Correspondence to: Xiangdong Li, MD, Department of Neurosurgery, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou 215006, People's Republic of China. E-mail: email@example.com and Jin Hu, MD, Department of Neurosurgery, Shanghai Huashan Hospital, Fudan University, 12 Middle Wulumuqi Road, Shanghai 200040, People's Republic of China. E-mail: firstname.lastname@example.org
Glioma is the most common primary intracranial malignant tumor and accounts for ∼40% of all primary malignant central nervous system tumors. Glioma is a morphologically diverse neoplasm with poor prognosis, median survival of about 12 months, despite of advances in combined treatment of surgical resection, radiotherapy, and chemotherapy (Miller and Perry, 2007). Given that glioma is usually located in the depths of the central nervous system, surgical removal or radiation of tumor tissues is very dangerous for the patients with glioma. Therefore, novel biological therapies that produce more efficacy by inhibiting tumor cell growth are urgent.
The discovery of MicroRNAs (miRNAs) has improved our understanding of the mechanisms underlying gene expression regulation. These small noncoding RNA molecules are recognized to participate in the regulation of gene expression during tumorigenesis, which may induce cleavage of their target mRNAs or repress translation (Gregory and Shiekhattar, 2005). There are accumulating data documenting many abnormal miRNA expression patterns in various human malignancies, some of which act as oncogenes and tumor suppressors. As exemplified in lung cancer, miR-let-7 was reported to be a tumor suppressor and loss of miR-let-7 expression was associated with poor prognosis in lung cancer patients (Takamizawa et al., 2004), and miR-206 was correlated with invasion of lung cancer (Wang et al., 2011). Besides, miR-21 was shown to be overexpressed in pancreatic cancer and used as a potential predictor of survival for patients with pancreatic cancer (Dillhoff et al., 2008).
Increased expressions of miR-21 and miR-221 have been reported in human glioma cells, which are suggested to act as oncogenes (Chan et al., 2005; Medina et al., 2008). However, miR-181 family numbers are downregulated and function as tumor suppressors in human glioma cells (Liu et al., 2007; Shi et al., 2008). These findings suggest that different miRNAs may play various roles in tumorigenesis in glioma cells. More recently, Xia et al. (2009) found that miR-15b expression was lower in glioma cell line U87 cells and the U87 cells transfected with miR-15b mimics had a higher percentage of cells in G0/G1 and lower in S phase. However, the expression and function of miR-16-1 (belongs to a cluster together with miR-15) remain unclear in glioma cells. Therefore, in the present study, we first examined the expression of miR-16-1 in normal brain tissues and glioma cell lines, and then we explored the functions of miR-16-1 that involves cell proliferation, apoptosis, motility, and invasion in glioma cells. Finally, we performed real-time PCR (RT-PCR) analysis to detect mRNA expression levels of Zyxin, one of putative target genes of miR-16-1, in U251 glioma cells after transfecting with miR-16-1 mimics.
MATERIALS AND METHODS
Cell Lines and Tissue Samples
Human glioma cell lines (U87 and U251) were purchased from Chinese Academy of Sciences Cell Bank. Fresh frozen human normal brain tissues were obtained after informed consent from patients with severe traumatic brain injury who needed post trauma surgery in Department of Neurosurgery at the Sixth Affiliated Hospital of Shanghai Jiao Tong University. All the normal brain tissues were surgically resected from part of temporal lobe.
Cell Culture and Materials
The U251 and U87 cell lines were maintained in a 37°C, 5% CO2 incubator (Thermo) in Dulbecco's modified Eagle medium (DMEM) and supplemented with 10% fetal bovine serum (FBS) (Hyclone), respectively. Hsa-miR-16-1 mimics were chemically synthesized by Shanghai GenePharma Company (Shanghai, China). Sequences were as follows: hsa-miR-16-1: sense, 5′-UAGCAGCACGUAAAUAUUGGCG-3′; antisense, 5′-CCAAUAUUUACGUGCUGCUAUU-3′; and nonspecific control miRNA: sense, 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense, 5′-ACGUGACACGUUCGGAGAATT-3′.
miRNA mimics were transfected into glioma cells by INTERFERin™ (Shanghai DAKEWEI) reagent according to the manufacturer's instructions. The U251 cells were transfected with 50-nmol/L fluorescein isothiocyanate (FITC)-conjugated miR-16-1, and the transfection efficiency was analyzed by flow cytometry 24 hr later.
RNA Isolation and Real-Time RT-PCR
Total RNA from tissues or cultured cells was isolated using mirVana™ miRNA Isolation Kit (Ambion) for miR-16-1 analyses. Synthesis of cDNA with reverse transcriptase was performed by NCode™ miRNA Quantitative RT-PCR (qRT-PCR) Kits (Invitrogen). For analysis of miRNA expression, RT-PCR analysis was carried out using SYBR® Green Reagents (Biosystems) according to the manual. Relative expression was calculated using the ΔΔCT (cycle threshold) method. RT-PCR was performed on a 7500 RT-PCR System. Primer sequences are as follows: human 18S rRNA, 5′-GTGAACCTGCGGAAGGATCA-3′; miR-16-1, 5′-CGCCAATATTTACGTGCTGCT-3′. Detection of Zyxin mRNA levels was performed using qRT-PCR as previously described by Wu et al. (2011).
