A novel TGFßR2 splice variant in patient with aortic aneurysm and family history for aortic dissection: a case report.

We report the clinical presentation and genetic screening of a 31-year-old man with dilatation of the aortic root and ascending aorta and a positive family history for aortic dissection and sudden death. A novel heterozygous variant in a splice acceptor site (c.1600-1G>T) of TGFßR2 gene was identified by using a targeted multi-gene panel analysis. Bioinformatics tools predicted that the c.1600-1G>T variant is pathogenic by altering acceptor splice site at - 1 position affecting pre-mRNA splicing. These data confirm that the diverging splicing in the TGF-ß pathway genes may be an important process in aneurismal disease and emphasize the utility of genetic sequencing in the identification of high-risk patients for a more patient's management able to improve outcomes and minimize costs for the care of patients with heritable thoracic aortic aneurysm and dissection.

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Circ_0070203 Promotes Epithelial-mesenchymal Transition in Ovarian Serous Cystadenocarcinoma through miR-370-3p/TGFßR2 Axis.

INTRODUCTION: Circular RNAs (circRNAs) are important biological molecules associated with the pathogenesis of multiple cancers. OBJECTIVE: This work aimed to investigate the function and molecular mechanism of circ_0070203 in high-grade serous ovarian cystadenocarcinoma (HGSOC). METHODS: circRNA microarray was conducted to detect the circ_0070203 expression in HGSOC tissues. Bioinformatics analysis was used to find the binding sites between circ_0070203, miR- 370-3p and TGFßR2. Real-time quantitative reverse transcription PCR (RT-qPCR) was executed to detect the expressions of circ_0070203, miR-370-3p and TGFßR2 in HGSOC tissues and SKOV3 cells. Dual-luciferase reporter gene assay was used to validate the relationships between miR-370-3p and circ_0070203 or TGFßR2. Besides, transwell assays were conducted to assess the migrative, invasive abilities of ovarian cancer (OC) cells. Western blotting was adopted to detect the expression of epithelial-mesenchymal transition (EMT)-related proteins. The related patents were also studied during the research. RESULTS: Circ_0070203 and TGFßR2 were upregulated, while miR-370-3p was downregulated in FIGO stage III-IV HGSOC tissues and SKOV-3 cell lines. circ_0070203 overexpression changed the expression of other EMT-related proteins and enhanced the migrative, invasive abilities of OC cells, while silencing circ_0070203 worked oppositely. Mechanistically, circ_0070203 could upregulate TGFßR2 expression in OC cells via sponging miR-370-3p. CONCLUSION: Circ_0070203 could promote the epithelial-mesenchymal transition, invasion, and metastasis of HGSOC via regulating the miR-370-3p/TGFßR2 axis. Our findings provided a potential biomarker for HGSOC therapy.

miR-186-5p targets TGFßR2 to inhibit RAW264.7 cell migration and proliferation during mouse skin wound healing.

BACKGROUND: Active peptides play a vital role in the development of new drugs and the identification and discovery of drug targets. As the first reported native peptide homodimer with pro-regenerative potency, OA-GP11d could potentially be used as a novel molecular probe to help elucidate the molecular mechanism of skin wound repair and provide new drug targets. METHODS: Bioinformatics analysis and luciferase assay were adopted to determine microRNAs (miRNAs) and its target. The prohealing potency of the miRNA was determined by MTS and a Transwell experiment against mouse macrophages. Enzyme-linked immunosorbent assay, realtime polymerase chain reaction, and western blotting were performed to explore the molecular mechanisms. RESULTS: In this study, OA-GP11d was shown to induce Mus musculus microRNA-186-5p (mmu-miR-186-5p) down-regulation. Results showed that miR-186-5p had a negative effect on macrophage migration and proliferation as well as a targeted and negative effect on TGF-ß type II receptor (TGFßR2) expression and an inhibitory effect on activation of the downstream SMAD family member 2 (Smad2) and protein-p38 kinase signaling pathways. Importantly, delivery of a miR-186-5p mimic delayed skin wound healing in mice. CONCLUSION: miR-186-5p regulated macrophage migration and proliferation to delay wound healing through the TGFßR2/Smad2/p38 molecular axes, thus providing a promising new pro-repair drug target.

Circ_CHMP5 aggravates oxidized low-density lipoprotein-induced damage to human umbilical vein endothelial cells through miR-516b-5p/TGFßR2 axis.

