Ginsenoside Rd Inhibits Glioblastoma Cell Proliferation by Up-Regulating the Expression of miR-144-5p

Abstract

miR-144-5p exhibits anti-tumor activities in various cancers. Although treatment for glioblastoma has progressed rapidly, novel targets for glioblastoma are insufficient, particularly those used in precision medicine. In the current study, we found that ginsenoside Rd reduced the proliferation and migration of glioblastoma cells. Ginsenoside Rd up-regulated the tumor-suppressive miR-144-5p in glioblastoma cells. Moreover, Toll-like receptor 2, which is a target of miR-144-5p, was down-regulated. After inhibition of miR-144-5p, the effect of Ginsenoside Rd on proliferation inhibition and down-regulation of Toll-like receptor 2 was reduced. These data demonstrated the ginsenoside Rd/miR-144-5p/Toll-like receptor 2 regulatory nexus that controls the glioblastoma pathogenesis of glioblastoma. Our work provided novel targets for glioblastoma diagnosis and treatment.

INTRODUCTION

In the human genome, miR-144-5p (passenger strand) resides in clustered microRNA (miRNA) sequences located within the 17q11.2 region. In recent years, the tumor-suppressive miRNA miR-144-5p has been found to exhibit anti-tumor activities by targeting cyclin E1/E2 (CCNE1/2) in bladder cancer, activating transcription factor 2 (ATF2) in non-small-cell lung cancer cells, syndecan-3 (SDC3) in renal cell carcinoma, the extracellular signal related kinase/MYC proto-oncogene (ERK/c-Myc) signaling pathway in ECa9706 esophageal cancer cells and neuronal calcium sensor 1 (NCS1) in advanced-stage lung squamous cell carcinoma.^1–5) The up-regulation of Toll-like receptor 2 (TLR2) is involved in the progression, migration, and poor clinical diagnosis of various cancers through multiple pathways.^6–9) In previous studies, TLR2 was validated as a target of miR-144-5p, and the expression of TLR2 can be inhibited by the overexpression of miR-144-5p.^10,11)

Ginsenoside Rd (GS-Rd) has shown strong anti-tumor activity through inhibiting epidermal growth factor receptor (EGFR) signaling, oxidative stress, and protein kinase B (Akt)/mammarian target of rapamycin kinase (mTOR)/ribosomal protein S6 kinase (p70S6K) (Akt/mTOR/p70S6K) signaling in various cancers.^12–15) Wang et al. first found that GS-Rd reduces breast cancer metastasis and that this mechanism of action implicates the down-regulation of SMAD family member 2 (Smad2) targeting by miR-18a.¹⁶⁾ This finding suggests a likely relationship between GS-Rd and miRNAs. Recently, Gu et al. confirmed that the Gs-Rd can remarkably inhibit the proliferation and promote cell apoptosis of human glioma U251 cells in vitro.¹⁷⁾

Therefore, the current study intends to explore the effect of GS-Rd on miR-144-5p targeting of TLR2 in human glioblastoma cells. Our findings would suggest that the GS-Rd/miR-144-5p/TLR2 axis may serve as a potential target for the treatment of glioblastoma.

MATERIALS AND METHODS

Cell and Drug

U251, H4 (HTB148), U87 MG (HTB-14, glioblastoma of unknown origin) cells and normal human astrocytes (NHA) were obtained from American Type Culture Collection (ATC C) (Manassas, VA, U.S.A.). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with 10% fetal bovine serum (ThermoFisher Gibco, Shanghai, China) in a humidified incubator at 37 °C and 5% CO₂. All works were performed in accordance with the ethical principles for medical research outlined in the Declaration of Helsinki 1964 and per subsequent revisions (https://www.wma.net/). GS-Rd (C₄₈H₈₂O₁₉, CAS No. 52705-93-8, Purity ≥98%) was purchased from ChemFaces (Wuhan, Hubei, China) and dissolved in ethanol at the stocking concentration of 500 µM for use, and its structure is shown in Fig. 1.

