR-374a in glioma cell lines (T98, U87, H4, and A172) and NHAs. It was observed that miR-374a levels were dramatically decreased in all glioma cell lines to different extent compared to NHAs cell line (Figure 1). Among these investigated glioma cell lines, U87 and A172 have the first and second lowest miR-374a expression levels (Figure 1). These results revealed that miR-374a may contribute the progression of glioma.

Figure 1.

miR-374a was downregulated in glioma cell lines. Expressions of miR-384 in several glioma cell lines (T98, U87, H4, and A172) and normal human astrocytes (NHAs) were detected by RT-qPCR. **P < .01; ***P < .001). miR-374a indicates microRNA-374a; RT-qPCR, quantitative real-time polymerase chain reaction.

Overexpression of miR-374a Inhibits Glioma Cell Proliferation and Invasion In Vitro

In order to manipulate the levels of miR-374a in glioma cells, synthetic miRNAs were transfected into these investigated cells. After transfected with miR-374a mimic or miR-374a inhibitor, the levels of miR-374a were significantly upregulated or downregulated compared to the miR-NC group (Figure 2A). The MTT assay revealed that ectopic expression of miR-374a inhibited cell proliferation (Figure 2B). Next, flow cytometry analysis results revealed that miR-374a overexpression arrested cell cycle at G0/G1 phase (Figure 2C). On the contrary, the transfection of miR-374a inhibitor induced cell proliferation and cell cycle progression compared to miR-NC group (Figure 2B and C). Subsequently, transwell invasion assay revealed that miR-374a mimic decreased cell invasion, while miR-374a inhibitor increased cell invasion (Figure 2D). Tube formation assay showed the tube formation ability was inhibited by miR-374a mimic but increased by miR-374a inhibitor (Figure 2E). Our results collectively revealed that miR-374a overexpression inhibited glioma cell proliferation, cell cycle progression, cell invasion, and tube formation in vitro.

Figure 2.

Inhibiting proliferation and invasion of glioma cells by miR-374a mimic. A, The expression of miR-374a in A172 and U87 transfected with miR-NC, miR-374a mimics, or miR-374a inhibitor were detected by RT-qPCR. B, The proliferations of A172 and U87 were measured by cell counting kit-8 (CCK-8) assay after transfection of miR-NC, miR-374a mimics, or miR-374a inhibitor. C, Cell cycle distribution of A172 and U87 were measured by flow cytometry after transfection of miR-NC or miR-374a mimics. D, The invasions of A172 and U87 were measured by transwell invasion assay after transfection of miR-NC, miR-374a mimics, or miR-374a inhibitor. E, Tumor formation ability of A172 and U87 were measured after transfection of miR-NC, miR-374a mimics, or miR-374a inhibitor. **P < .01; ***P < .001. miR-374a indicates microRNA-374a; ns, not significant; RT-qPCR, quantitative real-time polymerase chain reaction; miR-NC, negative control miRNA.

Micro-374a Directly Target Prok2 to Negatively Regulate Prok2 Expression

We found a binding site in the 3′-UTR of Prok2 for miR-374a through the TargetScan algorithm (Figure 3A). Introduction of miR-374a mimic reduced Prok2-wt luciferase activity but not the luciferase activity of cells transfected with Prok2-mut (Figure 3B). Next, we confirmed this prediction by Western blot. The results showed that transfection of miR-374a mimic significantly decreased Prok2 protein expression levels, while the miR-374a inhibitor increased Prok2 protein expression levels in glioma cells (Figure 3C). These results indicated that miR-374a could directly modulate the expression levels of Prok2.

Figure 3.

Prok2 was a potential target of miR-374a. A, Putative binding site for miR-374a in 3′-UTR of Prok2. B, Luciferase activities of Prok2 were detected in glioma cells transfected with Prok2-wt or Prok2-mut and miR-374a mimic or miR-NC. C, Proteins of Prok2 in A172 and U87 cells transfected with miR-374a mimic, miR-374a inhibitor or miR-NC. ***P < .001. miR-374a indicates microRNA-374a; miR-NC, negative control miRNA; mut, mutant; ns, not significant; Prok2, Prokineticin 2; UTR, untranslated region; wt, wild-type.

