Jian 等。 - 2019 - Down-regulated MicroRNA-30b-3p Inhibits Proliferat

Bioscience Reports (2019) 39 BSR20182226
https://doi.org/10.1042/BSR20182226
Research Article
Down-regulated microRNA-30b-3p inhibits

proliferation, invasion and migration of glioma cells
via inactivation of the AKT signaling pathway by
up-regulating RECK
Yan Jian, Chun-Hua Xu, You-Ping Li, Bin Tang, She-Hao Xie and Er-Ming Zeng
Department of Neurosurgery, The First Affiliated Hospital of Nanchang University, Nanchang 330006, P.R China
Correspondence: Er-Ming Zeng (zemzengerming@126.com)
microRNAs (miRNAs) have been found to affect various cancers, and expression of numer-
ous miRNAs is revealed in glioma. However, the role of microRNA-30b-3p (miR-30b-3p) in
glioma remains elusive. Therefore, the present study aims to explore the specific mechanism
by which miR-30b-3p influence the development of glioma in relation to the AKT signaling
pathway. First, glioma cell lines were collected with miR-30b-3p and reversion-inducing
cysteine-rich protein with kazal motifs (RECK) expression measured. The functional role
of miR-30b-3p and RECK in glioma was determined via gain- and loss-of-function ap-
proaches. Subsequently, the expression of invasion- and migration-related factors (MMP-2
and MMP-9) and the AKT signaling pathway-related factors (AKT, p-AKT and PI3K-p85)
was detected. Moreover, in vivo experiments were also conducted to investigate how
miR-30b-3p influences in vivo tumorigenesis. The results showed that miR-30b-3p was
up-regulated and RECK was down-regulated in glioma. RECK was a target gene of
miR-30b-3p. Decreased miR-30b-3p and overexpressed RECK led to decreased expres-
sion of MMP-2, MMP-9 and p-AKT. Overexpressed RECK and LY294002 could decrease
p-AKT and PI3K-p85 expression accompanied with unchanged expression of total protein
of AKT. Additionally, proliferation, migration and invasion of glioma cells and tumor forma-
tion in nude mice were repressed owing to reduced expression of miR-30b-3p or elevated
expression of RECK. In summary, miR-30b-3p inhibition suppresses metastasis of glioma
cells by inactivating the AKT signaling pathway via RECK up-regulation, providing a new
target for glioma treatment.
Introduction
Glioma is a common and severe brain tumor accompanied by high incidence and death rates [1]. Almost
5.26 out of 100000 people suffer from malignant glioma every year [2]. As one of the severest glioma,
glioblastoma multiforme treated with standard methods has an average survival of only ∼15 months [3].
The underlying molecular mechanism involving in both diagnosis and therapy of glioma has raised wide
Received: 29 November 2018
interest in recent years and has achieved great advancement [4]. However, despite a more profound molec-
Revised: 18 June 2019 ular insight of glioma, there has been no significant change in survival over the past few decades with
Accepted: 24 June 2019 unclear etiology [5,6]. Therefore, more effective biological markers are needed to offer a new treatment
for glioma [7].
Accepted Manuscript Online:
03 July 2019 As small non-coding RNAs with ∼22 nucleotides, microRNAs (miRNAs) regulate expression of genes
Version of Record published: at post-transcriptional level and are usually aberrantly expressed in various types of tumors [8,9]. More
13 August 2019 specifically, a prior study suggested that once being identified in human beings, many kinds of miRNAs
© 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution 1
License 4.0 (CC BY).
are found to be closely associated with the development of glioma [10]. Belonging to the microRNA-30 (miR-30)
family, miR-30b is reported to have different effects on tumor cells and immune cells which is caused by genetic
differences [11]. Moreover, results from a study found that microRNA-30b-3p (miR-30b-3p) was highly expressed in
glioblastoma microvascular proliferation [12]. miR-30b-3p was predicted to have binding site on reversion-inducing
cysteine-rich protein with kazal motifs (RECK) in biological prediction website microRNA.org. RECK is a plasma
membrane protein which plays a vital role in embryo and placental vascular remodeling [13]. As a tumor suppressor,
RECK is extensively demonstrated to be closely related to the biological processes of diverse cancers or tumors [14].
Besides, expression of RECK has been found in endothelial cells but not in tumor cells of glioma [15]. AKT, also
named as the serine/threonine protein kinase B, is a member belonging to the AGC family of protein kinases with
essential functions in signaling pathways [16]. AKT1, an isoform of AKT, is a target molecule of the transcriptional
factor glioma-related oncogene homolog 1 [17]. A previous study highlighted that the PI3K/AKT signaling pathway
could contribute to enhanced cell growth and invasion of glioma by inactivating the signals related to cell apoptosis
[18]. Based on these findings, it can be indicated that miR-30b-3p, RECK and AKT signaling pathway are closely
linked to glioma. Thus, the present study is going to investigate how miR-30b-3p influences development of glioma
by regulating RECK and AKT signaling pathway.
Materials and methods

Cell culture
Normal human astrocytes (NHA) and glioma cell lines SHG44, U251, U87 and A172 were purchased from the In-
stitute of Cell Research, Shanghai Academy of Sciences (Shanghai, China). Four kinds of glioma cells were cultured
in Dulbecco’s modified eagle medium (DMEM) (Gibco, Carlsbad, California, U.S.A.) supplemented with 10% fetal
bovine serum (FBS) (Sigma–Aldrich, SF, CA, U.S.A.) and NHA cells were cultured in DMEM/F12 medium imple-
mented with 2% FBS and 1% astrocyte growth factor. All the cells were cultured in an incubator at 37◦ C with 5% CO2
and saturated humidity. The culture medium was replaced every 2–3 days based on the growth condition of the cells,
and cells were passaged when cells settled 80–90% of the culture plates.
Dual luciferase reporter gene assay

