ASMCs were isolated and cultured from bronchial biopsies from healthy (n=6) and severe asthmatic (n=6) subjects as previously described.14 Briefly, bronchial biopsies were cut into small pieces and allowed to attach on culture plates in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 20μg/mL streptomycin (Thermo Fisher Scientific), 20U/L of penicillin (Thermo Fisher Scientific), 4mM L-glutamine (Thermo Fisher Scientific) and 2.5μg/mL amphotericin B (Thermo Fisher Scientific), to allow ASMC growth at 37˚C in a humidified incubator supplemented with 5% CO2. Written informed permission was obtained from the asthma patients and healthy donors. This study was approved by the Ethics Committee of the First Affiliated Hospital of Hebei North University. The human 293T cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and was maintained in DMEM supplemented with 10% FBS, 100U/ml penicillin, 100μg/ml streptomycin at 37˚C in a humidified incubator supplemented with 5% CO2.

Different concentration of dexamethasone (10−10, 10−9, 10−8, 10−7 and 10−6M, Sigma-Aldrich, St. Louis, MO, USA) were directly added into ASMC culture for 2h. For inducing cell proliferation and inflammatory factor secretion, TNF-α was added at 10ng/mL and then incubated for 24h.

CASC7 sequence was synthesized and sub-cloned into transfection plasmid to generate recombinant vector pcDNA3.1-CASC7 (CASC7), and the sequence was verified. miR-21 mimics, miR-21 inhibitor and the corresponding control mimics (mimics NC) or control inhibitor (inhibitor NC) were purchased from GenePharma (Shanghai, China). Cell transfection and co-transfection were conducted with Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) following the manufacture's instruction. After 48h, cells were harvested for subsequent experiments.

To construct dual luciferase reporter plasmids, the predicted binding sequence of miR-21 in CASC7 (WT-CASC7) and PTEN (WT-PTEN) and their mutated sequence (MUT-CASC7/PTEN) were separately cloned into pmirGLO vector (Promega, Madison, WI, USA). For luciferase assay, 293T cells were transfected with above constructs and co-transfected with miR-21 mimics, inhibitor or the corresponding control. After 48h, 293T cells were harvested, and the luciferase activities were measured using the Dual-Glo® Luciferase Assay System (Promega) according to the manufacturer's direction. The relative firefly luciferase activity was calculated by normalizing to renilla luciferase activity.

MTT assay was conducted to measure the proliferation of AMSCs. Briefly, AMSCs were seeded into 96-well plate with 1×104cells per well. After 24h, the medium was replaced with fresh culture medium. Then 10μL of MTT stock solution (Thermo Fisher Scientific) was added to the cells, and incubated at 37°C for 4h. Then, the supernatants were discarded carefully and 100μL dimethyl sulfoxide were added to dissolve the formazan crystals. The absorbance of each well at 450nm was recorded using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

The concentrations of CCL5, CCL11 and IL-6 in culture supernatant of ASMCs after TNF-α treatment and/or indicated transfection were determined using ELISA. In brief, AMSCs were treated or transfected differently, and the culture supernatant was collected. The concentrations of CCL5, CCL11 and IL-6 in the culture supernatant were determined using ELISA kits obtained from Thermo Fisher Scientific following the product manual.

Total RNA was extracted from ASMCs using TRIzol reagent (Thermo Fisher Scientific) and was reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's directions. For miR-21, miRNA was extracted from cells with mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific), and was reverse transcribed into cDNA using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA). For CASC7, high-capacity RNA to cDNA kit (Applied Biosystems) was used to synthesize cDNA after total RNA extraction. Expression levels of indicated genes, miR-21, CASC7 and PTEN, were detected using Applied Biosystems 7500 Real Time PCR System (Applied Biosystems) with a SYBR Green QPCR Master Mix (Thermo Fisher Scientific). Primers used in PCR were listed below: CASC7 forward 5′-ATCAACGTCAAGCTGGGAGG-3′, reverse 5′-CTTGTCCCCCGCTCGTTC-3′; miR-21 forward 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACG ACTCAACA-3′, reverse 5′-CGCGCTAGCTTATCAGACTGA-3′; PTEN forward 5′-TTACAGTTGGGCCCTGTACC-3′, reverse 5′-ATTTGATGCTGCCGGTAAAC-3′; U6 (internal control for miRNA) forward 5′-CTCGCTTCGGCAGCACA-3′, reverse 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH (internal control for mRNAs) forward 5′-CCAGGTGGTCTCCTCTGA-3′ and reverse 5′-GCTGTAGCCAAATCGTTGT-3′.The relative RNA levels were calculated by the 2−ΔΔCt method. U6 and GAPDH were used as internal controls.

