Overexpression of miR-1283 Inhibits Cell Proliferation and Invasion of Glioma Cells by Targeting ATF4.

It is well known that activating transcription factor 4 (ATF4) expression is closely associated with progression of many cancers. We found that miR-1283 could directly target ATF4. However, the precise mechanisms of miR-1283 in glioma have not been well clarified. Our study aimed to explore the interaction between ATF4 and miR-1283 in glioma. In this study, we found that the level of miR-1283 was dramatically decreased in glioma tissues and cell lines, the expression of ATF4 was significantly increased, and the low level of miR-1283 was closely associated with high expression of ATF4 in glioma tissues. Moreover, introduction of miR-1283 significantly inhibited proliferation and invasion of glioma cells. However, knockdown of miR-1283 promoted the proliferation and invasion in glioma cells. Bioinformatics analysis predicted that the ATF4 was a potential target gene of miR-1283. Luciferase reporter assay demonstrated that miR-1283 could directly target ATF4. In addition, knockdown of ATF4 had similar effects with miR-1283 overexpression on glioma cells. Upregulation of ATF4 in glioma cells partially reversed the inhibitory effects of miR-1283 mimic. Overexpression of miR-1283 inhibited cell proliferation and invasion of glioma cells by directly downregulating ATF4 expression.

INTRODUCTION

Glioma is the most common malignant tumor of the brain1. According to the World Health Organization (WHO) classification, which is based on histological criteria, human glioma is divided into three major histologic groups: well-differentiated low-grade astrocytomas (WHO grades I–II), anaplastic astrocytomas (WHO grade III), and glioblastoma multiforme (GBM; WHO grade IV)2. Despite the advances in therapeutic intervention, such as surgery, radiotherapy, chemotherapy, gene therapy, immunotherapy, and other novel biological therapies, prognosis and treatment of this tumor type continue to be dismal3. Thus, identifying novel molecular biomarkers that can inhibit the progression will be urgent for the development of novel therapeutic strategies in glioma.

MicroRNAs (miRNAs) are small, noncoding RNAs that contain 22 nucleotides and regulate gene expression posttranscriptionally through binding to various target mRNAs4–6. miRNAs, as considerable epigenetic regulators, are strongly associated with human disease processes. Several studies have disclosed that disordered miRNAs are significantly correlated to carcinogenesis and tumor progression7,8. Among gliomas, tumor prognosis and progression have been closely associated with expression levels of miR-21, miR-105, miR-143, and miR-1974,9–11. miRNAs disrupt carcinogenesis and can serve as oncogene suppressors or tumor inhibitors by downregulating expression of their target genes5. Therefore, exploration of disordered miRNA expression levels in glioma might result in the discovery of novel miRNA biomarkers and glioma therapy targets12.

Activating transcription factor 4 (ATF4) ubiquitously expressed in human organs belongs to a member of the CREB/ATF transcription factor family, and it can be activated in response to various stress signals, including anoxia, hypoxia, endoplasmic reticulum stress, amino acid deprivation, and oxidative stress13,14. The stress-induced ATF4 expression causes adaptive responses in cells via regulation of target gene expressions involved in angiogenesis, differentiation, metastasis, amino acid synthesis, and drug resistance15–17. Furthermore, high level of ATF4 has been reported to be closely associated with malignancy in human tumor pathologies18,19. Therefore, overexpression of ATF4 is considered to promote tumor progression. Mechanistically, ATF4 can regulate transcription of many essential genes involved in tumor cell proliferation18,20,21. Up to now, little has been known about the relationship between ATF4 and miRNA processing in tumor progression.

However, until now, the precise mechanism of miR-1283 in glioma has remained unclear. In this study, we found significant upregulation of miR-1283 in glioma tissues and cells for the first time. Overexpression of miR-1283 inhibited the proliferation and invasion of glioma cells. Furthermore, we found that ATF4 was a direct target of miR-1283 in glioma. Introduction of ATF4 reversed the inhibitory effects of miR-1283 overexpression. Therefore, our results showed critical roles for miR-1283 in the pathogenesis of glioma and suggested its possible application in tumor treatment.

