Smad2和Smad4在家猫睾丸发育和精子发生中的表达与定位

目的 研究转化生长因子β(TGF-β)超家族的下游信号转导分子Smad2和Smad4蛋白,在不同发育阶段家猫睾丸中的表达和定位,探索Smad2和Smad4蛋白与家猫睾丸发育和精子发生的关系. 方法 应用免疫组织化学技术,研究Smad2和Smad4蛋白在幼年(n=3)、青春期(n=3)和性成熟(n=18)睾丸中的定位,并通过Western blotting技术对免疫组织化学中所用抗体的特异性进行了检测. 结果 免疫组织化学结果显示,Smad2和Smad4蛋白定位于各发育阶段家猫睾丸的生殖细胞、支持细胞和间质细胞的胞质中;Western blotting结果显示,多克隆兔抗Smad2和Smad4抗体与家猫睾丸蛋白提取物中分子量约为58kD、66kD的蛋白条带发生免疫阳性反应. 结论 Smad2和Smad4蛋白在家猫睾丸发育和精子发生的各个阶段均有表达,提示其参与睾丸发育和精子发生的调节.     

Expression and localization of Smad2 and Smad4 proteins in the porcine ovary

The objective of the present study was to investigate the temporal and spatial expression of Smad2 and Smad4 proteins, the downstream signaling molecules of the transforming growth factor beta (TGF-β) superfamily, in the porcine ovary. Cellular localization of Smad2 and Smad4 proteins was examined using immunohistochemistry. The specificity of the antibodies was examined using Western blot assay. Western blot analyses demonstrated that 52 kDa Smad2 and 60 kDa Smad4 proteins were expressed in the porcine ovary. Immunohistochemistry revealed that Smad2 and Smad4 were widely expressed in the porcine ovary, mainly localized in the oocyte, granulosa and thecal cells at different stages of folliculogenesis. Within the primordial and primary follicles, Smad2 and Smad4 showed strong staining in oocytes and follicular cells. In the antral follicle, strong staining was observed in oocytes, granulosa and theca cells. These findings suggest that Smad2 and Smad4 may be a key regulator of follicular development and growth of oocytes in the porcine ovary.     

Smad4在人食管癌中的表达

目的探讨Smad4蛋白在食管癌中的表达及其意义。方法应用组织微阵列联合免疫组织化学方法检测45例食管癌及其癌旁正常组织中Smad4蛋白的表达情况;选择其中10例患者的肿瘤组织、癌前病变组织及正常组织的新鲜标本,采用Western blot检测Smad4表达。结果Smad4在食管正常组织中表达的阳性率高于癌组织(P<0.05),在没有淋巴结转移的食管癌组织中表达的阳性率明显高于(P<0.001)有淋巴结转移的癌组织。结论Smad4在食管癌和有淋巴结转移的食管癌低表达。     

Smad4对小鼠眼睑发育的影响

目的: 利用眼特异性 Smad4 基因敲除小鼠,观察其眼睑表型并探讨其眼睑发育异常的可能机制。方法: 选择 PAX6 第一启动子P0驱动的晶体外胚层特异性表达 Cre重组酶的转基因小鼠(Le-Cre)作为介导敲除的工具鼠,将其与 Smad4 条件基因小鼠( Smad4 ^fl/fl)交配获得 Le-Cre特异性Smad4 基因敲除小鼠,通过HE染色揭示其眼睑组织形态学的改变,采用免疫染色技术对某些关键蛋白的表达进行检测并与野生型小鼠进行比较,并检测其眼睑上皮细胞凋亡和增殖的改变。结果: Smad4 在眼睑的失活导致眼睑在发育过程中不能融合,生后眼睑保持开放;Smad4在眼睑的表达缺失不影响眼睑睑缘上皮细胞的增殖和凋亡,但导致上皮细胞内c-Jun磷酸化过程受损,表皮生长因子受体(EGFR)核转位受影响,引起细胞内肌动蛋白束装配异常而导致上皮细胞移行受损,出现眼睑发育时融合不能。结论: Smad4在眼睑发育中对于眼睑的闭合是必需的。     