BACKGROUND: Atherosclerosis (AS) was one of the main causes of death in the elderly, and lesions in human umbilical vein endothelial cells (HUVECs) could lead to AS. CircRNA-charged multivesicular body protein 5 (circ_CHMP5) was reported to participate in the progression of AS. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the levels of circ_CHMP5, miR-516b-5p, and transforming growth factor beta receptor 2 (TGFßR2) in AS patients or ox-LDL-induced HUVECs. 5-Ethynyl-2'-deoxyuridine and cell counting kit-8 assays were performed to detect cell proliferation. Proteins expression was assessed by western blot assay. Cell apoptosis was examined by flow cytometry. Tube formation assay was utilized to measure the tube formation ability of HUVCEs. The targeting relationships between miR-516b-5p and circ_CHMP5 or TGFßR2 were confirmed by dual-luciferase reporter assay and RNA-pull down assay. RESULTS: Circ_CHMP5 was enhanced in the serum of AS patients and ox-LDL-exposure HUVECs. Ox-LDL blocked proliferation and tube formation of HUVECs and induced cell apoptosis, and circ_CHMP5 knockdown reversed these effects. In addition, circ_CHMP5 regulated the growth of ox-LDL-induced HUVECs through miR-516b-5p and TGFßR2. Moreover, the effects of circ_CHMP5 knockdown on ox-LDL-induced HUVECs were obviously recovered by downregulation of miR-516b-5p, and overexpression of TGFßR2 restored the effects of miR-516b-5p upregulation on ox-LDL-stimulated HUVECs. CONCLUSION: Silence of circ_CHMP5 overturned ox-LDL-treated inhibition of HUVECs proliferation and angiogenesis by miR-516b-5p and TGFßR2. These results provided new solutions for the treatment of AS.

miR-17-5p slows progression of hepatocellular carcinoma by downregulating TGFßR2.

OBJECTIVE: Downregulation of miR-17-5p has been reported in several cancers, but whether and how miR-17-5p is downregulated in hepatocellular carcinoma (HCC) is unknown. Here, we examined whether miR-17-5p is downregulated in HCC and whether that affects expression of its target gene encoding transforming growth factor ß receptor 2 (TGFßR). METHODS: We screened for potential microRNAs (miRNAs) involved in HCC by analyzing published transcriptomes from HCC patients. Expression of miR-17-5p was measured in HCC cell lines and in tissues from HCC patients using quantitative real-time PCR. The in vitro effects of miR-17-5p on HCC cells were assessed by EdU proliferation assay, CCK-8 cell proliferation assay, colony-formation assay, transwell migration/invasion assay, wound healing assay, and flow cytometry. Effects of miR-17-5p were evaluated in vivo using mice with subcutaneous tumors. Effects of the miRNA on the epithelial-mesenchymal transition (EMT) were assessed, while its effects on TGFßR2 expression were analyzed using bioinformatics and a dual luciferase reporter assay. RESULTS: Patients with low miR-17-5p expression showed lower rates of overall and recurrence-free survival than patients with high miR-17-5p expression, and multivariate Cox regression identified low miR-17-5p expression as an independent predictor of poor overall survival in HCC patients. In vitro, miR-17-5p significantly inhibited HCC cell proliferation, migration, invasion, and the EMT, while promoting apoptosis. In vivo, it slowed the development of tumors. These protective effects of miR-17-5p were associated with downregulation of TGFßR2. CONCLUSION: The miRNA miR-17-5p can negatively regulate the expression of TGFßR2 and inhibit the EMT, thereby slowing tumor growth in HCC, suggesting a potential therapeutic approach against HCC.

hASCs-derived exosomal miR-155-5p targeting TGFßR2 promotes autophagy and reduces pyroptosis to alleviate intervertebral disc degeneration.

