Significant role of μ-calpain (CANP1) in proliferation/survival of bovine skeletal muscle satellite cells

Calpains are a family of Ca²⁺-dependent intracellular cysteine proteases, including the ubiquitously expressed μ-calpain (CANP1) and m-calpain (CANP2). The CANP1 has been found to play a central role in postmortem proteolysis and meat tenderization. However, the physiological roles of CANP1 in cattle skeletal satellite cells remain unclear. In this study, three small interference RNA sequences (siRNAs) targeting CANP1 gene were designed and ligated into pSilencer plasmid vector to construct shRNA expression constructs. Suppression of CANP1 in satellite cells was evaluated using these shRNA expressing constructs. Our results revealed that all three siRNAs could downregulate the expression of CANP1. Suppression of CANP1 significantly reduced cell viability in cell proliferation when compared with control cells. We found a crosstalk between CANP1 and caspase systems, particularly suppression of CANP1 resulted in an increase in the expressions of apoptotic caspases such as caspase-3, caspase-6, caspase-7, caspase-8, and caspase-9, as well as heat-shock protein (HSP) systems. Additionally, suppression of CANP1 led to the upregulation of other apoptosis and DNA damage-regulating genes whilst at the same time downregulating proliferation, migration, and differentiation-regulating genes. The results of our findings report for the first time that suppression of CANP1 resulted in the activation of caspase and HSP systems which might in turn regulate apoptosis through the caspase-dependent cell death pathway. This clearly demonstrates the key roles of CANP1 in regulation of cell proliferation and survival.     

Myostatin gene silencing by RNA interference in chicken embryo fibroblast cells

Myostatin (MSTN), a member of transforming growth factor-β (TGF-β) superfamily, is a negative regulator of the skeletal muscle growth, and suppresses the proliferation and differentiation of myoblast cells. Dysfunction of MSTN gene either by natural mutation or genetic manipulation (knockout or knockdown) has been reported to interrupt its proper function and to increase the muscle mass in many mammalian species. RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful tool for gene knockdown studies. In the present study transient silencing of MSTN gene in chicken embryo fibroblast cells was evaluated using five different shRNA expression constructs. We report here up to 68% silencing of myostatin mRNA using these shRNA constructs in transiently transfected fibroblasts (p<0.05). This was, however, associated with induction of interferon responsive genes (OAS1, IFN-β) (3.7-64 folds; p<0.05). Further work on stable expression of antimyostatin shRNA with minimum interferon induction will be of immense value to increase the muscle mass in the transgenic animals.     

Targeted suppression of μ‐calpain and caspase 9 expression and its effect on caspase 3 and caspase 7 in satellite cells of Korean Hanwoo cattle

The calpains play an important role in cell death and cell signalling. Caspases catalyse wholesale destruction of cellular proteins which is a major cause of cellular death. The current study looks at the function of μ‐calpain and caspase 9, using RNAi (RNA interference)‐mediated silencing, and to observe the mRNA expression level of caspase genes during satellite cell growth. The satellite cells were treated with siRNA (small interfering RNA) of μ‐calpain and caspase 9 separately. There was reduction of 16 and 24% in CAPN1 (calpain1)‐siRNA2 and CAPN1‐siRNA3 transfected cells respectively, whereas it was 60 and 56% in CAPN1‐siRNA1 and CAPN1‐siRNA4 transfected cells respectively. CAPN1‐siRNA4 and CAPN1‐siRNA1 treated cells showed more reduction in caspase 3 and 7 gene expression. CARD9 (caspase recruitment domain 9)‐siRNA1 and CARD9‐siRNA2‐treated cells showed reduction of 40 and 49% respectively. CARD9‐siRNA1 and CARD9‐siRNA2 showed an increase in caspase 3 gene expression, whereas CARD9‐siRNA2 showed reduction in caspase 7 gene expression. These results suggest a strong cross‐talk between μ‐calpain and the caspase enzyme systems. Suppression of target genes, such as μ‐calpain and caspase 9, might have genuine potential in the treatment of skeletal muscle atrophy.     

