MiR-664-3p suppresses osteoblast differentiation and impairs bone formation via targeting Smad4 and Osterix.

Osteoporosis is a metabolic disorder characterized by low bone mass and deteriorated microarchitecture, with an increased risk of fracture. Some miRNAs have been confirmed as potential modulators of osteoblast differentiation to maintain bone mass. Our miRNA sequencing results showed that miR-664-3p was significantly down-regulated during the osteogenic differentiation of the preosteoblast MC3T3-E1 cells. However, whether miR-664-3p has an impact on bone homeostasis remains unknown. In this study, we identified overexpression of miR-664-3p inhibited the osteoblast activity and matrix mineralization in vitro. Osteoblastic miR-664-3p transgenic mice exhibited reduced bone mass due to suppressed osteoblast function. Target prediction analysis and experimental validation confirmed Smad4 and Osterix (Osx) are the direct targets of miR-664-3p. Furthermore, specific inhibition of miR-664-3p by subperiosteal injection with miR-664-3p antagomir protected against ovariectomy-induced bone loss. In addition, miR-664-3p expression was markedly higher in the serum from patients with osteoporosis compared to that from normal subjects. Taken together, this study revealed that miR-664-3p suppressed osteogenesis and bone formation via targeting Smad4 and Osx. It also highlights the potential of miR-664-3p as a novel diagnostic and therapeutic target for osteoporotic patients.

Arginine methylation of R81 in Smad6 confines BMP-induced Smad1 signaling.

Bone morphogenetic proteins (BMPs) secreted by a variety of cell types are known to play essential roles in cell differentiation and matrix formation in the bone, cartilage, muscle, blood vessel, and neuronal tissue. BMPs activate intracellular effectors via C-terminal phosphorylation of Smad1, Smad5, and Smad9, which relay the signaling by forming a complex with Smad4 and translocate to the nucleus for transcriptional activation. Smad6 inhibits BMP signaling through diverse mechanisms operative at the membrane, cytosolic, and nuclear levels. However, the mechanistic underpinnings of Smad6 functional diversity remain unclear. Here, using a biochemical approach and cell differentiation systems, we report a cytosolic mechanism of action for Smad6 that requires arginine methylation at arginine 81 (R81) and functions through association with Smad1 and interference with the formation of Smad1-Smad4 complexes. By mutating the methylated arginine residue, R81, and by silencing the expression of protein arginine methyltransferase 1, we show that protein arginine methyltransferase 1 catalyzes R81 methylation of Smad6 upon BMP treatment, R81 methylation subsequently facilitates Smad6 interaction with the phosphorylated active Smad1, and R81 methylation facilitates Smad6-mediated interruption of Smad1-Smad4 complex formation and nuclear translocation. Furthermore, Smad6 WT but not the methylation-deficient R81A mutant inhibited BMP-responsive transcription, attenuated BMP-mediated osteogenic differentiation, and antagonized BMP-mediated inhibition of cell invasion. Taken together, our results suggest that R81 methylation plays an essential role in Smad6-mediated inhibition of BMP responses.

Structural based screening of potential inhibitors of SMAD4: a step towards personalized medicine for gall bladder and other associated cancers.

Gall bladder cancer (GBC) is an aggressive and most common malignancy of biliary tract lacking effective treatment due to unavailability of suitable biomarkers and therapeutics. SMAD4 is an essential mediator of transforming growth factor-ß pathway involved in various cellular processes like growth, differentiation and apoptosis and also recognized as therapeutic target for GBC and other gastrointestinal tract cancers. In the present study, 3D structure of SMAD4 mutants was optimized through molecular dynamics simulation (MDS) along with wildtype. Furthermore, binding site of protein was predicted through hybrid approach and structural based virtual screening against two drug libraries was performed followed by docking. MDS of top docking score protein-ligand complexes were carried, and binding free energy was rescored. Two potential inhibitors, namely ZINC2098840 and ZINC8789167, were screened that displayed higher binding affinity towards mutant proteins compared with wildtype and both hydrophilic as well as hydrophobic interactions play a crucial role during protein-ligand binding. Current study identified novel and potent inhibitors of SMAD4 mutant that could be used as a drug candidate for the development of personalized medicine for gall bladder and other associated cancers.

