Human female AML (n=41) samples were recruited at the Department of Pathology of Nanjing Drum Tower Hospital from January 2008 to December 2017 (age, 30–50 years). Human female LAM (n=4) and normal lung tissues (n=5) used in this study were collected from LAM patients and healthy donors at Wuxi People's Hospital from January 2008 to December 2017 (age, 30–50 years). This study was approved by the Scientific Research Ethics Committee of the Affiliated Drum Tower Hospital, Medical School of Nanjing University. Informed consents were obtained from all participants.

Fifteen 6-week-old male BALB/c-nu/nu were purchased from Nanjing Biomedical Research Institute (NBRI) of Nanjing University (Nanjing, China) (weight, 18–19 g), and were maintained on a normal 12-h light/dark schedule at a constant temperature and humidity. The housing conditions met the specific pathogen-free (SPF) standards. All mice were maintained on a regular chow diet and water ad libitum. Animals were sacrificed when the tumors reached 1.5 cm³. All animal procedures were carried out in accordance with the approval of the Animal Care and Use Committee at the Model Animal Research Center of Nanjing University.

TSC2 wild-type/p53-null and TSC2-null/p53-null mouse embryonic fibroblasts (generously provided by Dr John Blenis, Harvard University, Boston, MA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS). LAM patient-derived 621–101 cells (provided by Dr John Blenis) were cultured in IIA complete medium in a 37°C incubator with 5% CO₂ and a humidified atmosphere.

For the transfection of pcDNA3.3-RXRα and miR-124 mimics, cells were transfected with pcDNA3.3-RXRα (1/2 µg) and miR-124 mimics (final concentration of 20 nM) using Lipofectamine 2000 reagent (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The empty pcDNA3.3 and scramble sequence of miRNA mimics were used as the negative controls (NCs). Cells were treated with rapamycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 20 nM for 48 h. An equivalent volume of dimethyl sulphoxide (DMSO) was delivered in a vehicle control group. Cell morphology was observed under a fluorescence microscope (CKX41; Olympus Corp., Tokyo, Japan).

Inducible TSC2-null cells expressing miR-124 (designated as TSC2 Null-tet-124) were established. Each mouse (male BALB/c-nu/nu, 6-weeks-old) was subcutaneously injected in the lower back with 1×10⁷ TSC2 null-tet-124 cells diluted in 200 µl DMEM. After 7 days, the nude mice bearing tumors were randomly divided into 2 groups (5–7/group). One group received doxycycline (Dox) treatment (1 mg/ml Dox in drinking water) to induce the miR-124 expression. The other group was not treated with Dox and was set as the negative control group. The tumor volume was assessed every 4 days. Tumor-bearing mice were sacrificed under deep anesthesia with pentobarbital sodium at 31 days after inoculation, and then the tumors were dissected, weighed and kept at −80°C for further study.

Cells were harvested at 48 h after transfection and collected by 160 × g centrifugation. Cells were washed with phosphate-buffered saline (PBS) and resuspended in 500 µl binding buffer. Then, 5 µl of Annexin V-FITC and 5 µl of propidium iodide (PI) (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) were added. The cells were incubated in the dark for 10 min, and then subjected to flow cytometric analysis. Cell morphology was observed under a fluorescence microscope (Olympus CKX41; Olympus Corp.).

The cellular ATP level was determined using the ATP Determination kit (Invitrogen; Thermo Fisher Scientific, Inc.). All values were normalized to total protein levels.

Total RNA was extracted using TRIzol reagent and then reverse transcribed to cDNA using PrimeScript RT Master Mix (both from Takara Bio, Inc., Otsu, Japan). cDNA microarray analysis of TSC2 wild-type and TSC2-null cells transfected with miR-124 mimics was performed by Shanghai Biotechnology Corp. (Shanghai, China).