Effect of Co-transfection of Anti-myostatin shRNA Constructs in Caprine Fetal Fibroblast Cells

Knockdown of myostatin gene (MSTN), transforming growth factor-β superfamily, and a negative regulator of the skeletal muscle growth, by RNA interference (RNAi), has been reported to increase muscle mass in mammals. The current study was aimed to cotransfect two anti-MSTN short hairpin RNA (shRNA) constructs in caprine fetal fibroblast cells for transient silencing of MSTN gene. In the present investigation, approximately 89% MSTN silencing was achieved in transiently transfected caprine fetal fibroblast cells by cotransfection of two best out of four anti-MSTN shRNA constructs. Simultaneously, we also monitored the induction of IFN responsive genes (IFN), pro-apoptotic gene (caspase3) and anti-apoptotic gene (MCL-1) due to cotransfection of different anti-MSTN shRNA constructs. We observed induction of 0.66-19.12, 1.04-4.14, 0.50-3.43, and 0.42-1.98 for folds IFN-β, OAS1, caspase3, and MCL-1 genes, respectively (p < 0.05). This RNAi based cotransfection method could provide an alternative strategy of gene knockout and develop stable caprine fetal fibroblast cells. Furthermore, these stable cells can be used as a cell donor for the development of transgenic cloned embryos by somatic cell nuclear transfer (SCNT) technique.     

核受体相关因子1表达下调对多巴胺能细胞酪氨酸羟化酶和神经突起生长的影响

将合成的核受体相关因子1（nuclear receptor-related factor 1,Nurr1）特异性短发夹寡核苷酸（small-hairpin RNA,shRNA）序列插入真核表达载体pSilen Circle（pSC）,构建Nurr1基因特异性shRNA真核表达载体,转染体外培养多巴胺能神经前体细胞系MN9D,分别采用实时荧光定量PCR和Western blot方法检测其对MN9D细胞内源Nurr1的干扰作用及其对酪氨酸羟化酶（tyrosine hydroxylase,TH）表达的影响,并在倒置显微镜下观察MN9D细胞神经突起生长的情况,探讨Nurr1 shRNA表达载体对多巴胺能细胞表型标记物删和以神经突起延长为特征的细胞成熟的影响。结果表明,脂质体组细胞和转染阴性对照质粒的MN9D细胞内Nurr1、TH的表达正常,而转染Nurr1 shRNA真核表达载体（pSC-N1和pSC-N2）的MN9D细胞内Nurr1和TH的mRNA水平明显降低,Nurr1 mRNA的下降率分别为62．3％和45．6％,TH mRNA的下降率分别为76.3％和62．6％。同时Nurr1和TH蛋白的表达亦明显下调,Nurr1蛋白的下降率分别为57．4％和72．0％,TH蛋白的下降率分别为79．1％和70．1％。另外,转染Nurr1 shRNA真核表达质粒的MN9D细胞神经突起延长有所减少,但是与正常细胞无明显差异。结果提示：Nurr1 shRNA真核表达载体能显著下调MN9D细胞内源Nurr1和TH mRNA和蛋白的表达,同时可能对MN9D细胞的神经突起延长有一定的抑制作用。Nurr1 shRNA表达载体的成功构建为多巴胺能神经元发育以及帕金森病相关基因的功能研究奠定了基础。     

Androgen receptor enhances myogenin expression and accelerates differentiation

Animal and clinical studies indicated that the androgen-AR signaling pathway is required for appropriate development of sexually dimorphic skeletal muscles and increases lean muscle mass, muscle strength, and muscle protein synthesis. However, the detailed mechanisms by which the androgen-AR signaling pathway regulates skeletal muscle development need further study at the molecular level. C2C12 myoblast cells stably transfected with the Flag-tagged AR were used to analyze the role of androgen-AR signaling pathway in skeletal muscle development. The results indicate that the androgen-AR signaling pathway may suppress skeletal myoblast cell growth and accelerate myoblast cell differentiation via enhanced myogenin expression. This is a first report showing the role of androgen-AR signaling pathway in regulation of myoblast cell growth and myogenic regulatory factors.     

