A growing body of evidence suggests that microRNA-365 (miR-365) played crucial role in the initiation and development of many types of cancers. However, the biological role of miR-365 in human glioma remains unclear. Herein, the aims of this study were to investigate the role and underlying mechanisms of miR-365 in glioma by a series of
Glioma is the most common tumor type in the central nervous system with high morbidity and mortality (
MicroRNAs (miRNAs) are small, approximately 22 nucleotides in length, non-coding RNAs that negatively regulate gene expression at a post-translational level by binding to complementary sequences in the 3′UTRs of targeted mRNAs (
miR-365, a newly discovered miRNA, has been reported to be involved in tumor progression and development in several types of human cancers, such as lung cancer (
This study was approved by the Medical Ethics Committee of Jilin University (Changchun, China). All participating patients provided written informed consent for the use of surgical samples before surgery. All animals were treated in accordance with standard guidelines for the care and use of laboratory animals of Jilin University.
Thirty-six pairs of glioma tissues and their adjacent normal brain tissues were collected from patients undergoing surgery at the first of Hospital of Jilin University from July 2012 to December 2014. All the tissues were snap-frozen in liquid nitrogen immediately after resection and stored at −80°C until use.
Primary normal human astrocytes (NHA) and four human glioma cell lines (U251, U87, U118 and LN18) were from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma, St. Louis, MO, USA) at 37°C with 5% CO2 in a humidified atmosphere.
Total RNA was extracted from the glioma cell lines (2×106 cells) and glioma tissues (100 mg) with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The optical density of the RNA samples at 260 nM was quantified by a NanoDrop ND-2000 Spectrophotometer (NanoDrop Technologies, Houston, TX, USA). For identification of miR-365 expression, complementary DNA (cDNA) were synthesized using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). qRT-PCR was performed with the TaqMan MicroRNA Assay for miR-365 and U6 (Ambion, Austin, TX, USA) and TaqMan Universal Master Mix II without UNG (Ambion) under ABI PRISM 7900 Sequence Detection System (Applied Biosystems). For determination of
miR-365 mimic and corresponding negative control miRNA (miR-NC) were purchased from Shanghai GenePharma (Shanghai, China). PIK3R3 overexpression plasmid (pCDNA3.1) was a gift of Dr Peng Zhang (Jilin University). Transfection was performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions.
Cell proliferation assay was performed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma). Briefly, approx. 2000 transfected cells were seeded into each well of 96-well plates and cultured for 1–4 days. At the indicated time (24, 48, 72 and 96 h), 100 µl fresh medium containing MTT 0.5 mg/ml was added into each well and cultured for 4 h at 37°C, then the medium was replaced with 100 µl of dimethyl sulfoxide (DMSO, Sigma) and shaken at room temperature for 10 min. The absorption was measured at 490 nm with a microplate reader (Thermo Labsystems, Helsinki, Finland).
The invasive ability of glioma cells was determined using 24-well Transwell chambers coated with Matrigel (BD Biosciences, San Jose, CA, USA). In brief, 1×105 transfected cells in serum-free medium were seeded at in the top chamber coated with Matrigel and incubated at 37°C in a humidified incubator containing 5% CO2. The bottom chamber was filled with medium containing 10% FBS as a chemoattractant. After 24 h of incubation, the non-invaded cells on the upper surface of the membrane were removed with a cotton swab, cells that invaded to the underside of the membrane were fixed with 70% ethanol for 30 min and stained with 0.2% crystal violet for 10 min. Photographs were imaged, and the number was counted in five randomly selected fields under a light microscope (Olympus, Tokyo, Japan). For Transwell migration assays, glioma cells were determined using Transwell chambers without the Matrigel coating.
The wild-type 3′-UTR segment of PIK3R3, which contained a putative binding site for miR-365, was amplified from normal human genomic DNA by PCR and inserted downstream of the luciferase gene in pGL3-control vector (Promega, Madison, WI, USA), named as Wt-PIK3R3. A mutant 3-UTR of PIK3R3 contained a mutation in the complementary seed region of miR-365 was amplified by PCR and inserted downstream of the luciferase gene in pGL3-control vector, referred to as Mut-PIK3R3. U87 cells (1×105) were seeded in 24-well plates and grown to 60–70% confluence. Cells were then cotransfected with 200 ng Wt-PIK3R3 or Mut-PIK3R3 reporter plasmid, 50 nmol miR-365 mimic or miR-NC, and 20 ng pRL-TK
Five-week-old female BALB/c nude mice were purchased from the Animal Center of Jilin University (Changchun, China). All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. U87 cells stable expressing miR-365 or miR-NC were injected subcutaneously into each side of the posterior flank of the nude mouse (six per group, 2×106 cells for each mouse). Tumor growth was examined every five day after injection, and tumor volumes were calculated using the equation V=AxB2/2 (mm3), where A is the largest diameter and B is the perpendicular diameter. Five weeks after the implantation, the mice were sacrificed, and the xenograft tumors were excised, and weighted. Part of tumor tissues were stored for further analysis.
