Unbalanced apoptosis is a major cause of structural remodelling of vasculatures associated with PAH (pulmonary arterial hypertension), whereas the underlying mechanisms are still elusive. miRNAs (microRNAs) regulate the expression of several proteins that are important for cell fate, including differentiation, proliferation and apoptosis. It is possible that these regulatory RNA molecules play a role in the development of PAH. To test this hypothesis, we studied the effect of several miRNAs on the apoptosis of cultured PASMCs (pulmonary artery smooth muscle cells) and identified miR-138 to be an important player. miR-138 was expressed in PASMCs, and its expression was subjected to regulation by hypoxia. Expression of exogenous miR-138 suppressed PASMC apoptosis, prevented caspase activation and disrupted Bcl-2 signalling. The serine/threonine kinase Mst1, an amplifier of cell apoptosis, seemed to be a target of miR-138, and the activation of the Akt pathway was necessary for the anti-apoptotic effect of miR-138. Therefore the results of the present study suggest that miR-138 appears to be a negative regulator of PASMC apoptosis, and plays an important role in HPVR (hypoxic pulmonary vascular remodelling).
- hypoxic pulmonary vascular remodelling
- pulmonary arterial hypertension
PAH (pulmonary arterial hypertension) is a malignant pulmonary disease characterized by elevated pulmonary arterial pressures leading to right ventricular failure and death. A major pathogenic process in the development of PAH is the remodelling of pulmonary vessels involving apoptosis and regeneration of PASMCs [PA (pulmonary artery) SMCs (smooth muscle cells)]. Although these events also occur in healthy lungs, a subtle balance between them is normally achieved without permanent damage to the structure and function of pulmonary vasculature [1–3]. Unbalanced vascular remodelling can cause a structural change in the pulmonary vessels, persistent vasoconstriction and PAH [4–8].
Hypoxia, a well-known trigger event of PAH, has effects on pulmonary vascular remodelling. Although the mechanisms underlying hypoxic PAH are still not fully understood, experimental evidence suggests that hypoxia has an anti-apoptotic effect that can misbalance the remodelling of pulmonary vasculature [9,10]. The resulting excessive regeneration of PASMCs and prolongation of their viability may produce hypertrophy of vascular smooth muscles, thickening of the vascular wall, narrowing of vessel inner diameters and an increase in the perfusion resistance. These pathophysiological alterations in turn can worsen tissue hypoxia. Thus a cascade of events is triggered by hypoxia, leading to adverse vasoconstriction and PAH.
The molecular and cellular basis for the hypoxic vascular remodelling of PAs is unclear. One potential mechanism is the alteration of mRNA stability by miRNAs (microRNAs). miRNAs are endogenous non-coding small RNAs that negatively regulate gene expression by targeting mRNAs. Such post-transcriptional regulation affects a variety of physiological and pathological processes, including cell proliferation and apoptosis, underlying tumorigenesis and several cardiovascular diseases [11–18]
In a recent study, we screened a number of miRNAs in PAs with hypoxic exposure. We found seven miRNAs to be upregulated and two miRNAs (miR-328 and miR-290) to be down-regulated. The latter two were studied in detail, and our previous results suggested that miR-328 acts on PASMC remodelling and PA constriction . In contrast, how the other seven miRNAs function in hypoxia-induced PH (pulmonary hypertension) remains unclear. Therefore we performed the present study to investigate how these hypoxia-augmented miRNAs affect apoptosis of PASMCs after the cells were challenged with hypoxia, and which intracellular signalling mechanisms are critical.
MATERIALS AND METHODS
Animal care and use complied to the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23, revised 1996), and were approved by the Institutional Animal Care and Use Committee of Harbin Medical University [Protocol (2009)-11]. Adult male Wistar rats (180–200 g) were from the Experimental Animal Center of Harbin Medical University.
Induction of the HPVR (hypoxic pulmonary vascular remodelling) rat model and tissue collection
HPVR rat models were induced by raising the animals in a hypoxic environment (12% O2) for 9 days . After this time period, rats were anaesthetized with pentobarbital sodium (50 mg/kg of body weight intraperitoneally) . When animals were sufficiently anaesthetized, the chest was surgically opened. Then the heart and lungs were removed together and placed in ice-cold PBS solution for the preparation of tissue collection . The lungs were harvested and processed for immunohistochemistry and in situ hybridization. The PAs were collected for qRT (quantitative real-time reverse-transcription)-PCR and Western blot assay.