SiRNA‐Mediated Down‐Regulation of CLIC4 Gene Inhibits Cell Proliferation and Accelerates Cell Apoptosis of Mouse Liver Cancer Hca‐F and Hca‐P Cells

This study explored the effects involved in silencing CLIC4 on apoptosis and proliferation of mouse liver cancer Hca‐F and Hca‐P cells. A CLIC4‐target small interfering RNA (siRNA) was designed to compound into two individual complementary oligonucleotide chains. A process of annealing and connection to a pSilencer vector was followed by transfection with Hca‐F and Hca‐P cells. Quantitative real‐time polymerase chain reaction and Western blotting techniques were used to determine CLIC4 mRNA and protein expressions. CCK8 assay and flow cytometry were employed for analysis of the survival and apoptosis rate as well as the cell cycle in an octreotide‐induced apoptosis model. Expressions of caspase 3, caspase 9, and cleaved PARP were measured using Western blotting. The CLIC4 mRNA and protein expressions in Hca‐F and Hca‐P cells transfected by pSilencer‐CLIC4 siRNA plasmid in the blank group displayed remarkably decreased levels of expression, when compared with both the control and negative control (NC) groups. Decreased survival rates and cleaved PARP expression, increased cell apoptosis rate,expressions of caspase 3 and caspase 9 in Hca‐F and Hca‐P cells were detected in groups that had been cultured in a medium containing octreotide. The pSilencer‐CLIC4 siRNA‐2 group when compared with the control and NC groups exhibited decreased survival rates, cleaved PARP expression, increased cell apoptosis rates, and increased expressions of caspase 3 and caspase 9 of Hca‐F and Hca‐P cells. The results demonstrated that siRNA‐induced down‐regulation of CLIC4 could proliferation, while in turn promoting apoptosis of mouse liver cancer Hca‐F and Hca‐P cells. J. Cell. Biochem. 119: 659–668, 2018. © 2017 Wiley Periodicals, Inc.     

RNA干扰抑制麻疹病毒体外复制的实验研究

为了研究短发夹RNA(shRNA)介导的RNA干扰对麻疹病毒体外复制的抑制作用,构建靶向与麻疹病毒复制密切相关的宿主细胞基因Rab9 GTPase基因特异性shRNA表达载体,分别转染Vero-E6和B95a细胞后感染麻疹病毒Edmonston株和野生株。逆转录聚合酶链反应(RT-PCR)和免疫印迹技术(Western-blot)检测转染细胞内Rab9 GTPase基因表达水平;标准蚀斑试验测定麻疹病毒滴度。结果显示转染细胞内Rab9 GTPase mRNA和蛋白质的表达水平同对照组相比明显降低,标准蚀斑试验显示麻疹病毒的复制受到显著抑制,抑制率达到90%以上。结果表明载体介导的shRNAs能通过特异性下调Rab9 GTPase基因表达抑制麻疹病毒体外复制,Rab9 GTPase可能成为治疗麻疹病毒感染的RNA干扰靶。     

Hyperosmolarity‐mediated mitochondrial dysfunction requires Transglutaminase‐2 in human Corneal epithelial cells

Hyperosmolar‐induced ocular surface cell death is a key mitochondria‐mediated event in inflammatory eye diseases. Transglutaminase (TGM)‐2, a cross‐linking enzyme, is purported to mediate cell death, but its link to mitochondria is unclear. In the cornea, the integrity of the epithelial cells is important for maintaining transparency of the cornea and therefore functional vision. We evaluated the role of TGM‐2 and its involvement in hyperosmolarity‐stimulated mitochondrial cell death in human corneal epithelial (HCE‐T) cells. HCE‐T cell lines stably expressing either shRNA targeting TGM‐2 (shTG) or scrambled shRNA (shRNA) were constructed. Hyperosmolar conditions reduced viability and increased mitochondrial depolarization in shRNA cells. However, hyperosmolarity failed to induce mitochondrial depolarization to the same extent in shTG cells. Transient overexpression of TGM‐2 resulted in very high levels of TGM‐2 expression in shTG and shRNA cells. In the case of shTG cells after overexpression of TGM‐2, hyperosmolarity induced the same extent of mitochondrial depolarization as similarly treated shRNA cells. Overexpression of TGM‐2 also elevated transamidase activity and reduced viability. It also induced mitochondrial depolarization, increased caspase‐3/7 and ‐9 activity, and these increases were partially suppressed by pan‐caspase inhibitor Z‐VAD‐FMK. Corneal epithelial apoptosis via mitochondrial dysfunction after hyperosmolar stimulation is partially dependent on TGM‐2. This TGM‐2‐dependent mechanism occurs in part via caspase‐3/7 and ‐9. Protection against mitochondrial stress in the ocular surface targeting TGM‐2 may have important implications in the survival of cells in hyperosmolar stress. J. Cell. Physiol. 226: 693–699, 2011. © 2010 Wiley‐Liss, Inc.     