DNA extraction from human embryonic kidney (HEK)-293T cells (CRL-1415, Shanghai Xin Yu Biotech
Co., Ltd, Shanghai, China) was conducted according to the instructions of DNA extraction kits (TIANGEN
BIOTECHNOLOGY CO. LTD, Beijing, China). The RECK-3 -untranslated region (3 UTR)-wild-type (Wt) and the
RECK-3 -UTR-mutant type (Mut) without the miR-30b-3p-binding site were designed, and then the luciferase re-
porter vectors were constructed. Intracellular mature miR-30b-3p mimic sequence (miR-30b-3p mimic) and its
negative control (NC) sequence (mimic-NC) were co-transfected into HEK-293T cells with RECK-3 -UTR-Wt and
RECK-3 -UTR-Mut, respectively. Luciferase activity of samples was detected using a dual luciferase reporter assay
reagent (Promega, Madison, WI, U.S.A.). After 48 h of transfection, the original culture medium was removed, and
the samples were washed by phosphate buffer saline (PBS) twice. Afterward, with the addition of 100 μl of passive
lysis buffer (PLB), cells in each well were oscillated at room temperature for 20 min to collect the cell lysate. Pre-
pared LARIIStop&Glo® Reagent was added to luminescent tube or plate containing cell lysate (20 μl per sample)
and then detected in a bioluminescence detector (Modulus™, Turner BioSystems, Mary Ave Sunnyvale, CA, U.S.A.)
with prereading time set as 2 s, read value set as 10 s and sample size set as 100 μl/time.
Cell grouping and transfection

The glioma cell line U87 was cultured in vitro and the cells in the logarithmic growth phase were seeded into
six-well plates. When the cell confluence reached 60–80%, the cells were transfected in accordance with the in-
structions of lipofectamine 2000 (Invitrogen, Carlsbad, California, U.S.A.). The cells were grouped into mimic-NC
group (transfected with miR-30b-3p mimic NC sequence), inhibitor-NC group (transfected with miR-30b-3p in-
hibitor NC sequence), miR-30b-3p mimic group (transfected with miR-30b-3p mimic), miR-30b-3p inhibitor group
(transfected with miR-30b-3p inhibitor), RECK-NC group (transfected with RECK NC sequence), pcDNA3-RECK
group (transfected with pcDNA3-RECK), pcDNA3-RECK + mimic NC (transfected with pcDNA3-RECK and
miR-30b-3p mimic NC sequences), miR-30b-3p mimic + RECK-NC (transfected with miR-30b-3p mimic and RECK
NC sequence), pcDNA3-RECK + miR-30b-3p mimic group (transfected with pcDNA3-RECK and miR-30b-3p
mimic), pcDNA3-RECK + dimethyl sulfoxide (DMSO) (transfected with pcDNA3-RECK with the addition of
DMSO) and pcDNA3-RECK + 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) (transfected with
pcDNA3-RECK with the addition of LY294002, the inhibitor of the AKT signaling pathway). All the transfection
2 © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
Table 1 Sequences of transfected fragment
Target segments Sequences
Inhibitor-NC 5 -UCACAACCUCCUAGAAAGAGUAGA-3
miR-30b-3p inhibitor 5 -AGCUGAGUGUAGGAUGUUUACA-3
mimic-NC 5 -CAGUACUUUUGUGUAGUACAA-3
miR-30b-3p mimic 5 -UGUAAACAUCCUACACUCAGCU-3
Abbreviation: NC, negative control.
Table 2 Primer sequences for RT-qPCR

Target genes Primer sequences
miR-30b-3p F: 5 -UGUAAACAUCCUACACUCAGCU-3
R: 5 -ACAUUUGUAGGAUGUAGUCGA-3
RECK F: 5 -TGTTGACCTGTTTAGCGGATGT-3
R: 5 -GAAAAGTTCTGTTGGCCTGTTGT-3
GAPDH F: 5 -AGGCTGTTGGGAAAGTTCTTC-3
R: 5 -ACTGTTGGAACTCGGAATGC-3
U6 F: 5 -TGCGGGTGCTCGCTTCGGCAGC-3
R: 5 -CCAGTGCAGGGTCCGAGGT-3
Abbreviations: F, forward; R, reverse.
reagents were purchased from Shanghai GenePharma Co. Ltd. (Shanghai, China), and the transfected fragment se-
quences are shown in Table 1. Then each well was added with 800 μl of serum-free medium, and a mixture of mimic,
inhibitor or siRNA-RECK (dissolved by Opti-Minimum Essential Medium [MEM]) and lipo2000 (11668027, Thermo
Fisher Scientific, Massachusetts, U.S.A.) was added into a six-well plate. The cells were cultured for 6 h, and the origi-
nal medium was replaced by the complete culture medium. After further transfection for 48 h, the cells were observed
under a microscope and then collected with RNA and protein extracted for subsequent experimentations.
Plasmid construction
The forward and reverse primers were designed according to the RECK gene sequence in Gen Bank and synthesized
by GenePharma Ltd. Company (Shanghai, China). The double-stranded oligomer obtained by polymerase chain reac-
tion (PCR) amplification was ligated between KpnI and NotI sites of pcDNA3 after digestion. The reaction system (10
μl) contained 5 μl of 2× ligase buffer, 2 μl double-stranded oligomer, 1 μl pcDNA3 expression vector, 1 μl T4 ligase (1
U/μl) and 1 μl sterile water. These substances were mixed and incubated at 22◦ C for 3 h to construct pcDNA3-RECK
overexpression plasmids. The plasmids were then transformed into competent cell of Escherichia coli (DH5α) and
cultivated in a culture plate overnight at 37◦ C. The next day, the monoclonal colonies were selected for amplification.
Subsequently, the plasmids were extracted according to the instructions of kits for rapid extraction of plasmid prior
to double enzyme digestion with KpnI and NotI and identification by agarose gel electrophoresis.
Reverse transcription-quantitative polymerase chain reaction

The TRIzol method (15596026, Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) was used to lyse and extract total
RNA from glioma cells. RNA was then reversely transcribed into a complementary DNA (cDNA) template using PCR
reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using ABI7500 quantitative
PCR instrument (ABI Company, Oyster Bay, NY, U.S.A.). U6 was used as an internal reference for miR-30b-3p, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for RECK. The primers used in the system are shown in Table
2. According to 2−C t [19], the ratio of the expression of the target gene in the experimental group to the control
group was expressed as follows: C t = C t experimental group − C t control group , wherein C t = C t target gene − C t
internal reference gene . C t is the number of cycles of amplification that occurs when the real-time fluorescence intensity of
the reaction reaches a set threshold, at which point the amplification is logarithmic.
Western blot analysis

