Deletion mutational analysis of BMRP,a pro-apoptotic protein that binds to Bcl-2

Bcl-2 is an anti-apoptotic member of the Bcl-2 family of proteins that protects cells from apoptosis induced by a large variety of stimuli. The protein BMRP (MRPL41) was identified as a Bcl-2 binding partner and shown to have pro-apoptotic activity. We have performed deletion mutational analyses to identify the domain(s) of Bcl-2 and BMRP that are involved in the Bcl-2/BMRP interaction, and the region(s) of BMRP that mediate its pro-apoptotic activity. The results of these studies indicate that both the BH4 domain of Bcl-2 and its central region encompassing its BH1, BH2, and BH3 domains are required for its interaction with BMRP. The loop region and the transmembrane domain of Bcl-2 were found to be dispensable for this interaction. The Bcl-2 deletion mutants that do not interact with BMRP were previously shown to be functionally inactive. Deletion analyses of the BMRP protein delimited the region of BMRP needed for its interaction with Bcl-2 to the amino-terminal two-thirds of the protein (amino acid residues 1-92). Further deletions at either end of the BMRP(1-92) truncated protein resulted in lack of binding to Bcl-2. Functional studies performed with BMRP deletion mutants suggest that the cell death-inducing domains of the protein reside mainly within its amino-terminal two-thirds. The region of BMRP required for the interaction with Bcl-2 is very relevant for the cell death-inducing activity of the protein, suggesting that one possible mechanism by which BMRP induces cell death is by binding to and blocking the anti-apoptotic activity of Bcl-2.     

Phosphorylation and membrane association of the Rubella virus capsid protein is important for its anti‐apoptotic function

Rubella virus (RV), a member of Togaviridae, is an important human pathogen that can cause severe defects in the developing fetus. Compared to other togaviruses, RV replicates very slowly suggesting that it must employ effective mechanisms to delay the innate immune response. A recent study by our laboratory revealed that the capsid protein of RV is a potent inhibitor of apoptosis. A primary mechanism by which RV capsid interferes with programmed cell death appears to be through interaction with the pro‐apoptotic Bcl‐2 family member Bax. In the present study, we report that the capsid protein also blocks IRF3‐dependent apoptosis induced by the double‐strand RNA mimic polyinosinic‐polycytidylic acid. In addition, analyses of cis‐acting elements revealed that phosphorylation and membrane association are important for its anti‐apoptotic function. Finally, the observation that hypo‐phosphorylated capsid binds Bax just as well as wild‐type capsid protein suggests that interaction with this pro‐apoptotic host protein in and of itself is not sufficient to block programmed cell death. This provides additional evidence that this viral protein inhibits apoptosis through multiple mechanisms.     

Decreased expression of autophagic beclin 1 protein in idiopathic pulmonary fibrosis fibroblasts

Autophagy is the main cellular pathway for degradation of long‐lived proteins and organelles and regulates cell fate in response to stress. Beclin 1 is a key regulator of this process. In some settings autophagy and apoptosis seem to be interconnected. Recent reports indicate that fibroblasts in idiopathic pulmonary fibrosis (IPF) acquire resistance to apoptosis. Here, we examined the expression of beclin 1, and of the anti apoptotic protein Bcl‐2 in human IPF fibroblasts using immunohistochemistry and molecular biology in bioptic sections, in primary cultures of fibroblasts taken from patients with IPF and in fibroblast cell lines. Expression of beclin 1 in fibroblasts from IPF was down‐regulated in comparison with fibroblasts from normal lungs while the anti‐apoptotic protein Bcl‐2 expression was over‐expressed. Treatment of fibroblast cell cultures with cisplatin induced a significant increase in beclin 1 and caspase 3 protein levels but a reduction in Bcl‐2 expression. These observations were confirmed by the analysis of acid compartments and transmission electron microscopy. Our results demonstrate a modified expression of the apoptotic beclin 1 Bcl‐2 proteins in human IPF fibroblasts suggesting the existence of an autophagy/apoptosis system dysfunction. J. Cell. Physiol. 228: 1516–1524, 2013. © 2012 Wiley Periodicals, Inc.     

