ABSTRACT efficacy of the therapy effect against glioma. In

ABSTRACT

This work aimed to
scrutinize the inter-regulatory functions of hsa-mir-127 and replication
initiator 1 (REPIN1) on the proliferation and metastasis of glioma cells. The in-silico data on the implication hsa-mir-127
and REPIN1 in glioma were retrieved from The Cancer Genome Atlas (TCGA). The
expression levels of hsa-mir-127 and REPIN1 mRNA were determined by qRT-PCR
whereas western blot was used detection of REPIN1 protein expression in glioma
cell lines. The proliferation of glioma cells was determined by means of the MTT
assay, while the transwell assay was employed for assessing the extent of cell
migration and invasion. The interaction among REPIN1 and hsa-mir-127 was checked
using the luciferase reporter assay. The expression of hsa-mir-127 was markedly
increased in clinical data obtained from TCGA and in glioma cells compared with
normal tissues and control cells, respectively. Increased expression of
hsa-mir-127 and decreased expression of REPIN1 were both associated with poor
overall survival. Moreover, hsa-mir-127 overexpression noticeably promoted the proliferation,
inhibited apoptosis and increased the invasive and migratory capacities of
glioma cells. Inverse effects were found with hsa-mir-127 antisense inhibitor. Interestingly,
overexpressing hsa-mir-127 downregulated REPIN1 expression and luciferase
reporter assay displayed that the tumorigenesis effect of hsa-mir-127 requires,
in part, its direct targeting of REPIN1. Conclusively, the hsa-mir-127/REPIN1 pathway
is involved in gliomas and could be a potential therapeutic target.  

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Keywords: glioma, hsa-mir-127,
REPIN1, proliferation, invasion, migration

 

INTRODUCTION

Gliomas are the
most common brain tumors in children and adolescents (Ostrom et al., 2015).
Low-grade (benign) gliomas are more common in younger age groups, while
malignant gliomas affect older children or adolescents (de Groot, 2015).
These tumors include very good prognosis forms such as pilocytic astrocytoma
with types much more difficult to treat such as infiltrating glioma of the
brainstem. Surgery is usually used as a first-line treatment for malignant
astrocytomas whereas most of the treatment protocols for malignant glioma
include radiotherapy, chemotherapy, and new forms of treatment, such as
photoradiotherapy with porphyrins that are being assessed (Agulnik & Mason, 2006).
Nonetheless, the aggressive property of malignant gliomas thwarts the efficacy
of the therapy effect against glioma. In addition, the molecular mechanisms involved
in gliomas physiopathology are not fully elucidated, which is a key drawback to
the development of efficient anti-glioma drugs. Thus, scrutinizing the molecular
mechanisms governing the proliferation, migration and invasion of glioma cells
is of paramount significance.

Micro RNAs (miRNAs)
are approximately 21 nucleotides-length noncoding small RNAs that control the
regulation of their target genes. Increasing number of studies reveals the correlation
of miRNAs with human disorders including cancers. Hsa-mir-127 is a miRNA with
controversial roles in different type of human cancers. Indeed, some studies on
ovarian, gastric, esophageal, giant bone, pancreatic and hepatocellular cancers
have demonstrated that hsa-mir-127 exhibits a tumor suppressor function (Bi et al., 2016; Fellenberg et al., 2016; Gao et al.,
2016; Guo et al., 2013; Herr et al., 2017; Huan et al., 2016; Yu et al., 2016;
Zhang et al., 2016; Zhou et al., 2014)
whereas other studies indicated the oncogene role of this miRNA in
glioblastoma, lung, breast, and mucoepidermoid  carcinoma cells (Jiang et al., 2014; Shi et al., 2017; Shin et al.,
2013; Wang et al., 2014; Yan et al., 2008).
Though silencing of has-miR-127 in glioma cells was conveyed to counteract
Adriamycin resistance through mechanisms involving cell cycle arrest and
induction of cell apoptosis (Feng & Dong, 2015),
the functional role of has-miR-127 has not been fully investigated and the
potential molecular mechanisms need to be elucidated.