Cells and tissues were obtained, followed by total protein extraction. The total protein concentration was measured
by bicinchoninic acid (BCA) kits (Thermo Fisher Scientific, Massachusetts, U.S.A.). After protein separation by
12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), the protein was transferred onto a
polyvinylidene fluoride (PVDF) membrane. The membrane was sealed with skim milk for 2 h at room tempera-
ture and incubated overnight at 4◦ C with the addition of corresponding antibodies: rabbit anti-human antibodies
against RECK (1: 1000, ab115844, Abcam Inc., Cambridge, MA, U.S.A.), AKT (9272, Cell Signaling Technologies
(CST), Beverly, MA, U.S.A.), p-AKT (1:2000, ab64148, Abcam Inc., Cambridge, MA, U.S.A.), matrix metallopro-
teinase (MMP)-2 (1: 2000, ab37150, Abcam Inc., Cambridge, MA, U.S.A.), MMP-9 (1:2000, ab73733, Abcam Inc.,
Cambridge, MA, U.S.A.) and PI3K-p85 (1:1000, ab191606, Abcam Inc., Cambridge, MA, U.S.A.). Afterward, the
membrane was washed with Tris Buffered saline Tween (TBST) three times (10 min for each), followed by incuba-
tion with goat anti-rabbit secondary antibody (ab6721, 1: 2000, Abcam Inc., Cambridge, U.S.A.) at room temperature
for 1 h. Subsequently, the membrane was developed with the addition of enhanced chemiluminescence (ECL), fixed
and analyzed. GAPDH was set as the internal reference protein to calculate the relative expression of proteins, which
was expressed as the ratio of the gray value of the target protein to the internal reference protein.
5-ethynyl-2 -deoxyuridine assay

After 48 h of transfection, the cells were inoculated into the 96-well plate with 2 × 103 to 4 × 104 cells in each well and
cultured to normal growth stage. Each well was added with 100 μl of 5-ethynyl-2 -deoxyuridine (EdU) solution and
incubated for 2 h. With the medium discarded, the cells were washed twice with PBS, added with cell fixative (100
μl/well), and incubated for 30 min at room temperature. After the addition of 2 mg/ml glycine (100 μl/well), the cells
were incubated for 5 min, washed with PBS for 5 min, followed by incubation with penetrant (PBS containing 0.5%
Triton X-100, 100 μl/well) for 10 min, and PBS washing. Following that, each well was added with 1× Apollo staining
reaction solution for incubation in the dark for 30 min. After that, the cells were added with penetrant, washed with
methanol, added with 100 μl of 1× Hoechst 33342 reaction solution and incubated for 30 min in the dark at room
temperature. Each well was added with 100 μl anti-fluorescence quenching mounting medium after staining. Cells
were observed under a fluorescence microscope with six to ten visual fields randomly selected per well and images
were captured.
Scratch test
After 48 h of transfection, the cells were inoculated into a six-well plate with 5 × 105 cells in each well. When the
cell confluence reached nearly 90%, a thin wound was created along the center of each well using a sterile pipette
tip. After removal of the floating cells by PBS, the cells were continuously cultured with the addition of serum-free
medium. Images of the cells were captured at 0 and 24 h, respectively, and the cell migration distance was measured
using Image-Pro Plus Analysis software (Media Cybernetics Inc., Rockville, Maryland, U.S.A.) with the mean value
obtained.
Transwell assay
Matrigel (356234, Becton, Dickinson and Company, NJ, U.S.A.) was dissolved overnight at 4◦ C, diluted with the
serum-free medium at a ratio of 1:3, and added into the apical chamber of Transwell chamber at the density of 50
μl/well. Then the chamber was air-dried in an incubator for 4–5 h. The cells were diluted with serum-free medium,
and added to the apical chamber at the density rate of 1 × 105 cells/ml. The basolateral chamber was added with
culture medium containing 15% FBS. The number of cells that had transferred through the pores of the Matrigel to
the back of the chamber within 24 h was used as an index to evaluate its invasive ability. Cells transferred to the other
side of the membrane were observed and images were captured under the microscope (×200) with four visual fields
randomly selected, and the mean number of cells in each visual field was counted.
Xenograft tumor in nude mice

A total of 96 male specific pathogen-free (SPF) BALB/c nude mice (SCXK, Hubei, China, 2013) aging 4-week old
were allocated into eight groups with 12 mice in each: mimic-NC group, inhibitor-NC group, miR-30b-3p mimic
group, miR-30b-3p inhibitor group, RECK-NC group, pcDNA3-RECK group, pcDNA3-RECK + mimic NC group
and pcDNA3-RECK + miR-30b-3p mimic group. Nude mice in each group were inoculated subcutaneously with
transfected cells at 1 × 106 cells (200 μl) from the left and right thigh roots, reared in the same environment, and
observed once every 7 days. The length and width of the tumors were recorded in detail, with tumor volume calculated
Figure 1. miR-30b-3p is overexpressed in glioma cells

*, P<0.05, compared with the NHA cell line; the experiment was repeated three times; data among multiple groups were analyzed
by one-way ANOVA and expressed using mean + − SEM; Abbreviation: SEM, standard error of the mean.
according to the formula: volume = (length × width2 )/2. On the 35th day, the nude mice were killed, the tumor was
dissected and weighed and part of the tissue was taken for Western blot analysis.
Statistical analysis
The data were analyzed by SPSS 21.0 statistical software (IBM Corp. Armonk, N.Y., U.S.A.). The measured data
were expressed by mean + − standard deviation. Data between two groups were compared using the Student’s t test,
and comparison of data among multiple data was conducted using one-way analysis of variance (ANOVA). Data at
different time points were compared using repeated measurement ANOVA. The data normality test was performed
using the Kolmogorov-Smirnov method. The data in conformity with normal distribution among multiple groups
were compared by one-way ANOVA, and Tukey was used for post hoc test. The data with skewed distribution were
tested by nonparametric test Kruskal–Wallis with Dunn’s multiple comparison for data post hoc test. P<0.05 indicate
the difference is of statistical significance.
Results
miR-30b-3p is up-regulated in glioma cells
The level of miR-30b-3p in NHA cell line and SHG44, U251, U87 and A172 cell lines was detected using RT-qPCR.
The results (Figure 1) showed that compared with the NHA cell line, the expression of miR-30b-3p was increased in
SHG44, U251, U87 and A172 cell lines (all P<0.05), and U87 cell line exhibited the highest expression of miR-30b-3p
among the four glioma cell lines, so U87 cell line was selected for subsequent experiments. The above findings provide
evidence that glioma cells display elevated miR-30b-3p.
Down-regulated miR-30b-3p leads to inhibited proliferation, migration,

