Background
Glioma poses a tremendous threat to public health with an incidence of ~5 cases per 100,000 persons and its mortality is high around the world. Glioma is categorized into four histopathologic grades (I–IV) based on the degree of malignancy according to World Health Organization (WHO) classification [
1]. The majority patients suffered from glioma are diagnosed at the advanced stages and exhibit extremely poor prognosis. Combined with maximal safe surgical resection, standard radiation therapy and chemotherapy, the most advanced treatment is formed, while the most malignant glioma called gliobastoma multiform is lined with an average life expectancy of only 14 months [
2]. Fortunately, significant progress has been made in understanding the molecular mechanisms of glioma. Though clinical application is largely palliative, the fatality rate remains high in glioma patients. Therefore, it is extremely vital for us to elucidate new mechanisms associated with glioma development and establish potential therapeutic targets for the treatment of human glioma.
Just as the human genome project which delineated that only a small amount of mammalian genome is encoded proteins while the vast majority of mammalian genome are transcribed as non-coding RNAs (ncRNAs), such as long noncoding RNAs(lncRNAs) [
3,
4]. LncRNAs are a class of transcripts longer than 200 nucleotides in length with little functional protein-coding ability. Recently, many studies have demonstrated that lncRNAs could regulate gene expression at multiple levels, including transcriptional, post-transcriptional and epigenetic modulation [
5‐
7]. Numerous studies also indicated that dysregulated lncRNAs are involved in the biological process during cancer development and progression [
8,
9] Examples like lncRNAs include HOTAIR (HOX transcript antisense intergenic RNA) [
10], CRNDE(Colorectal neoplasia differentially expressed) [
11], MEG3 (Maternally Expressed Gene 3) [
12]. Yet, the biological roles of lncRNAs in glioma are largely unknown. Long noncoding RNA activated by TGF-β (lncRNA-ATB) was initially identified as a lncRNA in Hepatocellular carcinoma (HCC) and its expression was significantly up-regulated both in HCC cells and HCC samples [
13]. Furthermore, ATB was also overexpressed in other cancers, including breast cancer [
14] and colorectal cancer [
15]. Therefore, the aberrant expression level of ATB was involved in a series of cancer progression. However, functional mechanism and potential biological role of ATB in human glioma are still unknown.
MiRNAs are highly conserved among species, and play important roles in a variety of biological and pathological processes. A number of studies indicated that deviant expression of miRNAs contributes to tumorigenesis and plays a critical role in regulating the biological behaviors of tumor cells by modulating the protein or mRNA levels of its downstream target genes [
16,
17]. Dysregulation of miRNAs in glioma has also been reported, and certain miRNAs have been functionally involved in glioma. Previous studies have demonstrated that miR-200a as a member of the miR-200 family, which exerts as a tumor-suppressor gene and is down-regulated in many tumors, including glioma [
18]. However, the molecular mechanism of miR-200a deregulation and how such deregulation contributes to glioma tumorigenesis remains abstruse.
In the present study, we aimed to investigate the functional expression and clinical significance of ATB in human glioma. ATB was significantly up-regulated both in glioma tissues and cell lines, whereas knockdown of ATB diminished cell proliferation, migration and invasion in glioma. In addition, the interaction among ATB, miR-200a and TGF-β2 was also studied in order to reveal the underlying mechanisms. We identified that ATB may act as a ceRNA of miR-200a, which resulted in the derepression of TGF-β2. These findings will give a novel strategy for the treatment of glioma.
Methods
Human tissue samples
Seventy-nine glioma tissues and 15 normal brain tissues (NBTs) were obtained from the Department of Neurosurgery, the Second Affiliated Hospital of AnHui Medical University during 2011–2014. These glioma samples were from 50 males and 29 females with age ranging from 13 to 73 years (median, 47 years). All samples had confirmed pathological diagnosis and were divided into low grade (grade I–II) and high grade (grade III–IV) according to the WHO classification by neuropathologists. Informed consents were obtained from all patients, and this study was approved by the Clinical Research Ethics Committee at the Second Affiliated Hospital of AnHui Medical University.
Cell culture
The human glioma cell lines (U251 and A172) were purchased from the Chinese Academy of Sciences (Shanghai, China) and cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) with high glucose supplemented with 10 % fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and streptomycin (100 μg/ml), penicillin (100 U/ml). All cell lines were cultured at 37 °C in a humidified incubator with 5 % CO2.