Localization of miRNAs by in situ hybridization
In situ hybridization was performed using a detection kit from Boster Bio-engineering on sections of paraffin-embedded lung tissues. The digoxigenin-labelled probe for rno-miR-138 was designed and synthesized by Sangon Biotech. Morphometric analysis was analysed with Image software (Image Pro Plus).
Computational prediction of miRNA targets
We used three established miRNA target-prediction algorithms including TargetScan5.1, miRbase and miRGene prediction analysis to identify the candidate miRNAs that were potentially targeted by the apoptosis-related genes.
Cell isolation and cultivation
The intrapulmonary arteries were de-endothelialized and then gently digested with enzymatic solution: 0.15% type II collagenase (Worthington) and 0.15% BSA in PBS solution for 1 h at 37°C. The digested PASMCs were then cultured in complete DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) FBS (fetal bovine serum), 1% (w/v) streptomycin and 1% (w/v) penicillin for 3–5 days in a humidified incubator with 5% CO2 at 37°C. Passages 2–4 were used for further experiments. Before each experiment, the cells were incubated in serum-free low-glucose DMEM for 24 h to stop growth. For hypoxic cultivation, the cells were grown in a Tri-Gas incubator (HF100; Heal Force) providing an atmosphere of 92% N2/5% CO2/3% O2 for 24 or 48 h as described previously .
Induction of an apoptotic model of PASMCs
The apoptosis of PASMCs was induced by SD (serum deprivation) or H2O2 administration. Briefly, after transfection, cells were kept in SD and cultured under normoxic or hypoxic conditions for another 24 h before qRT-PCR or Western blot assay and 48 h before other apoptosis detection. For H2O2 administration, cells were transfected and switched to complete DMEM [10% (v/v) FBS] containing 100 μM H2O2 under normoxic conditions for the next 24–48 h and were prepared as above.
Transfection of oligonucleotides
After growth arrest, PASMCs were transfected with a 2 μg mixture of different groups of oligonucleotides using X-tremeGene siRNA (small interfering RNA) Transfection Reagent (Roche) according to the manufacturer's instructions. Subsequently, the cells underwent SD or were switched to complete DMEM [10% (v/v) FBS] under normoxic (21% O2/5% CO2/balance N2) or hypoxic (3% O2/5% CO2/balance N2) growth conditions for another 24–48 h [23–25].
Cell viability was measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, and mitochondrial membrane potential was measured with the JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) probe. Fragmented DNA of the apoptotic PASMCs was measured using the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assay, and chromatin condensation was examined using Hoechst 33258 staining. Caspase-3 activity and Western blotting were performed to evaluate the caspase cascade and activity of Bcl-2 family.
Analysis and quantification of miR-138 and Mst1 (serine/threonine kinase 4) mRNA expression levels using qRT-PCR were performed with high-capacity cDNA Reverse Transcription Kit and Fast SYBR® Green Master Mix (Applied Biosystems) following the manufacturer's instructions. Primers specific for miRNAs and Mst1, RNU6B and GAPDH (glyceraldehyde-3-phosphate dehydrogenase), were purchased from Sangon Biotech (Table 1). qRT-PCR was performed on a thermocycler ABI Prism 7500 fast (Applied Biosystems) for 40 cycles. The fold increase relative to control samples was determined by the 2−ΔΔCT method . Expression level of target gene mRNA was determined using qRT-PCR utilizing the total RNA from PAs or PASMCs. RNU6B and GAPDH were used as an internal control for miR-138 and Mst1 respectively.
Western blot assay
Proteins extracted from PASMCs and PAs were detected using a standard Western blotting protocol. An antibody specific for β-actin was used as an internal control.
Additional details and the other methods are supplied in the Supplementary Online Data (at http://www.biochemj.org/bj/452/bj4520281add.htm).
The composite data were expressed as means±S.E.M. Comparisons of data were accomplished by one-way ANOVA followed by Dunnett's test. The differences between means were considered significantly different at P≤0.05.
Effect of hypoxia on miR-138 expression and distribution in HPVR rat model
The rat model of HPVR was induced by consecutive hypoxic exposure for 9 days, and shown by the morphological change of PAs using H/E (haematoxylin and eosin) staining. Compared with normal rats, PAs obtained from rats exposed to hypoxia showed medial thickening by 36.0±0.1% (n=3 animals for each group) (Figures 1A and 1B).
