Glioma is a malignant brain tumor exhibiting high levels of proliferation and metastasis, and these have been related to its poor prognosis and high mortality rate. MicroRNA (miRNA)-325-3p exhibits tissue-specific expression profiles and is aberrantly expressed in multiple types of malignant tumors. Our research focuses on determining the function and mechanism of action of miR-325-3p in glioma. The relative expression levels of miR-325-3p in glioma tumor tissues and cell lines were verified by qRT-PCR. The effect of 325-3p on glioma tumor cell behavior was assessed using CCK-8 assays, EDU staining, colony formation assays, flow cytometry, transwell invasion assays, and a xenograft model. In addition, we searched for miR-325-3p targets, and their potential mechanism of action was demonstrated using a reporter assay and rescue experiments. Results showed that the expression levels of miR-325-3p in glioma cancer tissues and tumor cell lines were significantly lower than that of normal paired adjacent tissue or normal cell lines. Functional experiments illustrated that tumor proliferation, migration and invasion were suppressed via upregulation of miR-325-3p. To assess whether FOXM1 is a target of miR-325-3p, we examined this hypothesis using a luciferase report assay and then found that miR-325-3p could modulate the expression of FOXM1. Furthermore, the functional role of miR-325-3p was also confirmed in a xenograft model using nude mice. Together, our data demonstrated that in glioma, miR-325-3p may inhibit cancer cell growth through the suppression of FOXM1 and could be a promising new target for treating this type of brain cancer.
Glioma is one of the most malignant tumors and accounts for more than 80% of all primary malignant tumors of the central nervous system . The median overall survival rate is constant at 16 months, although huge progress has been made in therapy . At present, surgery, chemotherapy, and radiotherapy are the main strategies for glioma treatment . However, this disease is virtually incurable, and its five-year survival rate remains unsatisfactorily low . Thus, it is key to understand the molecular mechanisms for glioma’s occurrence and progression, which may help uncover new therapeutic targets. Recent studies have revealed many abnormally expressed genes, which may be involved in glioma progression . MiRNAs are endogenous, non-coding RNAs that regulate multiple physiological and pathological processes at the posttranscriptional level [6-8] and mounting evidence suggests the involvement of miRNAs in multiple cancers, including glioma. Here, we specifically focused on miR-325-3p, which was found to be aberrantly expressed in multiple types of malignant tumors. For example, miR-325-3p showed low expression levels in colorectal cancer (CRC), representing a key regulator of bone metastasis . In gastric cancer (GC), the miR-325-3p was decreased, and its low expression level was also associated with metastasis . Furthermore, miR-325-3p has an effective therapeutic function in treating lung and bladder cancer [11,12]. However, the exact functions of miR-325-3p in glioma have yet to be determined, and thus these data suggest that miR-325-3p could be a potential therapeutic target for malignant glioma (Fig. 1).
Schematic illustration of rationale to show the intersections between neuroscience and glioma biology.
Twenty-four glioma tissue samples and adjacent normal tissue samples were
obtained from Yongchuan Hospital of Chongqing Medical University, and all written
informed consent was obtained from the patients. At least two pathologists
diagnosed and confirmed all tissues had not been treated with radiotherapy or
chemotherapy before surgery. The tissues were collected and kept at –80
Normal human astrocytes (NHAs) and glioma cell lines SW1783, U87, and LN229 were
purchased from the Chinese Academy of Sciences cell bank. All Cells were cultured
using DMEM and RPPI-1640 (Gibco, Carlsbad, CA, USA), 10% fetal bovine serum
(Gibco) in an incubator at 37
Cells or tissues were collected, and total RNA was extracted with TRIZOL
reagent. Then total miRNAs were isolated using the mirVana
miR-325-3p, forward: 5
FOXM1, forward: 5
U6, forward: 5
GAPDH, forward: 5
For cell viability assays , a CCK-8 kit (C0038, Beyotime Biotechnology,
China) was used according to the manufacturer’s instructions. Transfected cells
were inoculated into 96-well plates and incubated overnight. Add CCK-8 reagent 10
mL to each well, and incubate at 37
Cells were plated in 6-well plates (500 per/well), and the medium was replaced every 3–4 days. When cell colonies were formed after 2 weeks, they were fixed and stained. Visible colonies were photographed and counted.