MATERIALS AND METHODS

Human Tissue Samples

Ten normal brain tissues (NBTs) and 30 high-grade glioma (HGG; WHO grades III and IV) samples were collected from the Yongchuan Hospital of Chongqing Medical University between 2015 and 2017. Both NBTs and glioma specimens were histologically confirmed. All samples were immediately frozen in liquid nitrogen for subsequent quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The Research Ethics Committee of Yongchuan Hospital of Chongqing Medical University approved our study. Written informed consent from each patient was received before participation.

Cell Culture

Human glioma cell lines such as U87, T98, LN229, H4, U118, U87, and A172 were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, P.R. China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, New York, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin and streptomycin (Gibco). Normal human astrocytes (NHAs) were obtained from Lonza (Basel, Switzerland) and cultured in the provided astrocyte growth media supplemented with recombinant human epidermal growth factor (EGF), insulin, ascorbic acid, GA-1000, l-glutamine (Gibco), and 5% FBS. All cells were cultured at 37°C in a humidified atmosphere of 5% on 0.1% gelatin-coated culture flasks.

Transient Transfection

The miR-1283 mimics, miR-1283 inhibitors, negative control (NC), siRNA for ATF4 (si-ATF4), and siRNA negative control (si-NC) were synthesized and purified by GenePharma (Shanghai, P.R. China). The ATF4 overexpression plasmid was generated by inserting ATF4 cDNA into a pcDNA3.1 vector. This plasmid was sequence confirmed by GenePharma. miR-1283 mimics (50 nM), miR-1283 inhibitors (100 nM), si-ATF4 (100 nM), and ATF4 overexpression plasmid (100 nM) were transfected using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocols. Total RNA and protein were collected 48 h after transfection.

RNA Isolation and qRT-PCR

Total RNA was isolated using TRIzol according to the manufacturer’s instructions, and analysis of mRNA levels was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with SYBR Green-based RT-PCR for all genes. For miRNA quantification, cDNA extraction was performed using the miRNA Extraction Kit (Tiangen, Beijing, P.R. China) and TaqMan miRNA assays (Life Technologies, Carlsbad, CA, USA), and reverse transcription RT-PCR was performed for miRNA quantifications according to the manufacturer’s instructions. Primer sequences used for ATF4, PCNA, p21, CDK2, cyclin E1, and GAPDH detection are shown in Table 1.

Table 1

Sequence of Primers for qRT-PCR

Gene	Primer Sequence
ATF4	F: 5′-ATGACCGAAATGAGCTTCCTG-3′
	R: 5′-GCTGGAGAACCCATGAGGT-3′
PCNA	F: 5′-CCTGCTGGGATATTAGCTCCA-3′
	R: 5′-CAGCGGTAGGTGTCGAAGC-3′
CDK2	F: 5′-TGTTTAACGACTTTGGACCGC-3′
	R: 5′-CCATCTCCTCTATGACTGACAGC-3′
Cyclin E1	F: 5′-AAGGAGCGGGACACCATGA-3′
	R: 5′-ACGGTCACGTTTGCCTTCC-3′
p21	F: 5′-TGTCCGTCAGAACCCATGC-3′
	R: 5′-AAAGTCGAAGTTCCATCGCTC-3′
U6	F: 5′-CTCGCTTCGGCAGCACA-3′
	F: 5′-AACGCTTCACGAATTTGCGT-3′
GAPDH	F: 5′-GAGTCAACGGATTTGGTCGTATTG-3′
	R: 5′-CCTGGAAGATGGTGATGGGATT-3′

Cell Viability Assay

Cell Counting Kit-8 (CCK-8) assay (Sigma-Aldrich, St. Louis, MO, USA) was used to detect the viability of U87 cells. U87 cells (1 × 104 cells/well) were seeded in 96-well plates overnight. After that, cells were transfected with miR-1283 mimic or inhibitor for 48 h. Cells were then incubated with WST-8 substrate at 37°C for 2 h. Absorbance (450 nm) of the medium was detected using a spectrophotometer by assessing the cell viability.