Background: Intervertebral disc degeneration (IDD) is a complex chronic disease involving nucleus pulposus cells (NPCs) senescence, apoptosis, autophagy and extracellular matrix (ECM) degradation. In this study, we aimed to investigate the role of human adipose tissue stem cells (hASCs)-derived exosomal miR-155-5p targeting TGFßR2 in IDD and the mechanisms involved. Then miRNA sequencing was performed, and hASCs-derived Exo (hASCs-Exo) was extracted and characterized. Methods: First, NPCs were treated with different concentrations of LPS. Then miRNA sequencing was performed, and hASCs-Exo was extracted and characterized. NPCs were treated with PBS or autophagy inhibitor 3-MA. NPCs were transfected with miR-155-5p mimic, si-TGFßR2 and negative control. Cell viability, apoptosis, ROS, caspase-1+PI, pyroptosis markers, inflammatory cytokines, autophagy markers, Aggrecan, MMP13, and Akt/mTOR pathway-related factors were measured. Bioinformatics prediction and dual-luciferase were performed to verify the binding sites of miR-155-5p to TGFßR2. Finally, we validated the role of hASCs-derived exosomal miR-155-5p on IDD in vivo. Results: LPS promoted pyroptosis of NPCs, and inhibited autophagy and ECM synthesis. MiR-155-5p was characterized as an inflammation-related miRNA in NPCs. HASCs-derived exosomal miR-155-5p inhibited pyroptosis of NPCs and promoted autophagy and ECM synthesis. After bioinformatics prediction and verification, it was found that miR-155-5p targeted TGFßR2. Moreover, miR-155-5p targeted TGFßR2 to promote autophagy and inhibit pyroptosis in NPCs. In vivo experiments revealed that hASCs-derived exosomal miR-155-5p alleviated IDD in rats. Conclusions: HASCs-derived exosomal miR-155-5p alleviated IDD by targeting TGFßR2 to promote autophagy and reduce pyroptosis. Our study may provide a new therapeutic target for IDD. Translational Potential of this Article: HASCs-derived exosomal miR-155-5p is expected to be a biomarker for clinical treatment of IDD. Our study may provide a new therapeutic target for IDD.

Exosomal lncRNA ATB Derived from Ovarian Cancer Cells Promotes Angiogenesis via Regulating miR-204-3p/TGFßR2 Axis.

BACKGROUND: Ovarian cancer is a life-threatening disease with a high mortality rate in women. Our previous work presented that long non-coding RNA (lncRNA) activated by transforming growth factor beta (TGF-ß) (lncRNA ATB) played a role of oncogene in ovarian cancer. However, whether exosomal lncRNA ATB from ovarian cancer cells could regulate the tumorigenesis of ovarian cancer remains unclear. METHODS: RT-qPCR assay was performed to evaluate the level of lncRNA ATB in cancer cells (SKOV3 and A2780). In addition, ovarian cancer cells-secreted exosomes were collected with ultracentrifugation. CCK8 assay was performed to detect the viability of ovarian cells and HUVECs. Meanwhile, Western blot was performed to detect the expression of mechanism related protein and tube formation assay was used to observe the angiogenesis of HUVECs. Finally, xenograft mice model was used to verify the role of ovarian cancer cell-derived exosomes in vivo. RESULTS: Ovarian cancer cells-derived exosomes promoted the viability, angiogenesis and migration of HUVECs; however, knockdown of lncRNA ATB in HUVECs reversed these phenomena. In addition, exosomal lncRNA ATB promoted the tumorigenesis of ovarian cancer via regulating miR-204-3p/TGFßR2 axis. Furthermore, ovarian cancer cells-secreted exosomal lncRNA ATB increased tumor growth in vivo. CONCLUSION: Exosomal lncRNA ATB derived from ovarian cancer cells could improve tumor microenvironment via regulating miR-204-3p/TGFßR2 axis. Thus, this study might provide new knowledge for the treatment of ovarian cancer.

TGF-Beta Receptor II Is Critical for Osteogenic Progenitor Cell Proliferation and Differentiation During Postnatal Alveolar Bone Formation.

Transforming growth factor beta (TGFß) signaling plays an important role during osteogenesis. However, most research in this area focuses on cortical and trabecular bone, whereas alveolar bone is largely overlooked. To address the role of TGFßR2 (the key receptor for TGFß signaling) during postnatal alveolar bone development, we conditionally deleted Tgfßr2 in early mesenchymal progenitors by crossing Gli1-Cre ERT2; Tgfßr2 flox/flox ; R26R tdTomato mice (named early cKO) or in osteoblasts by crossing 3.2kb Col1-Cre ERT2 ; Tgfßr2 flox/flox ; R26R tdTomato mice (named late cKO). Both cKO lines were induced at postnatal day 5 (P5) and mice were harvested at P28. Compared to the control littermates, early cKO mice exhibited significant reduction in alveolar bone mass and bone mineral density, with drastic defects in the periodontal ligament (PDL); conversely, the late cKO mice displayed very minor changes in alveolar bone. Mechanism studies showed a significant reduction in PCNA+ PDL cell numbers and OSX+ alveolar bone cell numbers, as well as disorganized PDL fibers with a great reduction in periostin (the most abundant extracellular matrix protein) on both mRNA and protein levels. We also showed a drastic reduction in ß-catenin in the early cKO PDL and a great increase in SOST (a potent inhibitor of Wnt signaling). Based on these findings, we conclude that TGFß signaling plays critical roles during early alveolar bone formation via the promotion of PDL mesenchymal progenitor proliferation and differentiation mechanisms.

