Suppression of miR-221 inhibits glioma cells proliferation and invasion via targeting SEMA3B
© Cai et al. 2015
Received: 16 April 2015
Accepted: 13 July 2015
Published: 22 July 2015
Gliomas are the most common primary tumors in the central nervous system. Due to complicated signaling pathways involved in glioma progression, effective targets for treatment and biomarkers for prognosis prediction are still scant.
In this study we revealed that a new microRNA (miR), the miR-221, was highly expressed in the glioma cells, and suppression of miR-221 resulted in decreased cellular proliferation, migration, and invasion in glioma cells. Mechanistic experiments validated that miR-221 participates in regulating glioma cells proliferation and invasion via suppression of a direct target gene, the Semaphorin 3B (SEMA3B). The rescue experiment with miR-221 and SEMA3B both knockdown results in significant reversion of miR-221 induced phenotypes.
Taken together, our findings highlight an unappreciated role for miR-221 and SEMA3B in glioma.
Glioma is one of the major human primary brain tumors , which is characterized by tumor cell local invasion and migration from remote distances . Though significant progress has been made in surgical and chemotherapy treatment, the prognosis of patients with glioma remains poor . Therefore, new therapeutic strategies for the management of glioma is therefore essential.
MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 22 nucleotides in length, which regulate gene expression post-transcriptionally. MiRNAs recognize and repress target mRNAs based on sequence complementarity between the miRNA and the target mRNA, and are critical in regulating a variety of biological processes, including cell cycle, differentiation, development, and metabolism, as well as diseases such as diabetes, neurodegenerative disorders, and cancers . Emerging literatures have found that aberrant miRNAs could act as oncogenes or tumor suppressors in glioma tumorigenesis . Dysregulation of these miRNAs contributes to tumorigenesis by stimulating glioma cells proliferation, angiogenesis and invasion [6, 7]. Among these miRNAs the miR-221/222 are attractive to researchers because of their bimodal function in the tumorigenesis of human cancers . They are encoded in tandem from a gene cluster located on chromosome X, and both tumor-suppressive and oncogenic roles of miR-221/222 have been reported in a series of cancer types . A couple of up-to-date studies have revealed collaborations between the expression of miR-221/222 and glioma carcinogenesis, since higher miR-221/222 expression correlated with poorer prognostic status of patients, which might be attributed to their attenuated sensitivity to chemotherapy and radiotherapy ; in addition Zhang et al.  reported plasma miR-221/222 family levels were significantly upregulated in glioma patients, and high positive plasma miR-221 and miR-222 were both correlated with poor survival rate. These studies suggested that detection of the miRNA-221/222 family should be considered as a new additional tool to better characterize glioma diagnostic and predictive value of miR-221/222 family for glioma. However, functions of miR-221/222 in glioma tumorigenesis and development are poorly understood.
In this study, we aimed at miR-221 as a candidate miRNA according to the RT-qPCR screening and validated its expression in glioma tissues and cell lines. Further researches to assess the effects of miR-221 on glioma cells proliferation and invasion were carried on, as well as the regulating mechanism on SEMA3B gene was explored.
miR-221 expression was elevated in glioma cells
Suppression of miR-221 inhibits cell growth in glioma cells
Suppression of miR-221 inhibits glioma cells migration and invasion
SEMA3B is the target for miR-221
Knockdown endogenous SEMA3B reversed the effect of miR-221 Inhibitor
Glioma is the most general primary tumor in nervous system with the highest mortality and mortality among endocranial tumors for its forceful malignant proliferation and invasion [13, 14]. An early diagnosis of glioma in patients greatly increases the chances for successful treatment, therefore a specific and sensitive marker is very important. Previous studies for identifying glioma biomarkers of glioma had mainly focused on proteins , however, miRNAs have absorbed more and more of attention from researchers for the advantage of easy and low cost of determination as new molecular markers . The aim of this study is to evaluate the function of miR-221 in human glioma and the potential as diagnostic and treatment marker. In the current study, we have found that miR-221 level in glioma is significantly higher than that in non-glioma tissues or cells; that miR-221 participates in glioma cell proliferation, migration and invasion; that miR-221 plays its biological roles via negatively regulating the SEMA3B gene in glioma. All the results suggested that miR-221 could be used as potential biomarkers in glioma identification, early diagnosis, and developing new therapeutic strategies.