Cell transfection
Short-hairpin RNA plasmid directed knock-down human ATBs (GenePharma, Shanghai, China), and was indicated as sh-ATB (sh-ATB sense:5'-GATCCGCCTGTCTGTATTTGCGAATACCTTTTTCAAGAGAAAAGGTATTCGCAAATACAGACAGGCTTTTTTG-3' and anti-sense: 5'-AATTCAAAAAAGCCTGTCTGTATTTGCGAATACCTTTTCTCTTGAAAAAGGTATTCGCAAATACAGACAGGCG-3') and its corresponding non-targeting sequence (sh-control) (sh-control sense: 5'-GATCCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAACTTTTTTG-3' and anti-sense: 5'-AATTCAAAAAAGTTCTCCGAACGTGTCACGTTCTCTTGA AACGTGACACGTTCGGAGAACG-3') plasmid (GenePharma, Shanghai, China) were transfected into U251 and A172 cells respectively by using Lipofectamine2000 (Invitrogen, USA) according to the manufacturer’s protocol. MiR-200a mimics, miR-200a inhibitors and miR-200a negative control (NC) were obtained from RiboBio (RiboBio, Guangzhou, China), and transfected into cell lines as the above described.
RNA extraction and quantitative Real-time PCR
Total RNAs were extracted from tissues and cultured cells using Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol. Using a Nanodrop Spectrophotometer (IMPLEN GmbH, Munich, Germany), RNA concentration and quality were determined by the 260/280 nm absorbance. Then RNA was reversely transcribed into High Capacity cDNA by using PrimeScript™ RT Master Mix (Perfect Real Time) (TaKaRa Biotechnology, Dalian, China). Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, USA) was used for quantitative Real-time PCR. The primers for genes were determined as follows: ATB forward 5′-ACAAGCTGTGCAGTCTCAGG-3′, reverse 5′- CTAGGCCCAAAGACAATGGA-3′; TGF-β2 forward 5′-CACCATAAAGACAGGAACCTG-3′, reverse 5′-GGAGGTGCCATCAATACCTGC-3′; GAPDH forward 5′-AGCAAGAGCACAAGAGGAAG-3′, reverse 5′-GGTTGAGCACAGGGTACTTT-3′. All-in-One™ miRNA First-Strand cDNA Synthesis Kit (Genecopoeia, Guangzhou, China) was used for miRNA reverse transcription and RT-QPCR was conducted using All-in-One™ miRNA qPCR Kit (Genecopoeia, Guangzhou, China) of miR-200a and U6 (miR-200a (Cat#HmiRQP0298) and U6 (Cat#HmiRQP9001), Genecopoeia, Guangzhou, China), respectively, by using the ABI 7100 (Applied Biosystems, Darmstadt, Germany). GAPDH and U6 were used as internal controls for ATB, TGF-β2 and miR-200a. PCR Cycling conditions for mRNA was 2 min at 50 °C, 10 min at 95 °C and followed by 45 cycles of 95 °C for 15 s, 60 °C for 60s; for miRNA was 10 min at 95 °C and followed by 45 cycles of 95 °C for 10s, 60 °C for 20s and 72 °C for 12 s. All qRT-PCR reactions were performed in triplicate. Relative quantification of gene expression was calculated by the 2−ΔΔCt method.
Cell proliferation assay
Cell Counting Kit-8 (CCK8, Beyotime Institute of Biotechnology, Jiangsu, China) was used to detect the cell,s proliferation ability. Cells were placed into 96-well plates at the density of 2000 cells/well. Approximately 20 μl of CCK8 regent was added to each well after transfection, and incubated at 37 °C for 2 h. Absorbance was measured at a wavelength of 450 nm by using a ST-360 micro-plate reader (KHB, Shanghai, China). Three replicate wells were set up in each group and experiments were repeated three times.
For the clone formation assay, approximately 200 viable cells were seeded in 6-well plates in triplicate and incubated at 37 °C with 10 % fetal bovine serum. After 12 days, cells were fixed with 4 % polyoxymethylene and stained with 1.5 % methylene blue. Colony formation ratio was calculated as (number of cells/initiative cell × 100 (%)).
Wound healing assay
Cells were cultured in 6-well plates. When 95 % confluency was reached, cell layers were wounded using a 10 μL tip to produce a gap, gently washed, and cultured with serum-free medium for 24 h. The wounded gaps were photographed with a light microscope (IX71; Olympus, Tokyo, Japan) at × 200 magnification. Lines were drawn along the leading edges of the cells, and the gap distances of migrating cells from five different areas for each wound were measured and analyzed.
Cell invasion assays
Cell invasion ability was tested by using 24-well chambers with 8 μm pore size (Corning, USA). 8 × 104 cells were resuspended in 150 μl serum-free medium and seeded into the upper chamber pre-coated with 500 ng/ml Matrigel solution (BD, USA) invasion assay, while 500 μl of 10 % FBS medium was placed in the lower chamber, After incubation at 37 °C for 48 h for invasion assay. Cells on the upper chamber membrane were scraped off by cotton swab. Cells on the lower chamber membrane were fixed with 4 % polyoxymethylene and stained with 0.1 % crystal violet. Five predetermined fields were counted under a microscope (×200). All assays were performed in triplicate.