Detection of Apoptosis by Annexin-V and Propidium Iodide Staining
Cells were transfected with miR-16-1 or nonspecific control miRNA (N.C.) at 50% confluence. After 48 hr, the adherent cells were harvested by trypsinization and were washed with phosphate buffer saline (PBS) for once and resuspended in 500 μL of 1× binding buffer (Annexin-V/FITC kit; Sigma). Then, 10-μL Annexin-V/FITC and 5-μL propidium iodide were added into the binding buffer, and tubes were incubated at room temperature for exactly 10 min in dark. Thus, the fluorescence of the cells was immediately determined with a flow cytometer.
Cellular Proliferation Assay
Cell counts were measured using Cell Counting Kit-8 (DOJINDO Japan). Two thousand cells per well were seeded in a 96-well plate and incubated for 24 hr, and the cells were transfected with miR-16-1 or nonspecific control miRNA at the final concentration of 50 nm/L. Ten microliters of Cell Counting Kit-8 was added to 100-μL cell culture media and incubated for 2 hr in CO2 incubator at 0, 24, 48, 72, 96, and 120 hr after transfection. Absorbance was measured at 450 nm. Three independent experiments were performed.
Biocoat Matrigel invasion chambers and wells (BD Biosciences, Bedford, MA) were rehydrated with 500-μL serum-free medium at 37°C for 2 hr. Cells transfected with miR-16-1 or nonspecific control miRNA for about 20 hr were trypsinized, quenched with PBS plus 0.1% bovine serum albumin, and counted. After removing rehydration medium, we added 750-μL medium plus 10% FBS to each well of the 24-well plate and then added immediately 1 × 105 cells in 500-μL serum-free medium plus the miR-16-1 (50 nM) or N.C. (50 nM) to each chamber, respectively. Plates were incubated at 37°C for 24 hr. Then, the inserts were removed and the noninvading cells on the upper surface were removed with a cotton swab after 24 hr. The cells on the lower surface of the membrane were fixed in 100% methanol for 15 min, air-dried, and stained with gentian violet stain for 15 min. The cells were recorded with a digital camera.
Wound Healing and Cell Motility Assay
U251 cells were seeded onto six-well plates. After 24 hr, glioma cells were transfected with miR-16-1 (50 nM) or nonspecific control miRNA (N.C., 50 nM) and were allowed to grow to confluence. A linear wound was created by scraping the wells with a 200-μL pipette tip. The floating cells were removed by gentle washes with culture medium. The healing process was dynamically examined and was recorded with a digital camera at 24 hr after the wound was created.
Data shown in the graphs represent the mean values ± SD of three independent experiments. Statistical analyses were performed using the student's t test. P values of less than 0.05 were considered statistically significant. All statistical analyses were performed by SigmaPlot 10 statistical analysis software (Systat Software, Inc).
Expression of miR-16-1 was lower in Human Glioma Cells than Normal Brain Tissues
To determine whether miR-16-1 plays a role in glioma tumorigenesis, we conducted real-time quantitative PCR to quantify mature miR-16-1 in two glioma cell lines, U251 and U87, and three independent normal brain tissues. The results showed that the expression levels of miR-16-1 were significantly lower in human glioma cells than normal brain tissues (Fig. 1).
Cellular Proliferation was significantly Reduced with miR-16-1 Expression
To determine whether overexpression of miR-16-1 has effect on growth of glioma cells, we performed cellular proliferation assays in U251 cells and found that cell proliferation was dramatically decreased in the cells after transfection with miR-16-1 for 48 hr (P < 0.05; Fig. 2A). Then, we carried out cell apoptosis assay to examine whether the reduction of cell proliferation was due to increased apoptosis induced by overexpression of miR-16-1, and found that there was no significant difference in cell apoptosis between U251 cells overexpressing of miR-16-1 and those with N.C. (P > 0.05; Fig. 2B).
MiR-16-1 decreased the Invasion and Migration Abilities of Glioma Cells
Cell migration and invasion are two essential processes of cancer metastasis. Thus, we first examined the invasive ability of glioma cells transfected with miR-16-1 and found that cells transfected with miR-16-1 presented less invasion ability than those with nonspecific control miRNA (Fig. 3A,B). As shown in Fig. 4, there was clear difference in the migration ability between glioma cells transfected with miR-16-1 and those with nonspecific miRNA, revealing that glioma cells transfected with miR-16-1 may close the wound more slowly when compared with those with nonspecific miRNA.
