Human glioma cell lines U-87MG (ATCC HTB-14 (RRID: CVCL_0022, glioblastoma of unknown origin), U251 (The Cell Bank of Type Culture Collection of Chinese Academy of Sciences; cat. no. TCHu 58), T98G [American Type Culture Collection (ATCC); cat. no. CRL-1690] and SNB19 (ATCC; cat. no. CRL-2219) and normal human astrocytes (NHAs; ScienCell Research Laboratories, Inc.; cat. no. 1820) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco™, 10564011) supplemented with 10% fetal bovine serum (FBS, Gibco™, 10099141) (both from Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37°C under a 5% CO₂ atmosphere. Glioma specimens were collected from 39 patients following prior approval and written informed consent. Ten normal brain tissue samples used as controls were collected by donations from individuals who died in traffic accidents. All clinical samples were collected and histologically examined by pathologists from July 2016 to May 2018 at The Second Affiliated Hospital of Harbin Medical University. The present study was approved by The Ethics Committee of The Second Affiliated Hospital of Harbin Medical University. Written informed consent was obtained from all enrolled subjects.

The miR-342 mimics, inhibitor and their corresponding miRNA negative control (miR-NC) were chemically synthesized by GenePharma (Shanghai, China) and transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Overexpression or knockdown plasmids were transfected using Lipofectamine 2000 following the manufacturer's instructions.

The longest transcript human genes of GPRC5A (NCBI Reference Sequence: NM_003979.3) were cloned into pcDNA 3.1 plasmids and then sequenced for validation. The siRNA duplexes targeting GPRC5A were obtained from Invitrogen; Thermo Fisher Scientific, Inc. In order to knock down GPRC5A expression in U87 cells, subconfluently cultured U87 cells were transfected with GPRC5A shRNA, or negative control shRNA using the RNAiMAX transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. GPRC5A expression was assessed following 3 days. The shRNAs were designed by Invitrogen (Invitrogen; Thermo Fisher Scientific, Inc.) and cloned using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Stable population transfection was obtained following selection with 1 µg/ml G418 (Amresco, LLC, Solon, OH, USA) for 2 weeks. All shRNA oligos were obtained from Invitrogen; Thermo Fisher Scientific, Inc.

Trizol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for total RNA extraction. Six microliters of the extracted RNA was reverse transcribed using the PrimeScript™ RT reagent kit with gDNA Eraser (Takara Bio, Inc., Otsu, Japan) according to the provider's protocol. Quantitative PCR was performed using SYBR^® Green Real-Time PCR Master Mix (Takara) in the StepOnePlus Real-Time System (Applied Biosystems™ ABI Prism 7500 Fast; Thermo Fisher Scientific, Inc.). The sequences of primers used were: GPRC5A forward 5′-CGCCACAAAGCAACGAA-3′ and reverse primer 5′-ATAGAGCGTGTCCCCTGTCT-3′; GAPDH forward 5′-GAAAGCCTGCCGGTGACTAA-3′ and reverse primer 5′-GCATCACCCGGAGGAGAAAT-3′; U6 small nuclear RNA forward 5′-CTCGCTTCGGCGCACA-3′ and reverse primer: 5′-AACGCTTCACGAATTTGCGT-3′; miR-342 forward 5′-AGGTGAGGGGTGCTATCTGT-3′ and reverse primer 5′-GGGTGCGATTTCTGTGTGAG-3′. All the samples were amplified in triplicate and each experiment was repeated three times. The conditions for the real-time fluorescent quantitative PCR were: 1 cycle at 95°C for 5 min in the holding stage; 40 cycles at 95°C for 15 sec and 60°C for 60 sec in the cycling stage; 1 cycle at 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec in the melt curve stage. Thermal cycling and real-time detection were conducted using the StepOnePlus Real-Time PCR Systems (ABI, Thermo Fisher Scientific, Inc.). The quantities of each mRNA were calculated using the comparative (2^−ΔΔCq) method (12).