Fig. 1. Structure of Ginsenoside Rd

Cell Viability

Cells were seeded at a density of 1 × 10⁴ cells (100 µL medium/well) in 96-well culture plates (Corning Inc., Corning, NY, U.S.A.). After 12 h, GS-Rd was added at final concentrations of 0, 5, 10, 50, 100, and 200 µM. After 24 h, 100 µL of medium was refreshed. Then 10 µL of the CCK-8 solution (MCE, HY-K0301, Shanghai, China) was added to each well of the plate at 37 °C for 4 h. The absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific Inc., Fremont, CA, U.S.A.). A concentration of 0 µM served as the control. Relative cell activity = (The absorbance of drug experimental group/The absorbance of control groups) × 100%.

Cell Migration

Cell migration was detected using Transwell chambers with an 8.0 µm pore size (Corning Inc.). For cell migration, 1 × 10⁵ cells/mL were seeded into the upper chamber in 300 µL of serum-free RPMI1640 medium, while the lower chamber was filled with RPMI-1640 containing 10% fetal bovine serum. After a 24 h culture, the cells were fixed in with 4% polyoxymethylene and stained with crystal violet. Stained cells were counted under a microscope. Experiments were performed in triplicates.

Transfection

The miR-144-5p mimics (forward: 5′-ggauaucaucauauacuguaag-3′, reverse: 5′-gaaugucauauacuacuauagg-3′), miR-Negative control (miR-NC) (5′-uucuccgaacgugucacgutt-3′), antagomir-144-5p (inhibitor: 5′-gaaugucauauacuacuauagg-3′) and antagomir negative control (i-NC: 5′-gaaugucauauacuacuauagg-3′) were purchased from RiboBio Co. (Guangzhou, Guangdong, China). Cells were seeded in six-well plates at a density of 10⁵ cells/mL and were transfected with 80 nM miR-144-5p mimic, miR-NC, inhibitor (i-miR) or i-NC using Lipofectamine2000 Reagent (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s protocol (RiboBio Co., Guangzhou, Guangdong, China). After 6 h, the medium was refreshed and the cells were incubated for another 48 h. To overexpress TLR2, cells were transfected with pcDNA3.1-LTR2 (Sigma-Aldrich, St. Louis, MO, U.S.A.) using Lipofectamine2000 Reagent, with pcDNA3.1-vector as the negative control.

Dual-Luciferase Reporter Assay

Potential targets of miR-144-5p were searched using with TargetScan (http://www.targetscan.org/), which revealed that TLR2 is a target of miR-144-5p. To verify TLR2 as a target of miR-144-5p, the wild-type sequence (5′-ugcaggauccucguggauaucaa-3′) at position 302–308 of the TLR2 3′UTR targeted by miR-144-5p (3′-gaaugucauauacuacuauagg-5′) was inserted into the Xhol-HindIII site of the pGL3 vector (Promega, Madison, WI, U.S.A.). Cells were cultured in 24-well plates and co-transfected with a miR-144-5p mimic, 250 ng of the constructed pGL3-TLR2-3′UTR vector, and 50 ng of pRL-TK inner control plasmids using Lipofectamine2000 transfection reagent (Invitrogen, Carlsbad, CA, U.S.A.). Cell lysates were collected 48 h after transfection and the firefly and renilla luciferase activities were measured using a Dual-Luciferase Reporter Assay kit according to the manufacturer’s protocol using a GLO-MAX 20/20 Luminometer (Promega, Shanghai, China). The relative renilla luciferase activity was then obtained.