Dimerization of the hepatitis C virus nonstructural protein 4B depends on the integrity of an aminoterminal basic leucine zipper

The hepatitis C virus (HCV) nonstructural (NS) protein 4B is known for protein–protein interactions with virus and host cell factors. Only little is known about the corresponding protein binding sites and underlying molecular mechanisms. Recently, we have predicted a putative basic leucine zipper (bZIP) motif within the aminoterminal part of NS4B. The aim of this study was to investigate the importance of this NS4B bZIP motif for specific protein–protein interactions. We applied in silico approaches for 3D‐structure modeling of NS4B‐homodimerization via the bZIP motif and identified crucial amino acid positions by multiple sequence analysis. The selected sites were used for site‐directed mutagenesis within the NS4B bZIP motif and subsequent co‐immunoprecipitation of wild‐type and mutant NS4B molecules. Respective interaction energies were calculated for wild‐type and mutant structural models. NS4B‐homodimerization with a gradual alleviation of dimer interaction from wild‐type towards the mutant‐dimers was observed. The putative bZIP motif was confirmed by a co‐immunoprecipitation assay and western blot analysis. NS4B‐NS4B interaction depends on the integrity of the bZIP hydrophobic core and can be abolished due to changes of crucial residues within NS4B. In conclusion, our data indicate NS4B‐homodimerization and that this interaction is facilitated by the aminoterminal part containing a bZIP motif.     

WT p53, but not tumor-derived mutants, bind to Bcl2 via the DNA binding domain and induce mitochondrial permeabilization

The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the anti-apoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component KD 535 +/- 24 nm. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.     

Potential hydrophobic interaction between two cysteines in interior hydrophobic region improves thermostability of a family 11 xylanase from Neocallimastix Patriciarum

In this study, we employed directed evolution and site‐directed mutagenesis to screen thermostable mutants of a family 11 xylanase from Neocallimastix patriciarum, and found that the thermostability and specific activity are both enhanced when mutations (G201C and C60A) take place in the interior hydrophobic region of the enzyme. Far‐ultraviolet circular dichroism analysis showed that the melting temperatures (T_m) of the G201C and C60A–G201C mutants are higher than that of the wild type by about 10 and 12°C, respectively. At 72°C, their specific activities are about 4 and 6 times as that of the wild type, respectively. Homology modeling and site‐directed mutagenesis demonstrated that the enhanced thermostability of the G201C and C60A–G201C mutants may be mainly attributed to a potential stronger hydrophobic interaction between the two well‐packed cysteines at sites 50 and 201, rather than the disulfide bond formation which was ruled out by thiol titration with dithionitrobenzoic acid (DTNB). And the strength of such interaction depends on the packing of the side‐chain and hydrophobicity of residues at these two sites. This suggests that cysteine could stabilize a protein not only by forming a disulfide bond, but also by the strong hydrophobicity itself. Biotechnol. Bioeng. 2010;105: 861–870. © 2009 Wiley Periodicals, Inc.     

Butylene fipronil induces apoptosis in PC12 murine nervous cells via activation of p16‐CDK4/6‐cyclin D1 and mitochondrial apoptotic pathway

Butylene fipronil (BFPN) is a phenylpyrazole insecticide, acting at the γ‐aminobutyric acid (GABA) receptor. Here, we show that BFPN inducedcytotoxicity in PC12 murinenervous cells, which lacks GABA receptor. Treatment with BFPN for 48 hours significantly enhanced G0/G1 arrest and induced apoptosis. BFPN decreased the expression of cyclin‐dependent kinase (CDK4 and CDK6) and increased P16 and cyclin D1. Simultaneously, Bcl‐2 protein was declined while Bax and cytochrome c were significantly enhanced in BFPN‐treated groups. The apoptotic enzymes caspase‐8, ‐9, and ‐3 were also activated by BFPN. Furthermore, treatment with BFPN significantly stimulated reactive oxygen species (ROS) generation, and pretreatment with antioxidant diphenyleneiodonium, substantially reduced cell death. Overall, these results suggest that BFPN is effective to induce G0/G1‐phase arrest and apoptosis in PC12 murine nervous cell. Stimulating ROS generation and activation of P16‐CDK4/6‐cyclin D1 and mitochondrial apoptotic pathway may participate in the cytotoxicity of BFPN.     