In our previous study, we identified seven miRNAs that were up-regulated by hypoxia in PAs . To determine their roles in HPH (hypoxic PH), we first tested their expression patterns in PASMCs under hypoxic conditions. Our results confirmed that only miR-138, miR-146a, miR-184 and miR-190 were expressed and up-regulated by hypoxia in PASMCs (Figure 1C).
Subsequently, we studied the function of these miRNAs in PASMC apoptosis under hypoxic conditions. They were overexpressed or inhibited by using the double-stranded Mims (miRNA mimics) and the Amos (antisense oligonucleotides) complementary to the mature miRNAs (Table 2). PASMC apoptosis was induced by SD after transfection with Mims or Amos. The MTT assay showed that a transfection with either miR-138 or Amo-138 significantly affected the cell viability under hypoxic conditions, whereas none of the other miRNAs had any significant effect (Figure 1D). Moreover, using algorithms based on miRNA–mRNA complementarity and its evolutionary conservation (TargetScan, miRBase and miRGene), we found that miR-138 targets candidate mRNAs known to be involved in regulating cell apoptosis in HPH. These findings suggest that miR-138 seems to play a role in HPH via an anti-apoptosis of PASMCs.
The precise location of miR-138 in PAs was further examined using in situ hybridization with digoxigenin-labelled miR-138 probes in lung tissue sections from normoxic and hypoxic rats. miR-138 was mainly expressed in PASMCs, and its expression level was augmented by hypoxia, as shown by the strength of staining in PASMCs from hypoxic rats (Supplementary Figure S1 at http://www.biochemj.org/bj/452/bj4520281add.htm).
miR-138 suppressed mitochondria-mediated caspase-dependent apoptosis in PASMCs
Our TUNEL assay, which measures DNA cleavage in apoptotic cells, showed that the number of TUNEL-positive cells was significantly increased after SD (Figure 2A). Such an effect was attenuated by an overexpression of miR-138 in the cells.
Under the SD conditions, apoptotic cells had a classical appearance of chromatin condensation, which is one of the most important criteria and is used to identify apoptotic cells. These nuclear morphological changes were also diminished in the miR-138-transfected cells compared with the SD groups. Meanwhile, co-transfection with Amo-138 partly reversed the effect of miR-138 on apoptosis (Figure 2B). To assess the components of the apoptotic cascade involved in the miR-138-mediated anti-apoptosis, we measured the enzymatic activity of caspase-3 and tested caspase activation, accompanied by a proteolytic cleavage of the unprocessed form (procaspase), using Western blot assay. We found that the miR-138 transfection reduced the SD-activated caspase-3 and -9 to a level similar to that in normal cells. Amo-138 co-transfection partially reversed the effect of miR-138 on caspase activities (Figure 3).
Caspase activity (such as caspase-9 activation) is known to induce mitochondrial damage during apoptosis. Therefore we measured mitochondrial potential in PASMCs using the JC-1 probe, which aggregates in the intact mitochondria in non-apoptotic cells emitting orange–red fluorescence and distributes widely in apoptotic cells emitting green fluorescence as the monomeric form at 488 nm. In SD-treated cells, the aggregated JC-1 within normal mitochondria was dispersed to the monomeric form (measured by green/red fluorescence intensity ratio). By miR-138 transfection, SD-induced mitochondrial collapse was relieved as shown in the fluorescent colour change from green to orange–red, and Amo-138 co-transfection induced an increase in green fluorescence, indicating a reduction of mitochondrial membrane potential (Figure 4A).
The Bcl-2 family proteins are important for the integrity of the mitochondria. Individual members (e.g. Bcl-2 and Bcl-xL) can suppress or promote [e.g. Bax and Bad (Bcl-2/Bcl-xL-antagonist, causing cell death)] apoptosis. Therefore we examined the effect of miR-138 on the activity of Bcl-2 and Bad by measuring their expression and phosphorylation using Western blot analysis. Compared with NC (negative control)-transfected SD groups, miR-138 transfection significantly increased the expression of Bcl-2 and its phosphorylation at Ser70 and down-regulated Bad with increased phosphorylation at Ser136. Meanwhile, Amo-138 co-transfection could reverse all of these effects on Bcl-2 family members (Figures 4B–4E), suggesting that miR-138 triggers a change in the balance between anti-apoptotic and pro-apoptotic factors, which might result in the observed decrease in active caspases.