Real-Time Quantitative PCR (RT-qPCR)

Cells were treated with drugs for 49 h. Total RNA was extracted from cells using the TRIzol reagent (Ambion, Thermo Fisher Scientific, Inc.). Then, to detect the miR-144-5p level, RT-qPCR was performed using a mirVana miRNA Isolation Kit (Ambion, Thermo Fisher Scientific, Inc.) and an EzOmics miRNA Q-PCR Detection Kit with stem-loop primer sets (Biomics Tech Inc., Nantong, Jiangsu, China). Briefly, small RNAs were isolated, after which 20 ng of small RNAs was reverse transcribed to cDNA under the following reaction conditions: 37 °C for 20 min and 95 °C for 10 min. The qPCR was performed in an Agilent Mx3000P PCR system (Agilent Technologies, Inc.) with the following amplification program: 95 °C for 10 min, 36 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 10 s. The qPCR primers sequences of miR-144-5p and U6 (Lot No. BK1010, Biomics Biotech Inc., Nantong, Jiangsu, China) were miR-144-5p, 5′-tac agt ctc tgg atg ata tcc-3′ (forward) and 5′-atc cag tgc agg gtc cga gg-3′ (reverse); U6, 5′-ctc gct tcg gca gca cat-3′ (forward) and 5′-ttt gcg tgt cat cct tgc g-3′ (reverse). The miR-144-5p levels were normalized to those of the reference gene U6.

To perform RT-PCR to detect TLR2 mRNA, 50 ng of total RNAs was reverse transcribed to cDNA using a FastQuant RT kit (Beijing Tiangen Biotech Co., Ltd., Beijing, China) under the following reaction conditions: 42 °C for 10 min and 95 °C for 2 min. qPCR was performed in a CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Shanghai, China) with an Ace Q qPCR SYBR Green Master Mix Kit (Vazyme, Nanjing, Jiangsu, China) under the following amplification program 95 °C for 2 min, 36 cycles of 95 °C for 10 s and 60 °C for 30 s, and 72 °C for 1 min. The qPCR primers for TLR2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized by Sangon (Shanghai, China) and their sequences were TLR, 5′-3′ ctt cac tca gga gca gca agc a (forward), 5′-3′ aca cca gtg ctg tcc tgt gac a (reverse); GAPDH, 5′-3′ gtc tcc tct gac ttc aac agc g (forward), 5′-3′ acc acc ctg ttg ctg tag cca a (reverse). The TLR2 mRNA levels were normalized to those of GAPDH. The fold change in the expression of genes of interest gene expression was equal to 2^−ΔΔCT. All results were presented as the mean ± standard deviation (S.D.) of three independent experiments.

Western Blotting

Cells were treated with drugs for 49 h. Cellular protein was extracted with cold RIPA buffer (Beyotime, Nanjing, Jiangsu, China) containing protease and phosphatase inhibitors (Millipore, Bedford, MA, U.S.A.). Lysates were centrifugated at 14000 rcf at 4 °C for 15 min. The protein concentration was detected using a bicinchoninic acid (BCA) assay (Thermo Fisher, Shanghai, China). Aliquots of protein (20 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after which the separated proteins were transferred onto 0.45 µm polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific). Membranes were blocked with 5% (w/v) non-fat milk in TBST (20 mM Tris–HCl, 150 mM NaCl, and 0.1% Tween 20, pH 7.5) and incubated with a rabbit polyclonal antibody to TLR2 (BS7380, Antibodies, Cambridge, U.K.) overnight at 4 °C. Subsequently, membranes were washed with TBST and incubated with horseradish peroxidase-labeled secondary antibody (ab6721, Abcam, U.K., 1 : 1000 diluted) at room temperature for 1 h. Membranes were then washed in TBST. The protein bands were detected using an ECL chemiluminescence system (Amersham, Shanghai, China) with filming. Blots were visualized and quantified using a UVP Imaging System (UVP, Inc., Upland, CA, U.S.A.).