Reactive oxygen species induced by Streptococcus pyogenes invasion trigger apoptotic cell death in infected epithelial cells

Streptococcus pyogenes (group A streptococcus, GAS), one of the most common pathogens of humans, attaches and invades into human pharyngeal or skin epithelial cells. We have previously reported that induction of apoptosis is associated with GAS invasion, which induces mitochondrial dysfunction and apoptotic cell death. We demonstrate here that GAS‐induced apoptosis is mediated by reactive oxygen species (ROS) production. Both the induction of apoptosis and ROS production markedly increased upon invasion of wild‐type GAS strain JRS4 into HeLa cells; however, the apoptotic response was not observed in fibronectin‐binding protein F1‐disrupted mutant SAM1‐infected cells. In Bcl‐2‐overexpressing HeLa cells (HBD98‐2‐4), the induction of apoptosis, ROS production and mitochondrial dysfunction were significantly suppressed, whereas the numbers of invaded GAS was not different between HeLa (mock cells) and the HeLa HBD98‐2‐4 cells. Whereas Rac1 activation occurred during GAS invasion, ROS production in GAS‐infected cells was clearly inhibited by transfection with the Rac1 mutants (L37 or V12L37), but not by the dominant active mutant (V12L61) or by the dominant negative mutant (N17). These observations indicate that GAS invasion triggers ROS production through Rac1 activation and generated ROS induced mitochondrial dysfunction leading to cellular apoptosis.     

BMRP is a Bcl-2 binding protein that induces apoptosis

Members of the Bcl-2 family of proteins play important roles in the regulation of cell death by apoptosis. The yeast Two-Hybrid system was utilized to identify a protein that interacts with the anti-apoptotic protein Bcl-2, designated BMRP. This protein corresponds to a previously known mitochondrial ribosomal protein (MRPL41). Binding experiments confirmed the interaction of BMRP to Bcl-2 in mammalian cells. Subcellular fractionation by differential centrifugation studies showed that both Bcl-2 and BMRP are localized to the same fractions (fractions that are rich in mitochondria). Northern blot analysis revealed a major bmrp mRNA band of approximately 0.8 kb in several human tissues. Additionally, a larger 2.2 kb mRNA species was also observed in some tissues. Western blot analysis showed that endogenous BMRP runs as a band of 16-17 kDa in SDS-PAGE. Overexpression of BMRP induced cell death in primary embryonic fibroblasts and NIH/3T3 cells. Transfection of BMRP showed similar effects to those observed by overexpression of the pro-apoptotic proteins Bax or Bad. BMRP-stimulated cell death was counteracted by co-expression of Bcl-2. The baculoviral caspase inhibitor p35 also protected cells from BMRP-induced cell death. These findings suggest that BMRP is a mitochondrial ribosomal protein involved in the regulation of cell death by apoptosis, probably affecting pathways mediated by Bcl-2 and caspases.     

Identification of two amino acids in activin A that are important for biological activity and binding to the activin type II receptors

Activins are members of the transforming growth factor-beta family of growth and differentiation factors. In this paper, we report the results of a structure-function analysis of activin A. The primary targets for directed mutagenesis were charged, individual amino acids located in accessible domains of the protein, concentrating on those that differ from transforming growth factor-beta2, the x-ray crystal structure of which is known. Based on the activities of the recombinant activin mutants in two bioassays, 4 out of 39 mutant proteins (D27K, K102A, K102E, and K102R) produced in a vaccinia virus system were selected for further investigation. After production in insect cells and purification of these four mutants to homogeneity, they were studied in bioassays and in cross-linking experiments involving transfected receptor combinations. Mutant D27K has a 2-fold higher specific bio-activity and binding affinity to an ActRIIA/ALK-4 activin receptor complex than wild type activin, whereas mutant K102E had no detectable biological activity and did not bind to any of the activin receptors. Mutant K102R and wild type activin bound to all the activin receptor combinations tested and were equipotent in bioassays. Our results with the Lys-102 mutants indicate that the positive charge of amino acid 102 is important for biological activity and type II receptor binding of activins.     

