Background
Glioma is one of the most frequent intracranial malignant tumors. Due to the highly invasive growth pattern and frequent resistance to therapies, the prognosis for patients with glioma is dismal [
1]. Glioma stem cells (GSCs), a neoplastic subpopulation within glioma, are responsible for therapy resistance [
2] and poor survival rate of glioma [
3]. GSCs have characteristics of neurosphere formation, self-renewal and multi-directional differentiation [
4]. Therefore, illustration of the molecular mechanisms underlying GSCs offers new orientation toward the gloma treatments.
Lately, long non-coding RNAs (lncRNAs), which are defined as non-coding RNAs more than 200 nucleotides in length, are involved in various genetic phenomena, including transcriptional, post-transcriptional and epi-genetic regulations [
5]. Moreover, lncRNAs have been identified as new modulators in the origination and progression of glioma [
6]. Mounting evidence has also demonstrated that lncRNAs involved in the regulation of gene expression in stem cell biology and in tumorigenesis [
7].
Long intergenic non-coding RNA 152 (Linc00152) was found aberrant expression in numerous cancers. Linc00152 was distinctly up-regulated in gastric cancer and its expression was positively correlated with tumor invasion depth, lymph node metastasis and poor survival [
8]. Moreover, linc00152 could modulate the expression of miR-193a-3p to increase disease recurrence through acting as a competing endogenous RNA in colon cancer [
9]. However, the expression and possible functional role of linc00152 in GSCs remains uncharted.
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by binding to the 3′-UTRs of mRNAs [
10]. MiRNAs are reported to be involved in a variety of human carcinogenesis, including glioma [
11]. MiR-103a-3p was illuminated to participate in the proliferation, migration and invasion process in ovarian carcinoma [
12]. It is predicted that miR-103a-3p has putative binding sites with linc00152 by Starbase (
http://starbase.sysu.edu.cn/). Moreover, miR-103a-3p was identified as prognostic biomarker of colon cancer, breast cancer and pleural mesothelioma [
13‐
15], etc. However, limited knowledge is available concerning whether miR-103a-3p affected the biological processes of GSCs.
Forebrain embryonic zinc finger protein 1(FEZF1) is highly conserved transcription factor that encoded a transcriptional repressor which contributed to the embryonic migration of gonadotropin-releasing hormone neurons into the brain [
16]. In addition, FEZF1 is required for olfactory development and knock down FEZF1 in mice, olfactory neurons fail to mature [
17]. We predicted FEZF1 as a presumed target of miR-103a-3p by miRNA target prediction software Target Scan (
http://www.targetscan.org/). Studies have shown that FEZF1 played a oncogenic role through DNA demethylation [
18] in gastric cancer. Nevertheless, the role of FEZF1 in GSCs remains ambiguous.
Cell division cycle 25A (CDC25A), a member of the CDC25 family of phosphatases, removes the inhibitory phosphorylation in cyclin-dependent kinases (CDKs) [
19], and regulates the progression from G1 to the S phase of the cell cycle [
20]. It is discovered that the promoter of CDC25A contains sequence of CnnCAnCn core which is a putative consensus FEZF1 binding site [
21,
22]. Furthermore, CDC25A was identified as an oncogene, and over-expression of CDC25A promoted tumorigenesis [
23].
The major purpose of this study was to explore the expression of linc00152, miR-103a-3p, FEZF1 and CDC25A in glioma tissues and GSCs. We also investigated the roles in regulating the malignant behavior of GSCs and the potential molecular pathways among them. Our findings will give a new direction for the treatment of glioma.
Dicussion
In this study, we elaborated that expression of the lncRNA linc00152 was elevated in glioma tissues and GSCs. Linc00152 could directly bind to miR-103a-3p and negatively regulated the expression of miR-103a-3p. This implied a significant mechanism of linc00152 pathway in the glioma intervention. Knockdown of linc00152 restrained proliferation, migration and invasion, and facilitated apoptosis in GSCs. On the contrary, miR-103a-3p manifested carcinostatic role in GSCs and suppressed the proliferation, migration and invasion, prompted apoptosis of GSCs. In addition, FEZF1 was identified as a direct target of miR-103a-3p and mediated the tumor-suppressive effects of miR-103a-3p. Moreover, inhibition of miR-103a-3p promoted the expression of CDC25A by up-regulating FEZF1, which positively control the promoter activities via binding to the promoter of CDC25A. Further, PI3K/AKT pathway was blocked when CDC25A was suppressed. Experiment in vivo showed that knockdown of linc00152 combined with over-expression of miR-103a-3p generated the smallest tumor and led to the longest survival time in nude mice.