Replication
initiator 1 (REPIN1) is a zinc finger DNA-binding protein that enables the
initiation of DNA replication. The physiological and regulatory roles of REPIN1
have not been extensively studied since its discovery but recent findings have suggested
its implications in obesity and related metabolic syndromes including glucose
transport, fatty acid transport, adipogenesis and
the formation and fusion of lipid droplets (Andrade Fde et al., 2015; Bahr et al., 2011a; Bahr et
al., 2011b; Heiker & Kloting, 2013; Hesselbarth et al., 2017; Kern et al.,
2014; Kloting et al., 2007; Kunath et al., 2016; Ruschke et al., 2010).
Given that REPIN1 is involved in DNA replication, its dysregulation may be a
potential mechanism involved in tumorigenesis. Up to now, only one study has
suggested the association of REPIN 1 in breast cancer (Rengasamy et al., 2017).
However, the functional role of REPIN1 and its probable regulation by miRNAs in
gliomas are still unknown and need to be deeply scrutinized.

Hence, in this
study, we aimed to explore the implication of has-miR-127 and REPIN1 in glioma
processes and the possible regulatory interaction among them. In silico studies
indicated that hsa-mir-127 high expression was associated with decreased
survival of glioma patients while the inverse was observed with REPIN1. The
effect of hsa-mir-127 on the proliferation, invasion and migration of glioma
cells was investigated and REPIN1 was proposed as the target gene of hsa-mir-127.

MATERIALS AND METHODS

Retrieval of has-mir-127 and REPIN1 from TCGA glioma data

The freely
available portal LinkedOmics (www.linkedomics.org) that contains
multi-omics data from the 32 TCGA Cancer types was adopted for uncovering the effect
of has-mir-127 and REPIN1 on the survival of glioma
patients and their corresponding expression profiles in different tissue types.
The LinkFinder analytical module was used to search for mRNA expression
signatures (from mRNAseq data) associated with has-mir-127. The Pearson
correlation analysis was applied to assess the correlation of has-mir-127 with mRNA
expression signatures. Analysis results were visualized by scatter plots, box
plots, or Kaplan-Meier plots.    

Cell lines and culture

The normal human
glial cell HEB and two glioma cell lines U87 and LN-229 were al purchased from Chinese
Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified eagle
medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone, South
Logan, UT, USA), 50 ug/mL streptomycin and 100 ug/mL penicillin at 37 °C in an
incubator containing 5% CO2.

Quantitative real-time PCR

Total RNA was isolated
with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). An aliquot of purified RNA
was reverse-transcribed into cDNA using the Reverse Transcription Kit (Takara
Bio, Inc., Otsu, Japan) in accordance with the manufacturer’s instructions. The
amplification of REPIN1 was performed on a Biorad Realtime PCR platform. The PCR
reaction cycling conditions for the 30 cycles were: 94 °C for 2 min, 94 °C for
30 s, 56 °C for 30 s, 72 °C for 1 min and 72 ? for 10 min. GAPDH was used as an
endogenous control. Primer sequences were as follows: for REPIN1, forward 5 GAT-CGG-GCC-TTT-TTG-TGC-TC-3?
and reverse 5?-CTT-GCG-AGT-GAG-CCA-TTT-CG-3?; for GAPDH, forward 5?-GCA-ACT-AGG-ATG-GTG-TGG-CT-3?
and reverse 5?-TCC-CAT-TCC-CCA-GCT-CTC-ATA -3?.

The expression
level of mature hsa-mir-127-5p was determined with a TaqMan miRNA assay kit
(Applied Biosystems). The miRNA was purified and reverse transcribed using the
TaqMan miRNA RT kit (Applied Biosystems) with miRNA-specific RT primers
(Applied Biosystems). RT-PCR experiment was carried out on the StepOnePlus
Real-time PCR platform (Applied Biosystems). The reaction mixture contained 0.2
?M TaqMan probe, 2 ?l RT product, 10 ?M forward and reverse primers and 5 ?l
TaqMan Universal PCR Master Mix. RNU6B was used as an endogenous control for hsa-mir-127-5p.
 