MiR-16-1 downregulate Zyxin Expression by causing mRNA Instability
We performed qRT-PCR analysis to examine mRNA expression levels of Zyxin, one of putative target genes of miR-16-1, in U251 glioma cells after transfecting with hsa-miR-16-1 mimics, nonspecific control of miRNA mimics, and mock. We found that U251 cells transfected with miR-16-1 showed significantly lower endogenous mRNA levels of Zyxin than those transfected with N.C. or mock (P < 0.05; Fig. 5) at 24-hrpost transfection. This finding suggested that miR-16-1 might downregulate Zyxin expression by causing mRNA instability.
There are a lot of studies focusing on the relationship between cancer and deregulated miRNA expression. Several miRNAs have been identified as oncogenes and tumor suppressors that are involved in glioma development. For instance, miR-21, miR-221, and miR-9 have been shown to be expressed in glioma and to increase cell growth and invasion (Corsten et al., 2007; Gillies and Lorimer, 2007; Chao et al., 2008). In contrast, decreased expression of miR-181 family and miR-7 may inhibit glioma cell proliferation and invasion (Liu et al., 2007; Kefas et al., 2008; Shi et al., 2008). MiR-15a and miR-16-1 were two miRNAs in the same gene cluster at chromosome 13q14, which were firstly linked to cancer. MiR-15a and miR-16 were reported to be either absent or downregulated in the majority of patients with B-cell chronic lymphocytic leukemia (B-CLL) (Calin et al., 2002). However, the expression and function of miR-16 were poorly understood in glioma, although Ciafrè et al. (2005) have previously detected miR-16 expression in primary glioma by microarray. In this study, we used qRT-PCR to detect the expression of miR-16-1 and found that miR-16-1 expression level is significantly lower in glioma cells U251 and U87 compared with human normal brain tissues. This is in consistent with the previous study that identified miR-16 to act as a tumor suppressor gene in CLL (Calin et al., 2002) and suggest that miR-16 might be involved in human glioma tumorigenesis (Calin et al., 2004).
A family of miRNAs sharing seed region identity with miR-16 may negatively regulate cell cycle progression in human colorectal carcinoma cells (Linsley et al., 2007). In addition, miR-16 was reported to induce apoptosis by targeting BCL2 in CLL (Cimmino et al., 2005), whereas BCL2 may be only partially or not involved in mitomycin C (MMC)-induced apoptosis by upregulating miR-16 in human gastric carcinoma cells (Xia et al., 2008). These findings suggested that miR-16 could contribute to tumor cell proliferation and apoptosis. To investigate the function of miR-16 in glioma cells, we exogenously overexpressed miR-16-1 in high-invasive glioma U251 cells to determine its influence on glioma cell growth and apoptosis. Our results indicate that overexpression of miR-16-1 may cause attenuation of cell growth but unchanged apoptosis in U251 cells. Compared with the findings in gastric cancer (Xia et al., 2008), miR-16 did not induce apoptosis by downregulating BCL2 in glioma cells.
Although several miRNAs such as miR-21 and miR-181 have been shown to influence the invasion of glioma cells (Ma and Weinberg, 2008), whether miR-16 is associated with migration and invasion remains unaddressed in glioma cells. In this study, the transwell assays showed that overexpression of miR-16-1 remarkably diminished the invasion ability of U251 cells and the wound-healing assay demonstrated that glioma cells overexpressing miR-16-1 presented lower migration than control cells. These results demonstrate that miR-16-1 may play a role in invasion and migration of glioma cells.
Indeed, each miRNA is predicted to regulate hundreds of mRNAs and may have different functions depending on the cellular context. The expression and functions of miR-16 have been demonstrated to be diverse in various tumors. For pituitary adenoma, miR-16-1 expression is reduced and inversely correlated with tumor diameter and with arginyl-tRNA synthetase expression (Bottoni et al., 2005). In contrast, miR-16 expression is increased in cervical cancer (Wang et al., 2008). Furthermore, exploring the target genes of miR-16 may improve our understanding of the regulation function of miR-16 in cancer. More recently, Rivas et al. (2012) have performed bioinformatical approach to search target genes of miR-16 and confirmed that the CCNE1 gene (encoding cyclin E1) is a direct target of miR-16 in breast cancer development. MiR-16 also targets Zyxin and promotes cell motility in human laryngeal cancer cell line (Wu et al., 2011). Bonci et al. (2008) reported that miR-16-1 could targets CCND1 (encoding cyclin D1) and WNT3A, which promotes tumor invasion. In this study, we found that miR-16-1 might downregulate Zyxin expression by causing mRNA instability in high-invasive glioma cells U251, suggesting that Zyxin may be one of putative target genes of miR-16-1.
In summary, we demonstrated that miR-16-1 expression was markedly decreased in human glioma cell lines, and for the first time, we described the roles of miR-16-1 in cellular proliferation, migration, and invasion abilities in high-invasive glioma cells. In addition, this study suggested that Zyxin may be one of putative target genes of miR-16-1.