Luciferase reporter assays
ATB fragment containing the predicted miR-200a binding site, the putative sequences of the binding site then cloned into a pmirGlO Dual-luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) to form the reporter vector pmiRGLO-ATB-wild-type (ATB-WT). To mutate the putative binding site of miR-200a in ATB gene, the sequence of putative binding site was replaced as indicated and was named as pmiRGLO-ATB-mutated-type (ATB-MUT). pmirGLO-ATB-WT or pmirGLO-ATB-MUT was cotransfected with miR-200a mimics or miR-200a NC into glioma cells by using Lipofectamie 2000 (Invitrogen, USA). After 48 h transfection for luciferase assay using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol.
Similarly the 3’-UTR of TGF-β2 containing the putative miR-200a binding site, the putative sequences of the binding site were cloned into a pmirGlo Dual-luciferase miRNA Target Expression Vector to form the reporter vector TGF-β2-wild-type (TGF-β2-WT) (GenePharma). To mutate the putative binding site of miR-200a in the 3′-UTR-containing vector, the sequence of putative binding site was replaced as indicated and was named as TGF-β2-mutated-type (TGF-β2-MUT). The transfection procedure and measurement of Luciferase activities were handled similarly as described above. All assays were independently performed in triplicate.
RNA immunoprecipitation
EZ-Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) was used in RNA immunoprecipitation (RIP). RIP was implemented to pull down endogenous miR-200a associated with ATB in glioma cell lines, and was performed following the manufacturer's protocol. U251 and A172 cells were lysed by RIP lysis buffer, 100 μL of cell lysate was incubated with RIP immunoprecipitation buffer containing magnetic beads conjugated with human anti-Argonaute2 (Ago2) antibody (Millipore) and normal mouse IgG (Millipore) was indicated as negative control. Samples were incubated with Proteinase K buffer and then target RNA was extracted. Purified RNA was subjected to RT-QPCR analysis for further study.
Western blot analysis
Total proteins were extracted from the cells using RIPA buffer with PMSF (Beyotime Institute of Biotechnology) on ice, subjected to SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were incubated in 5 % nonfat milk dissolved in Tris-buffered saline (TBS) containing 0.1 % Tween-20 for 1.5 h at room temperature and then incubated with primary antibodies as follows: TGF-β2 (1:1000, Abcam, EUGENE, USA), β-actin (1:1000, Santa Cruz Biotechnology). After incubation with secondary antibodies (Goat anti-rabbit or Goat anti-mouse, 1:5000 respectively; ZSGB-BIO, Beijing, China), immune complexes were visualized by SuperSignal® West Femto Trial Kit (Thermo Fisher, USA) and blot bands were scanned using Find-do × 6 Tanon (Tanon, Shanghai, China).
Four-week-old female BALB/C athymic nude mice were purchased from the National Laboratory Animal Center (Beijing, China). Experiments with nude mice were conducted strictly in accordance with a protocol approved by the Administrative Panel on Laboratory Animal Care of the Second Affiliated Hospital of AnHui Medical University. The animals were free to autoclaved food and water during the study. U251 cells transfected with sh-control or sh-ATB were collected, and 3 × 106 cells were subcutaneously injected into the left flank of the nude mice. Tumor volumes were examined every 5 days, and after 40 days, the mice were killed and tumor tissues were excised and weighed for further study.
Immunohistochemistry
Tumor tissues from subcutaneous implantation assay were fixed in 4 % paraformaldehyde, and then were dehydrated, embedded in paraffin, and cut. Consecutive 4 μm thick sections were analyzed by immunohistochemistry using antibodies against Ki-67 (Santa Cruz Biotechnology, USA).
Statistical analysis
Experimental data were presented as means ± standard deviation (SD). GraphPad Prism V5.0 (GraphPad Software, Inc., La Jolla, CA, USA) software was used for statistical analysis. Differences were analyzed by SPSS 17.0 statistical software with the Student’s t-test or one-way ANOVA. The relationship between the expression of ATB, miR-200a and TGF-β2 in tissues was analyzed with Pearson’s correlation. Survival analysis was performed using the log-rank test in GraphPad Prism 5. Differences were considered significant if P < 0.05. Corresponding significance levels are indicated in the figures.
Discussion
Nowadays, operation and chemoradiotherapy termed as comprehensive treatments are the main means for the treatment of glioma patients, although it can alleviate patients’
,suffering and prolong life-span, the prognosis is still poor [
20,
21]. Therefore, it is urgent for us to find new effective therapies for glioma patients. Recent studies have suggested that dysregulation of lncRNAs are involved in the tumorigenesis and progression of multiple cancers, including glioma [
22,
23]. Consequently, understanding the underlying mechanisms and biological functions of lncRNAs in glioma may give us a new direction for the treatment of glioma patients.