Glioblastoma (GBM) is one of the most common and lethal primary malignant brain tumor in adults [
31,
32]. GBM harbor a subpopulation of self-renewing, therapy-resistant cells, GSCs, which can flourish in the stresses present in an unfavorable tumor microenvironment [
33]. In consequence, therapies targeting GSCs have become a hotspot in treatment of glioma [
34]. A growing number of evidence has implied that lncRNAs play an important role in the occurrence and progression of tumors recently. Exploring the potential molecular mechanisms of lncRNAs in GSCs may provide promising therapeutic strategies in glioma.
Our present data revealed that linc00152 was positively correlated with the histopathological grade in human glioma tissues and elevated in GSCs. In addition, knockdown of linc00152 inhibited cell proliferation, migration and invasion, as well as promoted apoptosis in GSCs, which indicated that linc00152 may serve as an oncogene in GSCs. Previous researches also showed the same oncogenic function of linc00152. Cai Q reported linc00152 was up-regulated in gallbladder cancer and correlated negatively with the overall survival time in gallbladder cancer patients [
35]. In addition, linc00152 was identified as increased expression in gastric cancer [
36] and regarded as a novel biomarker for predicting gastric cancer [
37]. Our results illustrated that linc00152 acted as a oncogene in GSCs for the first time, but the underlying mechanisms need to be investigated.
LncRNAs finely regulate gene expression through a variety of ways, including transcriptional regulation, splicing and translation, post-transcriptional regulation and epitogenetic modulation, etc. [
38]. To detect the potential oncogenic mechanism of linc00152 in glioma, bioinformatics analysis software was applied to identify miR-103a-3p as a novel target of linc00152. MiR-103a-3p situated in 5q34, and was associate with tumor, Parkinson disease and pregnancy-related complications [
39‐
41]. Our study suggested that miR-103a-3p was declined in glioma tissues and GSCs compared with NBTs and glioma cells, respectively. Furthermore, over-expression of miR-103a-3p inhibited proliferation, migration and invasion, as well as promoted apoptosis in GSCs. This was consistent with the previous studies showing that miR-103a-3p was low expressed in gastric cancer [
42] as well as bladder carcinoma [
43], and played a crucial role in cancer suppression.
Mechanistically, miR-103a-3p was identified as a direct target of linc00152 by dual-luciferase assay, suggesting the potential reciprocal repression feedback loop between linc00152 and miR-103a-3p. In addition, our results showed that ablation of linc00152 inhibited cell proliferation, migration and invasion as well as inducing apoptosis in GSCs by elevating miR-103a-3p, implying that linc00152 knockdown impaired the malignant behavior of GSCs by up-regulating miR-103a-3p. Moreover, the in vivo studies showed that nude mice in the sh-linc00152 + pre-miR-103a-3p group manifested the smallest tumors and highest survival compared to the other groups, suggesting that these were likely to achieve synergistic anti-tumor effects. Collectively, linc00152 acted as an oncogenic gene by restraining miR-103a-3p in GSCs, but the further mechanisms under miR-103a-3p were blurred.
We discovered that the protein levels of FEZF1 had adverse changes to the alteration of miR-103a-3p. Luciferase reporter assays certified that miR-103a-3p regulated gene expression post-transcriptionally by binding to the 3’UTR of FEZF1. In addition, our research showed that FEZF1 expression was raised in glioma tissues as well as in GSCs. Over-expression of FEZF1 facilitated cell proliferation, migration and invasion, and inhibited cell apoptosis, implying that FEZF1 functions as an oncogene in GSCs. Consistent with our results, FEZF1 was reported over-expressed in gastric cancer tissues and played a significant role in the progression and metastasis of gastric cancer by activation of the K-ras oncogene [
44]. What’s more, miR-103a-3p and FEZF1 co-overexpression significantly rescued the inhibition effect induced by up regulating miR-103a-3p alone, revealing that miR-103a-3p inhibits the malignant behavior of GSCs by declined FEZF1 expression.