Xenograft Tumor Growth

To analyze tumor growth in vivo, U251 cells were suspended at the concentration of 1 × 10⁷/mL. Then, 200 µL of cells were injected subcutaneously into the forearm armpits of 40 female 8-week-old nude mice (Laboratory Animal Center of Jilin University, Changchun, Jilin, China). The diet, spirit, activity, and tumor conditions of nude mice were observed every day with free access to food and water. As the tumors grew up to be palpable, GS-Rd was administered intragastrically at a dose of 60 g/kg/d. The longest axis and shortest axis of the tumors were measured every two days and then the volume size was computed using the formula of a  ×  b²/2 [the longest (a) and the shortest (b)]. On 13 d after GS-Rd was administered, the mice were anesthetized with isoflurane inhalation and sacrificed by cervical dislocation. The xenografts were removed and weighed. Total RNAs were then extracted from the xenografts. Animal experiments were approved by the Institutional Animal Care and Use Committee of Jilin University (No. 162012).

Statistical Analysis

All experiments were repeated at least three times. Data were presented as the mean ± S.D. Statistical analysis was performed using SPSS 20.0 (IBM SPSS, Chicago, IL, U.S.A.). Group differences were explored by one-way ANOVA with Dunnett’s post hoc test. p < 0.05 was considered to indicate a statistically significant difference.

RESULTS

Expression of miR-144-5p in Human Glioblastoma and Normal Cells

Before GS-Rd was administered to glioblastoma cells, the expression of miR-144-5p in human glioblastoma and normal cells was analyzed by RT-qPCR. The results showed that the miR-144-5p levels in the glioblastoma cells lines U251, U118, U87, and H4 were significantly down-regulated compared with those in normal human astrocytes (NHA) (p < 0.05 for each) (Fig. 2A). These data suggested that miR-144-5p may be down-regulated as a tumor suppressor in human glioblastoma. U251 and U87 cells exhibited a down-regulation of miR-144-5p of approximately 0.5-folds, but this was not observed in U118 and H4 cells.

Fig. 2. Levels of miR-144-5p in Glioblastoma Cells, the Anti-tumor Effect of Ginsenoside Rd and Dual-Luciferase Reporter System Assay

A, Levels of miR-144-5p in glioblastoma cells (U251, U118, H4 and U-87) and normal human astrocytes by RT-qPCR analysis. B, Ginsenoside Rd at 0, 100 and 200 µM was administered to U251 and U87 cells, and miR-144-5p was up-regulated in a dose-dependent manner. C, The CCK8 assay was performed to determine the viability of U251 and U87 cells. D, Representation of the seed region between miR-144-5p and the 3′-UTR of target gene TLR2 as predicted by TargetScan. The relative firefly/renilla luciferase activity was determined by a pGL3-TLR2 3′-UTR-wt/pRL-TK dual-luciferase reporter system to confirm the interaction between miR-144-5p and the 3′-UTR of TLR2 gene in U251 glioblastoma cells. * p < 0.05, vs. NC, n = 3.

U251 and U87 cells were used to explore the effect of GS-Rd on miR-144-5p regulation in glioblastoma. After U251 and U87 cells were exposed to GS-Rd at 0, 100, and 200 µM, respectively, miR-144-5p was up-regulated in a GS-Rd dose-dependent manner in these two kinds of cells (Fig. 2B). These data suggested that GS-Rd was able to up-regulate miR-144-5p.

Cell viability of U251 and U87 was determined by the CCK8 assay. The results showed that GS-Rd decreased the viability of glioblastoma cells in a dose-dependent manner, particular at 100 and 200 µM (p < 0.05 for each) (Fig. 2C). For this reason, GS-Rd was used at a concentration of 100 µM in subsequent experiments. Although the dose of cell experiments may not guide the clinical treatment completely, the current paper discussed the anti-tumor activity of GS-Rd.