Western blot analysis was performed as previously described (
All data are presented as the mean ± SD (standard deviation) from at least three independent experiments. The SPSS software package (version 18.0, SPSS Inc.; Chicago, IL, USA) was used to perform the statistical analysis. A two tailed Student's t-test was used to evaluate the significance of differences between two groups. ANOVA was employed to analyze the significance of differences in more than two groups. The relationship between miR-365 and PIK3R3 expressions was tested using Spearman's correlation analysis. The significance level was set as P<0.05.
To examine levels of miR-365 in glioma, we first measured the expression of miR-365 in four human glioma cell lines (U251, U87, U118 and LN18) and normal human astrocytes (NHA) by qRT-PCR. As shown in
To explore the possible biological functions of miR-365 in glioma, we transfected miR-365 mimic or miR-NC into U87 cells, which has lower expression of miR-365 (
To understand how miR-365 suppresses glioma growth, migration and invasion, bioinformatics (miRTarBase and TargetScan) were used to identify the target of miR-365. PIK3R3 was chosen as a target of miR-365, since it has a binding sequence for miR-365 at position (67–73) of 3′UTR (
Further experiments were performed to investigate the expression of PIK3R3 in glioma tissues and adjacent normal tissues by qRT-PCR. The result showed that the PIK3R3 mRNA expression was upregulated in glioma tissues compared with adjacent normal tissues (
To further illustrate whether miR-365 affects human glioma cell proliferation, migration and invasion through PIK3R3, U87 cells were co-transfected with miR-365 or miR-NC and the overexpression PIK3R3 plasmid. Western blot analysis showed that miR-365 overexpression significantly decreased PIK3R3 protein expression, while overexpression PIK3R3 plasmid restored PIK3R3 expression (
To investigate the role of miR-365 in tumor growth
PIK3R3 has been showed to be involved in tumor progression by regulating AKT/mTOR signal pathway (
Recently number of miRNAs have been identified to function as a tumor suppressor or an oncogene in glioma by regulating their target molecule (
Aberrant expression of miR-365 has been found in various human cancers. In gastric cancer (
PIK3R3 (phosphoinositide-3-kinase regulatory subunit 3), a member of the phosphatidylinositol 3-kinase (PI3K) family, has been suggested to play crucial roles in diverse biological processes, such as cell proliferation, differentiation, carcinogenesis and tumor angiogenesis (
Our study provides evidence that miR-365 expression was downregulated in glioma tissues and cell lines, and that miR-365 suppressed glioma cell proliferation, migration, and invasion
miR-365 was decreased in glioma tissues and cell lines. (A) The expression of miR-365 was determined in four human glioma cell lines (U251, U87, U118 and LN18) and normal human astrocytes (NHA). (B) The expression of miR-365 was determined in 36 paired glioma tissues and adjacent normal tissues by qRT-PCR. **P<0.01.
miR-365 inhibits glioma cell proliferation, migration and invasion. (A) The expression of miR-365 in U87 cells transfected with miR-365 mimic or miR-NC were detected by qRT-PCR. (B-D) Cell proliferation, migration and invasion were determined in U87 cells transfected with miR-365 mimic or miR-NC. **P<0.01.
PIK3R3 is a candidate target of miR-365 in glioma cells. (A) Schematic construction of wild-type (Wt) and mutant (Mut) 3′-UTR of PIK3R3 according to miR-365 and its putative binding sequence in the 3′-UTR of PIK3R3 (position 67–73). (B) The luciferase activities were determined in U87 cells co-transfected with wild-type (Wt) or mutant-type (Mut) 3′-UTR of PIK3R3 reporter plasmid and miR-365 mimic or miR-NC. (C and D) The PIK3R3 mRNA expression and protein expression levels were determined in U87 cells transfected with miR-365 mimic or miR-NC by qRT-PCR and western blotting, respectively. GAPDH was used as a control. **P<0.01.
PIK3R3 mRNA expression level was upregulated in glioma tissues and inversely correlated with miR-365 expression. (A) PIK3R3 mRNA expression was detected in 36 paired glioma tissues and adjacent normal tissues by qRT-PCR. GAPDH was used as an internal control. (B) The inverse correlation between miR-365 and PIK3R3 was analyzed in clinical glioma samples by Spearman's correlation coefficient (n=36). **P<0.01.
Overexpression of PIK3R3 reverses the suppressive effect of miR-365 in glioma. (A) PIK3R3 protein expression was determined in U87 cells transfected with miR-365 with/without PIK3R3 overexpression plasmid. GAPDH was used as the internal control. (B-D) Cell proliferation, migration and invasion were determined in U87 cells transfected with miR-365 with/without PIK3R3 overexpression plasmid. *P<0.05, **P<0.01.
miR-365 suppresses tumor growth
miR-365 regulates AKT/mTOR signaling pathway. AKT, p-AKT, mTOR and p-mTOR protein expression were determined in U87 cells transfected with miR-365 or miR-NC and xenograft tumor tissues from nude mice by western blotting. GAPDH was used as an internal control. **P<0.01.