The cDNA was subjected to real-time PCR using the SYBR-Green PCR kit (Takara Bio, Inc.) with the ABI PRISM 7300 Sequence Detector under the following thermocycling conditions: 95°C for 5 min, then 35 cycles of 95°C for 15 sec, 58°C 15 sec, 72°C for 30 sec and a final extension at 72°C for 5 min. The following primers were used: RXRα (mouse) 5′-ATGGACACCAAACATTTCCTGC-3′ (forward), and 5′-CCAGTGGAGAGCCGATTCC-3′ (reverse); Acox1 (mouse) 5′-TAACTTCCTCACTCGAAGCCA-3′ (forward), and 5′-AGTTCCATGACCCATCTCTGTC-3′ (reverse); RXRα (human) 5′-ATGGACACCAAACATTTCCTGC-3′ (forward), and 5′-GGGAGCTGATGACCGAGAAAG-3′ (reverse); Acox1 (human) 5′-AATCGGGACCCATAAGCCTTT-3′ (forward), and 5′-GGGAATACGATGGTTGTCCATTT-3′ (reverse); 18S 5′-GTCTGTGATGCCCTTAGATG-3′ (forward) and 5′-AGCTTATGACCCGCACTTAC-3′ (reverse). For miR-124 detection, RNA was extracted using a miRNeasy Mini Kit (Qiagen China Co., Ltd., Shanghai, China) according to the manufacturer's instructions. cDNA was synthesized by reverse transcription (RT) with a miScript RT II kit (Qiagen China Co.). Quantitative RT-PCR was performed using a miScript SYBR-Green PCR kit (Qiagen China Co.), and primers for miR-124 (MS00029211 and MS00006622) and RNU6-2 (MS00033740; all from Qiagen China Co.) were used according to the manufacturer's instructions. Relative mRNA expression levels were determined by the ΔΔCq method using 18S/RNU6-2 expression levels for normalization (12).

Tissues were lysed in tissue lysis buffer [20 mM Tris/HCl (pH 7.5), 4 mM EDTA (pH 8.0), 2% SDS] and cells were lysed in RIPA [20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS] supplemented with protease inhibitors and phosphatase inhibitor cocktails (Roche Diagnostics, Indianapolis, IN, USA). Western blot analysis was performed according to routine procedure. Proteins were quantified using the BCA method. Samples containing 50 µg protein were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% Difco™ Skim Milk (cat. no. 232100; BD Difco, Franklin Lakes, NJ, USA) for 1 h at room temperature. Then, the membranes were incubated overnight at 4°C with the specific antibodies. Antibodies used were as follows: Anti-Stat3 (dilution 1:1,000; cat. no. 4904), anti-AKT (dilution 1:1,000; cat. no. 9272), anti-4E-BP1 (dilution 1:1,000; cat. no. 9644), anti-p70 S6K (dilution 1:1,000; cat. no. 2708) (all from Cell Signaling Technology, Inc., Beverly, MA, USA), anti-α-Tubulin (dilution 1:1,000; cat. no. Ab102; Vazyme, Nanjing, China) and anti-RXRα (dilution 1:1,000; cat. no. 21218; ProteinTech Group, Inc., Chicago, USA). Membranes were incubated with anti-mouse IgG, HRP-linked antibody (dilution 1:5,000; cat. no.7076P2; Cell Signaling Technology, Inc.) or anti-rabbit IgG, HRP-linked antibody (dilution 1:5,000; cat. no. 7074P2; Cell Signaling Technology, Inc.) for 1 h at room temperature. Immunocomplexes were visualized with ECL and visualized by Tanon Chemiluminescent Imaging System (Tanon Science and Technology Co., Shanghai, China).

293T cells were cultured in 96-well plates and co-transfected with 10 ng pMIR-RXRα 3′-UTR wild-type (WT), pMIR-RXRα 3′-UTR mutant type 1594–1600 (MT1) or pMIR-RXRα 3′-UTR mutant type 2700–2706 (MT2) and 5 pmol miR-124 mimics or negative control. After a 48-h incubation, Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay system (Promega Corp., Madison, WI, USA).

Sections were deparafnized, incubated with primary antibodies (RXRα, dilution 1:1,000; cat. no. 21218; Proteintech, IL, Chicago, USA). A subsequent reaction was performed with biotin-free HRP enzyme-labeled polymer from an EnVision Plus detection system (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). Positive reactions were visualized with diaminobenzidine (DAB) solution followed by counterstaining with hematoxylin. Sections were observed under light microscopy and the staining intensity scores were independently assessed by 2 pathologists.

All data are presented as the means ± SEM. Statistical calculations were performed using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Student's t-test and one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test were used, respectively. Pearson's χ² test was performed to analyze the correlation between p-S6 expression and RXRα. All statistical analysis was carried out using SAS (version 9.0; SAS Institute, Inc., Cary, NC, USA). Values of P<0.05 were considered to indicate a statistically significant result.

To verify whether miR-124 regulated LAM progression, a TSC2 wild-type and a TSC2-null stable cell line were used to mimic healthy and LAM cell models, respectively. At 48 h after transfection with miR-124 mimics, the apoptosis of TSC2-null cells was significantly induced (Fig. 1A, B and E). In comparison, miR-124 mimics slightly influenced TSC2 wild-type cells. To further validate our hypothesis, 621–101 cells, another TSC2-deficient cell line, were treated with miR-124 mimics and cell apoptosis was assessed. The results revealed that cell apoptosis was significantly induced (Fig. 1C, D and F). Collectively, TSC2-deficient cells were more sensitive to miR-124-induced cell apoptosis.