The effect of miR-138 on apoptosis was also detected in H2O2-induced apoptosis (Supplementary Figures S2–S4 at http://www.biochemj.org/bj/452/bj4520281add.htm), confirming the role of miR-138 in suppressing apoptosis of PASMCs. In subsequent experiments, we focused on the response of PASMCs under conditions of SD since it is one of the most widely used and reproducible inducer of apoptosis in non-transformed cells.
miR-138 targets Mst1 and regulates the Akt signalling pathway
Our computational analysis showed a 6-nt match to the miR-138 seed region in the 3′-UTR (3′-untranslated region) of the serine/threonine kinase Mst1 that is highly conserved among rat, human and mouse (Figure 5A). To test whether miR-138 directly targeted the 3′-UTR of Mst1, we performed luciferase assays using 3′-UTR sequence fragments containing the predicted target of miR-138 and its mutated version inserted downstream of a luciferase reporter (Supplementary Figures S5A and S5B at http://www.biochemj.org/bj/452/bj4520281add.htm). As shown in Figure 5(B), transient transfection of HEK (human embryonic kidney)-293 cells with Mst1 3′-UTR and miR-138 (Supplementary Figure S5C) resulted in down-regulation of luciferase activity compared with Mst1 3′-UTR and NC co-transfected cells, which was abrogated when the predicted miR-138 binding site was mutated. The miR-138 or NC co-transfected with empty vectors generated similar luciferase activities, proving the stable luciferase assay system (Figure 5B).
Western blot analysis showed that the expression pattern of Mst1 and its cleavage product in PAs from normoxic and hypoxic rats was decreased at the protein level coinciding with up-regulation of miR-138 (Figures 5C and 5D).
To further understand the regulation of Mst1 expression by miR-138, we examined lysates from PASMCs when miR-138 was either overexpressed or antagonized, and found that there was an inverse correlation between miR-138 and Mst1 protein expression. The expression of Mst1 and its cleavage product at the protein level were clearly suppressed in the miR-138-transfected PASMCs compared with control samples, and this reduction was efficiently prevented by co-transfection with Amo-138 (Figures 6A and 6B), suggesting that miR-138 mediated Mst1 repression via a step after translation initiation, or repressed translation by sequestering the mRNA into a complex where it was not accessible to the ribosomes.
As the Mst1 signalling pathway is known to link the activation of phospho-Akt/Akt with apoptotic signalling in some cell types, we examined the activation of Akt signalling. A marked increase in Akt phosphorylation at Ser473 was found in PASMCs overexpressing miR-138, which was blocked by co-transfection with Amo-138 (Figures 6A and 6C), suggesting that miR-138 activates Akt signalling by negatively regulating Mst1 at the post-transcriptional level.
We also found that the effect of miR-138 on activation of Akt can be blocked by LY294002, an antagonist of the Akt pathway, administration, providing a link between miR-138 and Akt (Figure 7A). Meanwhile, the miR-138-induced inhibition of the cleavage of procaspase-3 and up-regulation of Bcl-2 expression during apoptosis was also suppressed by LY294002 treatment (Figures 7B and 7C). These data strongly suggest that the Akt signalling pathway is involved in the suppression of PASMC apoptosis by miR-138.
To determine whether Mst1 mediated the miR-138-dependent suppression of caspase-dependent apoptosis, we inhibited Mst1 (si-Mst1) in Amo-138-transfected PASMCs under hypoxic SD conditions and measured apoptosis. The effect of Amo-138 on caspase-dependent apoptosis under this condition was determined using a caspase inhibitor, ZDK [Z-VAD-FMK (benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone)]. As predicted, ZDK or si-Mst1 partially rescued the decrease in cell viability caused by Amo-138 (Figure 8A). Also, no effect of ZDK on miR-138 and Mst1 expression was found in PASMCs, whereas si-Mst1 reversed the Amo-138-induced strengthening of Mst1 and its cleavage expression (Figures 8B and 8C). These data thus suggest that the miR-138-mediated suppression of caspase-dependent apoptosis appears to target at Mst1.
miR-138-mediated hypoxia-induced anti-apoptosis in PASMCs
As hypoxia is likely to lead to the suppression of VSMC (vascular smooth muscle cell) apoptosis, we examined further the effect of miR-138 on apoptosis under hypoxic conditions. As predicted, hypoxic exposure inhibited cell apoptosis (increased cell viability, decreased cleavage of procaspase-3, enhanced Bcl-2 expression and activated phospho-Akt) (Figure 9). Using SD, all these hypoxic effects were relieved and miR-138 overexpression could reverse these attenuations. Amo-138 also attenuated the effect of miR-138 on apoptosis (Figures 9A–9C). Without SD induction, Amo-138 still inhibited the effect of hypoxia on PASMCs (Figures 9D–9G), confirming the role of miR-138 in mediating this hypoxia-induced anti-apoptosis.