Molecular mechanism of a novel CD59‐binding peptide sp22 induced tumor cells apoptosis

Some short peptides discovered by phage display are found to be able to inhibit cancer growth and induce cancer cell apoptosis. In this study, a novel cancer‐targeting short peptide which was composed of 22 amino acids (ACHWPWCHGWHSACDLPMHPMC, abbreviated as sp22) and specifically bound to human CD59 was screened from a M13 phage display library so as to counteract tumor immune escape activity. The mechanism of exogenous sp22 peptide in inducing apoptosis of MCF‐7 cells was investigated. The results suggested that sp22 could lower CD59 expression level, downregulate Bcl‐2 expression, activate Fas and caspase‐3, and finally increase apoptotic cell numbers of MCF‐7 cells. However, sp22 had no obvious influence on normal human embryonic lung cells. In addition, the effects of endogenous sp22 gene on CD59 expression and NKM cell apoptosis were explored using the recombinant plasmid sp22‐PIRES. It showed that sp22 gene was efficiently expressed in transfected NKM cells. Compared with normal NKM cells, NKM cells transfected with sp22 displayed reduced mRNA and protein expression levels of CD59, increased sensitivity to complement‐mediated cytolysis, decreased cell survival ratio, changes of the expression of apoptosis associated proteins, increased number of apoptotic cells and the appearance of apoptotic morphology. The results suggested that sp22 protein could bind to CD59 and inhibit the expression of CD59. The cytolytic activity of complement on tumor cells strengthened and apoptosis signal was stepwise transferred which might be a potential way to kill tumor cells. J. Cell. Biochem. 113: 3810–3822, 2012. © 2012 Wiley Periodicals, Inc.     

IBRDC2, an IBR‐type E3 ubiquitin ligase,is a regulatory factor for Bax and apoptosis activation

Bax, a pro‐apoptotic protein from the Bcl‐2 family, is central to apoptosis regulation. To suppress spontaneous apoptosis, Bax must be under stringent control that may include regulation of Bax conformation and expression levels. We report that IBRDC2, an IBR‐type RING‐finger E3 ubiquitin ligase, regulates the levels of Bax and protects cells from unprompted Bax activation and cell death. Downregulation of IBRDC2 induces increased cellular levels and accumulation of the active form of Bax. The ubiquitination‐dependent regulation of Bax stability is suppressed by IBRDC2 downregulation and stimulated by IBRDC2 overexpression in both healthy and apoptotic cells. Although mostly cytosolic in healthy cells, upon induction of apoptosis, IBRDC2 accumulates in mitochondrial domains enriched with Bax. Mitochondrial accumulation of IBRDC2 occurs in parallel with Bax activation and also depends on the expression levels of Bcl‐xL. Furthermore, IBRDC2 physically interacts with activated Bax. By applying Bax mutants in HCT116 Bax^?/? cells, combined with the use of active Bax‐specific antibodies, we have established that both mitochondrial localization and apoptotic activation of Bax are required for IBRDC2 translocation to the mitochondria.     

Betulinic acid induces apoptosis and suppresses metastasis in hepatocellular carcinoma cell lines in vitro and in vivo

Hepatocellular carcinoma (HCC) is a high incidence and mortality malignant tumour globally. Betulinic acid (BA) is a pentacyclic triterpenoid with potential pro‐apoptotic activities which widely found in many plants. In this study, we determined the effects of BA on proliferation, apoptosis, invasion, and metastasis in HCC cell lines and on tumour growth and pulmonary metastasis in mice. The results suggested that BA could inhibit cell viability and proliferation of HCC cell lines including HepG2, LM3, and MHCC97H. In addition, BA induced apoptosis of HepG2 cells characterised condensed nuclei and nuclear fragmentation. Moreover, western blot analysis showed that BA‐induced apoptosis associated with increasing of pro‐apoptotic protein Bax and cleaved caspase‐3 and decreasing of anti‐apoptotic protein Bcl‐2. Meanwhile, BA also reduced the reactive oxygen species (ROS) level. Furthermore, BA also significantly inhibited HCC growth in vivo and blocked pulmonary metastasis of HCC by regulating the metastasis‐related proteins including MMP‐2, MMP‐9, and TIMP2 without obvious toxicity. In all, the present study suggested that BA might be a promising anti‐HCC drug candidate by inhibiting proliferation, inducing apoptosis, and blocking metastasis.     