Semaphorins families are a large number of cytoplasmic and membrane-bound proteins, including SEMA1–A4, B1–B3, and C1 in vertebrate . The SEMA3B gene is widely expressed in the mammary gland, colon, lung, kidney, and so on . The SEMA3B protein is an antagonist of receptors for neuropilins 1 and 2 (Np1 and Np2) that also act as receptors for several isoforms of vascular endothelial growth factor (VEGF), which is a general initiator of tumor angiogenesis, and thus SEMA3B suppresses vascular growth in tumors . Suppression in transcriptional activity of this gene in tumors indicate that SEMA3B may participate in cell growth suppression in the kidney, ovary, and colon cancers . In our study, the expression level of SEMA3B in glioma cells was found lower than normal glia cells, which is the first observation of SEMA3B in glioma cells to our knowledge and further result suggest that SEMA3B participates in the regulation of glioma cells proliferation and invasion. Though SEMA3B was verified for the target of miR-221 by us for the first time, there were still some other targets of miR-221 existence, which needed us to explore in future.
Mounting evidence implicates miRNAs as regulators of the tumor phenotype through their ability to modulate the expression of critical genes and signaling networks involved in tumorigenesis and downstream malignant processes . miR-221/222 act as oncogenic miRNAs that facilitate cell proliferation via down-regulation of p27Kip1 and/or p57Kip2, which negatively regulate cell cycle progression from G1- to S-phase [22–24]. Although various studies present the mechanism by which miR-221 functions in cancer cells, the oncogene or tumor suppression capability of miR-221 in glioma was poorly understood since there were only few papers before this study. Zhang et al.  demonstrated that high levels of miR-221/222 expression in gliomas confer highly aggressive invasion and poorer overall survival through by targeting TIMP3 and act as prognostic factors for glioma patients; Cristina et al. reported that overexpression of miR-221/222 produces an increase in sensitivity to temozolomide via a reduction in the level of MGMT. In addition, these miRNAs increase DNA damage, conferring oncogenic features to glioma cells . In this study, we found miR-221, but not miR-222 level is significantly high in the glioma tissues and cells, and manipulating of expression of miR-221 could result in glioma cell proliferation, migration and invasion ability change, indicating that miR-221 is a critical oncogene in glioma. However, exploring the detailed function of miR-221/222 and searching more targets of miR-221/222 in glioma cells are the problems to be solved.
In summary, we described that miR-221 expression level is associated with glioma tumorigenesis. The loss of function of miR-221 in glioma cell line suggests that knockdown of miR-221 inhibits cell proliferation and invasion. Mechanistically, SEMA3B is the direct target of miR-221, which acts as the tumor suppressor in glioma. These results suggest that miR-221 plays an important role in glioma tumorigenesis and could be served as a potential molecular marker for the diagnostic and treatment of glioma.
There were totally 102 patients involved at the initiation of this study in the Department of neurology, Beijing Friendship Hospital, Capital Medical University, and only 30 cases with surgical resection of brain glioma were collected according to the strict inclusive criteria: (1) no other types of tumor, viral hepatitis, or autoimmune diseases; (2) no preoperative chemotherapy or radiation therapy. The paracancerous tissue was defined as 2 cm away from lesions. All specimens were obtained under sterile conditions during surgery. All patients were given informed consent, and this study was approved by the Ethic Committee Board of Capital Medical University.
Cell preparation and culture
NHA cells were purchased from Lonza Company (CA, USA), U87MG, U251, A172, T98G and LN-18 glioma cell lines were purchased from ATCC Company (VA, USA). All the cells were maintained in DMEM medium with high glucose and sodium pyruvate, supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air.
Transfection of shRNA
The shRNA directed against Sema3b and the control shRNA were both chemically synthesized, the sequence of shRNA used for Sema3b were Sense: 5′-GGCCGACUGUACUAAAGUAUU-3′, Antisense: 5′-UACUUUAGUACAGUCGGCCUU-3′; the sequence of control shRNA were Sense: 5′-GAUAGACAAAUGACGAAUGCGUAUU-3′, Antisense: 5′-TCGCTTCGTCTTTGTCTTCUU-3′. When the cells reached 70% confluent, Oligofectamine reagent from Invitrogen (Life Technology, NY, USA) was employed to perform the in vitro transfection according to the manufacturer’s instructions, and cells were harvested 48 h after transfection for further experiments.