LncRNA NEAT1 Promotes Gastric Cancer Progression Through miR-17-5p/TGFßR2 Axis Up-Regulated Angiogenesis.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been indicated to play critical roles in gastric cancer (GC) tumorigenesis and progression. However, their roles in GC remain to be further elucidated. METHODS: RT-qPCR and fluorescence in situ hybridzation (FISH) were conducted to detect the expression of lncRNA NEAT1 in GC tissues and cell lines. Gene Set Enrichment Analysis (GSEA) was performed to screen out potential phenotypes and pathways that NEAT1 may participate in. NEAT1-silenced AGS and MGC803 cells were constructed and a series of functional experiments to investigate the roles of NEAT1 in GC angiogenesis both in vitro and in vivo. RNA pull down and luciferase reporter assays were utilized to illustrate the mechanisms underlying the functions of NEAT1 in GC. RESULTS: We observed that NEAT1 was upregulated in most GC specimens and cell lines. NEAT1 high was correlated with poor prognosis of GC patients. In vitro experiments showed that NEAT1 promoted GC angiogenesis by enhancing proliferation, migration, and tube formation ability of endothelial cells. Mechanism researches revealed that NEAT1 could competitively sponge miR-17-5p which targeted TGFßR2 directly. Subsequently, activate TGFß/Smad pathway by following with upregulation of a series of classical proangiogenic factors especially VEGF. CONCLUSION: The study unveiled that the LncRNA NEAT1/miR-17-5p/TGFßR2 axis is a novel mechanism in GC angiogenesis. Disrupting this axis may be a potential strategy for GC treatment.

Canonical TGFß Signaling and Its Contribution to Endometrial Cancer Development and Progression-Underestimated Target of Anticancer Strategies.

Endometrial cancer is one of the leading gynecological cancers diagnosed among women in their menopausal and postmenopausal age. Despite the progress in molecular biology and medicine, no efficient and powerful diagnostic and prognostic marker is dedicated to endometrial carcinogenesis. The canonical TGFß pathway is a pleiotropic signaling cascade orchestrating a variety of cellular and molecular processes, whose alterations are responsible for carcinogenesis that originates from different tissue types. This review covers the current knowledge concerning the canonical TGFß pathway (Smad-dependent) induced by prototypical TGFß isoforms and the involvement of pathway alterations in the development and progression of endometrial neoplastic lesions. Since Smad-dependent signalization governs opposed cellular processes, such as growth arrest, apoptosis, tumor cells growth and differentiation, as well as angiogenesis and metastasis, TGFß cascade may act both as a tumor suppressor or tumor promoter. However, the final effect of TGFß signaling on endometrial cancer cells depends on the cancer disease stage. The multifunctional role of the TGFß pathway indicates the possible utilization of alterations in the TGFß cascade as a potential target of novel anticancer strategies.

miR-211-5p inhibits the proliferation, migration, invasion, and induces apoptosis of human hypertrophic scar fibroblasts by regulating TGFßR2 expression.