invasion and metastasis of glioma cells
With the purpose to explore the potential role of miR-30b-3p in glioma cells, the level of miR-30b-3p in each group
after transfection was detected using RT-qPCR, the cell viability of glioma cells by EdU assay, the migration ability of
glioma cells by scratch test and the invasion ability of glioma cells by Transwell. As shown in Figure 2A, miR-30b-3p
expression in the miR-30b-3p mimic group was significantly up-regulated compared with the mimic-NC group
(P<0.05). miR-30b-3p expression in the miR-30b-3p inhibitor group was significantly decreased than that in the
inhibitor-NC group (P<0.05). In addition, overexpression of miR-30b-3p increased cell proliferation, migration and
invasion, while inhibition of miR-30b-3p suppressed cell proliferation, migration and invasion (Figure 2B–D). Sub-
sequently, the expression of metastasis-related genes was evaluated using Western blot analysis. The results revealed
that the expression of MMP-2 and MMP-9 was greatly increased in the miR-30b-3p mimic group compared with
the mimic-NC group (P<0.05). However, with the addition of miR-30b-3p inhibitor, the expression of MMP-2 and
Figure 2. Down-regulated miR-30b-3p contributes to inhibited proliferation, migration and invasion of glioma cells
(A) Expression of miR-30b-3p after alteration of miR-30b-3p was detected by RT-qPCR; (B) the viability of glioma cells after alter-
ation of miR-30b-3p was detected by EdU assay (×200); (C) migration ability of glioma cells after alteration of miR-30b-3p was
detected by scratch test; (D) invasion ability of glioma cells after alteration of miR-30b-3p was detected by Trasnwell assay (×100);
(E) expression of metastasis-associated genes was detected by Western blot analysis; *, P<0.05 compared with the mimic-NC
group; #, P<0.05 compared with the inhibitor-NC group; the experiment was repeated three times; the comparison among multiple
groups was analyzed by one-way ANOVA, and the data were expressed using mean + − SEM; Abbreviation: SEM, standard error of
the mean.
MMP-9 was decreased significantly (P<0.05; Figure 2E). All in all, the conclusion could be drawn that miR-30b-3p
reduction can contribute to repressed proliferation, migration and invasion abilities of glioma cells in vitro.
RECK is a direct target gene of miR-30b-3p

There was a specific binding region between the 3 UTR of RECK gene and the miR-30b-3p sequence, which was
predicted from the biological prediction site, microRNA.org, indicating RECK was the target gene of miR-30b-3p.
In order to confirm that RECK is a direct target gene of miR-30b-3p, dual luciferase reporter assay was performed,
whose results showed that compared with the NC group, the luciferase activity of RECK-Wt-3 UTR was inhibited by
miR-30b-3p (P<0.05), while the luciferase activity of the RECK-Mut-3 UTR was not suppressed (Figure 3A). Then,
RT-qPCR and Western blot analysis were performed to confirm the interaction between miR-30b-3p and RECK,
finding that mRNA and protein levels of RECK were decreased in glioma cells after overexpression of miR-30b-3p but
increased in glioma cells after depletion of miR-30b-3p (Figure 3B,C). These findings demonstrated that miR-30b-3p
could specifically bind to RECK and down-regulate its expression.
Figure 3. miR-30b-3p targets and down-regulates RECK

(A) Prediction of the binding site between miR-30b-3p and RECK 3 UTR and the target relationship between miR-30b-3p and
RECK detected by dual luciferase reporter assay; (B) RECK mRNA level after alteration of miR-30b-3p was detected by RT-qPCR;
(C) protein levels of RECK after alteration of miR-30b-3p was detected by Western blot analysis; *, P<0.05 compared with the
mimic-NC group; #, P<0.05 compared with the inhibitor-NC group; the experiment was repeated three times; the comparison
between two groups was analyzed by one-way ANOVA, and the data were expressed using mean + − SEM; Abbreviation: SEM,
standard error of the mean.
Figure 4. U87 cells transfected with pcDNA3-RECK plasmid exhibit overexpression of RECK
(A) Restriction endonuclease digestion of recombinant pcDNA3-RECK plasmid, wherein 1 is DNA Marker, 2 is empty plasmid
pcDNA3, 3 and 4 are recombinant plasmid pcDNA3-RECK and 5 is the result of double enzyme digestion of recombinant plasmid
pcDNA3-RECK; (B) the expression of RECK in U87 cells transfected with pcDNA3-RECK plasmid was detected by RT-qPCR; *,
P<0.05 compared with the RECK NC group; the experiment was repeated three times, and the comparison between groups was
analyzed by one-way ANOVA, and the data were expressed using mean +
− SEM; Abbreviation: SEM, standard error of the mean.
RECK is up-regulated in U87 cells transfected with pcDNA3-RECK

plasmid
The recombinant pcDNA3-RECK plasmid was transformed into DH5α competent cells. Positive clones were picked
for amplification culture and double enzyme digestion using KpnI and NotI with bacterial fluid as the template.
Agarose gel electrophoresis showed that two fragments of 5.4 and 4.4 kb were excised, and the results suggest
that (Figure 4) the recombinant pcDNA3-RECK plasmid was successfully constructed. Compared with the RECK
Figure 5. miR-30b-3p down-regulation suppresses proliferation, migration and invasion of glioma cells by enhancing RECK
expression
(A) Viability of glioma cells after alteration of miR-30b-3p and RECK was detected by EdU assay (×200); (B) migration ability
of glioma cells after alteration of miR-30b-3p and RECK was detected by scratch test; (C) invasion ability of glioma cells after
alteration of miR-30b-3p and RECK was detected by Trasnwell assay (×200); (D) protein levels of metastasis-associated genes
after alteration of miR-30b-3p and RECK was detected by Western blot analysis; *, P<0.05 compared with the RECK NC group;
#, P<0.05 compared with the pcDNA3-RECK + mimic-NC group; the experiment was repeated three times, and the comparison
among multiple groups was analyzed by one-way ANOVA; the data were expressed using mean + − SEM; Abbreviation: SEM,
standard error of the mean.
NC group, the expression of RECK in U87 cells transfected with pcDNA3-RECK plasmid was obviously elevated
(P<0.05).
Depletion of miR-30b-3p suppresses proliferation, migration and invasion