A new sesquiterpenoid from the shoots of Iranian Daphne mucronata Royle with selective inhibition of STAT3 and Smad3/4 cancer-related signaling pathways.

PURPOSE: Daphne mucronata Royle grown in Iran has shown anticancer activities against different cancer cell lines. Therefore, within this study, we investigate the phytochemical pattern of this plant. METHOD: Phytochemical investigation was done using standard column chromatography system: The structures were recognized by the interpretation of one and two-dimensional nuclear magnetic resonance (NMR) spectra and the help of High-Resolution Electrospray Ionization Mass spectroscopy (HR-ESIMS) and Infrared spectroscopy (IR) data. Stereochemistry was determined using 2D and 3D NOESY, and comparison of coupling constant values with literature. The absolute configuration was determined and confirmed using specific rotation and electronic circular dichroism experiments. Cytotoxicity was done against HeLa cells by standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Luciferase assay was used to check if the compounds can inhibit the activation of cancer-related signaling pathways. Molecular docking simulation was done for biological activity evaluation and to examine the interaction of the ligand with each of the proteins. RESULTS: A new sesquiterpenoid, 4,11(12)-guiadiene-1-ol-3-one (4), together with eight specialized metabolites, betulinic acid (1), coniferyl aldehyde (2), oleanolic acid (3), daphnetoxin (5), apigenin (7), syringin (8), and genkwanol A (9) were isolated and reported for the first time from the shoots of the plant. Compound 4 as an undescribed compound was submitted for cytotoxicity assay and showed moderate activity with the IC50 value of 51.3 ± 4.2 µM against HeLa cancer cells. It showed selective inhibition of Interleukin-6 mediated signal transducer and activator of transcription 3 pathway (STAT-3/ IL-6), and Smad protein / transforming growth factor beta (TGF-ß) transcription factors when screened through an array of cancer signaling pathways. Molecular docking confirmed biological tests and showed the interaction with STAT3 and Smad proteins. CONCLUSION: An undescribed sesquiterpenoid: 4,11(12)-guiadiene-1-ol-3-one in addition to eight known compounds were isolated. The new sesquiterpene was evaluated for the luciferase assay on 14 main cancer-related signaling pathways and showed selective inhibition of STAT3/IL6, and Smad/ TGF-ß transcription factors. Molecular docking simulation showed more interactions with STAT3 than Smad, which confirms better interaction of compound 4 with STAT3 than Smad proteins. Graphical abstract.

Effects of suppressing Smads expression on wound healing in Hyriopsis cumingii.

As a specific pearl mussel in China, Hyriopsis cumingii has enormous economic value. However, the organism damage caused by pearl insertion is immeasurable. TGF-ß/Smad signal transduction pathways are involved in all phases of wound healing. We have previously reported on two cytoplasmic signal transduction factors, Smad3 and Smad5 in mussel H. cumingii (named HcSmads), suggesting their involvements in wound healing. Here, Smad4 was cloned and described. The full length cDNA of HcSmad4 was 2543 bp encoded 515 amino acids. Deduced HcSmad4 protein possessed conserved MH1 and MH2 domains, nuclear location signals (NLS), nuclear exput signals (NES) and Smad activation domain (SAD). Transcripts of Smad3, 4 and 5 were constitutively expressed in all detected tissues, at highest levels in muscles. Furthermore, HcSmad4 mRNA levels were significantly increased at incision site post wounding, and expression of downstream target genes of Smad4, such as HcMMP1, HcMMP19, HcTIMP1 and HcTIMP2 were upregulated to a certain extent. Whatever knocked down HcSmad3/4 or treated by specific inhibitors of Smad 3 (SIS3), expression levels of these genes displayed a significantly downregulated tendency compared with the wound group. In addition, histological evaluation suggested that Smad3 knockdown or SIS3 treatment was accelerated wound healing, and then Smad4 knockdown delayed the process of wound healing in mussels. These data implicate that Smad3/4 play an important role in tissue repair in mollusks.

Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells.

As an important post-transcriptional regulatory machinery mediated by ∼21nt short-interfering double-stranded RNA (siRNA), RNA interference (RNAi) is a powerful tool to delineate gene functions and develop therapeutics. However, effective RNAi-mediated silencing requires multiple siRNAs for given genes, a time-consuming process to accomplish. Here, we developed a user-friendly system for single-vector-based multiplex siRNA expression by exploiting the unique feature of restriction endonuclease BstXI. Specifically, we engineered a BstXI-based shotgun cloning (BSG) system, which consists of three entry vectors with siRNA expression units (SiEUs) flanked with distinct BstXI sites, and a retroviral destination vector for shotgun SiEU assembly. For proof-of-principle studies, we constructed multiplex siRNA vectors silencing ß-catenin and/or Smad4 and assessed their functionalities in mesenchymal stem cells (MSCs). Pooled siRNA cassettes were effectively inserted into respective entry vectors in one-step, and shotgun seamless assembly of pooled BstXI-digested SiEU fragments into a retroviral destination vector followed. We found these multiplex siRNAs effectively silenced ß-catenin and/or Smad4, and inhibited Wnt3A- or BMP9-specific reporters and downstream target expression in MSCs. Furthermore, multiplex silencing of ß-catenin and/or Smad4 diminished Wnt3A and/or BMP9-induced osteogenic differentiation. Collectively, the BSG system is a user-friendly technology for single-vector-based multiplex siRNA expression to study gene functions and develop experimental therapeutics.

SMAD4 Prevents Flow Induced Arteriovenous Malformations by Inhibiting Casein Kinase 2.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is an inherited vascular disorder that causes arteriovenous malformations (AVMs). Mutations in the genes encoding Endoglin ( ENG) and activin-receptor-like kinase 1 ( AVCRL1 encoding ALK1) cause HHT type 1 and 2, respectively. Mutations in the SMAD4 gene are present in families with juvenile polyposis-HHT syndrome that involves AVMs. SMAD4 is a downstream effector of transforming growth factor-ß (TGFß)/bone morphogenetic protein (BMP) family ligands that signal via activin-like kinase receptors (ALKs). Ligand-neutralizing antibodies or inducible, endothelial-specific Alk1 deletion induce AVMs in mouse models as a result of increased PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) signaling. Here we addressed if SMAD4 was required for BMP9-ALK1 effects on PI3K/AKT pathway activation. METHODS: The authors generated tamoxifen-inducible, postnatal, endothelial-specific Smad4 mutant mice ( Smad4iΔEC). RESULTS: We found that loss of endothelial Smad4 resulted in AVM formation and lethality. AVMs formed in regions with high blood flow in developing retinas and other tissues. Mechanistically, BMP9 signaling antagonized flow-induced AKT activation in an ALK1- and SMAD4-dependent manner. Smad4iΔEC endothelial cells in AVMs displayed increased PI3K/AKT signaling, and pharmacological PI3K inhibitors or endothelial Akt1 deletion both rescued AVM formation in Smad4iΔEC mice. BMP9-induced SMAD4 inhibited casein kinase 2 ( CK2) transcription, in turn limiting PTEN phosphorylation and AKT activation. Consequently, CK2 inhibition prevented AVM formation in Smad4iΔEC mice. CONCLUSIONS: Our study reveals SMAD4 as an essential effector of BMP9-10/ALK1 signaling that affects AVM pathogenesis via regulation of CK2 expression and PI3K/AKT1 activation.