In Vitro Invasion Assay

According to a previous study22, Transwell invasion assay was carried out by adding 100 μl of Matrigel (BD Biosciences, Franklin Lakes, NJ) into the upper chamber of the Transwell and placing cells onto the Matrigel. Next, the noninvasive cells that were above the faces of the membranes were removed. The invasive cells were fixed with methanol for 15 min and then stained with 0.1% crystal violet for 20 min. Cells were imaged at least five grids per field. The membranes were then rinsed with 30% glacial acetic acid. Finally, the washing solution was examined at 540 nm for the counting of the number of glioma cells. All assays were independently repeated three times.

Protein Extraction and Western Blot Analysis

The protein used for Western blot was extracted using lysis buffer (Beyotime Biotechnology, Shanghai, P.R. China) containing protease inhibitors (Roche, Basle, Switzerland). The proteins were quantified using the BCA Protein Assay Kit (Beyotime Biotechnology). The Western blot system was established using a Bio-Rad Bis-Tris Gel system according to the manufacturer’s instructions. Primary antibodies of ATF4 (ab124777), MMP-2 (ab37150), MMP-9 (ab76003), TIMP-1 (ab109125; Abcam, Cambridge, UK), PCNA (#13110), CDK2 (#2546), cyclin E1 (#20808), and p21 (#2947) (Cell Signaling Technology, Danvers, MA, USA) were prepared in 5% blocking buffer at a dilution of 1:1,000. Primary antibodies were incubated with the membrane at 4°C overnight, followed by wash and incubation with secondary antibody (1:5,000; Abcam) marked by horseradish peroxidase for 1 h at room temperature. After rinsing, the polyvinylidene difluoride (PVDF) membrane carrying blots and antibodies was transferred into the Bio-Rad ChemiDoc™ XRS system, and then Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA) was added to cover the membrane surface. The signals were captured, and the intensity of the bands was quantified using Image Lab™ Software (Bio-Rad, Shanghai, P.R. China).

Measurement of MMP-2, MMP-9, and TIMP-1 Levels by ELISA Assay

According to a previous study23, the supernatants of U87 cells were collected after treatment, and the concentrations of MMP-2, MMP-9, and TIMP-1 were measured using a sandwich ELISA kit according to the manufacturer’s instruction (Roche). Briefly, the primary antibody was coated onto ELISA plates and incubated for 2 h at room temperature. Samples and standards were added to the wells and incubated for 1 h. Then the wells were washed and a biotinylated antibody was added for 1 h. The plates were washed again, and streptavidin conjugated to horseradish peroxidase was added for 10 min. After washing, tetramethylbenzidine was added for color development, and the reaction was terminated with 1 mol/L H2SO4. Absorbance was measured at 490 nm. Values were expressed as ng/ml.

Luciferase Reporter Assay

The 3′-UTR target site was generated by PCR, and the luciferase reporter constructs with the ATF4 3′-UTR carrying a putative miR-1283 binding site into pGL3 (Promega, Madison, WI, USA) luciferase report vector were amplified by PCR. Cells were cotransfected with the reporter construct, control vector, and miR-1283 mimic or corresponding controls using Lipofectamine 2000 (Invitrogen). After transfection for 48 h, the firefly and Renilla luciferase activities were measured by using the dual-luciferase assay system (Promega) following the manufacturer’s information. All experiments were performed in triplicate.

Statistical Analysis

The data were expressed as the mean ± standard error of the mean (SEM). The number of independent experiments was represented by n. The relationship between miR-1283 and the clinicopathological characteristics was tested by the chi-square test. Correlations between miR-1283 and ATF4 mRNA levels were analyzed using Pearson’s correlation coefficient. Multiple comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparison test. Other comparisons were analyzed using two-tailed Student’s t-test. A value of p < 0.05 was considered statistically significant.