miR-144-5p Targeted the 3′-UTR of TLR2 Gene

TargetScan predicted that the miR-144-5p sequence 3′-CUAUAG-5′ interferes with the 3′UTR sequence 5′-GAUAUC-3′ of TLR2 (Fig. 2D). The interaction of miR-144-5p and its target gene TLR2 was tested using the pGL3-TLR2-3′UTR/pRL-TK dual-luciferase reporter system. The results of the relative Firefly/Renilla luciferase activity assay are shown in Fig. 2D. The TLR2-associated luciferase activity was significantly reduced in the miR-144-5p mimic group significantly compared with the miR-NC control (p < 0.05). After the miR-144-5p inhibitor was transfected into glioblastoma cells, the TLR2-associated luciferase activity was apparently enhanced. These data indicated that miR-144-5p targeted TLR2 (Fig. 2D). The result was consistent with a previous study.¹¹⁾

Effect of GS-Rd and miR-144-5p on TLR2 Expression in U251 and U87 Cells

The GS-Rd/miR-144-5p/TLR2 nexus was investigated by RT-qPCR and Western blot in the following experiments. The TLR2 mRNA levels were found to be up-regulated in untreated U251 and U87 cells compared with normal human astrocytes (NHA) (Fig. 3A). Then U251 and U87 cells were treated with 100 µM of GS-Rd and miR-144-5p mimic, respectively. The results showed that both GS-Rd and miR mimic down-regulated the TLR2 mRNA levels (Fig. 3A). The miR-144-5p mimic increased miR-144-5p expression, the miR-144-5p inhibitor reduced miR-144-5p expression, and both inhibitor-NC and miR-NC had no impact on miR-144-5p expression (Fig. 3A). The abovementioned results suggested that the miR-144-5p targeted TLR2 (Fig. 2D), thus, when the cells were transfected with miR-144-5p inhibitor, the level of TLR2 was increased in both cells (Fig. 3B). This outcome illustrated the nexus of miR-144-5p and TLR2.

Fig. 3. TLR2 Expression as Detected by RT-qPCR and Western Blotting

A, The mRNA levels of TLR2 in normal human astrocytes (NHA), U251 and U87 glioma cells, ginsenoside Rd treated cells and miR-144-5p mimic treated cells. The miR-144-5p levels of mimic, inhibitor, miR NC and inhibitor NC. B, The mRNA levels of TLR2 in glioma cells treated with miR-144-5p inhibitor. C, The mRNA levels of TLR2 in ginsenoside Rd treated cells that are subsequently transfected with miR mimic or miR inhibitor. D, E, The protein levels of TLR2 in U251 cells and U87 cells, respectively; the i-miR is miR inhibitor, +TLR2 is TLR2 overexpression. F, G, The semi-quantitative analysis based on Western blot results. * p < 0.05. vs. NC, n = 3.

To investigate the relationship among TLR2, GS-Rd and miR-144-5p, three groups, Rd+ miR-144-5p (mimic)/miR-144-5p inhibitor/miR-144-5p NC were compared in GS-RD treated cells (Fig. 3C). After deprivation of miR-144-5p using a miR-inhibitor, TLR2 levels were up-regulated apparently (Fig. 3C). The Western blot was also performed to investigate the relationship among TLR2, GS-Rd, and miR-144-5p in U251 cells (Fig. 3D) and U87 cells (Fig. 3E). The results at protein levels were consistent with those at RNA levels. These results suggested that GS-Rd could act on TLR2 indirectly via miR-144-5p targeting TLR2. These data demonstrated the Rd/miR-144-5p/TLR2 regulatory nexus.

Cell Migration and Cell Viability

When cell migration was tested by Transwell assay, the results showed that overexpression of TLR2 significantly promoted the cell migration ability significantly compared with the normal control (p < 0.05). GS-Rd (100 µM) reduced the cell migration compared with the normal control (p < 0.05), and a miR-144-5p mimic further reduced the cell migration compared with the GS-Rd group (p < 0.05) (Figs. 4A, B). As the miR-144-5p was knocked down with an miR inhibitor (i-miR), the cell migration ability of the Rd + miR inhibitor (i-miR) group was restored to some degree compared with the GS-Rd alone group. This result suggested that the lack of miR-144-5p attenuated the effect of GS-Rd on glioblastoma cell death because the suppression of TLR2 was decreased. In addition, knockdown of miR-144-5p did not completely block the anti-tumor effect of GS-Rd, which suggests that GS-Rd would also exert its anti-tumor activity through other pathways in glioblastoma cells.