Smad4/Smad7 balance: A role of tumorigenesis in gastric cancer

Smad signaling pathway plays an important role in tumorigenesis and progression in cancer (Halder, S.K., Rachakonda, G., Deane, N.G., Datta, P.K., 2008. Smad7 induces hepatic metastasis in colorectal cancer. Br. J. Cancer 99, 957-965). The protein level of Smad is associated with growth, inhibition, and metastasis in different cancers. It is unclear if the differentiation, metastasis and apoptosis are reduced by Smad expression pattern in gastric cancer. To determine the effect of Smad on gastric cancer cells, we investigated the relationship of Smad4/Smad7 expression, and differentiation, metastasis, and apoptosis in different gastric cancer. The results show that Smad4 expression in the gastric cancer tissue was dramatically lower than that in the peritumoral tissue. A lower expression of Samd4 was significantly lower in the poorly differentiated tissue than that in the well and middle differentiated tissues (P < 0.01). In contrast, Smad7 expression in gastric cancer tissues was significantly higher than that in the peritumoral tissue. Smad7 was overexpressed in poorly differentiated tissue, also higher than those in the middle, and well differentiated tissues (P < 0.05). The Smad4 or Smad7 expression obviously related with the lymphatic metastasis in gastric cancer. There were 45 cases with lymphatic metastasis in all 78 patients. Smad4 expression in the cases with lymphatic metastasis was lower than the cases without metastasis (P < 0.01), whereas Smad7 expression in the cases with lymphatic metastasis was much higher than the case without metastasis (P < 0.01). To better understand the mechanisms involved in tumorigenesis of gastric cancer, we established SGC7901 gastric cancer cell lines transduced with Smad4 or Smad7 plasmid DNA. Apoptosis and survival of cancer cells was induced after Smad4 and Smad7 transduction. This effect is concentration and time dependent. Thus, this study provides a mechanism by which a balance between Smad4 and Smad7 in human gastric cancer is critical for differentiation, metastasis, and apoptosis of tumor cells.     

miR-431 promotes differentiation and regeneration of old skeletal muscle by targeting Smad4

The myogenic capacity of myoblasts decreases in skeletal muscle with age. In addition to environmental factors, intrinsic factors are important for maintaining the regenerative potential of muscle progenitor cells, but their identities are largely unknown. Here, comparative analysis of microRNA (miRNA) expression profiles in young and old myoblasts uncovered miR-431 as a novel miRNA showing markedly reduced abundance in aged myoblasts. Importantly, elevating miR-431 improved the myogenic capacity of old myoblasts, while inhibiting endogenous miR-431 lowered myogenesis. Bioinformatic and biochemical analyses revealed that miR-431 directly interacted with the 3′ untranslated region (UTR) of Smad4 mRNA, which encodes one of the downstream effectors of TGF-β signaling. In keeping with the low levels of miR-431 in old myoblasts, SMAD4 levels increased in this myoblast population. Interestingly, in an in vivo model of muscle regeneration following cardiotoxin injury, ectopic miR-431 injection greatly improved muscle regeneration and reduced SMAD4 levels. Consistent with the finding that the mouse miR-431 seed sequence in the Smad4 3′ UTR is conserved in the human SMAD4 3′ UTR, inhibition of miR-431 also repressed the myogenic capacity of human skeletal myoblasts. Taken together, our results suggest that the age-associated miR-431 plays a key role in maintaining the myogenic ability of skeletal muscle with age.     

转化生长因子β及其信号转导分子Smad4在肾小球硬化中的作用研究

目的：探讨转化生长因子β（TGF-β1）及其信号转导分子Smad4在肾小球肾炎中的表达及其意义。 方法： 以20例正常肾组织为对照组,对38例各种不同组织学类型肾小球肾炎的肾活检标本,分别行免疫组化方法观察TGF-β1、Smad4、胶原Ⅰ（collagen type Ⅰ）在肾小球中的染色强度,并对其检测结果作图像分析处理;以体外培养的人系膜细胞为对象，观察TGFβ1对系膜细胞表达Smad4、胶原Ⅰ的影响，Smad4、胶原Ⅰ的基因表达用RT-PCR检测，蛋白表达用Western blotting检测。 结果： 所有增生、硬化病理类型肾炎中病变肾小球TGFβ1、Smad4、胶原Ⅰ蛋白表达均高于对照组（P<0.05）,且TGFβ1、Smad4两者在肾小球中的表达与胶原Ⅰ在肾小球中的堆积呈多重相关（P<0.05）；外源性TGFβ1刺激人肾系膜细胞后，细胞的Smad4基因表达和蛋白表达增高（P<0.05）；胶原Ⅰ的基因表达和蛋白表达也相应增高（P<0.05）。 结论： TGFβ1及其信号转导分子Smad4可能参与病变肾小球ECM的过量堆积，在肾小球硬化过程中起重要作用。     