MiR-378 and BMP-Smad can influence the proliferation of sheep myoblast.

MicroRNA (miRNA) is a sort of endogenous ~20-25 nt non-coding RNAs, and it can regulate a variety of biological events. We found the miR-378 may involve in regulating the muscle development of sheep during our previous research. However, the molecular mechanism of miR-378 regulating myoblast proliferation is still unclear. In this research, we predicted that BMP2 (Bone morphogenetic protein 2) was the target gene of miR-378 and the BMP-Smad signal pathway that BMP2 participated in playing an important role in the muscle development. Therefore, we tried to determine whether miR-378 influence myoblast proliferation of sheep through the BMP-Smad signal pathway. The results indicated that inhibit BMP-Smad signal pathway by interfering Smad4 to promote proliferation of sheep myoblasts; promote BMP-Smad signal pathway by interfering Smad7 to inhibit proliferation of sheep myoblasts; over-expression miR-378 promotes BMP-Smad signal pathway and myoblast proliferation in sheep; interfering miR-378 inhibits BMP-Smad signal pathway and myoblast proliferation in sheep. However, when both of which functioned at the myoblast, miR-378 could not fully depend on BMP-Smad signal pathway to regulate myoblast proliferation. In sum, both miR-378 and BMP-Smad can influence the proliferation of myoblast, but miR-378 does not target the 3' UTR of sheep BMP2.

TGF-ß1 Promotes Hepatocellular Carcinoma Invasion and Metastasis via ERK Pathway-Mediated FGFR4 Expression.

BACKGROUND/AIMS: TGF-ß1 is beneficial during early liver disease but is tumor-progressive during late stages especially for hepatocellular carcinoma (HCC). Thus, exploring the underlying mechanisms may provide information about a potentially therapeutic role of TGF-ß1 in HCC. METHODS: Western blot and real-time quantitative PCR were used to quantify FGFR4 expression in HCC cell lines and a normal liver cell line. After constructing the best silencing FGFR4 expression vector, migration and invasiveness of TGF-ß1 in HCC was studied in vitro and in vivo. Western blot was used to study the mechanism of TGF-ß1 induction on FGFR4 expression with various inhibitors. RESULTS: HepG2 cell lines had the most FGFR4 expression, and data show that silencing FGFR4 suppressed cell proliferation, invasion and migration in HCC induced by TGF-ß1 in vitro and in vivo. Moreover, TGF-ß1 induced FGFR4 expression through the ERK pathway. CONCLUSION: Promoting FGFR4 expression via the ERK pathway, TGF-ß1 contributes to HCC invasion and metastasis.

Nemopilema nomurai jellyfish venom exerts an anti-metastatic effect by inhibiting Smad- and NF-κB-mediated epithelial-mesenchymal transition in HepG2 cells.

Epithelial-mesenchymal transition (EMT) is a key initial step in metastasis for malignant cancer cells to obtain invasive and motile properties. Inhibiting EMT has become a new strategy for cancer therapy. In our previous in vivo study, Nemopilema nomurai jellyfish venom (NnV) -treated HepG2 xenograft mice group showed that E-cadherin expression was strongly detected compared with non-treated groups. Therefore, this study aimed to determine whether NnV could inhibit the invasive and migratory abilities of HepG2 human hepatocellular carcinoma cells and to examine its effect on EMT. Our results revealed that transforming growth factor (TGF)-ß1 induced cell morphological changes and downregulated E-cadherin and ß-catenin expression, but upregulated N-cadherin and vimentin expression through the Smad and NF-κB pathways in HepG2 cells. Treatment of TGF-ß1-stimulated HepG2 cells with NnV reversed the EMT-related marker expression, thereby inhibiting cell migration and invasion. NnV also significantly suppressed the activation of p-Smad3, Smad4, and p-NF-κB in a dose-dependent manner. These data indicated that NnV can significantly suppress cell migration and invasion by inhibiting EMT in HepG2 cells, and therefore might be a promising target for hepatocellular carcinoma therapeutics.