Fig. 4. Cell Migration and Viability of U251 and U87 Cells Were Assayed Using the Transwell and CCK-8 Methods

The i-miR is miR inhibitor, +TLR2 is TLR2 overexpression. * p < 0.05, vs. NC; ^# p < 0.05, vs. ginsenoside Rd, n = 3.

The cell viability of U251 and U87 cells were then assayed. The change in cell viability was consistent with the results of the cell migration experiment (Figs. 4A, B). These data suggested that TLR2 promoted glioblastoma cell viability and migration and that miR-144-5p inhibited glioblastoma cell migration and viability by down-regulating TLR2. This was confirmed by doing a rescue experiment by overexpressing TLR2 (+TLR2) in miR-144-5p treated cells (Figs. 4A, B). Thus, GS-Rd inhibited the viability and migration of U251 and U87 glioblastoma cells likely via the miR-144-5p/TLR2 nexus regulation.

GS-Rd Inhibited Xenograft Tumor Growth

Next, we explored the role of GS-Rd in xenografts growth. We inoculated the U251 cells into nude mice and measured the tumor volume sizes every two days. There was only one tumor per mouse. On 13 d, the volume of the largest tumor was 1072 mm³ for the NC group and 751 mm³ for the GS-Rd group (Fig. 5A). These mice were sacrificed at this time point and their tumor mass was removed and weighed (Fig. 5B). Our data showed that the addition of GS-Rd caused a significant decrease in tumor volume and weights (Figs. 5A, B), which indicates that GS-Rd can inhibit U251 cell proliferation in vivo. Moreover, we further analyzed the expression of miR-144-5p and TLR2 in the xenografts of nude mice. Interestingly, we found that miR-144-5p was significantly up-regulated and that TLR2 was significantly down-regulated in xenografts treated with GS-Rd (Figs. 5C, D).

Fig. 5. Nude Mouse Xenograft Experiment

A, The tumor volume was analyzed. B, The tumor weights were measured. C, The level of miR-144-5p in tumors was detected. D, RT-qPCR analysis was performed to examine the mRNA levels of TLR2 in tumors. * p < 0.05, vs. NC, n = 5.

Overall, our data revealed an anti-tumor effect of GS-Rd on U251 cells. In addition, we found that GS-Rd up-regulated the tumor suppressor miR-144-5p, which targets TLR2; TLR2 in turn controls the proliferation and migration of U251 cells (Fig. 6).

Fig. 6. The Mechanistic Explanation

By the regulatory nexus of ginsenoside Rd/miR-144-5p upregulation/TLR2 downregulation, ginsenoside Rd inhibited glioblastoma cell viability and metastasis.

