HFF obtained from the cell bank of the chinese academy of sciences (Shanghai, China) were incubated in Dulbecco's modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% sodium pyruvate (Invitrogen) in a humidified incubator with at 37 °C with 5% CO2. After being grown to 80% confluence, cells in the logarithmic phase of growth were inoculated into 96-well plates (104 cells/well) and further cultured for 24 h. Then, the culture medium was removed and replaced with DMEM containing 1% FBS. After 24 h of incubation, 0 or 200 μM H2O2 was added to the medium and maintained for 6 h to establish control HFF or H2O2-induced HFF. All experiments were performed in triplicate.

MiR-181a inhibitor and miR-NC were purchased from GenePharma (Shanghai, China) and were transfected into HFF (50 nM) for inhibiting miR-181a. Short hairpin RNA specifically targeting PDIA6 (sh-PDIA6), and control sh-NC also purchased from GenePharma were transfected into HFF (2 μg/μL) for downregulating PDIA6. Cell transfection was achieved by lipofectamine 2000 (Invitrogen) following the manufacturer’s recommendations. After 48 h, the transfection efficiency was assessed by RT-qPCR. All experiments were performed in triplicate.

After H2O2 treatment and/or indicated transfection, 10 μL of CCK-8 solution (Dojindo, Kumamoto, Japan) was added to the medium, and HFF were cultured at 37 °C for another 2 h. Then, a microplate reader (Molecular Devices, Shanghai, China) was utilized for measuring the absorbance at 450 nm according to the manufacturer’s instructions. All experiments were performed thrice.

Total RNA was isolated from HFF using TRIzol reagent (Invitrogen). Synthesis of cDNA was achieved by reverse transcription of total RNA using PrimeScript™ RT reagent Kit (Takara, Dalian, China). RT-qPCR was implemented using SYBR® Premix Ex Taq™ II (Takara) on a CFX96™ Real-Time System (Bio-Rad, Hercules, CA, USA). The relative expression levels of miR-181a and its downstream targets were calculated with the 2−ΔΔCt method, with U6 and GAPDH as normalization, respectively. All experiments were performed in triplicate. Primer sequences are listed in Table 1.

HFF were lysed in RIPA buffer (Cell Signaling, Danvers, MA, USA) and the concentration of proteins was measured by a BCA kit (Bio-Rad). Protein samples (20 μg) were separated by 10% SDS-PAGE gels, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and blocked with 5% non-fat milk. Afterwards, the membranes were incubated at 4 °C overnight with primary antibodies as follows: anti-p21 (ab109520, 1:1000), anti-p53 (ab32389, 1:10000), anti-SMP30 (ab233007, 1:400), anti-beta-actin (ab115777, 1:200), anti-PDIA6 (ab154820, 1:1000) (all from Abcam, Cambridge, MA, USA) followed by incubation with secondary antibody (ab97080, Abcam) for 1 h at room temperature. Beta-actin was used as a loading control. The proteins were visualized with an ECL system (Bio-Rad) and quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA). All experiments were performed in triplicate.

HFF were washed with PBS and fixed in 3% formaldehyde at room temperature for 15 min. After washing with PBS twice, HFF were incubated overnight at 37 °C with β-galactosidase staining reagents (1 mg/mL X-gal, 2 mM MgCl2, 150 mM NaCl, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide and 40 nM citric acid/sodium phosphate: Beyotime, Shanghai, China). Eventually, stained HFF were imaged with a light microscope (Nikon, Tokyo, Japan) at 50× magnification, and the percentage of positive cells were analyzed with Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA). All experiments were performed three times.