BACKGROUND: Hypertrophic-scar (HS) is the most common pathological healing phenomenon after trauma, especially after deep burns. We aimed to investigate the expression and role of microRNA-211-5p (miR-211-5p) in HS and explore its underlying mechanism. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-211-5p in 15 cases of HS tissues and normal skin tissues, as well as its expression in human hypertrophic scar fibroblasts (hHSFs) and normal fibroblasts. At the same time, the cell counting kit-8 (CCK-8), scratch test, cell invasion test, and flow cytometry were used to determine cell proliferation, migration, invasion, and apoptosis, respectively. Western blot assay was used to determine the expression of proteins. TargetScan was performed to predict the potential binding sites between miR-211-5p and TGFßR2, which was then verified by western blotting and luciferase reporter gene experiments. Also, co-transfection of plasmids that overexpress miR-211-5p and TGFßR2 were used to observe the reversal effect of miR-211-5p. RESULTS: The level of miR-211-5p in HS tissues and hHSFs cells was significantly down-regulated (both P<0.05). The TGFßR2/Smad3 signaling pathway was activated (both P<0.05). Furthermore, the overexpression of miR-211-5p could inhibit the proliferation (P<0.05), migration (P<0.05), and invasion (P<0.05) of hHSFs cells, and induce their apoptosis (P<0.05), and could also regulate the expression of related proteins (all P<0.05). Moreover, the overexpression of miR-211-5p could also inhibit the accumulation of ECM and the activation of the TGF-ßR2/Smad3 pathway (all P<0.05), while the opposite effect (all P<0.05) was observed when the level of miR-211-5p was interfered with. Finally, it was confirmed that miR-211-5p could target TGFßR2 (all P<0.05), and when hHSFs cells simultaneously overexpressed miR-211-5p and TGFßR2, the promotion effect of TGFßR2 on cells was reversed by miR-211-5p (all P<0.05). CONCLUSIONS: miR-211-5p can inhibit the activation of the TGF-ßR2/Smad3 signaling pathway by targeting TGFßR2, thereby suppressing the proliferation, migration, invasion, and ECM production of hHSFs, and inducing their apoptosis, suggesting that miR-211-5p can become a potential target for the treatment of HS.

miRNAs through ß-ARR2/p-ERK1/2 pathway regulate the VSMC proliferation and migration.

BACKGROUND: miRNAs are involved in plaque formation of atherosclerosis and vessel restenosis. In this study, we investigated the effects of miR-599, miR-204, and miR-181b on VSMC proliferation, and migration through TGFß receptor 2 (TGFßR2), ß-arrestin 2 (ß-ARR2), SMAD2/p-SMAD2, and ERK1/2/p-ERK1/2. MATERIALS & METHODS: Genes and miRNAs were predicted by bioinformatics tools and were transfected by PEI-miRNAs (miR-599, miR-204, and miR-181b) complexes into VSMCs. The gene and protein expression levels were evaluated by real-time RT-PCR and western blotting techniques, respectively. The VSMC proliferation and migration were studied by MTT and scratch assay, respectively. RESULTS: The miR-181b and miR-204 downregulated significantly ß-ARR2 gene and protein expression levels and p-ERK1/2 values. Moreover, TGFßR2 gene and protein expression levels and p-SMAD2 values were not significantly affected by miR-181b and miR-204. The VSMC proliferation (p = 0.0019, p = 0.0054, respectively) and migration (p < 0.0001, p < 0.0001, respectively) were inhibited by the miR-181b and miR-204. The miR-599 inhibited VSMC proliferation (p = 0.044) and migration (p = 0.0055) but it did not affect significantly the ß-ARR2 and TGFßR2 gene and protein expression levels. CONCLUSION: The results suggested that the inhibitory effects of miR-181b and miR-204 on VSMC proliferation and migration are mediated by the ß-ARR2/p-ERK1/2 pathway. Since VSMC proliferation and migration are involved in plaque growth, therefore this pathway can be a therapeutic target for atherosclerosis.

miR-373-3p inhibits epithelial-mesenchymal transition via regulation of TGFßR2 in choriocarcinoma.

AIM: Previous studies have indicated that early metastasis is a major cause of mortality in patients with choriocarcinoma. However, what determines whether early metastasis of choriocarcinoma has occurred is unknown. The emerging role of miRNA in regulating cancer development and progression has been recognized. miR-373 has been shown to play pivotal roles in tumorigenesis and metastasis. However, whether miR-373 functions to promote choriocarcinoma metastasis is not clear. The purpose of this study is to determine the function of miR-373-3p in the progression of this cancer. METHODS: In this study, we first compared epithelial-mesenchymal transition (EMT)-related markers, which were inversely correlated with miR-373-3p expression in trophoblast and choriocarcinoma cell lines. Using PCR and Western blot, upregulation of miR-373-3p was observed to inhibit EMT progression. Similarly, gain- and loss-of-function studies revealed that ectopic miR-373-3p overexpression inhibited the migration by transwell methods of choriocarcinoma cells. RESULTS: Our results revealed that miR-373-3p acted as an EMT inhibitor in JEG-3 and JAR cells; this was due to its mediation of the transforming growth factor-ß (TGFß) signaling pathway, which was responsible for EMT. miRNA microarray analysis demonstrated that miR-373-3p interacted with the 3' untranslated region of TGFßR2 mRNA, and then Western blot and dual-luciferase reporter gene assays verified this interaction. CONCLUSION: Our findings suggest that miR-373-3p upregulation partly accounts for TGFßR2 downregulation and leads to a restraint of EMT and migration. miR-373-3p may therefore serve as a valuable potential target in the treatment of choriocarcinoma.