of glioma cells by elevating RECK
To investigate the regulatory role of miR-30b-3p in glioma cell biological processes with the involvement of RECK,
glioma cells were treated with pcDNA3-RECK and miR-30b-3p mimic. Results of EdU assay showed that compared
with the RECK NC group, overexpression of RECK inhibited the viability of glioma cells, while transfection of both
overexpressed RECK and overexpressed miR-30b-3p at the same time restored viability of glioma cells (Figure 5A).
The migration ability was detected using the scratch test, and it was shown that overexpressed RECK led to re-
pressed migration of glioma cells in comparison with the RECK NC group, while overexpression of both RECK
and miR-30b-3p rescued migration ability of glioma cells (Figure 5B). Subsequently, Transwell assay was used to de-
tect invasion ability of glioma cells. The results suggested that when compared with the RECK NC group, invasion
of glioma cells was repressed with the overexpression of RECK, which was rescued with the transfection of both
overexpressed RECK and overexpressed miR-30b-3p (Figure 5C). Next, the expression of metastasis-related genes
was determined using Western blot analysis. It was also found that when overexpressing RECK, the protein levels of
MMP-2 and MMP-9 were remarkably reduced. However, with overexpression of both RECK and miR-30b-3p, protein
levels of MMP-2 and MMP-9 were markedly elevated in contrast to that of the pcDNA3-RECK + mimic NC group
(Figure 5D). Taken together, miR-30b-3p down-regulation can result in up-regulation of RECK, thus repressing the
proliferation, migration and invasion of glioma cells.
miR-30b-3p inhibition elevates RECK to suppress tumorigenesis in

glioma cells in vivo
The above in vitro findings were further confirmed in xenograft tumor in nude mice. The results (Figure 6A–C)
indicated that the size of tumor was increased obviously and tumor growth rate was the fastest in mice with enforced
miR-30b-3p; while overexpression of RECK or down-regulation of miR-30b-3p resulted in smaller tumor size and
reduced tumor growth rate. However, the tumor size and growth rate were restored in mice with overexpression of
both RECK and miR-30b-3p. Afterward, the expression of metastasis-associated gene and the extent of AKT phos-
phorylation was detected by Western blot analysis; and the results showed that (Figure 6D) after overexpression of
miR-30b-3p, the expression of MMP-2 and MMP-9 as well as the extent of AKT phosphorylation was enhanced
significantly, and the expression of RECK was dramatically decreased, overexpressing RECK or down-regulating
miR-30b-3p contributed to decreased expression of MMP-2 and MMP-9 as well as the extent of AKT phosphory-
lation while increased expression of RECK. Cells transfected with overexpression of both RECK and miR-30b-3p
exhibited up-regulated MMP-2 and MMP-9 as well as the extent of AKT phosphorylation but down-regulated RECK
in comparison with the pcDNA3-RECK + mimic NC group. The expression of the total protein of AKT in each
group was almost consistent. Taken together, the poor expression of miR-30b-3p decreases the tumorigenic ability
and metastatic ability of glioma cells by increasing RECK.
Down-regulated miR-30b-3p inactivates the AKT signaling pathway by