Ursolic acid overcomes Bcl‐2‐mediated resistance to apoptosis in prostate cancer cells involving activation of JNK‐induced Bcl‐2 phosphorylation and degradation

Androgen‐independent prostate cancers express high levels of Bcl‐2, and this over‐expression of Bcl‐2 protects prostate cancer cells from undergoing apoptosis. Ursolic acid (UA) has demonstrated an anti‐proliferative effect in various tumor types. The aim of this study is to evaluate the difference between UA‐induced apoptosis in androgen‐dependent prostate cancer cell line LNCaP cells and androgen‐independent prostate cancer cell line LNCaP‐AI cells and to reveal the molecular mechanisms underlying the apoptosis. We found that UA treatment in vitro can effectively induce apoptosis in LNCaP and LNCaP‐AI cells. UA can overcome Bcl‐2‐mediated resistance to apoptosis in LNCaP‐AI cells. Intrinsic apoptotic pathways can be triggered by UA treatment because c‐Jun N‐terminal kinase (JNK) is activated and subsequently provokes Bcl‐2 phosphorylation and degradation, inducing activation of caspase‐9. Although further evaluation is clearly needed, the present results suggest the potential utility of UA as a novel therapeutic agent in advanced prostate cancer. J. Cell. Biochem. 109: 764–773, 2010. © 2009 Wiley‐Liss, Inc.     

Pro‐apoptotic activity of mBNIP‐21 depends on its BNIP‐2 and Cdc42GAP homology (BCH) domain and is enhanced by coxsackievirus B3 infection

Our previous study reported that mouse BNIP‐21 (mBNIP‐21) induces apoptosis through a mitochondria‐dependent pathway. To map the functional domains of mBNIP‐21, we performed mutational analyses and demonstrated that the BNIP‐2 and Cdc42GAP homology (BCH) domain is required for apoptosis induction by mBNIP‐21 targeting the mitochondria and inducing cytochrome c release. This pro‐apoptotic activity was enhanced by coxsackievirus infection. However, deletion of the Bcl‐2 homology 3 (BH3)‐like domain, a well‐known cell ‘death domain’ in proapoptotic Bcl‐2 family proteins, did not affect the activity of mBNIP‐21. These data were further supported by transfection of a mouse Bax (mBax) mutant, whose BH3 was replaced by the mBNIP‐21 BH3‐like domain. This replacement significantly reduced the pro‐apoptotic activity of mBax. We also found that the predicted calcium binding domain has no contribution to the mBNIP‐21‐induced apoptosis. Further mapping of the motifs of BCH domain demonstrated that deletion of the hydrophobic motif proximal to the C‐terminal of the BCH significantly reduced its proapoptotic activity. These findings suggest that mBNIP‐21, as a member of the BNIP subgroup of the Bcl‐2‐related proteins, functions without need of BH3 but its BCH domain is critical for its activity in inducing cell elongation, membrane protrusions and apoptotic cell death.     

Mutagenesis of the regulatory subunit of yeast cAMP-dependent protein kinase. Isolation of site-directed mutants with altered binding affinity for catalytic subunit

Oligonucleotide-directed mutagenesis was used to produce mutants in the hinge region of the regulatory subunit (R) of the Saccharomyces cerevisiae cAMP-dependent protein kinase. The mutant proteins were expressed in Escherichia coli, purified, urea treated to produce cAMP-free regulatory (R), and analyzed in vitro for catalytic (C) subunit inhibitory activity in the presence and absence of cAMP. When assayed in the absence of cAMP, wild type R dimer inhibited C with an IC50 of 40 nM. Replacement of amino acid residue Ser-145 (the autophosphorylation site of yeast R) with Ala or Gly produced mutants which were 2-10-fold better inhibitors of C, while replacement with Glu, Asp, Lys, or Thr produced mutants which were 2-5-fold worse inhibitors of C relative to wild type R. When assayed in the presence of cAMP, all R subunits had a decreased affinity for C subunit, with Ser-145 and Thr-145 undergoing autophosphorylation. These results suggest that the amino acid at position 145 of R contributes to R-C interaction and therefore influences the equilibrium of yeast protein kinase subunits in vitro.     