ASMCs lysates were prepared in RIPA buffer (Thermo Fisher Scientific, MA, USA), and protein concentrations were determined using BCA protein quantitation kit (Thermo Fisher Scientific). 30μg cell lysates were subjected to 10% SDS-PAGE electrophoresis per lane and transferred to PVDF membrane (GE Healthcare, Pittsburgh, PA, USA). The blots were blocked with 5% non-fat milk for 1h, and probed with primary antibody against phosphorylated GR (1:1000), GR (1:1000), GAPDH (1:5000), PTEN (1:2000), p-AKT (1:1000) and AKT (1:1000) overnight at 4°C. All primary antibodies were ordered from Cell Signaling Technology (Boston, MA, USA). Then, the membranes were washed three times with TBST buffer prior to incubation with HRP linked secondary antibodies (Thermo Fisher Scientific), and were visualized using an ECL detection system (Thermo Fisher Scientific). The relative densities of bands were analyzed with NIH image J software.

All experiments were conducted at least three times in triplicate, and one representative experiment was shown. All data shown were expressed as mean±standard deviation (SD). Statistical analysis was performed using the two-sided Student's t-test for comparison between two independent groups or one-way analysis of variance (ANOVA) followed by Tukey post hoc test for multiple comparison with Graphpad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). p-Values <0.05 were considered statistically significant.

To investigate the corticosteroid sensitivity of ASMCs, we isolated and cultured ASMCs from healthy and severe asthmatic subjects separately. After TNF-α treatment, ELISA assays were used to analyze the production of chemokines and inflammatory cytokines, and all these factors (CCL5, CCL11 and IL-6) were up-regulated, especially in severe asthma patient cells with more induction of these factors (Fig. 1A–C). Then we treated ASMCs at different concentration of dexamethasone after TNF-α treatment, and we found that dexamethasone effectively inhibited the secretion of CCL5, CCL11 and IL-6 in normal ASMCs in concentration dependent manner, but not in the ASMCs from severe asthma patients, indicating the corticosteroid sensitivity was impaired in ASMCs from severe asthma patients (Fig. 1D–F). As GRs become hyper-phosphorylated upon glucocorticoid treatment, we examined the phosphorylation level of GR in ASMCs after treatment. As shown in Fig. 1G&H, dexamethasone markedly promoted the phosphorylation of GR in normal ASMCs compared with ASMCs from severe asthma patients was impaired. These results suggested ASMCs from severe asthma patients respond poorly to dexamethasone treatment.

Figure 1.

ASMCs from severe asthma patients respond poorly to dexamethasone treatment. The secretion of CCL5 (A), CCL11 (B) and IL-6 (C) was detected by ELISA assay in ASMCs culture supernatant from healthy or severe asthmatic subjects after TNF-α treatment. The secretion of CCL5 (D), CCL11 (E) and IL-6 (F) was measured by ELISA assay in ASMCs culture supernatant after dexamethasone and TNF-α treatment. (G) The protein level of p-GR and GR was detected by western blot analysis in ASMCs treated with different concentration dexamethasone. GAPDH was used a normalization control. (H) The p-GR/GR ratio was quantified. All the results were shown as mean±SD (n=3), and two-sided Student's t-test was used for statistical analysis. *p<0.05 and **p<0.01.

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Next, we examined the expression level of CASC7, miR-21 and PTEN in ASMCs from severe asthma patients. We found that CASC7 was down-regulated in ASMCs from severe asthma patients compared with normal ASMCs (Fig. 2A), providing the possible link between CASC7 and corticosteroid insensitivity. Furthermore, the expression of miR-21 was significantly up-regulated in severe asthma ASMCs, which was negatively correlated with CASC7 expression (Fig. 2B). Because miR-21 was reported to modulate PI3K/AKT activity via regulating PTEN expression, we analyzed PTEN expression and AKT activity. PTEN expression was decreased in severe asthma ASMCs at both mRNA and protein levels (Fig. 2C–E), and AKT phosphorylation was increased in severe asthma ASMCs (Fig. 2D&E). These observations implied that CASC7 might be involved in maintaining corticosteroid sensitivity possibly via regulating miR-21/PTEN axis and the PI3K/AKT signaling pathway.

Figure 2.

CASC7 and PTEN were down-regulated while miR-21 was up-regulated in ASMCs from severe asthma patients. The expression level of CASC7 (A), miR-21 (B) and PTEN (C) was detected by qRT-PCR in ASMCs from healthy or severe asthmatic subjects. (D) The protein levels of PTEN, p-AKT and AKT were assessed by western blot analysis in ASMCs from healthy or severe asthmatic subjects. GAPDH was used a normalization control. (E) The ratio of PTEN/GAPDH and p-AKT/AKT was quantified. All the results were shown as mean±SD (n=3), and two-sided Student's t-test was used for statistical analysis. *p<0.05 and **p<0.01.