人同源盒基因NKX3.1对前列腺癌细胞的诱导凋亡作用

构建人同源盒基因NKX3.1 cDNA真核表达载体,研究其在前列腺癌细胞PC-3、LNCaP 中的表达及对细胞的促凋亡作用.以人前列腺癌细胞LNCaP细胞中的总RNA为模板,RT-PCR扩增NKX3.1基因全长编码片段,将NKX3.1 cDNA重组到真核表达载体pcDNA3.1（+）中; 将pcDNA3.1-NKX3.1表达载体瞬时转染前列腺癌细胞PC-3和LNCaP 细胞,用RT-PCR和Western印迹检测NKX3.1 cDNA在转录水平和蛋白水平的表达;绘制细胞生长曲线,观察NKX3.1对前列腺癌细胞增殖的抑制作用;用DNA/ladder和流式细胞术检测NKX3.1对前列腺癌细胞凋亡的影响,进一步用RT PCR检测凋亡相关基因caspase3、caspase8、caspase9、Apaf1、survivin和Bcl2表达的变化.人同源盒基因NKX3.1 cDNA真核表达载体pcDNA3.1-NKX3.1经酶切及测序鉴定正确. pcDNA3.1-NKX3.1转染PC-3和LNCaP细胞后,经RT-PCR和Western印迹证明能有效表达NKX3.1.生长曲线显示,前列腺癌细胞转染NKX3.1 cDNA后细胞增殖受到抑制;前列腺癌细胞转染NKX3.1 cDNA 48 h后,DNA电泳呈现具有凋亡特征的DNA ladder;流式细胞术检测出现明显凋亡峰;RT-PCR检测凋亡相关基因.结果显示,caspase3、caspase8、caspase9基因表达明显增加,Bcl2基因表达明显减少.本研究成功构建了真核表达载体pcDNA3.1 NKX3.1, 转染PC3和LNCaP细胞后能有效表达,并对细胞具有诱导凋亡作用     

RNA interference-mediated silencing of laminin receptor 1 (LR1) suppresses migration and invasion and down-regulates matrix metalloproteinase (MMP)-2 and MMP-9 in trophoblast cells: implication in the pathogenesis of preeclampsia

Shallow trophoblast invasion is a common pathological feature of preeclampsia. The 67 kDa laminin receptor 1 (LR1) is a laminin-binding protein that has been reported to be down-regulated in preeclamptic placentas. The aim of the present study was to determine the functional role of LR1 in the migration and invasion of the trophoblast cell line, JEG3 cells. RNA interference mediated by plasmid expressing LR1 short hairpin RNA (shRNA) was utilized to knockdown LR1 expression in JEG3 cells. We found that the mRNA and protein expression levels of LR1 were significantly reduced in LR1-specific shRNA transfected cells compared with the untransfected and control shRNA transfected cells. The wound healing and Transwell invasion assays demonstrated that LR1 knockdown remarkably suppressed the migration and invasion potential of JEG3 cells. The gelatin zymography assay showed that LR1 knockdown greatly reduced matrix metalloproteinase (MMP)-2 and MMP-9 activities in the culture supernatants. Western blot analysis showed that LR1 shRNA significantly decreased expression levels of MMP-2, MMP-9 and phospho-extracellular signal-regulated kinase, but increased expression levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in comparison to the control vector-transfected cells. In conclusion, our data support an important role for LR1 in regulating trophoblast invasion and migration, and suggest a possible pathological mechanism of preeclampsia.     