RESULTS

High Expression of ATF4 Was in Glioma Specimens and its Effects on Cell Proliferation and Invasion of Glioma Cells

It has been reported that ATF4 expression is closely associated with many kinds of cancers18,19. In this study, we detected the mRNA and protein levels of ATF4 in glioma tissues. Our results showed that the mRNA level of ATF4 was significantly increased in HGG tissues compared with NBTs (Fig. 1A). To investigate the functional roles of ATF4 in glioma, several glioma cell lines were determined. Subsequently, we also determined the levels of ATF4 and miR-1283 in several glioma cell lines including U87, T98, LN229, H4, U118, U251, A172, and NHAs. Compared with NHAs, the level of ATF4 in U87 cells was higher than that in other glioma cell lines (Fig. 1B). We used U87 cells in the following experiments for further study, because its ATF4 activity is exceptionally high.

Figure 1

The expressions of activating transcription factor 4 (ATF4) and its effects in glioma tissues and cell lines. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of ATF4 expression in 10 normal brain tissues (NBTs) and 30 high-grade glioma (HGG) tissues. Transcript levels were normalized by GAPDH expression. (B) Relative ATF4 expression analyzed by qRT-PCR in seven glioma cell lines were normalized with GAPDH (n = 6). U87 cells were transfected with siRNA-ATF4 (si-ATF4) or siRNA-negative control (si-NC). (C) The protein expression of ATF4 was determined by Western blot. (D) Cell proliferation was assessed by the Cell Counting Kit-8 (CCK-8) assay. (E) The protein and mRNA expressions of PCNA, CDK2, cyclin E1, and p21 were determined by Western blot and qRT-PCR, respectively. (F) The invasion of glioma cells was assessed by Transwell assay. (G) Total secretions of MMP-2, MMP-9, and TIMP-1 in the culture supernatants were detected by ELISA. All data are presented as mean ± SEM, n = 6. **p < 0.01, ***p < 0.001 versus NBTs or normal human astrocytes (NHAs); ##p < 0.01, ###p < 0.001 versus si-NC.

Next, U87 cells were transfected with si-NC or si-ATF4 for 48 h, and then cell proliferation and invasion of glioma cells were detected. Western blot analysis showed that the ATF4 expression was significantly decreased in U87 cells transfected with si-ATF4 for 48 h compared to the si-NC group (Fig. 1C). The CCK-8 assay indicated that knockdown of ATF4 could significantly suppress the proliferation of glioma cells (Fig. 1D), and the qRT-PCR assay showed that downregulation of ATF4 decreased the protein and mRNA levels of PCNA, CDK2, and cyclin E1 and increased the expressions of p21 (Fig. 1E). Furthermore, the Transwell assay suggested that decreased ATF4 expression inhibited invasive ability of glioma cells (Fig. 1F) and dramatically downregulated the expressions of MMP-2 and MMP-9 and upregulated the expression of TIMP-1 (Fig. 1G).

miR-1283 Directly Targeted ATF4 3′-UTR

To further study the ATF4 expression regulated by miRNA, we detected the levels of miR-1283 predicted by online database TargetScan 6.2 in glioma tissues. We found that miR-1283 levels were significantly downregulated in HGG tissues (Fig. 2A) and U87 cells (Fig. 2B) compared with the adjacent tissues and NHAs. To determine whether the expression of ATF4 was associated with miR-1283 in glioma, Pearson’s correlation analysis revealed a significant inverse correlation between ATF4 and miR-1283 in HGG (Fig. 2C). Based on the online database microRNA.org, we found a binding site of miR-1283 in the 3′-UTR of ATF4 (Fig. 2D). To demonstrate that ATF4 is a direct target of miR-1283, luciferase plasmids containing the potential ATF4 miR-1283 binding sites (WT) or a mutated ATF4 3′-UTR were constructed. Upregulation of miR-1283 inhibited WT ATF4 reporter activity but not the activity of the mutated reporter construct in U87 cells, demonstrating that miR-1283 could specifically target the ATF4 3′-UTR by binding to the seed sequence (Fig. 2E). Next, we confirmed the results at the protein level. Introduction of miR-1283 could significantly decrease the expression of ATF4, whereas knockdown of miR-1283 increased ATF4 expression in U87 cells (Fig. 2F). These data indicated that miR-1283 directly regulated ATF4 expression in glioma cells through 3′-UTR sequence binding.