CDC25A is an identified cell division cycle protein, which is required to enter S time [
45], and the over-expression of this phosphatase accelerates the entrance to S time [
46]. Our research showed that expression of CDC25A was remarkably increased in GSCs. Moreover, over-expression of CDC25A facilitated cell proliferation, migration and invasion, while inhibited apoptosis in GSCs. Consistent with our study, Wang XQ [
47] reported expression of CDC25A elevated in hepatocellular carcinoma, which was significantly correlated with HCC tumor-node-metastasis staging and venous invasion. In addition, CDC25A was reported over-expressed and suppressed by miR-21 through a defined sequence in its 3′-UTR in colon cancer [
48].
Moreover, we discovered that CDC25A was involved in a FEZF1-induced oncogenic effect in GSCs. Over-expression of FEZF1 could elevate CDC25A expression, while FEZF1 knockdown showed contrary effects. Luciferase assays and ChIP assays showed that FEZ1 increased promoter activity and bound to the promoter region of CDC25A, indicated that CDC25A was involved in FEZF1 regulation in GSCs.
Taken together, we revealed that the linc00152-miR-103a-3p-FEZF1-CDC25A axis manifested an important role in human glioma. Briefly, the miR-103a-3p over-expression induced by the knockdown of linc00152 could down-regulate the expression FEZF1, decreased FEZF1 hindered the promoter activities of CDC25A and inhibited malignant biological behavior in GSCs.
We further explored the molecular mechanisms of CDC25A oncogenic functions. Recent studies showed that CDC25A promoted PKM2-dependent β-catenin transactivation, which promotes the Warburg effect and tumorigenesis [
49]. In addition, CDC25A was reported up-regulated in chondrosarcoma cells and played an important role in cell cycle progression and tumorigenesis of chondrosarcoma as an activator of CDK complexes [
50]. Moreover, the activation of PI3K/AKT pathway participated in the glioma progression and various biological effects including proliferation, migration, invasion and apoptosis [
51,
52]. The PI3K/AKT pathway inhibitors were discovered to exhibit efficacy against glioblastoma in clinically relevant mouse models [
53]. In this work, we revealed that CDC25A over-expression activated the PI3K/AKT pathways in GSCs and knockdown of CDC25A manifested the opposite effects. Therefore, the reduced CDC25A induced by FEZF1 knocked down could attenuate the activity of PI3K/AKT pathways to inhibit the malignant behaviors of GCSs. The mechanism underlying tumor suppressive function of human glioma cells by linc00152 knockdown is schematically presented in Fig.
8e.
Methods
Patients and glioma specimens
Grades of glioma were identified by neuropathologists according to WHO classification. Glioma specimens were divided into two groups: grade I–II glioma group (n = 10) and grade III–IV glioma group (n = 10). Normal brain tissues(NBTs) were used as the negative control group (n = 30). They were acquired from patients’ fresh autopsy material (donation from individuals who died in traffic accident and confirmed to be free of any prior pathologically detectable conditions).
Cell culture
Human glioma cell lines (U87, U251) and human embryonic kidney (HEK) 293 T cells were purchased from the Chinese Academy of Medical Sciences (Beijing, China). U87 glioma cells and HEK-293 cells were cultured in Dulbecco’s modified Eagle medium (DMEM)/high glucose with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), U251 cells were cultured in DMEM/F12 medium with 10% FBS. All cells were maintained in a humidified incubator at 37 °C with 5% CO2.