As previously described, cell invasion assays were performed using the Bio-Coat Cell Migration Chamber
(BD Biosciences, MA, USA) . Then a 24-well plate
containing an 8
After the sections were rehydrated as above , heat-induced antigen retrieval
was performed in sodium citrate for 15 minutes in a 95
The interactions between miR-325-3p and FOXM1 were assessed using TargetScan tools. The 3’-UTR. FOXM1 fragment was cloned full-length into a pmirGLO expression vector (Promega, WI, USA). The luciferase vector and miR-325-3p mimics were cotransfected into U87 cells the and the luciferase activity was measured with a Dual-Luciferase Assay System (Promega, Madison, WI, USA) 48 h later.
Total protein was extracted with RIPA reagent (P0013B, Beyotime Biotechnology,
China), and the protein concentration was measured using a BCA protein detection
kit (P0012, Beyotime Biotechnology, China). Equal amounts of total protein 30
The Animal Care Committee approved the animal experiments of Yongchuan Hospital
of Chongqing Medical University. All experiments involving mice were conducted in
accordance with the guidelines for animal welfare formulated by the laboratory
animal center at Yongchuan Hospital of Chongqing Medical University. The nude
mice aged 6 weeks were purchased from Chongqing Medical University Experimental
Animal Center. Each mouse was injected subcutaneously with 6
Data were assessed using Student’s t-test or one-way ANOVA in GraphPad
Prism 7.0 (GraphPad, CA, USA) software and expressed as mean
To determine the expression profile of miR-325-3p in glioma tissues, more than
twenty-paired surgical glioblastoma and RT-qPCR analyzed para-carcinoma tissues.
It can be seen that miR-325-3p expression was significantly decreased in glioma
tissues compared to the adjacent normal tissues (Fig. 2A, p
Decreased miR-325-3p expression in glioma tissue and cell lines.
(A) Results from RT-qPCR analysis showed that miR-325-3p was decreased in human
glioma tissues. (B) Decreased miR-325-3p levels were also detected in glioma
cells. Data are presented as the mean
As an association between miR-325-3p and glioma has been confirmed, we next investigated the functional roles of miR-325-3p in glioma cell growth. MiRNA-mimics were specifically and effectively used to upregulate miR-325-3p expression in SW1783 and U87 glioma cell lines (Fig. 3A). Furthermore, the CCK8 assay showed that upregulation of miR-325-3p inhibited the proliferation rate of SW1783 and U87 cells (Fig. 3B). Using the EdU and colony formation assays, we also found that upregulation of miR-325-3p inhibited the growth of SW1783 and U87 cells (Fig. 3C,D). These data showed that miR-325-3p upregulation could effectively inhibit glioma cell proliferation.
In vitro upregulation of miR-325-3p inhibits cell
growth. (A) The transfection efficiency of miR-325-3p was evaluated using RT-qPCR.
(B) Cell viability of SW1783 and U87 cells transfected with the miR-325-3p-mimic
was evaluated by CCK8 assay. (C) EDU assay was conducted to examine
proliferation. (D) Colony formation assay for the detection of proliferation.
Data are presented as the mean
Metastasis is a major risk for patients with glioma, and therefore, to further determine the function of miR-325-3p in glioma cell migration and invasion in vitro, transwell assays were employed. We found that upregulation of miR-325-3p significantly inhibited the migration of SW1783 and U87 cells (Fig. 4A,B). The transwell assay also consistently indicated that overexpression of miR-325-3p also decreased their invasive capability (Fig. 4C,D). Such findings suggested that miR-325-3p suppressed the malignant behaviors of glioma cells.
Upregulation of miR-325-3p suppressed glioma cell migration and
invasion. (A,B) Transwell assay revealed that upregulation of miR-325-3p
decreased the migration capability of SW1783 and U87 cells. (C,D) Transwell assay
revealed that upregulation of miR-325-3p decreased the invasion capability of
SW1783 and U87 cells. Data are presented as the mean
Cumulative evidence indicates that FOXM1 serves as a tumor promoter or suppressor in multiple tumors, regulating a wide range of biologic processes [16-18]. Through bioinformatics software TargetScan (http://www.targetscan.org) analysis, we predicted the potential targets of miR-325-3p and found that FOXM1 may represent such a target. FOXM1 does not interact with any known miR-325-3p targets [9-12]. After the histological analysis, immunohistochemistry revealed that FOXM1 expression was increased in glioma tissue (Fig. 5A), and as shown in Fig. 5B, FOXM1 expression was inversely correlated with miR-325-3p. Thus, both wild-type (WT) and mutant (Mut) luciferase reporters for FOXM1 were generated. Results showed that miR-325-3p suppressed activity in the Foxm1-WT group, but the opposite result was seen for U87 cells (Fig. 5C), thus confirming an interaction. Moreover, western blot analysis revealed that transfection with the miR-325-3p mimic or inhibitor significantly increased/decreased FOXM1 protein levels’ expression compared to the control (Fig. 5D,E). These results confirmed a role for miR-325-3p in the regulation of FOXM1 in glioma cell lines.