The activities of SOD, GPx and CAT were measured as previously described.21 Briefly, one unit of SOD was defined as the amount of enzyme that suppressed 50% of the formazan/min. Xanthine and xanthine oxidase were utilized to produce superoxide anions. The reaction between superoxide anions and tetrasodium chloride formed yellow formazan whose absorption was evaluated at 450 nm. For GPx, enzymatic reactions in tubes containing reduced glutathione, glutathione reductase and NADPH, were initiated by adding cumene hydroperoxide. GPx activity was measured with a wavelength of 340 nm. For CAT activity, one unit of CAT was defined as the amount of enzyme that decomposed 1 M of H2O2/min. The rate of decomposition of H2O2 was assessed at 570 nm. The SOD, GPx and CAT assay kits (Jiancheng Bioengineering Institute, Nanjing, China) were used for spectrophotometrically detecting the enzyme activities which were expressed as U/mg of protein. All experiments were performed in triplicate.

The complementary binding site of miR-181a on PDIA6 3’UTR was predicted by TargetScan (http://www.targetscan.org/vert_71/). The wild-type or mutant sequences of PDIA6 3’UTR were subcloned into pmirGLO vectors (Promega, Madison, WI, USA) to establishPDIA6-Wt/Mut. Afterward, these vectors were co-transfected into HFF with miR-181a inhibitor or miR-NC using Lipofectamine 2000 (Invitrogen). After 48 h of transfection, the luciferase activity was evaluated by a dual-luciferase reporter kit (Promega), normalized to Renilla luciferase activity. All experiments were performed in triplicate.

SPSS 18.0 (SPSS, Chicago, IL, USA) was utilized for statistical analysis. Data are presented as the mean ± standard deviation. Differences between two groups were analyzed by Student’s t-test while those among more than two groups were assessed by analysis of variance (ANOVA) followed by Tukey’s post-hoc analysis. Each experiment was repeated at least three times. The value of p < 0.05 was considered significant.

First, the authors detected miR-181a levels in HFF by RT-qPCR. Relative to the control group, miR-181a level was increased in H2O2-treated HFF and was reduced after transfection of miR-181a inhibitor (Fig. 1A). Next, the impact of miR-181a on the viability of HFF was assessed. As revealed by CCK-8 assay, H2O2 treatment markedly reduced the viability of HFF, while miR-181a depletion attenuated this effect (Fig. 1B), suggesting that downregulated miR-181a might protect HFF against H2O2-induced cell damage. Moreover, detection of SA-β-gal activity displayed that miR-181a inhibition led to a reduction in the H2O2-induced increased percentage of SA-β-gal positive cells (Fig. 1C). A similar trend was observed in the results of western blotting. Protein levels of senescence markers (p21, p53 and SMP30) were significantly raised by H2O2 treatment in HFF compared to the control groups and were then decreased by miR-181a inhibitor (Fig. 1D‒G). The above results indicated that downregulated miR-181a alleviates H2O2-induced cellular senescence in HFF. Subsequently, the authors tested whether miR-181a had an impact on H2O2-induced oxidative stress. As shown by Figure 1 H‒J, H2O2 treatment markedly elevated the activity of SOD, GPx, and CAT, which was partially reversed after the downregulation of miR-181a in HFF. The results suggested that the antioxidant enzymes actively respond to oxidative stress and miR-181a inhibition can relieve H2O2-induced oxidative stress.

Figure 1.

MiR-181a depletion mitigates H2O2-induced cellular senescence and oxidative stress. Assays were conducted in HFF with different treatments (with or without H2O2; transfection of miR-NC or miR-181a inhibitor). (A) RT-qPCR analysis of miR-181a level. (B) CCK-8 assay for evaluating cell viability. (C) SA-β-gal staining for assessing SA-β-gal activity. Cell pictures were imaged at 50× magnification. (D‒G) Western blotting for measuring protein levels of senescence markers. (H‒J) Measurement of the activities of SOD, GPx and CAT in HFF. *p < 0.05, **p < 0.01, ***p < 0.001.