Hedyotis diffusa Willd suppresses metastasis in 5­fluorouracil­resistant colorectal cancer cells by regulating the TGF­ß signaling pathway.

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive tract, and threatens the survival and health of patients with CRC. Chemotherapy remains one of the main therapeutic approaches for patients with CRC; however, drug resistance limits the long­term use. CRC cells with multi­drug resistance (MDR) exhibit increased survival times and metastatic potential, which may lead to the recurrence and metastasis of CRC. In addition, MDR is one of the major causes of chemotherapy failure in clinical treatment. Hedyotis diffusa Willd (HDW) has been used in the treatment of inflammation­associated diseases and malignant tumors, including CRC. The authors previously demonstrated that HDW could reverse MDR in CRC cells; however, its underlying mechanism, particularly in MDR­associated metastasis, remains to be elucidated. In the present study, the drug­resistant CRC cell line HCT­8/5­fluorouracil (5­FU) was used to investigate the effect of HDW on the growth and metastasis of cancer cells. Cell viability was assessed using the MTT assay. Cell adhesion potential was evaluated using adhesion experiments. Cell migration was assessed using wound healing and Transwell assays. The mRNA and protein expression levels of crucial factors in the transforming growth factor­ß (TGF­ß) signaling pathway, including TGF­ß, Mothers against decapentaplegic homolog 4 (SMAD4), neural (N)­cadherin, and epithelial (E)­cadherin, were analyzed using the reverse transcription­semi­quantitative polymerase chain reaction and western blotting, respectively. The results demonstrated that the HCT­8/5­FU cell line was more resistant to 5­FU and thus can be used as the resistant cell model. HDW was able to inhibit the viability, and adhesive, migratory and invasion potential of the HCT­8/5­FU cells. In addition, HDW was able to downregulate the expression of TGF­ß, SMAD4 and N­cadherin, and upregulate E­cadherin, at the gene and protein level. In conclusion, the results demonstrated that HDW may suppress the metastasis of 5­FU­resistant CRC cells via regulation of the TGF­ß signaling pathway, which was also considered to be one of the underlying mechanisms of its anti­CRC effect.

MicroRNA-124 inhibits the proliferation of C6 glioma cells by targeting Smad4.

MicroRNA-124 (miR-124) has been shown to be downregulated in glioma; however, its biological functions in glioma are not yet fully understood. The aim of this study was to examine the Smad4­dependent effects of miR­124 on C6 glioma cell proliferation. In this study, the level of miR­124 was found to be enhanced in C6 cells upon transfection with miR­124 mimics, and the mechanisms of action of miR­124 in C6 cells were investigated by reverse transcriptase-quantitative polymerase chain reaction, MTT assay, western blot analysis and luciferase reporter assays in vitro. The results revealed that miR­124 expression was significantly lower in the C6 cells than in either normal rat brain tissue or astrocytes. Upon the overexpression of miR­124, the proliferation of the C6 cells decreased and Smad4 expression was significantly suppressed. Smad4 was identified as a direct target of miR­124 through luciferase reporter assays. Furthermore, miR­124 was found to modulate signal transducer and activator of transcription 3 (Stat3) by downregulating Smad4 expression. Using small interfering RNA targeting Smad4 mRNA, we also confirmed that miR­124 downregulated c­Myc by modulating Smad4 expression. In addition, caspase­3 expression was induced by miR­124 overexpression, but not via Smad4 downregulation. On the whole, our results demonstrate that miR­124 upregulation inhibits the growth of C6 glioma cells by targeting Smad4 directly. These findings may be clinically useful for the development of therapeutic strategies directed toward miR­124 function in patients with glioma.

The BMP4-Smad signaling pathway regulates hyperandrogenism development in a female mouse model.