Isolation and identification of GSCs
GSCs were seperated as described previously [
54,
55]. In a nutshell, GSCs were cultured in DMEM/F-12 medium (Life Technologies Corporation, Grand Island, NY, USA) appended to basic fibroblast growth factor (bFGF, 20 ng/mL, Life Technologies Corporation, Carlsbad, CA, USA), epidermal growth factor (EGF, 20 ng/mL, Life Technologies Corporation, Gaithersburg, MD, USA) and 2% B27 (Life Technologies Corporation, Grand Island, NY, USA). The serum-free medium was replaced every 2 days until the spheres were visible under microscopy. The spheres were centrifuged for 3 min at 1000 r.p.m. and harvested, then trypsinized into single-cell suspensions and plated into a 96-well plate for the limiting dilution assay and colon sphere formation by limiting dilution as described previously [
56,
57]. For differentiation assay, sphere cells were plated onto glass coverslips coated with poly-L-ornithine (BD Biosciences, Franklin Lakes, NJ, USA) in medium containing 10% FBS for the differentiation assay. For immunostaining of undifferentiated spheres, cells were incubated with antibodies against Nestin and CD133 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA). For immunostaining of differentiated spheres, cells were stained with antibodies against GFAP (1:100, Abcam, Cambridge, MA, USA) and beta-tubulin III (1:100, Santa Cruz Biotechnology). The primary antibody complexes were visualized with anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 555 secondary antibodies (Beyotime Institute of Biotechnology, Jiangsu, China). Nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI).
Cell transfection and generation of stable transfected cells
Short-hairpin RNA directed against human linc00152 plasmid (sh-linc00152), short-hairpin RNA directed against human FEZF1 plasmid (FEZF1(−)), short-hairpin RNA directed against human CDC25A plasmid (CDC25A(−)) and their respective non-targeting sequence (negative control, NC) were synthesized (GenePharma, Shanghai, China). FEZF1 full length (FEZF1(+)) plasmid, CDC25A full length (CDC25A(+)) plasmid, and their respective non-targeting sequence (negative control, NC) were synthesized (GenScript, Piscataway, NJ, USA). Glioma cells were transfected in 24-well plates using Opti-MEM and Lipofectamine 3000 reagents (Invitrogen, CA, USA) according to the manufacturer’s instructions. Stable cell lines were selected applying Geneticin (G418; Invitrogen, CA, USA). Four weeks later, G418-resistant cell clones were established. The over-expression and the silence efficiencies were evaluated by quantitative real-time PCR (qRT-PCR). And then GSCs were isolated as previously described. To investigate the effect of linc00152 on GSCs, cells were divided into three groups: control group, sh-NC group (tansfected with empty plasmid) and sh-linc00152 group. To investigate the effect of FEZF1 on GSCs, cells were divided into five groups: control group, FEZF1(+)-NC group, FEZF1(+) group, FEZF1(−)-NC group and FEZF1(−) group. To investigate the effect of CDC25A on GSCs, cells were divided into five groups: control group, CDC25A(+)-NC group, CDC25A(+) group, CDC25A(−)-NC group and CDC25A(−) group.
Cell transfection of miRNAs
MiR-103a-3p agomir (pre-miR-103a-3p), miR-103a-3p antagomir (anti-miR-103a-3p) and their respective negative control molecules (pre-NC and anti-NC) were synthesized (GenePharma, Shanghai, China). To investigate the effect of miR-103a-3p on GSCs, cells were divided into five groups: control group, pre-NC group, pre-miR-103a-3p group, anti-NC group and anti-miR-103a-3p group. To investigate whether linc00152 mediated regulation of miR-103a-3p expression could regulate the behavior of GSCs, cells were divided into five groups: control group, sh-NC + pre-NC group (sh-NC stable transfected cells co-transfected with pre-NC), sh-linc00152 + pre-miR-103a-3p group (sh-linc00152 stable transfected cells co-transfected with pre-miR-103a-3p), sh-NC + anti-NC group (sh-NC stable transfected cells co-transfected with anti-NC) and sh-linc00152 + anti-miR-103a-3p group (sh-linc00152 stable transfected cells co-transfected with anti-miR-103a-3p). To investigate the mechanism of FEZF1 regulated by miR-103a-3p to effect the behavior of GSCs, cells were divided into five groups: control group, pre-NC + FEZF1-NC group (FEZF1-NC stable transfected cells co-transfected with pre-NC), pre-miR-103a-3p + FEZF1-NC group (FEZF1-NC stable transfected cells co-transfected with pre-miR-103a-3p), pre-NC + FEZF1 group (FEZF1 stable transfected cells co-transfected with pre-NC) and pre-miR-103a-3p + FEZF1 group (FEZF1 stable transfected cells co-transfected with miR-103a-3p).