FOXM1 is a direct target for miR-325-3p in glioma. (A) Tissue
comparison using H&E staining and IHC, showing higher levels of FOXM1 in glioma
tissues. (B) An inverse relationship between FOXM1 and miR-325-3p in glioma
tissues. (C) FOXM1 was predicted to be a target for miR-325-3p, based on the
Targets can database, and the WT and Mut luciferase reporter plasmids were
generated to perform dual-luciferase reporter assays. (D,E) The expression of
FOXM protein after transfection with NC, mimic, or inhibitor in U87 cells. Data
are presented as the mean
A series of rescue assays were performed to determine whether FOXM1 expression accounted for miR-325-3p-mediated migration and invasion in glioma cells. U87 cells were cotransfected with miR-325-3p-NC or miR-325-3p-mimic and pcDNA3.0 plasmid containing control or FOXM1 plasmid, and our western blot results confirmed the effectiveness of this rescue strategy (Fig. 6A). As expected, overexpression of FOXM1 attenuated the inhibitory effect of miR-325-3p on migration (Fig. 6B,C) and invasion (Fig. 6D,E). Furthermore, cancer cell invasion and migration are usually associated with epithelial-mesenchymal transition (EMT). For this reason, we further explored proteins associated with the EMT pathway. We found that FOXM1 overexpression reversed the effect of miR-325-3p upregulation, causing a decrease in E-cadherin expressions and an increase in N-cadherin, Vimentin, and Fibronectin in U87 cells (Fig. 6F). Remarkably, FOXM1 overexpression reversed the suppressive effect induced by miR-325-3p upregulation in U87 cells.
MiR-325-3p regulates migration and invasion in U87 cells by
targeting FOXM1 expression. (A) Western blot analysis showed that upregulation of
miR-325-3p decreased the expression of FOXM1, and this effect was reversed by
transfection with pcDNA3.0-FOXM1 in U87 cells. (B–E) Functional rescue
experiments with Transwell assays showed that pcDNA3.0-FOXM1 transfection
restored the migration and invasion abilities of U87 cells transfected with
miR-325-3p-mimic. (F) The expressions of EMT-related genes were detected after
FOXM1 was overexpressed in U87 cells via western blot. Data are presented as the
To validate our results in vivo, nude BALB/c mice were injected in the left flanks with NC or miR-325-3p-overexpressing U87 cells. Tumor volumes and weights in the miR-325-3p-overexpressing group were significantly reduced compared to the NC group (Fig. 7A,B). Furthermore, H&E staining and immunohistochemistry for the proliferation marker, Ki67, indicated that upregulation of miR-325-3p inhibited glioma proliferation in vivo (Fig. 6C). IHC staining showed that the EMT-associated marker N-cadherin was significantly lower in the miR-325-3p-overexpressing group when compared to the NC group, which is consistent with our in vitro results (Fig. 7C). Thus, these results demonstrated that miR-325-3p could inhibit glioma cell growth in vivo.
MiR-325-3p repressed glioma growth in vivo. (A) Tumor
volumes from the U87 cell xenograft model from the miR-325-3p-overexpressing
group were significantly reduced. (B) Representative tumor images from the
xenograft mice. (C) As indicated, representative H&E staining and IHC images of
Ki67 and N-cadherin in subcutaneous xenografts derived from cells. Scale
Glioma is one of the most aggressive and terminal diseases associated with the central nervous system and is associated with a very poor median survival of fifteen months. Therefore, there is an urgent need to uncover more biomarkers to enable clinicians to predict when and what therapy to deploy and help determine prognosis . A growing number of studies have focused on the role of miRNAs in tumorigenesis, as they are important for many biological processes, such as cell proliferation, differentiation and invasion in a variety of cancer types. The loss of miR-325-3p expression has been reported in CRC and gastric cancer [9,10].