Polycystic ovary syndrome is a common endocrine disorder and a major cause of anovulatory sterility in women at reproductive age. Most patients with polycystic ovary syndrome have hyperandrogenism, caused by excess androgen synthesis. Bone morphogenetic protein 4 (BMP4) is an essential regulator of embryonic development and organ formation, and recent studies have also shown that BMP4 may be involved in female steroidogenesis process. However, the effect of BMP4 on hyperandrogenism remains unknown. Here, using a female mouse model of hyperandrogenism, we found that ovarian BMP4 levels were significantly decreased in hyperandrogenism. Elevated androgens inhibited BMP4 expression via activation of androgen receptors. Moreover, BMP4 treatment suppressed androgen synthesis in theca cells and promoted estrogen production in granulosa cells by regulating the expression of steroidogenic enzymes, including CYP11A, HSD3B2, CYP17A1, and CYP19A1 Consistently, knockdown of BMP4 augmented androgen levels and inhibited estrogen levels. Mechanistically, Smad signaling rather than the p38 MAPK pathway regulated androgen and estrogen formation, thereby mediating the effect of BMP4. Of note, BMP4-transgenic mice were protected against hyperandrogenism. Our observations clarify a vital role of BMP4 in controlling sex hormone levels and offer new insights into intervention for managing hyperandrogenism by targeting the BMP4-Smad signaling pathway.

Smad inhibitor induces CSC differentiation for effective chemosensitization in cyclin D1- and TGF-ß/Smad-regulated liver cancer stem cell-like cells.

Understanding cancer stem cell (CSC) maintenance pathways is critical for the development of CSC-targeting therapy. Here, we investigated the functional role of the cyclin D1-dependent activation of Smad2/3 and Smad4 in hepatocellular carcinoma (HCC) CSCs and in HCC primary tumors. Cyclin D1 sphere-derived xenograft tumor models were employed to evaluate the therapeutic effects of a Smad inhibitor in combination with chemotherapy. Cyclin D1 overexpression confers stemness properties by enhancing single sphere formation, enhancing the CD90+ and EpCAM+ population, increasing stemness gene expression, and increasing chemoresistance. Cyclin D1 interacts with and activates Smad2/3 and Smad4 to result in cyclin D1-Smad2/3-Smad4 signaling-regulated liver CSC self-renewal. The cyclin D1-dependent activation of Smad2/3 and Smad4 is also found in HCC patients and predicts disease progression. A Smad inhibitor impaired cyclin D1-Smad-mediated self-renewal, resulting in the chemosensitization. Thus, pretreatment with a Smad inhibitor followed by chemotherapy not only successfully suppressed tumor growth but also eliminated 57% of the tumors in a cyclin D1 sphere-derived xenograft model. Together, The cyclin D1-mediated activation of Smad2/3 and Smad4 is an important regulatory mechanism in liver CSC self-renewal and stemness. Accordingly, a Smad inhibitor induced CSC differentiation and consequently significant chemosensitization, which could be an effective strategy to target CSCs.

Upregulation of microRNA-574-3p in a human gastric cancer cell line AGS by TGF-ß1.

The mechanisms that regulate miR-574-3p expression in cells remain elusive. In the present study, we used real-time PCR assay to demonstrate TGF-ß1-induced miR-574-3p upregulation in AGS cells, which was inhibited by TGF-ß receptor I inhibitor SB431542. We used a computer search to identify Smad binding sites upstream of the miR-574-3p precursor sequence. We demonstrated that silencing Smad4, but not Smad2 or Smad3, significantly inhibited the TGF-ß1-induced miR-574-3p upregulation in AGS cells. Furthermore, TGF-ß1 significantly increased the activity of a dual-luciferase reporter that contains the Smad binding sites upstream of the miR-574 precursor sequence. Silencing Smad4 significantly inhibited the TGF-ß1-induced increase in the activity of the reporter in AGS cells. ChIP assay showed that Smad4 directly bound to the promoter of miR-574-3p. MiR-574-3p inhibition was effective in eliminating the inhibition of AGS cell proliferation induced by TGF-ß1, suggesting that TGF-ß1 inducing upregulation of miR-574-3p is functionally significant.