Engineered NADH-dependent GRE2 from Saccharomyces cerevisiae by directed enzyme evolution enhances HMF reduction using additional cofactor NADPH

Furfural and 5-hydroxymethylfurfural (HMF) are inhibitors generated by lignocellulosic biomass pretreatment such as dilute acid hydrolysis that inhibit microbial growth and interfere with subsequent fermentation. It is possible to in situ detoxify these inhibitory compounds by aldehyde reductions using tolerant Saccharomyces cerevisiae. YOL151W (GRE2) is a commonly recognized up-regulated gene expressed under stress conditions that encodes reductase activities toward furfural and HMF using cofactor NADH. Applying a directed enzyme evolution approach, we altered the genetic code of GRE2 yielding two mutants with amino acid substitutions of Gln261 to Arg261 and Phe283 to Leu283; and Ile107 to Val107, Gln261 to Arg261, and Val285 to Asp285 for strain Y62-C11 and Y62-G6, respectively. Clones of these mutants showed faster growth rates and were able to establish viable cultures under 30 mM HMF challenges when compared with a wild type GRE2 clone when inoculated into synthetic medium containing this inhibitor. Compared with the wild type control, crude cell extracts of the two mutants showed 3- to 4-fold and 3- to 9-fold increased specific enzyme activity using NADH toward HMF and furfural reduction, respectively. While retaining its aldehyde reductase activities using the cofactor NADH, mutant Y62-G6 displayed significantly greater reductase activities using NADPH as the cofactor with 13- and 15-fold increase toward furfural and HMF, respectively, as measured by its partially purified protein. Using reverse engineering and site directed mutagenesis methods, we were able to confirm that the amino acid substitution of the Asp285 is responsible for the increased aldehyde reductase activities by utilizing the additional cofactor NADPH.     

野生型和突变型p16在H460细胞株的表达

应用ＰＣＲ体外定点突为技术,构建了ｐ１６－Ｐ４８Ｌ和ｐ１６－Ｄ７４ｎ突变体。野生型和突变型ｐ１６ｃＤＮＡ克隆地ｐｃＤＮＡ３真核表达载体,导入纯合缺失ｐ１６基因的人肺癌细胞株Ｈ４６０,经ＲＮＡ点杂交初筛吴Ｇ４１８抗性的细胞株,再用Ｎｏｒｔｈｅｒｎ印迹证实外源ｐ１６表达。     

MicroRNA‐181c targets Bcl‐2 and regulates mitochondrial morphology in myocardial cells

Apoptosis is an important mechanism for the development of heart failure. Mitochondria are central to the execution of apoptosis in the intrinsic pathway. The main regulator of mitochondrial pathway of apoptosis is Bcl‐2 family which includes pro‐ and anti‐apoptotic proteins. MicroRNAs are small noncoding RNA molecules that regulate gene expression by inhibiting mRNA translation and/or inducing mRNA degradation. It has been proposed that microRNAs play critical roles in the cardiovascular physiology and pathogenesis of cardiovascular diseases. Our previous study has found that microRNA‐181c, a miRNA expressed in the myocardial cells, plays an important role in the development of heart failure. With bioinformatics analysis, we predicted that miR‐181c could target the 3′ untranslated region of Bcl‐2, one of the anti‐apoptotic members of the Bcl‐2 family. Thus, we have suggested that miR‐181c was involved in regulation of Bcl‐2. In this study, we investigated this hypothesis using the Dual‐Luciferase Reporter Assay System. Cultured myocardial cells were transfected with the mimic or inhibitor of miR‐181c. We found that the level of miR‐181c was inversely correlated with the Bcl‐2 protein level and that transfection of myocardial cells with the mimic or inhibitor of miR‐181c resulted in significant changes in the levels of caspases, Bcl‐2 and cytochrome C in these cells. The increased level of Bcl‐2 caused by the decrease in miR‐181c protected mitochondrial morphology from the tumour necrosis factor alpha‐induced apoptosis.