miR-183 inhibits invasion of gastric cancer by targeting Ezrin

miR-183, a member of an evolutionarily conserved miRNA cluster (miR-96, miR-182, and miR-183), has been demonstrated to act as both a tumor suppressor and oncogene in various type of human cancer. However, the biological role of miR-183 in gastric cancer (GC) still remains unclear. In the present study, miR-183 expression was significantly decreased in gastric cancer tissues compared with its’ adjacent normal tissues, and down-regulation of miR-183 was significantly associated with lymph node metastasis and pathological TNM stage. Furthermore, Erzin, which was reported to be up-regulated in gastric cancer, was identified as an efficient target of miR-183. Overexpression of miR-183 markedly suppressed cells invasion by downregulation of Ezrin expression. However, miR-183 expression didn’t affect cells proliferation and cell cycle distribution of GC. In conclusion, our study demonstrated that miR-183 acts as a tumor suppressor in GC, partially at least via regulation of Ezrin. Therefore, miR-183 may be a potential target for the treatment of gastric cancer.     

Cell autonomous requirement of endocardial Smad4 during atrioventricular cushion development in mouse embryos

Atrioventricular (AV) cushions are the precursors of AV septum and valves. In this study, we examined roles of Smad4 during AV cushion development using a conditional gene inactivation approach. We found that endothelial/endocardial inactivation of Smad4 led to the hypocellular AV cushion defect and that both reduced cell proliferation and increased apoptosis contributed to the defect. Expression of multiple genes critical for cushion development was down‐regulated in mutant embryos. In collagen gel assays, the number of mesenchymal cells formed is significantly reduced in mutant AV explants compared to that in control explants, suggesting that the reduction of cushion mesenchyme formation in mutants is unlikely secondary to their gross vasculature abnormalities. Using a previously developed immortal endocardial cell line, we showed that Smad4 is required for BMP signaling‐ stimulated upregulation of Tbx20 and Gata4. Therefore, our data collectively support the cell‐autonomous requirement of endocardial Smad4 in regulating AV cushion development. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

MiR-3653 inhibits the metastasis and epithelial-mesenchymal transition of colon cancer by targeting Zeb2

Aberrant expression of microRNAs (miRNAs) has been widely recognized to play critical roles in the pathogenic processes of colon cancer. However, the expression and functions of miR-3653 in colon cancer remain uncovered. This study revealed for the first time that miR-3653 expression was significantly decreased in colon cancer tissues and cell lines. MiR-3653 overexpression led to decreased migration and invasion of HCT116 cells while miR-3653 knockdown resulted in opposite influence of the metastatic behaviors of HT29 cells. qRT-PCR and western blot demonstrated that miR-3653 suppressed the epithelial-mesenchymal transition (EMT) of colon cancer cells using both gain- and loss- of function assay. Mechanically, miR-3653 was found to interact with the 3′-UTR of Zeb2 through the complementary sequences and inhibited the expression of Zeb2 in colon cancer cells. Rescue experiments demonstrated that the inhibitory effect of miR-3653 overexpression on cell metastasis and EMT was abrogated by forced expression of Zeb2. This study demonstrates that miR-3653 suppresses the metastasis and EMT of colon cells by targeting Zeb2, and serves as a promising biomarker and therapeutic target in colon cancer.     

Immunolocalization of Smad4 protein in the testis of domestic fowl (Gallus domesticus) during postnatal development

The transforming growth factor beta (TGF-β) superfamily exerts a wide range of effects on biological events, including spermatogenesis. Smad proteins are downstream signal mediators, which transduce TGF-β signals from the cell surface to the nucleus. Smad4 protein is the common transducer of the TGF-β superfamily that participates in the signaling of all the members of TGF-β superfamily. Smad4 is expressed in the mammalian testis and is believed to play an important role during testicular development and spermatogenesis. Information about Smad4 distribution and function in the testis of birds, including the domestic fowl, is still unclear. In the current study, our objective was to clarify the signal transduction pathway of the TGF-β superfamily in the regulation of testicular development and spermatogenesis by investigating the expression of Smad4 protein in the testis of newborn, prepuberty, puberty and adult domestic fowl. Cellular localization of Smad4 was determined by immunohistochemistry. Our study revealed that the Smad4 was widely expressed in the fowl testis, mainly immunolocalized in the cytoplasm of Sertoli cells, Leydig cells and germ cells. The presence of Smad4 protein in these testicular cells provides molecular and morphological evidence for TGF-β signal transduction during testicular development and spermatogenesis.     