A number of studies have demonstrated that lncRNAs play considerable functional roles in human malignant tumor [
24‐
27]. Long non-coding RNA ATB was overexpressed in hepatocellular carcinoma, which promotes HCC cells invasion and tumor growth [
13]. Furthermore, ATB is also found as a novel biomarker of lncRNA, indicative of a poor prognosis in gastric cancer and colon cancer patients [
15,
28]. In the present study, our results confirmed that ATB was highly expressed in glioma, and high ATB expression was associated with a poor clinical outcome in glioma patients. Knockdown of ATB suppressed the proliferation, migration and invasion of glioma cells. In addition, the in vivo experiments also supported the above findings, suggesting that ATB exerted as an oncogene in human glioma. Moreover, overexpression of ATB in breast cancer cells promotes the invasiveness, which is consistent with the results of our study [
14].
MiRNAs may function as oncogenes and/or tumor suppressors in many cellular processes during cancer initiation and progression [
29]. Accumulating evidences have showed that lncRNAs can antagonize miRNA function by competing with miRNAs for binding to shared target mRNAs, and then to silence target mRNAs [
30]. Long noncoding RNA ANRIL promotes tumor growth by epigenetically repressing of miR-99a/miR-449a in human gastric cancer [
31]. Gas5 suppresses the malignant biological characteristics by down-regulating miR-222 in human glioma [
32]. Growing number of present work confirmed the common existence of a widespread interaction network of competitive endogenous RNAs (ceRNAs), in which lncRNAs may exert functions through targeting miRNAs and regulating their function role [
19,
33]. ATB could promote the invasion-metastasis cascade in HCC by negative regulating of miR-200 family [
13]. Previous study showed that down-regulation of miR-200a promoted glioma malignancy by up-regulating SIM2-s [
18]. Our present study confirmed that miR-200a was significantly decreased in glioma tissues and inversely correlated with ATB. To explore whether ATB has miR-200a binding site in glioma cells, and was confirmed by luciferase reporter assays and RIP. We verified that ATB is directly bound to miR-200a on ATB transcript. RNA-IP assay showed that the expression of ATB immunoprecipitated with in the miR-200a overexpression group was significantly increased. Meanwhile, we found that ATB exerted the function of anti-tumor by miR-200a in glioma cells. Taken all together, these data strongly suggested that ATB directly targets miR-200a and affects the biological characteristic of glioma cells by negatively regulating miR-200a.
The transforming growth factor-β2 (TGF-β2) belongs to TGF-β family, functioned as an oncogene in several cancer types, which promote cancer cells
, malignant behaviors [
34]. Recent study has reported that miR-200a suppresses renal cell carcinoma development by directly targeting TGF-β2 [
35] and miR-200a prevents renal fibrogenesis by suppressing of TGF-β2 [
36]. Consistent with previous studies, we confirmed that miR-200a regulated the expression of TGF-β2 via targeting the 3′UTR of TGF-β2.
Emerging evidences indicated that lncRNAs play a critical role in a variety of cellar biological processes by acting as a ceRNA or a molecular sponge in modulating the role and functions of miRNAs [
30,
37]. These lncRNAs act as a natural miRNA sponge to control endogenous miRNAs by using shared miRNAs responsive elements (MREs) and then modulating the derepression of these miRNAs targets via post-transcriptional regulation [
38]. For instance, HOTAIR, a well-known lncRNA could inhibit the expression of FGF1 by regulating miR-326 in human glioma [
39], and also functioned as a competing endogenous RNA to regulate HER2 expression by sponging miR-331-3p in promoting gastric cancer [
40]. To further explore whether TGF-β2 is involved in the ATB acts as a ceRNA in regulating the biological characteristics of glioma by modulating miR-200a. We demonstrated that miR-200a reversed the reduction of TGF-β2 mediated by ATB knockdown. In addition, we found that TGF-β2 expression was negatively correlated with miR-200a, but positively associated with ATB in glioma tissues. These results suggested that ATB functions as a ceRNA via decreasing miR-200a, up-regulating TGF-β2 in human glioma.
Abbreviations
ceRNAs, competing endogenous RNAs; CRNDE, Colorectal neoplasia differentially expressed; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HCC, Hepatocellular carcinoma; HOTAIR, HOX transcript antisense intergenic RNA; LncRNA-ATB, long noncoding RNA activated by TGF-β; lncRNAs, long non-coding RNAs; MEG3, Maternally Expressed Gene 3; miRNAs, microRNAs; MREs, miRNAs responsive elements; NBTs, normal brain tissues; ncRNAs, non-coding RNAs; RIP, RNA immunoprecipitation; TGF-β2, transforming growth factor-β 2