RNA and miRNA isolation and quantitative RT-PCR
Total RNA was isolated from the glioma tissues, NBTs and cells using Trizol reagent (Life Technologies Corporation, Carlsbad, CA, USA). The RNA concentration was determined by 260/280 nm absorbance using a Nanodrop Spectrophotometer (ND-100, Thermo, USA). One-Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) (Takara Bio, Inc., Japan) was used for qRT-PCR detection of linc00152 and mRNA of FEZF1 and CDC25A. cDNA from miRNAs was generated by using a TaqMan miRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). qRT-PCR was carried out by using TaqMan Universal Master Mix II with Taq-Man microRNA assays of miR-103a-3p and U6 (Applied Biosystems, Foster City, CA, USA). U6 and GAPDH were employed as endogenous controls for miRNA and gene expression detection. Expressions were normalized to endogenous controls and relative quantification (2−ΔΔCt) method was used for fold changes’ calculating.
Cell proliferation assay
Cell proliferation was measured by conducting Cell Counting Kit-8 (CCK-8, Beyotime Institute of Biotechnology, Jiangsu, China) assay. After transfection efficacy was measured, GSCs were dissociated with Accutase (Life Technologies Corporation, Carlsbad, CA, USA), resuspended, and seeded in 96-well plates at 2000 cells per well. Cells per well were added 10 μl CCK-8 solution and cultured for 2 h at 37 °C. The absorbance was recorded at 450 nm on a SpectraMax M5 microplate reader (Molecular Devices, USA).
Limiting dilution assay
Different amounts (2, 5, 10, 20, 50, 100, 200, 500) of GSCs were seeded into a 96-well plate. Serum-free conditional culture medium (25 μl) was added to each well every 2 days. The percentage of wells without neurospheres was enumerated. Then, a linear regression model was used to compare the slope of each group.
Cell migration and invasion assay
Cells were resuspended in 200 μL serum-free medium at a density of 2 × 105 cells/ml and placed in the upper chamber (or precoated with 80 μL of Matrigel solution (BD, Franklin Lakes, NJ, USA) for cell invasion assay) of the 24-well insert with 8 mm pore size. 600 μL of 10% FBS medium was added to the lower chamber. After incubation for 24 h, Cells had migrated or invaded to the lower side of the membrane were fixed and stained with 20% Giemsa. Stained cells were counted under a microscope in five randomly chosen fields and the average number was calculated.
Apoptosis detection
Apoptosis was detected by Annexin V-APC/7-AAD (BD Biosciences). Cells were harvested added allophycocyanin (APC) and 7-aminoactinomycin D (AAD) following the instructions of the manufacturer. Cell samples were analyzed by flow cytometry (FACScan, BD Biosciences), and apoptotic fractions were recorded.
Western blot analysis
Total proteins were isolated from cells with RIPA buffer on ice and were further analysed by SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. After non-specific binding was prevented by 5% nonfat milk at room temperature for 2 h, membranes were embraced overnight at 4 °C by primary antibodies as below: FEZF1 (1:200, Santa Cruz Biotechnology),CDC25A(1:1000, Abcam, EUGENE, USA), PI3K, p-PI3K, AKT, p-AKT (1:1000, Cell Signaling Technology, Beverly, MA, USA) and GAPDH (1:1000, Santa Cruz Biotechnology). Then, incubated 2 h at room temperature with HRP-conjugated secondary antibodies. Immunoblots were visualized by ECL chemiluminescence detection system and the relative integrated density values were calculated by FLuor Chem2.0 software.
Dual-luciferase reporter assays
Potential miR-103a-3p binding sites of linc00152 and FEZF1 3′-UTR were predicted by bioinformatics tool Starbase (
http://starbase.sysu.edu.cn/) and Target Scan (
http://www.targetscan.org/). In brief, the fragment of linc00152 possessing the assumptive miR-103a-3p binding sites was cloned into a pmirGLO Dual-Luciferase Vector (Promega, Madison, WI, USA) to construct the reporter vector linc00152-wild-type (linc00152-Wt) (GenePharma, Shanghai, China). Analogously, the corresponding mutant of hypothetic miR-103a-3p binding sites was manufactured to form the reporter vector linc00152-mutated-types (linc00152-Mut) (GenePharma, Shanghai, China). The 3′-UTR fragment of FEZF1 gene and its mutant of the theoretical miR-103a-3p binding site were cloned into a pmirGLO Dual-Luciferase Vector to form the reporter vector FEZF1-3′-UTR-wild-type (FEZF1-3′-UTR-Wt) and FEZF1-3′-UTR-mutated-type (FEZF1-3′-UTR-Mut) (GenePharma, Shanghai,China), respectively. The pmirGLO vector(wild type fragments or mutated type fragments) and indicated miRNAs were transfected into HEK 293 T cells using Lipofectamine 3000. Relative luciferase activities were measured 48 h after transfection and firefly luciferase activity was normalized by renilla luciferase activity.