大黄素通过上调Smad4蛋白表达抑制胰腺癌细胞

目的以Jab1为作用靶点探讨大黄素抗胰腺癌作用及可能的机制。方法 293T细胞随机分为对照组(不处理)、大黄素组(20μmol/L大黄素处理)、Jab1组(转染HA-Jab1质粒),用免疫印迹实验(Western blot)法测定胰腺癌PANC-1和As PC-1细胞系中Jab1与Smad4的蛋白表达。用免疫共沉淀(IP)测定大黄素组及Jab1对β-Tr CP1与Smad4结合的影响。采用Western blot法检测对照组及大黄素干预组PANC-1和As PC-1细胞系中Smad4蛋白表达。结果胰腺癌PANC-1细胞和As PC-1细胞中Jab1呈高表达,Smad4呈低表达,Jab1与Smad4呈负相关关系(P0.01)。293T细胞中Jab1能促使β-Tr CP1与Smad4结合,促进Smad4蛋白降解;而大黄素通过抑制β-Tr CP1与Smad4结合,增加Smad4的表达水平(均P0.05)。结论与Jab1的作用相反,大黄素可能通过抑制泛素化酶途径,抑制Smad4的降解,从而起到抗肿瘤作用。     

Smad4蛋白及mRNA在卵巢不同发育阶段的表达

目的：探讨Smad4在不同发育阶段大鼠卵巢中蛋白及mRNA的表达。 方法： 选择不同发育时期大鼠卵巢，运用免疫组化方法检测卵巢中Smad4蛋白表达，并进行图像分析；采用半定量逆转录聚合酶链反应(RT-PCR)方法检测Smad4 mRNA在卵巢中的表达。 结果： 免疫组化结果显示Smad4主要表达在各级卵泡中，在卵巢发育早期，Smad4主要在原始卵泡和窦前卵泡中表达；随着卵巢的发育成熟，Smad4在窦状及成熟卵泡颗粒细胞和卵泡膜细胞的表达与间质细胞比较无显著差异（P>0.05）。Smad4在卵泡中的表达强度也发生了变化：随着卵泡的发育，Smad4在窦状及成熟卵泡卵母细胞的表达与窦前卵泡卵母细胞比较明显减弱（P<0.05，P<0.01）；在卵泡膜细胞的表达逐渐增强（P<0.01），而在各级卵泡颗粒细胞中的表达无显著差异（P>0.05）。RT-PCR结果显示各阶段卵巢均有mRNA的表达，从第3周起Smad4 mRNA的表达明显增强，与生后1 d比较差异显著（P<0.05）。 结论： 卵巢内存在Smad4，提示TGF-β家族对卵泡发育的调节很可能是通过Smad信号转导模式实现的。     

miR-203a-3p promotes loureirin A-induced hair follicle stem cells differentiation by targeting Smad1

MicroRNAs (miRNAs) participate in the repair of skin trauma. Our previous study indicated that loureirin A promoted hair follicle stem cells (HFSCs) to repair skin epidermis. However, the mechanism of miRNA-mediated regulation of loureirin A-induced HFSC differentiation remained to be explored. In the present study, HFSCs from rat vibrissa were identified by immunofluorescence in vitro. Microarray and quantitative real time polymerase chain reaction analyses demonstrated that miR-203a-3p was upregulated in differentiated HFSCs induced by loureirin A. The expression of cytoskeletal keratin (CK) 5 and involucrin was promoted by miR-203a-3p mimics while repressed by a miR-203a-3p inhibitor. Smad1 was identified as a key target of miR-203a-3p using target prediction tools. Luciferase reporter gene test confirmed a special target association between miR-203a-3p and Smad1. Short interfering Smad1 was transfected into HFSCs, and the expression levels of CK5 and involucrin were upregulated. Thus, it can be inferred that miR-203a-3p negatively regulated the expression of Smad1 and promoted the differentiation of loureirin A-induced HFSCs. Bone morphogenetic protein (BMP) signal inhibition and Wnt activation coregulate skin injury repair. BMP/Smad1 signaling is involved in maintaining the characteristics of HFSCs and inhibiting their differentiation. Our results showed that miR-203a-3p reduces Smad1 to release BMP inhibition. Taken together, miR-203a-3p/Smad1 is a potential therapeutic molecular target in skin wound healing, and may play an active role in wound repair and regenerative medicine.     