These results have highlighted miR-325-3p as a potential new tumor regulatory molecule. However, the mechanism involved in the regulation of glioma growth by miR-325-3p remains unknown. We firstly detected the downregulation of miR-325-3p in glioma tissues and cells. We then confirmed the inhibitory effect of miR-325-3p on proliferation and invasion in glioma U87 and SW1783 cells using in vitro assays. These results provided elementary evidence for a role for miR-325-3p as a tumor-suppressor in glioma.
Similarly, it has been demonstrated that overexpression of miR-325-3p Inhibits proliferation and metastasis of bladder cancer cells  and miR-325 can also inhibit proliferation but induce apoptosis of T cells in acute lymphoblastic leukemia . In hepatocellular carcinoma cells, miR-325-3p can inhibit cell proliferation and induce apoptosis in hepatitis B virus-related hepatocellular carcinoma . The performance of miR-325-3p in regulating glioma cells migration and invasion implied an essential role for this miRNA in the mediation of glioma oncogenesis and tumor behavior, which is similar to previous studies mentioned above.
We further explored the potential mechanism by which miR-325-3p inhibited the invasion and migration in glioma and found FOXM1 as a potential target for miR-325-3p according to microRNA target databases. We found that FOXM1 was upregulated in glioma tissues and glioma cell lines compared to the control tissues and cells. Furthermore, statistical analysis showed a clear negative correlation between miR-325-3p and FOXM1. Our western blot data showed that miR-325-3p could reduce FOXM1 protein expression in vitro, and our dual-luciferase reporter assay confirmed an interaction between miR-325-3p and FOXM1 mRNA by directly targeting its 3’-UTR. Furthermore, in our rescue experiments, overexpression of FOXM1 countered the miR-325-3p-induced inhibitory effect on invasion and migration and metastasis-associated EMT protein expression, further supporting an association between miR-325-3p and FOXM1. Functionally FOXM1 as a typical proliferation-associated transcription factor is required to execute the mitotic program and chromosome stability . It is also significantly elevated in most human tumors and promotes tumorigenesis in many tissues, including pancreatic, esophageal, gastric, ovarian, and breast cancers [17,18,22,23]. Tissue microarray analysis revealed that FOXM1 expression was significantly higher in high-grade glioma than low-grade astrocytomas. The expression level of FOXM1 protein is directly related to the advanced grade, metastasis, and is negatively correlated with patient survival [24-26].
Several studies have also demonstrated that overexpression of FOXM1 promotes tumorigenicity, invasion, and angiogenesis of glioma cells [27-29]. However, in FOXM1 transgenic mice, no spontaneous brain tumors were observed , suggesting that FOXM1 overexpression alone does not induce gliomas or the presence of upstream regulatory mechanisms. Thus, it was important to determine the upstream miRNAs involved in the direct regulation of FOXM1, as these could represent potential biomarkers or therapeutical targets. We first confirmed the downregulation of miR-325-3p in glioma tissue and glioma cell lines. Therefore, upregulation of miR-325-3p could potently inhibit glioma cell proliferation and metastasis both in vitro and in vivo. Mechanistically, we found that the miR-325-3p/FOXM1 signaling pathway revealed a novel molecular mechanism for glioma progression suggesting that miR-325-3p could be a potential therapeutic target for malignant glioma. Finally, in our in vivo xenografted study, we confirmed again that miR-325-3p negatively inhibited tumor growth in mice.
MiR-325-3p was demonstrated to function as a tumor suppressor in glioma, at least in part, by targeting FOXM1. These findings may further elucidate the molecular mechanisms underlying glioma progression and provide a novel target and a better theoretical basis for new potential mechanisms involving the pathogenesis and molecular therapeutic strategy for glioma.
miRNA, MicroRNA; CRC, colorectal cancer; GC, gastric cancer; EMT, epithelialmesenchymal transition.
HS conceived and designed the present study. HS and QJX performed the experiments and analyzed the data. HS and QJX interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.
Animals were raised and handled at the QJX at the Laboratory Animal Center of the Chongqing Medical University. All animal experiments were carried out in accordance with the guidelines of the current institutional guidelines for the care and use of experimental animals and approved by the Animal Care Committee of Yongchuan Hospital of Chongqing Medical University. The glioma tissues were obtained with all participants’ informed consent, and the research conducted was approved by the Ethics Committee of Yongchuan Hospital of Chongqing Medical University (2020108).
We thank two anonymous reviewers for their excellent criticism of the article.
This work was supported by funding from Yongchuan Hospital Affiliated to Chongqing Medical University (YJZQN201525).
The authors declare no conflict of interest.