LncRNA NORAD regulates scar hypertrophy via miRNA-26a mediating the regulation of TGFßR1/2.

BACKGROUND: Transforming growth factor-ß (TGF-ß) pathway presents dysregulation in pathological scarring and mediates hypertrophic scar (HS) formation. OBJECTIVES: The study aims to analyze the potential mechanism of long non-coding RNA NORAD (LncRNA NORAD) and microRNA (miR-26a) regulation of the TGF-ß pathway in hypertrophic scar fibroblasts (HSFs). MATERIAL AND METHODS: Hypertrophic scar tissues were collected and assayed for LncRNA NORAD, miR-26a, transforming growth factor ß receptor I (TGF-ßR1) and TGF-ßR2, with enzyme-linked immunosorbent assay (ELISA) or qualitative polymerase chain reaction (qPCR). LncRNA NORAD interfering plasmids were transfected into HSFs and induced with TGF-ß1. Cell Counting Kit-8 (CCK-8) assays were performed to assess HSF proliferation, and flow cytometry to analyze apoptosis and the cell cycle. TGF-ßR1, TGF-ßR2, Smad2, and p-Smad2 levels were detected using western blot (WB). The related proteins (p21, cyclin D1 and cyclin-dependent kinase 4 (CDK4)) regulating the cell cycle, and apoptosis-related proteins (caspase-3 and Bcl-2) were also detected using WB. The binding sites of miRNA-26a and LncRNA NORAD, TGF-ßR2, or UBE3A were predicted using Starbase and confirmed with dual luciferase reporter assay. RNA immunoprecipitation (RIP) was utilized to explore the interplay of miR-26a with its target genes. RESULTS: LncRNA NORAD is decreased, miR-26a is increased and TGF-ß receptors show abnormal expression in scar tissue. LncRNA NORAD knockdown inhibits proliferation of HSF cells induced by TGF-ß1 treatment. In addition, cell apoptotic levels are markedly increased and cell numbers in G0/G1 phase are increased. Moreover, the TGF-ß/Smad pathway is regulated by decreasing endogenous LncRNA NORAD levels, possibly by affecting the relative levels of TGF-ßR1. p21 is notably upregulated, while cyclin D1 and CDK4 are downregulated. Apoptosis-related proteins are significantly affected. LncRNA NORAD may act as a sponge, binding miR-26a and changing its expression. Finally, RIP shows that miR-26a targets the 3'UTRs of TGF-ßR2 and UBE3A. CONCLUSIONS: LncRNA NORAD regulates HSF proliferation via miR-26a mediating the regulation of TGF-ßR2/R1. LncRNA NORAD/miR-26a could be a potential target for treating HS.

Retinal pigment epithelial cells secrete miR-202-5p-containing exosomes to protect against proliferative diabetic retinopathy.

Previous studies have reported that endothelial-to-mesenchymal transition (EndoMT) contributes to pathological fibrosis in proliferative diabetic retinopathy (PDR). The hypothesis of our study was that exosomes from high glucose (HG)-treated ARPE19 cells reprogram endothelial cell behavior in HG conditions by transferring their genetic contents. Our study showed that ARPE19-derived exosomes were internalized by human umbilical vein endothelial cells (HUVECs). Additionally, miR-202-5p, a miRNA known to target TGFßR2, was enriched in ARPE19-derived exosomes. A dual luciferase reporter assay, qPCR, and western blotting were used to characterize the expression of miR-202-5p and phosphorylation of the TGF/Smad pathway proteins. We showed that miR-202-5p-containing exosomes suppressed HUVEC cell growth, migration, and tube formation. Furthermore, TGFßR2 was confirmed as the target of miR-202-5p. A dual luciferase reporter assay showed that TGFßR2 expression was negatively regulated by miR-202-5p. We also showed that miR-202-5p-containing exosomes suppressed HG-induced EndoMT. These collective results suggested that ARPE-derived exosomes may serve as significant mediators of cell-to-cell crosstalk to suppress EndoMT by transferring miR-202-5p through the TGF/Smad pathway, and may be a potential treatment for PDR patients.