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To investigate the regulatory relationship among CASC7, miR-21 and PTEN, AMSCs were transfected with pcDNA3.1-CASC7. We found CASC7 and PTEN were highly up-regulated, and miR-21 expression was obviously decreased in ASMCs transfected with pcDNA3.1-CASC7 (Fig. 3A). As shown in Fig. 3B, miR-21 mimics resulted in highly expression of miR-21 and markedly reduced expression of PTEN. Conversely, miR-21 expression was suppressed and PTEN expression was up-regulated in ASMCs transfected with miR-21 inhibitor (Fig. 3B). These data indicated that CASC7 was very likely to regulated PTEN expression via modulating miR-21 expression. To further validate the interaction among CASC7, miR-21 and PTEN, we predicted that there were binding sites of miR-21 in CASC7 and the 3′-UTR of PTEN by bioinformatic analysis (Fig. 3C&D). Dual-luciferase reporter assays showed that the luciferase activity was markedly decreased by co-transfection with miR-21 mimics and wild type CASC7 construct (CASC7-WT), while the enhanced luciferase activity was observed by co-transfection with miR-21 inhibitor and WT-CASC7, suggesting that miR-21 was the direct target of CASC7 (Fig. 3E). The luciferase activity was not changed with mutated CASC7 construct (CASC7-MUT) (Fig. 3E). Similar results were also observed in PTEN-WT and PTEN-MUT (Fig. 3F). These observations demonstrated that CASC7 could directly sponge miR-21 to counteract its suppression on PTEN, serving as a positive regulator of PTEN.

Figure 3.

CASC7 increased PTEN expression via targeting miR-21. (A) The expression level of CASC7, miR-21 and PTEN was detected by qRT-PCR in ASMCs transfected with pcDNA3.1-CASC7. (B) The expression level of miR-21 and PTEN was assessed by qRT-PCR in ASMCs transfected with miR-21 mimics or miR-21 inhibitor. (C) The predicted miR-21 binding site within CASC7. (D) The predicted binding of miR-21 and 3′-UTR of PTEN. The luciferase reporter containing the predicted binding sties of miR-21 within CASC7 (E) and PTEN (F) were co-transfected with miR-21 mimic or inhibitor, and then the luciferase activity was measured by dual luciferase assay. All the results were shown as mean±SD (n=3), and ANOVA was conducted for statistical analysis. *p<0.05 and **p<0.01.

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After ASMCs were transfected with pcDAN3.1-CASC7 and miR-21 mimics, the effects of dexamethasone on cell proliferation and cytokine secretion were examined by MTT and ELISA assay, respectively. Compared with control group, overexpression of CASC7 promoted dexamethasone-induced ASMCs proliferation inhibition, but was reversed by miR-21 mimics transfection (Fig. 4A). The secretion of CCL5, CCL11 and IL-6 was suppressed by overexpressing CASC7, which were also partially reversed by miR-21 mimics transfection (Fig. 4B–D). In addition, overexpression of CASC7 promoted the phosphorylation of GR induced by dexamethasone, and it was restored by miR-21 mimics (Fig. 4E&F). These results indicated that CASC7 could suppress the proliferation and the secretion of CCL5, CCL11 and IL-6 by repressing miR-21 expression, thereby increasing the sensitivity of ASMCs to corticosteroids.

Figure 4.

Overexpression of CASC7 promoted dexamethasone induced the inhibition of cytokine release and GR phosphorylation via decreasing miR-21. (A) Cell proliferation was detected by MTT assay in ASMCs. The secretion of CCL5 (B), CCL11 (C) and IL-6 (D) was assessed by ELISA assay in ASMC culture supernatant. (E) The protein level of p-GR and GR was measured by western blot analysis in ASMCs. GAPDH was used a normalization control. (F) The ratio of p-GR/GR was quantified. All the results were shown as mean±SD (n=3), and ANOVA was conducted for statistical analysis. *p<0.05 and **p<0.01.

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Since PTEN was identified to block the activity of the PI3K/AKT signaling by dephosphorylating,12 we examined whether overexpression of CASC7 affected the PI3K/AKT signaling pathway by western blotting. In ASMCs, TNF-α treatment could decrease PTEN expression while dexamethasone could inhibit TNF-α induced PTEN down-regulation (Fig. 5A&B). Overexpression of CASC7 facilitated the above inhibition of dexamethasone by directly down-regulating miR-21 (Fig. 5A&B). In contrast with the expression pattern of PTEN, dexamethasone could suppress the phosphorylation of AKT induced by TNF-α, which was promoted by overexpressing CASC7 (Fig. 5A&B). Moreover, miR-21 mimics restored the high level of AKT phosphorylation (Fig. 5A&B). These results provided the evidence that CASC7 might attenuate corticosteroid resistance by targeting miR-21 to inhibit the PI3K/AKT signaling pathway.

Figure 5.

CASC7 facilitated inhibition of the PI3K/AKT signaling pathway by dexamethasone via inhibiting miR-21. (A) The protein level of PTEN, p-AKT and AKT was measured by western blot analysis in ASMCs. GAPDH was used a normalization control. (B) The ratio of PTEN/GAPDH and p-AKT/AKT was quantified. All the results were shown as mean±SD (n=3), and ANOVA was conducted for statistical analysis. *p<0.05 and **p<0.01.

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