Protein was isolated from the cells and tissues with RIPA lysis buffer containing 1% protease inhibitor cocktails (Pierce Biotechnology, Inc.; Thermo Fisher Scientific, Inc.). After sample buffer was added to the proteins (each well, 30 µg per sample), proteins were boiled at 95°C for 10 min. Then, the proteins were separated using 10% polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to polyvinylidene fluoride (PVDF) membranes with 100 V transfer-molded voltage lasting for 45 to 70 min. After determination of the protein concentration, primary antibodies for western blotting were applied which included anti-GPRC5A (dilution, 1:1,000; PAB14597; Abnova, Taipei, Taiwan) and anti-GAPDH (dilution, 1:2,000; ab8245; Abcam, Cambridge, UK). HRP-conjugated IgG (1:5,000) antibody was used as the secondary antibody. After which membranes were washed 3 times (5 min/time). Development was completed with chemiluminescence reagents. GAPDH was used as an internal reference. Bands were visualized with a Bio-Rad Gel Doc EZ imager (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The specific bands were visualized with a chemiluminescence system (Millipore), and then visualized with Quantity One software 4.6.2 (Bio-Rad Laboratories, Inc.).

Cells in the logarithmic growth phase were digested with trypsin and seeded on 96-well plates at 100 µl of medium containing 1×10⁴ cells per well. Then we measured the cell proliferation rate at 0, 24, 48, and 72 h after transfection. Ten microliters of CCK-8 reagent was added into each well following another 2-h incubation at 37°C. The absorbance value was determined by using the XT-96DJ ELISA analyzer at a wavelength of 490 nm.

Wild-type and mutated GPRC5A 3′-UTR containing the putative binding site of miR-342 were synthesized and sequenced. Cells were seeded in 24-well plates and transfected with reporter vectors together with miR-342 mimics, miR-342 inhibitor or the corresponding miR-NC. The firefly luciferase activity was measured and normalized to Renilla signals at 48 h post-transfection.

In total, 16 BALB/c male nude mice (specific pathogen-free grade; weight, 16–18 g; age, 4–6 weeks) were purchased from the Laboratory Animal Center of Harbin Medical University. Lentiviral-mediated stable GAPC5A, GAPC5A+miR-342, miR-342 mimic cells and miR-NC cells were resuspended in Hank's buffer and mixed with an equal volume of Matrigel (BD Biosciences) at a concentration of 5×10⁶ cells/ml. The cells were subcutaneously injected into the flanks of nude mice (n=4 in each group). Subsequently, the mice were maintained in a specific pathogen-free grade laboratory, under the following conditions: Controlled temperature, 23±2°C; humidity, 40–70%; 12-h light/dark cycle) at the Laboratory Animal Center in our hospital with ad libitum access to food and water for 4 weeks. The volume of xenograft tumors was monitored every 3 days by measuring the length and width (Volume=length × width × width/2). The animal study was conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines, and was approved by the Experimental Animal Ethics Committee of The Second Affiliated Hospital of Harbin Medical University.

All values are expressed as mean ± SEM and were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test among groups using Statistical Product and Service Solutions (SPSS) (version 17.0) (SPSS, Inc., Chicago, IL, USA). Pearson's correlation analysis was performed to study the correlation between the expression of miR-342 and GPRC5A in cancer tissues. A P-value <0.05 was considered to indicate a statistically significant difference between groups.

Downregulation of miR-342 was observed in the glioma tissues (P<0.01; Fig. 1A) and cell lines (P<0.05; Fig. 1B) when compared with that noted in the normal human prostate tissues and the normal human astrocytes (NHAs). Meanwhile, the western blot results showed that the protein expression of GPRC5A was significantly upregulated in human glioma tissues (Fig. 1C) and cell lines (Fig. 1D). According to the results of RT-qPCR, GPRC5A expression in the U87 cell line was the highest, therefore, we chose this cell line for further experiments. The correlation analysis confirmed that the expression of miR-342 and GPRC5A was significantly negatively correlated (r=−0.4559, P=0.0035; Fig. 1E).