Smad4 protein expression correlates with grade, stage, and DNA ploidy in prostatic adenocarcinomas

The tumor suppressor gene Smad4 (DPC4) has been localized to chromosome 18q21.1 and is a member of the Smad family that mediates the transforming growth factor beta signaling pathway suppressing epithelial cell growth. However, variable expression of this protein has been reported, with a loss in some cancers and increased expression in others. Given both the variability and lack of consensus reported regarding Smad4 expression in prostate cancer, we assessed Smad4 immunoreactivity in prostatic adenocarcinomas (PACs). Formalin-fixed, paraffin-embedded tissue sections from 133 PACs were immunostained by a manual method using indirect biotin streptavidin horseradish peroxidase and diaminobenzidine detection using a monoclonal mouse antihuman Smad4 antibody (sc-7966; Santa Cruz Biotechnology Inc, Santa Cruz, Calif). Nuclear immunoreactivity and cytoplasmic immunoreactivity were each semiquantitatively scored based on intensity and percentage of positive cells. Deoxyribonucelic acid ploidy was determined on Feulgen-stained tissue sections by static image analysis. Results were correlated with morphological and prognostic variables. Variable nuclear and cytoplasmic Smad4 positivity was noted in the adjacent benign glands in all cases. Of 133 PACs, 64 (48%) featured increased nuclear and 68 (51%) featured increased cytoplasmic protein expression. Nuclear Smad4 overexpression correlated with tumor grade (P = .02), stage (P = .04), and DNA ploidy (P = .04). Cytoplasmic overexpression correlated with tumor grade (P = .04) and DNA ploidy (P = .04) while showing a trend for correlation with tumor stage (P = .08). Neither nuclear nor cytoplasmic Smad4 overexpression correlated with postsurgical biochemical disease recurrence. Smad4 protein expression persists in PACs compared with benign glands, with both nuclear and cytoplasmic overexpression correlating with prognostic variables indicative of aggressive tumor behavior. Given the significant reported variability of Smad4 in several different cancers, further studies in prostate and other tumors are warranted to elucidate its role in tumorigenesis.     

MiR-125b promotes migration and invasion by targeting the vitamin D receptor in renal cell carcinoma

Purpose: To investigate the expression of miR-125b and vitamin D receptor (VDR) in renal cell carcinoma (RCC) and assess the biological function of miR-125b in RCC.Methods: We used quantitative real-time polymerase chain reaction (RT-PCR) to detect the expression of nucleic acids and western blotting to analyze the protein abundance in RCC cell lines. MiR-125b mimic and inhibitor were employed to investigate the function and behavior of miR-125b in RCC cell lines. The relationship between miR-125 and VDR was verified using luciferase assays.Results: Overexpression of miR-125b promoted migration and invasion and prevent cell apoptosis in ACHN cells. In contrast, miR-125b deficiency suppressed migration and invasion and induced cell apoptosis in 786-O cells. Luciferase assays indicated the interaction between miR-125b and VDR. In collected samples, miR-125b was significantly higher in RCC tissues and negatively correlated to VDR (r=-0.444, p=0.04).Conclusion: MiR-125b displays an oncogene profile in RCC, patients with high expression of miR-125b should be a more frequent follow-up. MiR-125B may be a potential therapeutic target for RCC.     

MiR-195 inhibits proliferation and growth and induces apoptosis of endometrial stromal cells by targeting FKN

MiR-195, which exhibits a proliferation-inhibiting role in different tumors, has been reported to be down-regulated in the ectopic endometrium. The aim of this study was to determine the impact of miR-195 on the biological characteristic of the endometrial stromal cells (ESCs). MiR-195 has been presumed to target the 3’-untranslated regions (3’-UTR) of Fractalkine (FKN), which also plays important roles in endometriosis. Fluorescence reporter assays showed that miR-195 effectively binds to the 3’-UTR of FKN. The normal ESCs showed a significant higher miR-195 expression than that of eutopic and ectopic ESCs associated with endometriosis, while the FKN expression showed opposite results. MiR-195 mimics inhibited proliferation and growth and induced apoptosis of eutopic ESCs, and these effects were abolished by FKN-siRNA. miR-195 could decrease the expression of survivin, matrix metalloproteinase-9 (MMP9) and up-regulate the expression of CD82, tissue inhibitor of metalloproteinase 1 (TIMP1) and TIMP2 of eutopic ESCs by targeting FKN. Our study has demonstrated for the first time that miR-195 plays important roles in regulating the functions of ESCs through targeting FKN. The information may be useful for developing a new therapeutic strategy for endometriosis.