To explain why miR-124 selectively induced the apoptosis of TSC2-deficient cells, we treated TSC2-null cells with rapamycin to reduce mTORC1 activity. The results revealed that the apoptosis of TSC2-null cells treated with or without rapamycin was not markedly changed, indicating that rapamycin could not prevent cell apoptosis of TSC2-null cells induced by miR-124 (Fig. 2A-C). Recent studies (6,13,14) have reported that STAT3 and AKT are targets of miR-124 in colorectal cancer and hepatocellular carcinoma. We therefore detected the expression of STAT3, AKT, 4E-BP1 and p70 S6K under miR-124 overexpression. As revealed in Fig. 2D and E, null cells exhibited no significant difference in their expression. The aforementioned results demonstrated that miR-124 induced TSC2-null cell apoptosis via an mTORC1-independent manner.

Then, we compared the transcriptome between TSC2 wild-type and null cells. The cDNA microarray analyses revealed 3,272 genes with a fold change >3 in TSC2-null cells compared with that in TSC2 wild-type cells when miR-124 was overexpressed (Fig. 3A). Combination with TargetScan prediction (http://www.targetscan.org/) and the annotation of Gene Ontology Biological Function (http://geneontology.org/), 12 candidates involved in cell growth were further investigated (Fig. 3B). Among these genes, RXRα mRNA was significantly downregulated by miR-124 in TSC2 wild-type and null cells (Fig. 3C). Additionally, restoration of RXRα reversed miR-124-induced apoptosis of TSC2-null cells (Fig. 3D and E), implying that miR-124 causes the apoptosis of TSC2-null cells via RXRα.

To further validate whether RXRα was directly regulated by miR-124, we introduced WT) or MT 3′-UTR (1594–1600 and 2700–2706) of RXRα into luciferase reporter plasmids. The WT and MT binding sequences are presented in Fig. 3F. Reporter assays revealed that miR-124 significantly decreased the luciferase activity of WT RXRα 3′-UTR, but not that of 2 MT RXRα 3′-UTR (Fig. 3G), indicating specific binding between miR-124 and RXRα 3′-UTR at both sites. Collectively, these data supported our hypothesis that RXRα is a direct target of miR-124.

The RXRα/PPARα heterodimer is required for PPARα transcriptional activity on fatty acid oxidation genes such as Acox1 (Acyl-CoA oxidase 1) (15). Thus, we detected the expression of Acox1 in TSC2-null cells. The results revealed that Acox1 was suppressed when miR-124 was overexpressed, which was reversed by RXRα reconstitution (Fig. 3H). Additionally, ATP levels exhibited a similar tendency (Fig. 3I), further implying that miR-124/RXRα may mediate fatty acid oxidation of TSC2-null cells.

Next, we studied the effects of miR-124 on the growth of TSC2-null cells in vivo. The TSC2-null cells (referred to TSC2-null-tet-124) with a Tet-On system to overexpress miR-124 were injected into nude mice (Fig. 4A). At 7 days after injection, the expression of miR-124 was induced with Dox (Dox⁺) or not induced (Dox⁻). We found that compared with Dox⁻ group, mice in the Dox⁺ group revealed significantly reduced tumor volume and weight at 31 days after injection (Fig. 4B-D). However, body weights of Dox^+/− mice revealed no significant difference (data not shown). We further examined the expression of miR-124 and RXRα in a xenograft tumor. As expected, Dox-treated xenograft tumors expressed significantly higher miR-124 levels and lower RXRα/Acox1 levels compared to those in the control group (Fig. 4E, F and I). Western blot assays also revealed that RXRα exhibited a markedly reduced level in miR-124-overexpressing xenograft tumors (Fig. 4G and H). Therefore, these findings indicated that the miR-124/RXRα axis regulated the growth of TSC2-null cells in vivo.

To further address the significance of the miR-124/RXRα pathway, we performed quantitative real-time PCR to examine the expression of miR-124 and RXRα in LAM-patient-derived tissues (n=4). The results revealed a significant decrease of miR-124 expression in LAM samples compared to that in healthy controls (Fig. 5A), whereas the mRNA levels of RXRα and Acox1 were significantly upregulated (Fig. 5B and D). However, linear regression analysis of miR-124 and RXRα (Fig. 5C) was also performed. The results revealed a negative correlation between miR-124 and RXRα, further indicating that RXRα is an important target of miR-124 in LAM. The expression of RXRα in AML tissues was further analyzed and it was revealed that, the marker of mTORC1 activation, phosphorylation of S6 was negatively correlated with the expression of RXRα (Fig. 5E and Table I). Patients with higher mTORC1 activity expressed more RXRα. These results indicated that miR-124/RXRα may serve as a promising biomarker for LAM patients.