up-regulating RECK in glioma cells
In addition, Western blot analysis was performed to measure the effect of miR-30b-3p and RECK on the AKT signal-
ing pathway-related factors AKT, p-AKT and PI3K-p85. The results showed that with medium added with DMSO or
with glioma cells as NC, it was found that extent of AKT phosphorylation and PI3K-p85 expression were reduced by
LY294002 (Figure 7A). And glioma cells simultaneously overexpressed with miR-30b-3p and added with exogenous
RECK exhibited no great difference of the total protein expression of AKT, while the extent of AKT phosphorylation
and PI3K-p85 was significantly down-regulated compared with cells overexpressed with miR-30b-3p only. How-
ever, when administrated with exogenous RECK followed by the addition of the inhibitor LY294002, glioma cells
displayed unchanged total protein expression of AKT and obviously down-regulated extent of AKT phosphoryla-
tion and PI3K-p85 in comparison with the cells added with the exogenous RECK alone (Figure 7B). It suggests that
miR-30b-3p down-regulates RECK to activate the AKT signaling pathway in glioma cells.
Discussion
As the most prevalent malignant brain cancer, glioma is graded to five levels with glioblastomas as the most malignant
[20]. Although a lot of progress in the treatment of glioma including surgical resection, radiation and chemotherapy
have been made in recent years, the results are not optimistic [21]. The primary barrier of the treatment of glioma
is believed to be the metastasis of glioma cells [22]. Thus, it is in urgent need to find out novel and effective treat-
ment approaches for glioma. The purpose of the present study was to determine the roles of miR-30b-3p, RECK and
the AKT signaling pathway in glioma and the findings of the present study demonstrated that down-regulation of
miR-30b-3p could inhibit proliferation, migration and invasion of glioma cells and tumor formation in nude mice
via the inactivation of the AKT signaling pathway by up-regulating the expression of RECK.
At first, our results provided evidence that the glioma cells exhibited down-regulated RECK but up-regulated
miR-30b-3p. RECK, which is considered an inhibitor of cancers, is often down-regulated in various malignant tu-
mors [23]. Consistently, the expression of RECK was found to be reduced when glioma enters a higher level [24].
Besides, both miR-30b-3p and miR-344b-3p were found to be highly expressed in nephropathy triggered by adri-
amycin [25]. More specifically, accumulating evidences suggested that there was an increase of miR-30b expression
in glioblastomas tissues, cell lines and TRAIL-resistant glioma cells, and patients with overexpression of miR-30b had
shortened survival time [26, 27]. Then, we further found out that RECK, as a target gene of miR-30b-3p, was nega-
tively correlated with the expression of miR-30b-3p. As shown in a previous study, it has been proved that RECK is a
Figure 6. Inhibition of miR-30b-3p repressed tumorigenesis and metastasis in glioma cells in vivo by up-regulating RECK
(A) Tumor volume after alteration of miR-30b-3p and RECK; (B) tumor weight after alteration of miR-30b-3p and RECK on the 35th
day; (C) tumor size after alteration of miR-30b-3p and RECK on the 35th day; (D) protein expression of the metastasis-associated
genes and the extent of AKT phosphorylation after alteration of miR-30b-3p and RECK detected by Western blot analysis; *, P<0.05
compared with the mimic-NC group; #, P<0.05 compared with the inhibitor-NC group; and P<0.05 compared with the RECK NC
group; 䉭, P<0.05 compared with the pcDNA3-RECK + miR-30b-3p NC group; n=12; the comparison among multiple groups was
analyzed by one-way ANOVA, and comparison of data at different time points was analyzed using repeated measures analysis of
variance; the data were expressed using mean +
− SEM; Abbreviation: SEM, standard error of the mean.
Figure 7. miR-30b-3p activates the AKT signaling pathway in glioma cells via RECK down-regulation
(A) Protein expression of AKT signaling pathway-related genes of glioma cells after interference of LY294002 by Western blot anal-
ysis. (B) Protein expression of AKT signaling pathway-related genes of glioma cells after interference of miR-30b-3p and RECK
by Western blot analysis; *, P<0.05; the experiment was repeated three times; the comparison among multiple groups was ana-
lyzed by one-way ANOVA and the data were expressed using mean + − SEM; AKT, Phosphoinositide 3-Kinase/Protein Kinase B;
Abbreviation: SEM, standard error of the mean.
target gene of miR-21, and miR-21 can modulate invasion of glioma cells via regulation of RECK [28]. Moreover, it
has also been found that miR-30b-3p can target the Rho GTPase-activating protein 26 gene [29].
In addition, our study also found that down-regulation of miR-30b-3p could suppress proliferation, migration
and invasion of glioma cells and tumor formation in nude mice through inactivation of AKT signaling pathway
by increasing the expression of RECK, which was indicated by reduced MMP-2, MMP-9 and p-AKT. A previous
study revealed that the levels of p-AKT and phosphorylated S6 ribosomal protein were increased in higher level of
gliomas [30]. Moreover, it has also been proved that when p-AKT was up-regulated by SPARC, the survival of glioma
cells can be promoted [31]. With down-regulation of miR-17-92, AKT level can be decreased in human mantle cell
lymphoma cells [32]. Besides, AKT was found to be closely related to many signaling pathways which play a vital role
in cell proliferation [33]. And as shown in a previous study, MMP-2 and MMP-9 are matrix metalloproteinases which
are associated with the progression, invasion and metastasis of malignant tumors [34]. More specifically, previous
findings suggest that knockdown of Proteolipid protein 2 can inhibit cell proliferation, migration and invasion of
glioma with decrease of MMP-2 and MMP-9 [35]. Furthermore, elevated expression of miR-30b found in renal cell
carcinoma (RCC) cells was demonstrated to promote RCC cell proliferation, invasion and migration but inhibit cell
apoptosis [36]. In line with our results, accumulating evidences show that RECK can inhibit tumor angiogenesis,