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As shown in Figure 2A, four downstream targets of miR-181a were screened out by ENCORI (https://starbase.sysu.edu.cn/) with the condition of a number of supported AGO CLIP-seq experiments (AgoExpNum) ≥40. Results from RT-qPCR showed that after inhibiting miR-181a in HFF, only the PDIA6 level was significantly enhanced (Fig. 2B). Likewise, western blotting displayed that miR-181a inhibitor increased the protein level of PDIA6 in HFF (Fig. 2C). The existence of putative complementary site between miR-181a and PDIA6 was predicted by TargetScan (Fig. 2D). Moreover, the luciferase activity of PDIA6-Wt was upregulated in HFF after transfection of miR-181a inhibitor, while that of PDIA6-Mut was not significantly influenced, as shown by luciferase reporter assay (Fig. 2E). Notably, RT-qPCR and western blotting demonstrated that mRNA and protein expression of PDIA6 was markedly downregulated in H2O2-treated HFF compared with the control group (Fig. 2F‒H). Collectively, PDIA6 is a target for miR-181a in HFF.

Figure 2.

MiR-181a binds with PDIA6. (A) Four downstream targets of miR-181a predicted by ENCORI. (B) RT-qPCR analysis for assessing these mRNA levels in HFF transfected with miR-181a inhibitor. (C) Western blotting of PDIA6 protein expression in miR-181a inhibitor-transfected HFF. (D) The binding site of miR-181a on PDIA6 3’UTR predicted by TargetScan. (E) Luciferase reporter assay for elucidating the binding relation between PDIA6 and miR-181a. (F) RT-qPCR analysis of PDIA6 level in H2O2-treated HFF. G–H. Western blotting of PDIA6 protein level in H2O2-treated HFF and control group. **p < 0.01, ***p < 0.001.

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To investigate the impact of PDIA6 on cellular senescence in HFF, the authors first transfected sh-PDIA6 into HFF. The mRNA and protein expression levels of PDIA6 were decreased in HFF transfected with sh-PDIA6, as displayed by RT-qPCR and western blotting, respectively (Fig. 3A‒C). CCK-8 assay revealed that miR-181a inhibitor promoted the viability of HFF while co-transfection of sh-PDIA6 attenuated this effect (Fig. 3D). Additionally, results from SA-β-gal staining displayed that depletion of miR-181a significantly reduced the percentage of SA-β-gal positive cells, which was partially reversed by knocking down miR-181a and PDIA6 simultaneously (Fig. 3E‒F). This was consistent with western blotting which showed that knocking down PDIA6 rescued the reduction in senescence marker protein levels caused by miR-181a inhibitor in HFF (Fig. 3G‒J). Hence, it was suggested by the above results that downregulated miR-181a alleviates H2O2-mediated cellular senescence by targeting PDIA6.

Figure 3.

PDIA6 knockdown reverses miR-181a inhibitor-mediated suppressive impact on cellular senescence and oxidative stress. (A) RT-qPCR analysis of PDIA6 expression in sh-PDIA6-transfected HFF. (B‒C) Western blotting of PDIA6 protein expression in transfected HFF. (D) CCK-8 assay for evaluating the viability of HFF with transfection of miR-181a inhibitor, miR-181a inhibitor + sh-PDIA6 or miR-NC. (E‒F) SA-β-gal staining for assessing the percentage of SA-β-gal positive cells of HFF with above transfection. Cell pictures were imaged at 50× magnification. (G‒J) Western blotting for detecting concentration of senescence markers in HFF with above transfection. (K‒M) Measurement of the activities of SOD, GPx and CAT in HFF transfected with miR-181a inhibitor, miR-181a inhibitor + sh-PDIA6 or miR-NC. *p < 0.05, **p < 0.01, ***p < 0.001.

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Then, the authors tested whether miR-181a exerted its influence on oxidative stress by regulating PDIA6. As exhibited by the results, knocking down PDIA6 alleviated the suppressive effect on SOD, GPx and CAT activities in HFF resulting from miR-181a inhibitor (Fig. 3K‒M). This revealed that downregulated miR-181a alleviates H2O2-induced oxidative stress by targeting PDIA6.