Transactivation of miR-202-5p by Steroidogenic Factor 1 (SF1) Induces Apoptosis in Goat Granulosa Cells by Targeting TGFßR2.

MicroRNAs play key roles during ovary development, with emerging evidence suggesting that miR-202-5p is specifically expressed in female animal gonads. Granulosa cells (GCs) are somatic cells that are closely related to the development of female gametes in mammalian ovaries. However, the biological roles of miR-202-5p in GCs remain unknown. Here, we show that miR-202-5p is specifically expressed in GCs and accumulates in extracellular vesicles (EVs) from large growth follicles in goat ovaries. In vitro assays showed that miR-202-5p induced apoptosis and suppressed the proliferation of goat GCs. We further revealed that miR-202-5p is a functional miRNA that targets the transforming growth factor-beta type II receptor (TGFßR2). MiR-202-5p attenuated TGF-ß/SMAD signaling through the degradation of TGFßR2 at both the mRNA and protein level, decreasing p-SMAD3 levels in GCs. Moreover, we verified that steroidogenic factor 1 (SF1) is a transcriptional factor that binds to the promoters of miR-202 and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) through luciferase reporter and chromatin immunoprecipitation (ChIP) assays. That contributed to positive correlation between miR-202-5p and CYP19A1 expression and estradiol (E2) release. Furthermore, SF1 repressed TGFßR2 and p-SMAD3 levels in GCs through the transactivation of miR-202-5p. Taken together, these results suggest a mechanism by which miR-202-5p regulates canonical TGF-ß/SMAD signaling through targeting TGFßR2 in GCs. This provides insight into the transcriptional regulation of miR-202 and CYP19A1 during goat ovarian follicular development.

Circular RNA circ_0003204 inhibits proliferation, migration and tube formation of endothelial cell in atherosclerosis via miR-370-3p/TGFßR2/phosph-SMAD3 axis.

BACKGROUND: Circular RNAs (circRNAs) represent a class of non-coding RNAs (ncRNAs) which are widely expressed in mammals and tissue-specific, of which some could act as critical regulators in the atherogenesis of cerebrovascular disease. However, the underlying mechanisms by which circRNA regulates the ectopic phenotype of endothelial cells (ECs) in atherosclerosis remain largely elusive. METHODS: CCK-8, transwell, wound healing and Matrigel assays were used to assess cell viability, migration and tube formation. QRT-qPCR and Immunoblotting were used to examine targeted gene expression in different groups. The binding sites of miR-370-3p (miR-370) with TGFßR2 or hsa_circ_0003204 (circ_0003204) were predicted using a series of bioinformatic tools, and validated using dual luciferase assay and RNA immunoprecipitation (RIP) assay. The localization of circ_0003204 and miR-370 in ECs were investigated by fluorescence in situ hybridization (FISH). Gene function and pathways were enriched through Metascape and gene set enrichment analysis (GSEA). The association of circ_0003204 and miR-370 in extracellular vesicles (EVs) with clinical characteristics of patients were investigated using multiple statistical analysis. RESULTS: Circ_0003204, mainly located in the cytoplasm of human aorta endothelial cells (HAECs), was upregulated in the ox-LDL-induced HAECs. Functionally, the ectopic expression of circ_0003204 inhibited proliferation, migration and tube formation of HAECs exposed to ox-LDL. Mechanically, circ_0003204 could promote protein expression of TGFßR2 and its downstream phosph-SMAD3 through sponging miR-370, and miR-370 targeted the 3' untranslated region (UTR) of TGFßR2. Furthermore, the expression of circ_0003204 in plasma EVs was upregulated in the patients with cerebral atherosclerosis, and represented a potential biomarker for diangnosis and prognosis of cerebrovascular atherogenesis. CONCLUSIONS: Circ_0003204 could act as a novel stimulator for ectopic endothelial inactivation in atherosclerosis and a potential biomarker for cerebral atherosclerosis.

GTF2IRD1 on chromosome 7 is a novel oncogene regulating the tumor-suppressor gene TGFßR2 in colorectal cancer.