Cell transfection

Cells in
logarithmic phase were collected and trypsinized. The mature-type of hsa-miR-127-5p,
hsa-miR-127-5p antisense inhibitor (anti–miR-127-5p) and nonspecific microRNA
(miR-Control) were acquired from Dharmacon (Dharmacon, Lafayette, CO, USA).
These oligonucleotides were transfected into glioma cells by using the Lipfactomine
TM 2000 (Gibco BRL, Burlington, Ont., Canada) following the vendor-recommended transfection
method. The transfection efficiency was assessed by performing a qRT-PCR
experiment for the miRNA as described above.

MTT assay

The MTT assay was used
to evaluate the viability of cells. The stock solution was prepared by
dissolving 5 mg MTT reagent (Cell Proliferation Kit I, Roche, USA) in 1 mL PBS,
followed by filtering in 0.22 ?m membrane. Cells were cultured for 48 h in
96-well plates.  Then, 20 ?L of stock
solution (5 mg/mL, Sigma-Aldrich, St Louis, MO, USA) was added to each well and
further incubated for an additional 4 h. After removal of supernatants, the plates
were added with 150 ?L of DMSO (Sigma-Aldrich) for solubilization of the
formazan crystals and the absorbance at 490 nm wavelength was measured using
the microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Transwell migration and invasion assays

To detect the
extent of cell migration and invasion, transwell assay was performed. Matrigel-coated
(invasion assay) or Matrigel-uncoated (migration assay) membranes were placed
into the upper chambers at 37 °C overnight. Following lysis using trypsin, around
1×105 cells serum-free medium was employed for preparing dilutions.
Next, cells were transferred into the upper chamber while the medium
supplemented with 5 mg/L fibronectin and 10% FBS (Invitrogen) was added to the lower
chamber. Following incubation in 5% CO2 incubator at 37
°C for 24 h, cells that passed through the membranes into the lower chamber
were fixed for about 20 min using methyl alcohol and subsequently stained with
crystal violet (0.1%) for 10 min. Finally, cells were examined using an optical
microscope (Nikon, Japan) and counted.

Luciferase reporter assay

The Phusion
Site-Directed Mutagenesis Kit (Thermo Fisher Scientific) was used to induce mutagenesis
in REPIN1 3’UTR. Next, the wild-type and mutated REPIN1 3’UTR were inserted in
the psiCHECK TM-2 vector (Promega, Madison, WI, USA) and the obtained
recombinant psiCHECK TM-2 vectors were cotransfected into glioma cells with hsa-mir-127-mimics,
hsa-mir-127 inhibitor or psiCHECK empty vector. Finally, the relative
luciferase activity was determined after 48 h post-transfection with the
Luciferase Assay Reagent II (Promega, Madison, WI, USA).

Western blot

Total protein was extracted
using the radio-immunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich,
St Louis, Mo) and separated using SDS–PAGE (Bio-Rad, Hercules, CA, USA)
approach. Next, proteins were transferred to polyvinylidene difluoride (PVDF)
membranes and blocked with 5% skimmed milk for 1h. Then, the membrane was
incubated with primary antibodies against REPIN1 (Thermo Fisher Scientific) and
?-actin (Thermo Fisher Scientific) at 4 °C overnight. Next, after incubation
with anti-APS IgG-HRP (BOSTER) secondary antibodies, immune complexes were revealed
by an enhanced chemiluminescent (ECL, Thermo Scientific). The relative protein
expression was determined using a densitometric approach with Image J software.

Statistical analysis

Statistical
analysis was achieved using the GraphPad Prism V6.01 software (GraphPad
Software, Inc., La Jolla, CA, USA). The experiments were performed in
triplicate and data expressed as average ± standard deviation (SD). The
intergroup differences were evaluated using one-way ANOVA or two-way ANOVA
followed by Tukey’s multiple comparison posttests. P

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