Hypoxic induction of miR-138 in PASMCs was HIF (hypoxiainducible factor)-1α-dependent
HIFs are a family of transcription factors that are activated in response to hypoxia, regulating gene expression and the cellular response to hypoxia . We asked whether the induced expression of miR-138 was indeed triggered by HIF. We used HIF1α and HIF2α siRNA oligonucleotides to suppress the HIF pathway and test the expression levels of miR-138 under these conditions. After stable knockdown of HIF1α and HIF2α (Figures 10A and 10B), the PASMCs transfected with siRNA scramble controls had a robust induction of miR-138 expression under hypoxic conditions. However, this induction was abolished not by siRNAs against HIF2α, but by siRNAs against HIF1α (Figure 10C), implying a HIF-1α-dependent regulatory mechanism.
In the present study, we have shown evidence for the role for miR-138 in regulating PASMC apoptosis in HPH, and found the underlying mechanisms. The importance of miRNAs in pathological processes is being recognised, especially in cardiovascular disease. A number of miRNAs have been implicated in signal transduction pathways relevant to PH. For example, miR-21 in PH has been shown to target proteins that regulate Bcl-2 and Akt signalling pathways and subsequent cell proliferation and apoptosis . miR-221 and miR-204 have been shown to participate in PH by regulating cell-cycle inhibitors and the Src/Stat3 (signal transducer and activator of transcription 3) cascade [29,30]. There is evidence suggesting the role of miR-328 and miR-214 in regulating the expression of ion channels (CACNA1C and CACNB1) and eNOS (endothelial nitric oxide synthase), candidate regulators of vascular tones [31,32]. Moreover, Albinsson et al.  have shown that, in the Dicer-depleted mouse line, the loss of miRNAs in SMCs results in a dramatic decrease in blood pressure due to loss of contractile function, phenotypic modulation of SMCs and vascular remodelling, indicating an important role of miRNAs in differentiation of VSMCs. All these previous studies suggest the importance of miRNAs in vascular cell fate and the consequential effect on PH. In the present study, we have focused on miR-138, and shown its effect on PASMC viability under hypoxic conditions.
The results of the present study indicate that miR-138 suppresses caspase-dependent cell apoptosis and attenuates mitochondrial depolarization in apoptotic PASMCs. This effect exists under hypoxic conditions. According to bioinformatics-based analysis and luciferase assay, Mst1 has been identified to be a direct target of miR-138 involvement in HPVR. Mst1 is a 487-amino-acid protein that contains two cleavage sites between the regulatory and catalytic domains, which may be selectively cleaved to generate catalytically active enzymes of 36 kDa . It has been shown that the activation of Mst1, in both cleaved and full-length forms, is associated with the mechanotransduction pathway for cell apoptosis. Activated Mst1 and its cleavage products have been proved to be novel inhibitors of Akt through binding to Akt1 in the cytoplasm and the nucleus following activation of an apoptotic signal [35–38]. Thus Mst1 may suppress the PI3K (phosphoinositide 3-kinase)/Akt pathway during apoptosis by inhibiting Akt . In the present study, hypoxia and miR-138 can decrease the expression of both cleaved and full-length Mst1. Moreover, both ZDK and si-Mst1 can partially rescue the decrease of cell viability caused by Amo-138. Although lacking direct imaging analysis, the results of the MTT assay suggest that the miR-138-induced suppression of PASMC apoptosis is mediated by targeting Mst1. The luciferase reporter and Western blot assays further validated Mst1 to be a direct target of miR-138 at the post-transcriptional level.
Previous studies have shown the effects of miR-138 on cell viability in carcinogenicity. The deregulation of miR-138 is frequently associated with a variety of cancers, including HCC (hepatocellular carcinoma), ATC (anaplastic thyroid carcinoma) and CML (chronic myeloid leukaemia) [40–42]. Thus the regulation of the expression of miR-138 may be a potential tumour suppression mechanism in different carcinomas.
In the present study, we have examined the signalling pathway underlying the miR-138-regulated anti-apoptosis of PASMCs. We found that miR-138 induces Akt phosphorylation that leads to the cleavage of procaspase-3 and up-regulation of Bcl-2.