Next, we explored the potential role of miR-342 in glioma. The transfection efficiency was determined according to the level of miR-342 using RT-qPCR. As shown in Fig. 2A, a significantly increased expression of miR-342 was achieved after miR-342 mimic transfection and a significantly decreased expression of miR-342 was achieved after miR-342 inhibitor transfection. Moreover, upregulation of miR-342 resulted in greater suppression of cell proliferation than the control (mimics NC), whereas downregulation of miR-342 promoted cell proliferation (Fig. 2B) as determined using the CCK-8 assay. These results indicated that miR-342 inhibited the proliferation of U87 and U251 cells and downregulation of miR-342 promoted the proliferation of cells.

In order to determine the mechanism of miR-342 in cell proliferation, G-protein coupled receptor family C group 5 member A (GPRC5A) was found to be a putative target of miR-342 (Fig. 3A). At the protein and mRNA levels, overexpression or knockdown of miR-342 resulted in a significant decrease or increase in the expression level of GPRC5A, respectively (Fig. 3B and C). In addition, luciferase reporter assays were performed to ascertain whether miR-342 targets GPRC5A by binding to its 3′UTR. The results from the luciferase reporter assay indicated that upregulated expression of miR-342 significantly inhibited the activity of the reporter gene, whereas miR-342 inhibitor significantly increased it (Fig. 3D). The results indicate that GPRC5A is a direct target of miR-342.

In order to investigate the cellular function of GPRC5A, the expression level of GPRC5A in U87 cells was manipulated by transfection with an overexpression (OE) or shRNA-mediated knockdown plasmids. The mRNA and protein levels of GPRC5A were determined in the transfected cells for which the expression levels of GPRC5A were markedly increased in the presence of overexpression plasmids or decreased in the absence of plasmids or silenced by shRNA. The inhibitory effect of each shRNA (sh1, sh2, sh3) was not significantly different while sh2 had the highest inhibition rate of GPRC5A, thus this shRNA was selected for further experiments (Fig. 4A and B). Moreover, CCK-8 assay results showed that overexpression of GPRC5A significantly promoted cell growth, while knockdown of GPRC5A suppressed it (Fig. 4C). The observation indicated GPRC5A expression associated with cell proliferation in vitro.

Based on the above results, it was proposed that miR-342 suppresses cell proliferation via up-regulation of GPRC5A. Considering the low expression level of miR-342, rescue experiments were performed by co-transfecting the miR-342 mimics with or without GPRC5A followed by determination of the cell proliferation of U87 cells. Growth curves obtained by CCK-8 assay showed that upregulation of the expression of miR-342 alone significantly inhibited cell growth whereas overexpression of GPRC5A alone significantly promoted cell growth; co-expression of GPRC5A with miR-342 abrogated the inhibitory effects of miR-342 mimics on the cell proliferation (Fig. 5A). Based on the in vitro data, the effect of miR-342 and GPRC5A on tumor growth was further evaluated in vivo. Lentivirus of miR-342 and GPRC5A were either used to infect cells alone or simultaneously. We found that tumor xenografts derived from cells infected with miR-342 alone were significantly smaller than the negative control while those infected with GPRC5A showed an opposite trend; co-infection of GPRC5A with miR-342 abrogated the inhibitory effects of miR-342 (Fig. 5B). The expression level of GPRC5A in tumor xenografts was also assessed. The expression level of GPRC5A was much lower in the miR-342 mimic group and higher in the GPRC5A overexpression or co-infected groups, compared with their counterparts in the negative control (NC) group (Fig. 5C). The in vitro and in vivo observations suggest that miR-342 targets the 3′UTR of GPRC5A directly and inhibits U87 cell proliferation via GPRC5A up-regulation.