Cells were seeded in 96-well plates at confluence of 2,000 cells per well. The absorptions of the cells were measured using CCK-8 kit (Dojindo Laboratories, Japan) based on the manufacturer’s instruction at indicated time points.
Transwell membranes were coated with matrigel for 6 h for invasion assay, or without matrigel for migration assay. 5 × 104 cells were seeded onto the upper chamber, and 800 μl medium with 10% fetal bovine serum was added to the lower chamber. After incubation for 24 h, cells adhering to the upper surface of the membrane were removed with a cotton swab. The invasion or migration cells, which adhered to the lower surface, were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The cells were finally extracted with 33% acetic acid and detected quantitatively using microplate reader (at 570 nm). Data were obtained from three independent experiments.
Quantitative real-time PCR (qPCR)
RNAs were isolated using an RNAvzol reagent (Vigorous Biotechnology, Beijing, China) according to the manufacturer’s protocol. cDNA was synthesized with M-MLV reverse transcriptase (Promega, WI, USA) in a 20 μl reaction mixture (2 µg total RNA, 2 µl 10 × RT buffer, 1 µl M-MLV enzyme, 1 µl dNTP, 1 µl miR primer, 1 µl random primer, and ddH2O to 20 µl) at 37°C for 2 h. RT-qPCR was performed in the ABI 7500 real-time RT-PCR system with reagents from the SYBR® Green Real-time PCR Master Mix (TOYOBO, Japan) and the appropriate primers in a 20 µl reaction system (10 µl Master mixture, 1 µl cDNA, 1 µl forward primer and 1 µl reverse primer, and 7 µl ddH2O). A 10 min “hot start” was carried on firstly, and then 40 cycles were run with each cycle including a denaturation step at 95°C for 10 s, annealing starting at 60°C for 20 s, and amplification at 72°C for 30 s. For quantification of the target gene, the ΔΔCt algorithm was used. The primers for miR-221, miR-222 and U6 were all purchased from RiboBio Company. All experiments were performed at least three times and the mean values were used.
Western blot analysis
Cells were washed twice in ice-cold PBS and lysed in 1 × lysis buffer (Cell Signal Technology, CA, USA), the protein concentration of each sample was detected using BCA assay before loading, and then whole cell extracts (50 μg protein) was analyzed by 12% SDS PAGE and subsequently transferred onto nitrocellulose membranes. For immunoblot experiments, membranes were blocked for 1 h with 5% skimmed milk powder in TBS containing 0.1% Tween-20, and incubated with primary antibody at 4°C overnight. The rabbit polyclonal anti-SEMA3B antibody (code: sc-21204-R, 1:500 dilution) and the goat polyclonal anti-GAPDH antibody (code: sc-48167, 1:2,000 dilution) were both purchased from Santa Cruz Biotechnologies (CA, USA). The secondary antibodies were all purchased from Zhongshanjinqiao Company (Beijing, China), bound antibodies were visualized with an enhanced chemiluminescence system (DuPont, Boston, MA). The densitometric analysis was been carried on using the ImageJ software (Fiji Software), and the percentage of inhibition was normalized as SEMA3B/GAPDH.
Luciferase reporter assay
The luciferase reporter containing the 3′ UTR of the human SEMA3B gene was purchased from RiboBio Company (Guangzhou, China). HeLa cells were co-transfected with miR-221 mimics (100 pM) with luciferase reporter for 48 h, and Renilla luciferase expression construct. Cells were harvested 36 h post-transfection and assayed with Dual Luciferase Assay (Promega, WI, USA) according to the manufacturer’s instructions. Luciferase activity was measured and normalized to Renilla luciferase activity. Three independent experiments were performed in triplicate.
The data are expressed as mean ± standard deviation (SD). Comparisons between groups were analyzed using Student’s t test or ANOVA. Differences were considered to be statistically significant at p < 0.05.
CGL carried out the molecular studies, and drafted the manuscript. QSS participated in the design of the study and performed the statistical analysis. CK conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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