migration, invasion and metastasis in glioma [37–39].
© 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons 11
Attribution License 4.0 (CC BY).
Conclusions
In summary, the current study came to the conclusion that down-regulation of miR-30b-3p inhibited the proliferation,
migration and invasion of glioma cells and tumor formation in nude mice by inactivating the AKT signaling pathway
through up-regulation of RECK. These findings indicate miR-30b-3p as potential biomarkers for treatment of glioma.
Acknowledgments
We acknowledge and appreciate our colleagues for their valuable efforts and comments in the present paper.
Ethics statement
The present study was carried out strictly conforming to the recommendations in the Guide for the Care and Use of Laboratory
Animals, and was approved by the Animal Ethics Committee of The First Affiliated Hospital of Nanchang University.
Funding
The study was supported by Jiangxi Nature Science Fund [grant number 20114BAB205059] and Jiangxi Provincial Department of
Education Research Project [grant number GJJ12055].
Competing Interests
The authors declare that there are no competing interests associated with the manuscript.
Author Contribution
Yan Jian and Chun-Hua Xu designed the study. You-Ping Li and Bin Tang collated the data, carried out data analyses and pro-
duced the initial draft of the manuscript. She-Hao Xie and Er-Ming Zeng contributed to drafting the manuscript. All authors have
read and approved the final submitted manuscript.
Abbreviations
ANOVA, analysis of variance; BCA, bicinchoninic acid; cDNA, complementary DNA; CST, Cell Signaling Technology;
DEPC, diethyl phosphorocyanidate; DMEM, Dulbecco’s modified eagle medium; DMSO, dimethyl sulfoxide; EdU,
5-ethynyl-2 -deoxyuridine; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embry-
onic kidney; MEM, minimum essential medium; miRNAs, microRNA; miR-30b-3p, microRNA-30b-3p; miR-30, microRNA-30;
MMP, matrix metalloproteinase; Mut, mutant type; NC, negative control; NHA, normal human astrocytes; PBS, phosphate
buffer saline; PCR, polymerase chain reaction; PLB, passive lysis buffer; PVDF, polyvinylidene fluoride; RCC, renal cell car-
cinoma; RECK, reversion-inducing cysteine-rich protein with kazal motif; RT-qPCR, reverse transcription-quantitative PCR;
SDS/PAGE, sodium dodecyl sulfate/polyacrylamide gel electrophoresis; SPF, specific pathogen-free; TBST, Tris-buffered saline
Tween; TRAIL, tumor necrosis factor-alpha-related apoptosis-inducing ligand; Wt, wild-type.
References
1 Pereira, S., Pinto, A., Alves, V. and Silva, C.A. (2016) Brain tumor segmentation using convolutional neural networks in MRI images. IEEE Trans. Med.
Imaging 35, 1240–1251, https://doi.org/10.1109/TMI.2016.2538465
2 Omuro, A. and DeAngelis, L.M. (2013) Glioblastoma and other malignant gliomas: a clinical review. JAMA 310, 1842–1850,
https://doi.org/10.1001/jama.2013.280319
3 Seliger, C., Luber, C., Gerken, M., Schaertl, J., Proescholdt, M., Riemenschneider, M.J. et al. (2018) Use of metformin and survival of patients with
high-grade glioma. Int. J. Cancer 144, 273–280
4 Diamandis, P. and Aldape, K.D. (2017) Insights from molecular profiling of adult glioma. J. Clin. Oncol. 35, 2386–2393,
https://doi.org/10.1200/JCO.2017.73.9516
5 Tong, B., Pantazopoulou, V., Johansson, E. and Pietras, A. (2018) The p75 neurotrophin receptor enhances HIF-dependent signaling in glioma. Exp. Cell
Res. 371, 122–129, https://doi.org/10.1016/j.yexcr.2018.08.002
6 Goodenberger, M.L. and Jenkins, R.B. (2012) Genetics of adult glioma. Cancer Genet. 205, 613–621,
https://doi.org/10.1016/j.cancergen.2012.10.009
7 Zhou, Q., Liu, J., Quan, J., Liu, W., Tan, H. and Li, W. (2018) lncRNAs as potential molecular biomarkers for the clinicopathology and prognosis of
glioma: a systematic review and meta-analysis. Gene 668, 77–86, https://doi.org/10.1016/j.gene.2018.05.054
8 Kalla, R., Ventham, N.T., Kennedy, N.A., Quintana, J.F., Nimmo, E.R., Buck, A.H. et al. (2015) MicroRNAs: new players in IBD. Gut 64, 504–517,
https://doi.org/10.1136/gutjnl-2014-307891
9 Rohan, T., Ye, K., Wang, Y., Glass, A.G., Ginsberg, M. and Loudig, O. (2018) MicroRNA expression in benign breast tissue and risk of subsequent
invasive breast cancer. PLoS ONE 13, e0191814, https://doi.org/10.1371/journal.pone.0191814
10 Ye, X., Wei, W., Zhang, Z., He, C., Yang, R., Zhang, J. et al. (2017) Identification of microRNAs associated with glioma diagnosis and prognosis.
Oncotarget 8, 26394–26403
12 © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons
Attribution License 4.0 (CC BY).
11 Duan, Z.Q., Shi, J.D., Wu, M.N., Hu, N.Z. and Hu, Y.Z. (2016) Influence of miR-30b regulating humoral immune response by genetic difference.
Immunol. Res. 64, 181–190, https://doi.org/10.1007/s12026-015-8736-z
12 Xu, G. and Li, J.Y. (2016) Differential expression of PDGFRB and EGFR in microvascular proliferation in glioblastoma. Tumour Biol. 37, 10577–10586,
https://doi.org/10.1007/s13277-016-4968-3
13 Cho, C., Smallwood, P.M. and Nathans, J. (2017) Reck and Gpr124 are essential receptor cofactors for Wnt7a/Wnt7b-specific signaling in mammalian
CNS angiogenesis and blood-brain barrier regulation. Neuron 95, 1056–1073.e1055, https://doi.org/10.1016/j.neuron.2017.08.032
14 Liu, Y., Li, L., Liu, Y., Geng, P., Li, G., Yang, Y. et al. (2018) RECK inhibits cervical cancer cell migration and invasion by promoting p53 signaling
pathway. J. Cell. Biochem. 119, 3058–3066, https://doi.org/10.1002/jcb.26441
15 Rahmah, N.N., Sakai, K., Sano, K. and Hongo, K. (2012) Expression of RECK in endothelial cells of glioma: comparison with CD34 and VEGF
expressions. J. Neurooncol. 107, 559–564, https://doi.org/10.1007/s11060-011-0778-z
16 Fayard, E., Xue, G., Parcellier, A., Bozulic, L. and Hemmings, B.A. (2010) Protein kinase B (PKB/Akt), a key mediator of the PI3K signaling pathway. Curr.
Top. Microbiol. Immunol. 346, 31–56
17 Agarwal, N.K., Qu, C., Kunkalla, K., Liu, Y. and Vega, F. (2013) Transcriptional regulation of serine/threonine protein kinase (AKT) genes by
glioma-associated oncogene homolog 1. J. Biol. Chem. 288, 15390–15401, https://doi.org/10.1074/jbc.M112.425249