DISCUSSION

Accumulating evidence confirms that the long non-coding RNA (lncRNA) HOXA transcript at the distal tip (HOTTIP) was aberrantly down-regulated in U87, U251, U118 glioblastoma cell lines.¹⁶⁾ Furthermore, deletions of 1p and 19q are particularly common in oligodendrogliomas. For instance, U87, U251, and H4 cell lines were found to have a deletion of 1p36.3 or 1p36.2 by loss of heterozygosity (LOH), comparative genomic hybridization to arrayed BAC (CGHa), and fluorescence in situ hybridization (FISH).¹⁸⁾ What particularly appealed to us is that miR-144-5p is a tumor suppressor that plays an important role in various cancers.^1–5) TLR2 promotes multiple cancers though the TLR2/MYD88 innate immune signal transduction adaptor (MyD88)/tumor necrosis factor (TNF) receptor associated factor 6 (TRAF66)/Jun proto-oncogene (Jun)/p38 MARK (p38)/mitogen-activated protein kinase (MAPK) (TLR2/MyD88/TRAF6/Jun/p38/MAPK) pathways.^6–9) The current study explored the ability of miR-144-5p to target TLR2 in glioblastoma cells after GS-Rd was administration. miR-144-5p was down-regulated in 4 types of glioblastoma cells (Fig. 2A), which implies that the tumor suppressor miR-144-5p was inhibited by some mechanism in glioblastoma. After U251 and U87 glioblastoma cells were exposed to GS-Rd, miR-144-5p was up-regulated in a dose-dependent manner (Figs. 3F, G), which indicates that GS-Rd promoted miR-144-5p by some unknown mechanism. The GS-Rd and miR-18a nexus have been reported in the literature,¹⁹⁾ But our results revealed a new GS-Rd/miR-144-5p nexus.

TargetScan predicted a target gene, TLR2, and the dual-luciferase reporter system was utilized to confirm the interaction between miR-144-5p and TLR2. Our results showed that miR-144-5p targeted TLR2 by binding to specific sequences (Fig. 2) to induce the down-regulation of TLR2, which in turn inhibited the promotive action of TLR2 on glioblastoma cells. These results were consistent with previous studies, which reported that TLR2 was a direct target of miR-144-5p and miR-144-5p down-regulated TLR2 expression.^10,11)

Herein, the GS-Rd/miR-144-5p/TLR2 axis was established. Subsequently, this axis was verified by TLR2 expression, as detected by Western blotting and RT-qPCR. Our data showed that TLR2 expression was up-regulated in glioblastoma cells, that GS-Rd to promoted miR-144-5p, which inhibited TLR2 expression, and that miR-144-5p increased the inhibitory action of GS-Rd on TLR2. Additionally, GS-Rd could not regulate TLR2 directly, but rather, GS-Rd regulated TLR2 indirectly via miR-144-5p (Fig. 3). The existing literature shows that GS is the main substance that exerts the pharmacological effects of ginseng. In rat neural stem cells cultured in vitro, GS-Rd significantly increased the size and number of neurospheres but did not affect the differentiation of neural stem cells into astrocytes and neurons.²⁰⁾ In addition, in cultured astrocyte cells, GS-Rd increased the expression of glutamate transporter (GTL-1) both in mRNA and protein levels, which can be inhibited by Akt or ERK1/2 inhibitor.²¹⁾ The mechanism of the GS-Rd/miR-144-5p/TLR2 axis was explained in Fig. 6.

miR-144-5p can suppress certain cancers as described in previous literatures^1–5) and GS-Rd promotes miR-144-5p in glioblastoma cells as our data shows. We expected that both GS-Rd and miR-144-5p would inhibit glioblastoma cell migration and viability. Accordingly, the results demonstrated that GS-Rd and the miR-144-5p mimic inhibited the viability and migration of glioblastoma cells (Fig. 4). The anti-tumor activity of GS-Rd was found to be related to the regulatory action of miR-144-5p on TLR2. The results of animal xenograft tumor studies confirmed again that GS-Rd exerted an anti-tumor effect that consisted of miR-144-5p up-regulation and TLR2 down-regulation.

In conclusion, our data illustrated that GS-Rd can promote the targeting of TLR2 by miR-144-5p in glioblastoma cells. Our study revealed the GS-Rd/miR-144-5p/TLR2 regulatory nexus, which controls the glioblastoma pathogenesis and provided a novel target for the diagnosis and treatment of glioblastoma.

Acknowledgments

This research was funded by Project of Jilin Provincial Development and Reform Commission, Jilin Province of China (Grant Nos. 3J115AK93429; 2019C045-2, 2019C045-5); Project of Jilin Provincial Sci&Tech Department, China (Grant No. 20191102008YY).

Conflict of Interest

The authors declare no conflict of interest.

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