鼻咽癌联合基因治疗的体外研究

目的:探讨人端粒酶逆转录酶(hTERT),癌基因蛋白(C-myc),存活素(Survivin),血管内皮生长因子(VEGF)基因对人类鼻咽癌细胞生长的影响,以及同时编码四个基因的短发夹重组质粒对人鼻咽癌细胞生长抑制作用及机制。方法:利用基因重组技术构建一个同时靶向作用四个基因的短发夹双链RNA(shRNA)真核表达载体和靶向单独作用hTERTmRNA的shRNA真核表达载体,脂质体法转染人CNE-2Z细胞;试验分组:空白对照组BC组(不进行干扰),阴性对照组NC组(加入阴性质粒),A组(hTERT单基因质粒组),B组(多基因联合质粒组)。激光共聚焦显微镜观察转染情况;MTT法检测细胞增殖活性;RT-PCR和Western blot法分别检测转染后细胞内各基因mRNA和蛋白表达情况。结果:MTT法检测,与BC相,NC组相比,A组和B组的细胞增殖活性均降低,与A组相比,B组的增殖活性降低更显著;RT-PCR,Western blot法,A组和B组mRNA和蛋白表达水平均降低,B组降低更显著。结论:四个基因共同参与了鼻咽癌细胞的发生和发展。多个基因的联合干扰与单基因干扰相比,能更高效下调各基因蛋白在鼻咽癌细胞的表达水平,更好抑制鼻咽癌细胞的增殖。     

Inhibition of phosphodiestrase 9 induces cGMP accumulation and apoptosis in human breast cancer cell lines, MCF-7 and MDA-MB-468

Objectives

Phosphodiesterase 9 (PDE9) is a major isoform of phosphodiesterase hydrolysing cGMP and plays a key role in proliferation of cells, their differentiation and apoptosis, via intracellular cGMP signalling. The study described here was designed to investigate expression, activity and apoptotic effect of PDE9 on human breast cancer cell lines, MCF‐7 and MDA‐MB‐468.

Materials and methods

Activity and expression of PDE9 were examined using colorimetric cyclic nucleotide phosphodiesterase assay and real‐time RT‐PCR methods respectively; cGMP concentration was also measured. MTT viability test, annexin V‐FITC staining, Hoechst 33258 staining and caspase3 activity assay were used to detect apoptosis.

Results

Treatment of both cell lines with BAY 73‐6691 lead to reduction in PDE9 mRNA expression, PDE9 cGMP‐hydrolytic activity and elevation of the intracellular cGMP response. BAY 73‐6691 significantly reduced cell proliferation in a dose‐ and time‐dependent manner and caused marked increase in apoptosis through caspase3 activation.

Conclusion

Our results revealed that BAY 73‐6691 induced apoptosis in these breast cancer cell lines through the cGMP pathway. These data suggest that BAY 73‐6691 could be utilized as an agent in treatment of breast cancer.     

Cell adhesion to collagen and decreased myogenic gene expression implicated in the control of myogenesis by transforming growth factor beta

Transforming growth factor beta 1 (TGF-beta 1) is an inhibitor of skeletal muscle myoblast differentiation. Myoblast differentiation is dependent on the expression of certain myogenic differentiation genes and is affected by cell interaction with the extracellular matrix. We have searched for events in the differentiation process of L6E9 rat myoblasts that may be involved in the inhibitory action of TGF-beta 1. Elevated expression of the myogenic differentiation gene, myogenin, which is thought to play a central role in the differentiation process, occurs 10 h after the shift of L6E9 myoblasts to differentiation medium. Elevation of myogenin mRNA is blocked by TGF-beta 1 added at the time of the shift. This effect is preceded by, and might be related to, a rapid up-regulation of junB mRNA observed in TGF-beta 1-treated L6E9 myoblasts. However, TGF-beta 1 can also block myogenic differentiation in cells transfected with the myogenin gene under the control of a constitutive SV40 viral promoter. The nature of a mechanism that could mediate the inhibitory action of TGF-beta 1 without blocking myogenin mRNA expression is suggested by the observations that (a) TGF-beta 1 upregulates type I collagen expression and deposition in L6E9 myoblasts, (b) a fibrillar type I collagen layer inhibits L6E9 myoblast differentiation, and (c) inhibition of L6E9 myoblast differentiation by a type I collagen layer occurs without a block in myogenin expression. Thus, the data suggest that inhibition of L6E9 myoblast differentiation by TGF-beta 1 may be accomplished by at least two mechanisms acting in concert. One mechanism leads to a block in the expression of myogenin whereas the other mechanism may involve TGF-beta 1-induced changes in cell adhesion that either block the action of myogenic differentiation gene products or prevent the function of other as yet unknown components of the myogenic differentiation pathway.