Figure 2

ATF4 was a direct target of miR-1283. U87 cells were transfected with miR-1283 mimic or inhibitor for 48 h. (A) qRT-PCR analysis of miR-1283 level in 10 NBTs and 30 HGG tissues. Transcript levels were normalized by U6 level. (B) qRT-PCR analysis of miR-1283 level in NHAs and U87 cells. (C) Pearson’s correlation analysis of the relative expression levels of miR-1283 and the relative ATF4 mRNA levels in HGG tissues. (D) Schematic representation of ATF4 3′-UTRs showing putative miRNA target site. (E) The analysis of the relative luciferase activities of ATF4-WT and ATF4-MUT. (F) The protein expression of ATF4 was determined by Western blot. All data are presented as mean ± SEM, n = 6. *p < 0.05, **p < 0.01, ***p < 0.001 versus NBTs or NHAs; ##p < 0.01 versus NC.

Upregulation of miR-1283 Inhibited Cell Proliferation in Glioma Cells

Next, we studied the effects of miR-1283 on the proliferation and invasion of glioma cells. After transfection with miR-1283 mimic or inhibitor, the qRT-PCR analysis showed that the level of miR-1283 was significantly upregulated or downregulated in the miR-1283 mimic or inhibitor group compared to the NC group (Fig. 3A), respectively. To study the effect of miR-1283 on the proliferation of glioma cells, the results from CCK-8 assay showed that increased level of miR-1283 markedly inhibited the proliferation of U87 cells (Fig. 3B). However, overexpression of miR-1283 promoted the cell proliferation of U87 cells (Fig. 3B).

Figure 3

Effects of miR-1283 on the proliferation and related gene expressions in glioma cells. U87 cells were transfected with miR-1283 mimic or inhibitor for 48 h. (A) The levels of miR-1283 in glioma cells were determined by qRT-PCR. (B) Cell proliferation was assessed by CCK-8 assay. (C) The mRNA and protein expressions of PCNA, CDK2, cyclin E1, and p21 were determined by qRT-PCR and Western blot, respectively. All data are presented as mean ± SEM, n = 6. #p < 0.05, ##p < 0.01, ###p < 0.001 versus NC.

To further confirm the above results, we detected the effects of miR-1283 on several proliferation and cell cycle-related genes. Overexpression of miR-1283 decreased the mRNA levels of PCNA, CDK2, and cyclin E1 and increased the mRNA levels of p21 in glioma cells (Fig. 3C). In addition, knockdown of miR-1283 had the opposite effects of miR-1283 inhibitor (Fig. 3C).

The Effects of miR-1283 on the Invasion in Glioma Cells

Next, we transfected miR-1283 mimic or inhibitor into U87 cells, and the invasive ability of glioma cells was assessed by Transwell assay. Increased miR-1283 level significantly reduced the number of invading glioma cells compared to the NC group (Fig. 4A). However, cell invasion ability in the miR-1283 inhibitor group was stronger than that of the NC group (Fig. 4A). In addition, the expressions of MMP-2, MMP-9, and TIMP-1 were determined. Our Western blot and ELISA assays demonstrated that protein expressions of MMP-2 and MMP-9 and total secretion of MMP-2 and MMP-9 in the culture supernatants were markedly decreased by increasing the miR-1283 level in U87 cells (Fig. 4B and C), whereas expression of TIMP-1 was dramatically increased by increasing the miR-1283 level (Fig. 4B and C). However, downregulation of miR-1283 had the reversed effects on the expressions of MMP-2, MMP-9, and TIMP-1 (Fig. 4B and C).