Magnolol Attenuates Concanavalin A-induced Hepatic Fibrosis, Inhibits CD4+ T Helper 17 (Th17) Cell Differentiation and Suppresses Hepatic Stellate Cell Activation: Blockade of Smad3/Smad4 Signalling.

Magnolol is a pharmacological biphenolic compound extracted from Chinese herb Magnolia officinalis, which displays anti-inflammatory and antioxidant effects. This study was aimed at exploring the potential effect of magnolol on immune-related liver fibrosis. Herein, BALB/c mice were injected with concanavalin A (ConA, 8 mg/kg/week) up to 6 weeks to establish hepatic fibrosis, and magnolol (10, 20, 30 mg/kg/day) was given to these mice orally throughout the whole experiment. We found that magnolol preserved liver function and attenuated liver fibrotic injury in vivo. In response to ConA stimulation, the CD4+ T cells preferred to polarizing towards CD4+ T helper 17 (Th17) cells in liver. Magnolol was observed to inhibit Th17 cell differentiation in ConA-treated liver in addition to suppressing interleukin (IL)-17A generation. Hepatic stellate cells were activated in fibrotic liver as demonstrated by increased alpha smooth muscle actin (α-SMA) and desmin. More transforming growth factor (TGF)-ß1 and activin A were secreted into the serum. Magnolol suppressed this abnormal HSC activation. Furthermore, the phosphorylation of Smad3 in its linker area (Thr179, Ser 204/208/213) was inhibited by magnolol. In vitro, the recombinant IL-17A plus TGF-ß1 or activin A induced activation of human LX2 HSCs and promoted their collagen production. Smad3/Smad4 signalling pathway was activated in LX2 cells exposed to the fibrotic stimuli, as illustrated by the up-regulated phospho-Smad3 and the enhanced interaction between Smad3 and Smad4. These alterations were suppressed by magnolol. Collectively, our study reveals a novel antifibrotic effect of magnolol on Th17 cell-mediated fibrosis.

Repression of Smad4 by miR­205 moderates TGF-ß-induced epithelial-mesenchymal transition in A549 cell lines.

The TGF-ß/Smad signaling pathway plays important roles in cancer cell proliferation, apoptosis, differentiation, angiogenesis and epithelial-mesenchymal transition (EMT), which is the key event in the early stages of cancer metastasis and enhances the capability of cell migration and invasion. Smad4 acts as the only Co-Smad of TGF/Smad signaling pathway and plays the key role in TGF-ß-mediated EMT. Nevertheless, the mRNA regulation mechanisms of Smad4 in human non-small cell lung cancer (NSCLC) remains largely unclear. Computational algorithms predicted that the 3'-UTR of Smad4 is a target of miR­205. Here, we validated that miR­205 could directly bind to 3'-UTR of Smad4 by luciferase assays. Moreover, we investigated the functional roles of miR­205 and its molecular link to Smad4 in lung cancer cells. In this study, we confirmed that overexpression of miR­205 suppressed the expression of Smad4, in turn, weakened the TGF-ß/Smad signaling pathway and inhibited TGF-ß/Smad4-induced EMT, invasion and migration ultimately. Furthermore, this study shows that miR­205 can serve as a promising therapeutic target of highly aggressive NSCLC.

MicroRNA-20a-5p promotes colorectal cancer invasion and metastasis by downregulating Smad4.