Overexpression of Prok2 Enhanced Glioma Cell Proliferation and Invasion In Vitro

To investigate whether miR-374a targets Prok2 was responsible for the effects of miR-374a on glioma cell proliferation and invasion, rescue experiments were conducted by cotransfecting Prok2 construct and miR-374a mimic into the glioma cells. These results showed that Prok2 construct enhanced Prok2 expression in the miR-374a mimic transfected glioma cells (Figure 4A). Cell proliferation assay showed that coexpression of miR-374a and Prok2 abolished the inhibitory effect of miR-374a (Figure 4B). Cell cycle analysis revealed that introduction of Prok2 construct attenuated the effects of miR-374a mimic on cell cycle progression (Figure 4C). Moreover, overexpression of Prok2 also reversed the inhibitory effects of miR-374a mimic on glioma cell invasion and tube formation (Figure 4D and E).

Figure 4.

Prok2 overexpression promotes cell proliferation and invasion. A, Proteins of Prok2 in U87 cells transfected with miR-374a mimic and Prok2 construct. B, The proliferations of U87 cells were measured by cell counting kit-8 (CCK-8) assay after transfection of miR-374a mimic and Prok2 construct. C, Cell cycle distribution of U87 cells were measured by flow cytometry after transfection of miR-374a mimic and Prok2 construct. D, The invasions of U87 cells were measured by transwell invasion assay after transfection of miR-374a mimic and Prok2 construct. E, Tube formation of U87 cells were measured after transfection of miR-374a mimic and Prok2 construct. **P < .01; ***P < .001. miR-374a indicates microRNA-374a; ns, not significant; Prok2, Prokineticin 2.

Discussion

Extensive investigations on the mechanisms of cancer development over the past decades have improved our understanding on the molecular basis of tumor initiation and progression.[7-16,18,19] Numerous oncogenes, tumor suppressor genes, and signaling pathways closely related to the pathogenesis of tumor have been identified and led to the emergence of novel molecular-targeted therapy methods.[7-16,18] Targeting the specific oncogenes that are overexpressed in human cancers is therefore possible and will be an effective way to control human cancer.[19] Therefore, the identification of genes abnormal expressed during the progression of human cancers will advance the development of novel therapeutic measures.

MicroiR-374a was previously found significantly downregulated in several human cancers and was regarded to function as tumor suppressor in these cancer types.[14-16] However, to the best of our knowledge, the understanding of miR-374a in the pathogenesis of glioma remains limited. In this study, we for the first time demonstrated the levels of miR-374a were significantly downregulated in 4 glioma cell lines investigated compared to that in NHAs cell line and that such an alteration in expression levels will result in aberrant cell behaviors. Subsequently, a series of in vitro experiments including cell proliferation assay, cell cycle assay, cell invasion assay, and tube formation assay were conducted. Our results indicated that miR-374a overexpression inhibited cell proliferation, invasion, tumor formation, and arrested cell cycle at G0/G1 phase. These results implied miR-374a functions as a tumor suppressor to participate in the progression of glioma.

The Prok2 is a cysteine-rich secreted protein that contains a conserved N-terminal sequence of AVITGA and 10 cysteines.[20] Recent studies have shown Prok2 is related to drug resistance of breast cancer and has the potential to be used as prognostic predictor for the progression of colorectal cancer, which suggested the oncogenic role of Prok2 in human cancers.[21,22] We showed that Prok2 was a direct target of miR-374a through online prediction, luciferase activity reporter assay, and Western blot assay. Rescue functional assays revealed that Prok2 overexpression could impair the inhibitory effects of miR-374a on glioma cell malignancy behaviors. Nevertheless, further studies are needed to investigate the clinical significance of miR-374a in patients with glioma and to further explore the downstream targets of miR-374a in glioma to validate miR-374a as a target for cancer treatment.

In summary, miR-374a exerts the tumor suppressive effects through targeting the expression of Prok2 in glioma. Moreover, for the first time, our study sheds light on the association between miR-374a and Prok2 and revealed miR-374a could negatively regulate Prok2 expression. Our finding advanced our understanding of the molecular mechanisms underlying glioma progression.