Figure 4

The effects of miR-1283 on invasion and related molecules in glioma cells. U87 cells were transfected with miR-1283 mimic or inhibitor for 48 h. (A) The invasion of glioma cells was assessed by Transwell assay. (B) Protein expressions of MMP-2, MMP-9, and TIMP-1 were determined by Western blot. (C) Total secretions of MMP-2, MMP-9, and TIMP-1 in the culture supernatants were detected by ELISA assays. All data are presented as mean ± SEM, n = 6. #p < 0.05, ##p < 0.01 versus NC.

Overexpression of ATF4 Significantly Blocked the Effects of miR-1283 Mimic on Cell Proliferation and Invasion in Glioma Cells

To determine whether miR-1283 targeting ATF4 was responsible for inhibition of the proliferation and invasion of glioma cells, we constructed an expression vector that encoded the entire ATF4 coding sequence but lacked the 3′-UTR. Then we cotransfected this vector (pcDNA-ATF4) or its NC (pcDNA3.1) with miR-1283 mimic or NC into U87 cells (Fig. 5A). Cell proliferation assay data showed that concomitant overexpression of miR-1283 and ATF4 abrogated the inhibitory effect of miR-1283 mimic (Fig. 5B). The protein and mRNA levels of PCNA, CDK2, and cyclin E1 were increased, and the levels of p21 were decreased in miR-1283-overexpressing glioma cells after exogenous introduction of ATF4 (Fig. 5C). Next, we found that increased ATF4 expression could reverse the inhibitory effect of the miR-1283 mimic on invasion of glioma cells (Fig. 5D), enhance the expressions of MMP-2 and MMP-9 inhibited by miR-1283 mimic, and reduce the expression of TIMP-1 upregulated by miR-1283 mimic (Fig. 5E). Therefore, the inhibitory effects of miR-1283 were reversed by ATF4 overexpression. Taken together, all above results clearly confirmed that miR-1283 inhibited cell proliferation and invasion in glioma cells by downregulation of ATF4, and miR-1283 targeting ATF4 was responsible for inhibition of the proliferation and invasion of glioma cells.

Figure 5

Overexpression of ATF4 partially promoted cell proliferation and invasion in miR-1283-overexpressing glioma cells. U87 cells were transfected with either miR-1283 mimic with or without pcDNA-ATF4 vector. (A) The protein expression of ATF4 was determined by Western blot. (B) Cell proliferation was assessed by CCK-8 assay. (C) The mRNA and protein expressions of PCNA, CDK2, cyclin E1, and p21 were determined by qRT-PCR and Western blot, respectively. (D) The invasion of glioma cells was assessed by Transwell assay. (E) Total secretions of MMP-2, MMP-9, and TIMP-1 in the culture supernatants were detected by ELISA assays. All data are presented as mean ± SEM, n = 6. #p < 0.05, ##p < 0.01 versus miR-1283 mimic + pcDNA3.1.

DISCUSSION

As the most common intracranial tumor, it is difficult to evaluate the prognosis of patients with glioma in clinical practice because of the heterogeneity of patients. Moreover, glioma has the characteristics of high proliferation and invasion potential, which lead to a poor prognosis24. It was reported that the survival time of most patients diagnosed as having glioblastoma was less than 1 year25. Therefore, identification and confirmation of appropriate biomarkers are still urgent for evaluating the prognosis and guiding tumor treatment of glioma. Up to now, previous studies have demonstrated that ATF4 expression is closely associated with multiple cancers, and all of the studies consider ATF4 as a pro-oncogene26–28. In the present study, mRNA levels of ATF4 were significantly higher in glioma tissues and cells compared to those in noncancerous tissues and normal cells. However, the effect and mechanism of ATF4 on glioma are still poorly understood. According to our data, we demonstrated that knockdown of ATF4 significantly inhibited the proliferation and invasion of glioma cells.