18 Chaudhuri, S., Singh, M.K., Bhattacharya, D., Datta, A., Hazra, I., Mondal, S. et al. (2018) T11TS immunotherapy repairs PI3K-AKT signaling in T-cells:
clues toward enhanced T-cell survival in rat glioma model. J. Cell. Physiol. 233, 759–770, https://doi.org/10.1002/jcp.26047
19 Xu, J., Lu, M.X., Cui, Y.D. and Du, Y.Z. (2017) Selection and evaluation of reference genes for expression analysis using qRT-PCR in Chilo suppressalis
(Lepidoptera: Pyralidae). J. Econ. Entomol. 110, 683–691
20 Sumiyoshi, K., Koso, H. and Watanabe, S. (2018) Spontaneous development of intratumoral heterogeneity in a transposon-induced mouse model of
glioma. Cancer Sci. 109, 1513–1523, https://doi.org/10.1111/cas.13579
21 Yu, F., Asghar, S., Zhang, M., Zhang, J., Ping, Q. and Xiao, Y. (2018) Local strategies and delivery systems for the treatment of malignant gliomas. J.
Drug Target. 27, 367–378
22 Lin, Y. and Wu, Z. (2018) MicroRNA-128 inhibits proliferation and invasion of glioma cells by targeting COX-2. Gene 658, 63–69,
https://doi.org/10.1016/j.gene.2018.03.020
23 Chen, Y. and Tseng, S.H. (2012) The potential of RECK inducers as antitumor agents for glioma. Anticancer Res. 32, 2991–2998
24 Chen, Y., Tsai, Y.H. and Tseng, S.H. (2012) Valproic acid affected the survival and invasiveness of human glioma cells through diverse mechanisms. J.
Neurooncol. 109, 23–33, https://doi.org/10.1007/s11060-012-0871-y
25 Jiang, C.B., Wei, M.G., Tu, Y., Zhu, H., Li, C.Q., Jing, W.M. et al. (2015) Triptolide attenuates podocyte injury by regulating expression of miRNA-344b-3p
and miRNA-30b-3p in rats with adriamycin-induced nephropathy. Evid. Based Complement. Alternat. Med. 2015, 107814,
https://doi.org/10.1155/2015/107814
26 Li, Z., Guo, J., Ma, Y., Zhang, L. and Lin, Z. (2018) Oncogenic role of MicroRNA-30b-5p in glioblastoma through targeting proline-rich transmembrane
protein 2. Oncol. Res. 26, 219–230, https://doi.org/10.3727/096504017X14944585873659
27 Quintavalle, C., Donnarumma, E., Iaboni, M., Roscigno, G., Garofalo, M., Romano, G. et al. (2013) Effect of miR-21 and miR-30b/c on TRAIL-induced
apoptosis in glioma cells. Oncogene 32, 4001–4008, https://doi.org/10.1038/onc.2012.410
28 Han, L., Yue, X., Zhou, X., Lan, F.M., You, G., Zhang, W. et al. (2012) MicroRNA-21 expression is regulated by beta-catenin/STAT3 pathway and
promotes glioma cell invasion by direct targeting RECK. CNS Neurosci. Ther. 18, 573–583, https://doi.org/10.1111/j.1755-5949.2012.00344.x
29 Wang, Q., Hui, H., Guo, Z., Zhang, W., Hu, Y., He, T. et al. (2013) ADAR1 regulates ARHGAP26 gene expression through RNA editing by disrupting
miR-30b-3p and miR-573 binding. RNA 19, 1525–1536, https://doi.org/10.1261/rna.041533.113
30 Yang, J., Liao, D., Wang, Z., Liu, F. and Wu, G. (2011) Mammalian target of rapamycin signaling pathway contributes to glioma progression and
patients’ prognosis. J. Surg. Res. 168, 97–102, https://doi.org/10.1016/j.jss.2009.06.025
31 Schultz, C.R., Golembieski, W.A., King, D.A., Brown, S.L., Brodie, C. and Rempel, S.A. (2012) Inhibition of HSP27 alone or in combination with pAKT
inhibition as therapeutic approaches to target SPARC-induced glioma cell survival. Mol. Cancer 11, 20, https://doi.org/10.1186/1476-4598-11-20
32 Jiang, P., Rao, E.Y., Meng, N., Zhao, Y. and Wang, J.J. (2010) MicroRNA-17-92 significantly enhances radioresistance in human mantle cell lymphoma
cells. Radiat. Oncol. 5, 100, https://doi.org/10.1186/1748-717X-5-100
33 Chen, A., Xiong, L.J., Tong, Y. and Mao, M. (2013) Neuroprotective effect of brain-derived neurotrophic factor mediated by autophagy through the
PI3K/Akt/mTOR pathway. Mol. Med. Rep. 8, 1011–1016, https://doi.org/10.3892/mmr.2013.1628
34 Huang, C.F., Teng, Y.H., Lu, F.J., Hsu, W.H., Lin, C.L., Hung, C.C. et al. (2017) beta-mangostin suppresses human hepatocellular carcinoma cell invasion
through inhibition of MMP-2 and MMP-9 expression and activating the ERK and JNK pathways. Environ. Toxicol. 32, 2360–2370,
https://doi.org/10.1002/tox.22449
35 Chen, Y.H., Hueng, D.Y. and Tsai, W.C. (2018) Proteolipid protein 2 overexpression indicates aggressive tumor behavior and adverse prognosis in human
gliomas. Int. J. Mol. Sci. 19, pii: E3353, https://doi.org/10.3390/ijms19113353
36 Jin, L., Li, Y., He, T., Hu, J., Liu, J., Chen, M. et al. (2017) Identification of miR30b as an oncogene in renal cell carcinoma. Mol. Med. Rep. 15,
1837–1846, https://doi.org/10.3892/mmr.2017.6197
37 Rahmah, N.N., Sakai, K., Li, Y., Sano, K. and Hongo, K. (2012) Comparison of manual and digital microvascular density counting of RECK expression in
glioma. Neuropathology 32, 245–251, https://doi.org/10.1111/j.1440-1789.2011.01265.x
38 Zhang, C., Ling, Y., Zhang, C., Xu, Y., Gao, L., Li, R. et al. (2012) The silencing of RECK gene is associated with promoter hypermethylation and poor
survival in hepatocellular carcinoma. Int. J. Biol. Sci. 8, 451–458, https://doi.org/10.7150/ijbs.4038
39 Lee, H.N., Mitra, M., Bosompra, O., Corney, D.C., Johnson, E.L., Rashed, N. et al. (2018) RECK isoforms have opposing effects on cell migration. Mol.
Biol. Cell 29, 1825–1838, https://doi.org/10.1091/mbc.E17-12-0708

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Bioscience Reports (2019) 39 BSR20182226

Down-regulated microRNA-30b-3p inhibits

Materials and methods

Dual luciferase reporter gene assay

Cell grouping and transfection

Table 1 Sequences of transfected fragment

Target segments Sequences

Abbreviation: NC, negative control.

Table 2 Primer sequences for RT-qPCR

Abbreviations: F, forward; R, reverse.

Reverse transcription-quantitative polymerase chain reaction

Western blot analysis

5-ethynyl-2 -deoxyuridine assay

Xenograft tumor in nude mice

Figure 1. miR-30b-3p is overexpressed in glioma cells

Down-regulated miR-30b-3p leads to inhibited proliferation, migration,

RECK is a direct target gene of miR-30b-3p

Figure 3. miR-30b-3p targets and down-regulates RECK

RECK is up-regulated in U87 cells transfected with pcDNA3-RECK

Depletion of miR-30b-3p suppresses proliferation, migration and invasion

miR-30b-3p inhibition elevates RECK to suppress tumorigenesis in

Down-regulated miR-30b-3p inactivates the AKT signaling pathway by

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