BACKGROUND: Tumor metastasis is one of the leading causes of poor prognosis for colorectal cancer (CRC) patients. Loss of Smad4 contributes to aggression process in many human cancers. However, the underlying precise mechanism of aberrant Smad4 expression in CRC development is still little known. RESULTS: miR-20a-5p negatively regulated Smad4 by directly targeting its 3'UTR in human colorectal cancer cells. miR-20a-5p not only promoted CRC cells aggression capacity in vitro and liver metastasis in vivo, but also promoted the epithelial-to-mesenchymal transition process by downregulating Smad4 expression. In addition, tissue microarray analysis obtained from 544 CRC patients' clinical characters showed that miR-20a-5p was upregulated in human CRC tissues, especially in the tissues with metastasis. High level of miR-20a-5p predicted poor prognosis in CRC patients. METHODS: Five miRNA target prediction programs were applied to identify potential miRNA(s) that target(s) Smad4 in CRC. Luciferase reporter assay and transfection technique were used to validate the correlation between miR-20a-5p and Smad4 in CRC. Wound healing, transwell and tumorigenesis assays were used to explore the function of miR-20a-5p and Smad4 in CRC progression in vitro and in vivo. The association between miR-20a-5p expression and the prognosis of CRC patients was evaluated by Kaplan-Meier analysis and multivariate cox proportional hazard analyses based on tissue microarray data. CONCLUSIONS: miR-20a-5p, as an onco-miRNA, promoted the invasion and metastasis ability by suppressing Smad4 expression in CRC cells, and high miR-20a-5p predicted poor prognosis for CRC patients, providing a novel and promising therapeutic target in human colorectal cancer.

A comprehensive transcriptomic view on the role of SMAD4 gene by RNAi-mediated knockdown in porcine follicular granulosa cells.

As a key mediator of the transforming growth factor-beta (TGF-ß) signaling pathway, which plays a pivotal role in regulating mammalian reproductive performance, Sma- and Mad-related protein 4 (SMAD4) is closely associated with the development of ovarian follicular. However, current knowledge of the genome-wide view on the role of SMAD4 gene in mammalian follicular granulosa cells (GCs) is still largely unknown. In the present study, RNA-Seq was performed to investigate the effects of SMAD4 knockdown by RNA interference (SMAD4-siRNA) in porcine follicular GCs. A total of 1025 differentially expressed genes (DEGs), including 530 upregulated genes and 495 downregulated genes, were identified in SMAD4-siRNA treated GCs compared with that treated with NC-siRNA. Furthermore, functional enrichment analysis indicated that upregulated DEGs in SMAD4-siRNA treated cells were mainly enriched in cell-cycle related processes, interferon signaling pathway, and immune system process, while downregulated DEGs in SMAD4-siRNA treated cells were mainly involved in extracellular matrix organization/disassembly, pathogenesis, and cell adhesion. In particular, cell cycle and TGF-ß signaling pathway were discovered as the canonical pathways changed under SMAD4-silencing. Taken together, our data reveals SMAD4 knockdown alters the expression of numerous genes involved in key biological processes of the development of follicular GCs and provides a novel global clue of the role of SMAD4 gene in porcine follicular GCs, thus improving our understanding of regulatory mechanisms of SMAD4 gene in follicular development.

Large-Scale Profiling of Kinase Dependencies in Cancer Cell Lines.

One approach to identifying cancer-specific vulnerabilities and therapeutic targets is to profile genetic dependencies in cancer cell lines. Here, we describe data from a series of siRNA screens that identify the kinase genetic dependencies in 117 cancer cell lines from ten cancer types. By integrating the siRNA screen data with molecular profiling data, including exome sequencing data, we show how vulnerabilities/genetic dependencies that are associated with mutations in specific cancer driver genes can be identified. By integrating additional data sets into this analysis, including protein-protein interaction data, we also demonstrate that the genetic dependencies associated with many cancer driver genes form dense connections on functional interaction networks. We demonstrate the utility of this resource by using it to predict the drug sensitivity of genetically or histologically defined subsets of tumor cell lines, including an increased sensitivity of osteosarcoma cell lines to FGFR inhibitors and SMAD4 mutant tumor cells to mitotic inhibitors.