More and more evidence has reported that dysregulated miRNAs are closely associated with malignant biological behaviors including proliferation, cell cycle progression, migration, invasion, EMT, apoptosis, angiogenesis, and chemoresistance29. For example, overexpression of miR-544a promotes migration and invasion of breast cancer cells by reducing cadherin 1 expression30. miR-20b downregulates HIF-1α to inhibit the proliferation and invasion of osteosarcoma cells31. miR-146a inhibits proliferation and enhances chemosensitivity in epithelial ovarian cancer cells through downregulation of SOD232. However, the expression and effect of miR-1283 in glioma are still unknown. For the first time we found that the levels of miR-1283 were higher in HGG tissues and cells compared to those in noncancerous tissues and normal cells. To test the biological function of miR-1283 in glioma, we overexpressed or knocked down miR-1283 in U87 cells by transfecting them with miR-1283 mimics or inhibitors, respectively. We demonstrated that miR-1283 overexpression significantly inhibited the proliferation of glioma cells. To confirm this result, we found that introduction of miR-1283 decreased the mRNA levels of PCNA, CDK2, and cyclin E1 and increased the mRNA levels of p21 in glioma cells.

Invasion is one process of metastasis. In the present study, the Transwell assay showed that overexpression or knockdown of miR-1283 significantly suppressed or promoted the invasive ability of U87 cells compared with the NC group, respectively. In addition, degradation of extracellular matrix (ECM) components by proteolytic enzymes is very important for invasion of cancer cells33, and MMPs degrading the ECM process is closely associated with invasion, metastasis, and angiogenesis of cancer cells34–36. Particularly, both MMP-2 and MMP-9 are responsible for the invasion and EMT of malignant tumors by degrading components of the basement membrane35,37,38. A previous study showed that the imbalance between MMP activity and the specific inhibitor TIMPs in glioma tissues might be a significant factor in the process of tumor invasion and metastasis39. It has been demonstrated that the expressions of MMP-2 and MMP-9 in glioma tissues were significantly higher40, but TIMP-2 was significantly lower than that in normal tissues41. We found that expressions of MMP-2 and MMP-9 were significantly decreased and TIMP-1 expression was dramatically increased in U87 cells after transfection with miR-1283 mimics, whereas miR-1283 inhibitor could markedly enhance the MMP-2 and MMP-9 expressions and reduce the TIMP-1 expression. Collectively, our results suggested that increased miR-1283 level inhibited invasion of glioma cells, potentially leading to the suppression of metastasis of glioma.

It has been reported that miR-214 can directly target ATF442,43. For example, miR-214 targets ATF4 to inhibit bone formation42. miR-214 suppresses gluconeogenesis by targeting ATF443. However, no studies have demonstrated the relationship between miR-1283 and ATF4 in glioma. Based on previous studies and our findings, we hypothesized that the overexpression of miR-1283 reduced the expression of ATF4 and inhibited cancerous signals such as proliferation and invasion. In our study, the overexpression of miR-1283 in U87 cells enhanced the inhibition of ATF4, and such an effect was replicated by the knockdown of ATF4 in U87 cells. Besides, restoration of ATF4 reversed the inhibitory effects of miR-1283, indicating that ATF4 might play critical roles in the progression and metastasis of glioma.

In conclusion, our results have shown that ATF4 expression was significantly upregulated and miR-1283 level was dramatically downregulated in glioma tissues and cells. Moreover, our findings revealed a significant inverse correlation between ATF4 and miR-1283 in glioma tissues. Overexpression of miR-1283 inhibited proliferation and invasion of glioma cells through directly downregulating ATF4 expression. Therefore, our study provides functional evidence, which fully supports that miR-1283 and ATF4 were the prognostic factors for glioma.