The present study has implicated a central role of the transcriptional factor HIF in regulating gene expression under hypoxic conditions [43,44]. Also, previous studies have shown that regulation of hypoxia-induced miRNAs is HIF-dependent [45,46]. As hypoxic HIF activity is controlled primarily through post-translational modification and stabilization of HIF1α and HIF2α subunits , therefore we used HIF1α and HIF2α siRNA to expose the mechanisms underlying this hypoxia-induced miR-138 modulation. Our results show that the hypoxic regulatory mechanism of miR-138 is transcriptionally mediated via HIF-1α. Further studies are still needed to reveal how HIF-1α regulates miR-138 expression under hypoxic conditions. Precisely analysing the location of the HIF DNA-binding sites or adjacent transcripts regulated by HIF-1α conferring miR-138 regulation will be required. There are more than 300 predicted targets of miR-138, some of which are relevant to the apoptotic process [e.g. CCNL2 (cyclin L2), ROCK (Rho kinase) and RARA (retinoic acid receptor α)]. Obviously, other unidentified miR-138 targets may contribute to the pathological process of HPVR. Nevertheless, the aberrant expression of the global miRNAs in HPH suggests that post-transcriptional gene regulation by miRNAs is an important step in HPH pathogenesis.
A number of miRNAs induced during hypoxia have been identified. Interestingly, reports about these altered miRNAs are different. It has been reported that miR-210 is strongly induced by HIF-1α and has multiple effects in different cell types. For example, in HUVECs (human umbilical vein endothelial cells), miR-210 expression results in increased tubulogenesis and increased cell migration through repression of Ephrin-A3. In stromal cells, miR-210 increases osteoblastic differentiation by repression of AcvR1b while promoting cell migration and invasion in human HCC cells under hypoxic conditions [48–50]. In addition, the roles of other hypoxia-inducible miRNAs (e.g. miR-34a, miR-200b, miR-20a etc.) are indicated in different cellular types [51–53]. Although progress has been made regarding the role of hypoxia-induced miRNAs, their roles in HPH have not been fully understood. Therefore the results from the present study illustrating the role of hypoxia-induced miR-138 in PASMCs constitute a significant step towards the understanding of the molecular mechanisms of HPH.
In conclusion, the present study provides new evidence showing that miR-138 plays an important role in the vascular remodelling of HPH. miR-138 represses Mst1 expression, which, in turn, results in the activation of the Akt signalling pathway functioning as a negative regulator of PASMC apoptosis via a mitochondria-mediated caspase-dependent mechanism. Therefore the stabilization of the miR-138 level may be a novel strategy for clinical treatment of HPH in the future.
Shanshan Li conceived and designed the project, and performed experiments, data analysis and interpretation, wrote the paper and provided financial support. Yajuan Ran conceived and designed the project, and performed experiments, data analysis and interpretation. Dandan Zhang, Jianguo Chen and Shuzhen Li performed experiments, data analysis and interpretation. Daling Zhu conceived and designed the project, and performed experiments, data analysis and interpretation, and provided financial support.
We thank Dr Chun Jiang at Georgia State University, Atlanta, GA, U.S.A. for his comments on this paper prior to submission.
This study was supported by the National Natural Science Foundation of China [grant numbers 30370578 and 31071007]; the Science and Technique Foundation of Harbin [grant numbers 2008AA3AS097 and 2006RFXXS029 (to D.L.Z.)], the National Natural Science Foundation of China [grant number 81100036] and the Science Foundation of Health Department of Heilongjiang Provence [grant number 2009-250 (to S.S.L.)].
Abbreviations: Amo, antisense oligonucleotide; Bad, Bcl-2/Bcl-xL-antagonist, causing cell death; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; H/E, haematoxylin and eosin; HEK, human embryonic kidney; HIF, hypoxia-inducible factor; HPH, hypoxic pulmonary hypertension; HPVR, hypoxic pulmonary vascular remodelling; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; Mim, miRNA mimic; miRNA, (mIR), microRNA; Mst1, serine/threonine kinase 4; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NC, negative control; PA, pulmonary artery; PAH, pulmonary arterial hypertension; PASMC, pulmonary artery smooth muscle cell; PH, pulmonary hypertension; qRT, quantitative real-time reverse-transcription; SD, serum deprivation; siRNA, small interfering RNA; SMC, smooth muscle cell; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling; 3′-UTR, 3′-untranslated region; VSMC, vascular smooth muscle cell; ZDK, Z-VAD-FMK (benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone)
- © The Authors Journal compilation © 2013 Biochemical Society