Chromosome 7q (Ch.7q) is clonally amplified in colorectal cancer (CRC). We aimed to identify oncogenes on Ch.7q that are overexpressed through DNA copy number amplification and determine the biological and clinical significance of these oncogenes in CRC. We identified general transcription factor 2I repeat domain-containing protein 1 (GTF2IRD1) as a potential oncogene using a CRC dataset from The Cancer Genome Atlas with a bioinformatics approach. We measured the expression of GTF2IRD1 in 98 patients with CRC using immunohistochemistry and RT-quantitative PCR (RT-qPCR). The biological effects of GTF2IRD1 expression were explored by gene set enrichment analysis (GSEA). Next, we undertook in vitro cell proliferation and cell cycle assays using siGTF2IRD1-transfected CRC cells. We further investigated the oncogenic mechanisms through which GTF2IRD1 promoted CRC progression. Finally, we assessed the clinical significance of GTF2IRD1 expression by RT-qPCR. GTF2IRD1 was overexpressed in tumor cells and liver metastatic lesions. The GSEA revealed a positive correlation between GTF2IRD1 expression and cell cycle progression-related genes. GTF2IRD1 knockdown inhibited cell proliferation and induced cell cycle arrest in Smad4-mutated CRC. GTF2IRD1 downregulated the expression of the gene encoding transforming growth factor ß receptor 2 (TGFßR2), a tumor-suppressor gene in Smad4-mutated CRC. On multivariate analysis, high GTF2IRD1 expression was an independent poor prognostic factor. Clinicopathological analysis showed that GTF2IRD1 expression was positively correlated with liver metastasis. In conclusion, GTF2IRD1 promoted CRC progression by downregulating TGFßR2 and could be a prognostic biomarker on Ch.7q in CRC. GTF2IRD1 could also be a novel oncogene in CRC.

Downregulation of oncogenic gene TGFßR2 by miRNA-107 suppresses non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer cases with a low 5-year survival rate. MicroRNA may be targeted in the clinical treatment of the disease. In this study, miR-107 showed low expression in NSCLC serum samples, and it could suppress cell proliferation, migration and arrest cell cycle in NSCLC cell lines. Results revealed that miR-107 could inhibit the expression of transforming growth factor ß receptor 2 (TGFßR2) via targeting TGFßR2. Downregulation of TGFßR2 also suppressed cell proliferation, migration and cell cycle in NSCLC cell lines. Our data suggested that miR-107 could inhibit the progression of NSCLC by targeting TGFßR2.

MiR-181a, a new regulator of TGF-ß signaling, can promote cell migration and proliferation in gastric cancer.

Transforming growth factor-beta (TGF-ß) signaling pathway plays pivotal roles in various types of cancer. TGF-ß receptor 2 (TGFßR2) contains a kinase domain that phosphorylates and activates the downstream of the TGF-ß signaling pathway. Our previous microarray analysis revealed marked changes in miR-181a expression in gastric cancers, and the bioinformatics analysis suggested that miR-181a negatively regulated TGFßR2. In order to verify the effect of miR-181a on TGFßR2 and clarify the influence of miR-181a on the migration and proliferation of gastric cancer, studies in gastric cancer cell lines and xenograft mouse models were carried out. We found that a reduced expression of TGFßR2 and an increased expression miR-181a in gastric cancer tissues compared to adjacent noncancerous tissues. A luciferase reporter assay confirmed that TGFßR2 was a target of miR-181a. In addition, we found that miR-181a mimics, which increased the level of miR-181a, downregulated the expression of TGFßR2 in the gastric cancer cell line SGC-7901. Moreover, both the overexpression of miR-181a and the downregulation of TGFßR2 promoted the migration and proliferation of SGC-7901 cells. Conversely, SGC-7901 cell migration and proliferation were inhibited by the downregulation of miR-181a and the overexpression of TGFßR2. Furthermore, the increased expression of miR-181a and the decreased expression of TGFßR2 also enhanced the tumor growth in mice bearing gastric cancer. Our results herein indicated that miR-181a promoted the migration and proliferation of gastric cancer cells by downregulating TGFßR2 at the posttranscriptional level. The present study suggests that miR-181a is a novel negative regulator of TGFßR2 in the TGF-ß signaling pathway and thus represents a potential new therapeutic target for gastric cancer.