Chromatin immunoprecipitation (Chip) assay
The Chip assay was carried out using the Simple Chip Enzymatic Chromatin IP kit (Cell Signaling Technology, Danvers, MA) following the manufacturer’s instructions. Cells were cross-linked with 1% formaldehyde for 10 min and then dealed with glycine for 5 min at room temperature. Cells were lysed with cold buffer containing PMSF and resuspended with cold PBS. Chromatin was digested by micrococcal nuclease and incubated for 20 min at 37 °C with frequent mixture. Lysates (2%) were employed as an input reference. Other immunoprecipitation samples were incubated overnight with normal rabbit IgG or anti-FEZF1 antibodies (Santa Cruz Biotechnology, CA, USA) at 4 °C with vibration. Protein G agarose beads were used to collect the chromatinimmune complex, and beads were scoured with low salt chip buffer and high salt chip buffer. DNA crosslinks were reversed by 5 mol/l NaCl and proteinase K at 65 °C for 2 h to purify. DNA was amplified by PCR applying the following DNA fragments: putative binding site 1 using the primers 5′-GGCTAAGAAGTGGTGGGAAAG-3′ and 5′-AGGGAGGGAATGCAATGAC-3′, generating a 211 bp product; putative binding site 2 using the primers 5′-GCTTTCTTCTTCCCCTCTCA-3′ and 5′-CCGACCTACACCTCTTACCC-3′, generating a 195 bp product; control using the primers 5′-TCTACCTCCTTCAGGGCTCA-3′ and 5′-TTTGGCCTTATCCTGTGGAC-3′, generating a 171 bp product.
Tumor xenografts in nude mice
In vivo study, the stable expressing cells were applied. Lentivirus encoding miR-103a-3p and its non-targeting sequence (negative control, NC) were generated using pLenti6.3/V5eDEST Gateway Vector Kit (Life Technologies Corporation, Carlsbad, CA, USA). The miR-103a-3p and short-hairpin RNA targeting human linc00152 were ligated into the pLenti6.3/V5eDEST vector and LV3-CMV-GFP-Puro vector (GenePharma, Shanghai, China), respectively. And then pLenti6.3/V5eDEST-miR-103a-3p and LV3-CMV-GFPPuro-sh-linc00152 vectors were generated. The ViraPower Packaging Mix was used to generate Lentivirus in 293FT cells. After infection, the stable expressing cells of miR-103a-3p (miR-103a-3p(+)), sh-linc00152 (linc00152(−)) were picked. The lentiviruses of miR-103a-3p(+) were transduced in linc00152(−) stably transfected cells to generate linc00152(−) + miR-103a-3p(+) cells.
The nude mice were divided into five groups: control group, linc00152(−)-NC + miR-103a-3p(+)-NC group, linc00152(−) group, miR-103a-3p(+) group and linc00152(−) + miR-103a-3p(+) group.
For subcutaneous implantation, every mouse was subcutaneously injected with 3 × 105 cells in the right flank. Tumor volume was measured every 4 days using the formula: volume (mm3) = length × width2/2. The subcutaneous tumor-bearing mice were executed 40 days after injection. For survival analysis in orthotopic transplantation, 3 × 105 cells were stereotactically transplanted into the right striatum of the mice. The number of survived nude mice was recorded every day and survival analysis was conducted applying KaplaneMeier survival curve.
Statistical analysis
SPSS 18.0 statistical software was used for statistical analysis. All data are presented as the mean ± standard deviation (SD) from at least three independent replicates. Statistical analysis of data was performed using the Student’s t-test. Differences were considered to be statistically significant when P < 0.05.
Acknowledgements
Not applicable.