CircCOL5A1 inhibits proliferation,migration, invasion,and extracellular matrix production of keloid fibroblasts by regulating the miR-877-5p/EGR1 axis

BackgroundCircular RNA (circRNA) has been proved to mediate the biological functions of fibroblasts to participate in the regulation of keloid formation. However, the role of circCOL5A1 in keloid formation remains to be further confirmed.MethodsPrimary keloid fibroblasts were isolated form keloid tissues. The expression of circCOL5A1, microRNA (miR)? 877–5p, and early growth response 1 (EGR1) were determined by quantitative real-time PCR. Transfection experiments were carried out to explore the effects of circCOL5A1, miR-877–5p, and EGR1 on cell functions. Cell proliferation, migration, invasion and apoptosis were detected using cell counting kit 8 assay, colony formation assay, transwell assay and flow cytometry. The protein levels of apoptosis markers, extracellular matrix (ECM) markers and EGR1 were measured by western blot analysis. The mechanism of circCOL5A1 was confirmed by RNA pull-down assay, dual-luciferase reporter assay and RIP assay.ResultsOur data showed that circCOL5A1 was upregulated in keloid tissues and fibroblasts. Silencing of circCOL5A1 had an inhibition effect on proliferation, migration, invasion and ECM production, while had a promotion effect on apoptosis in keloid fibroblasts. MiR-877–5p could be sponged by circCOL5A1, and its overexpression could repress the biological functions of keloid fibroblasts. The rescue experiments showed that miR-877–5p inhibitor could reverse the suppressive effect of circCOL5A1 knockdown on the biological functions of keloid fibroblasts. In addition, EGR1 was a target of miR-877–5p, and its expression was positively regulated by circCOL5A1. The inhibition effect of miR-877–5p on the biological functions of keloid fibroblasts could be abolished by EGR1 overexpression.ConclusionIn summary, circCOL5A1 facilitates keloid fibroblast proliferation, migration, invasion and ECM production through the miR-877–5p/EGR1 axis, thereby potentially promoting keloid formation.     

Long non-coding RNA HOXA11-AS contributes to the formation of keloid by relieving the inhibition of miR-182-5p on ZNF217

BackgroundLong non-coding RNA (lncRNA) dysregulation is demonstrated to be associated with disease progression. Mounting studies show that lncRNA promotes or inhibits the development of keloid. We aimed to disclose the role of homebox A11 antisense RNA (HOXA11-AS) in the formation of keloid.MethodsQuantitative real-time PCR (qPCR) was adopted for expression analysis of HOXA11-AS, miR-182-5p and zinc finger protein 217 (ZNF217) mRNA, and the expression of ZNF protein and marker proteins was detected by western blot. Cell proliferation, cell migration and cell apoptosis were investigated using CCK-8 assay, wound healing assay and flow cytometry assay, respectively. The potential interplay between miR-182-5p and HOXA11-AS or ZNF217 was verified by dual-luciferase reporter assay, RIP assay and pull-down assay. The role of HOXA11 in vivo was studied by establishing animal models.ResultsHOXA11-AS was highly expressed in tissues and fibroblasts of keloid. Deficiency of HOXA11-AS blocked the proliferation and migration of keloid fibroblasts and induced fibroblast apoptosis. HOXA11-AS directly combined to miR-182-5p whose downregulation reversed the effects of HOXA11-AS knockdown. ZNF217 was a target of miR-182-5p, and HOXA11-AS indirectly promoted ZNF217 expression by binding to miR-182-5p. MiR-182-5p enrichment also blocked keloid fibroblast proliferation, survival and migration, while further ZNF217 overexpression abolished these effects. HOXA11-AS knockdown also hindered the growth of keloid in mouse models.ConclusionHigh expression of HOXA11-AS promoted the formation and growth of keloid through the upregulation of ZNF217 by targeting miR-182-5p, and the inhibition of HOXA11-AS might be a novel strategy to prevent keloid development.     

Role and mechanism of Circ-PDE7B in the formation of keloid

The excessive proliferation of keloid fibroblasts is one of the important reasons leading to the formation of keloids. Circular RNA (circRNA) is an important regulator that regulates the biological functions of cells. However, the role and mechanism of circ-PDE7B in keloid formation have not been studied yet. QRT-PCR was used to detect the circ-PDE7B, miR-331-3p and cyclin-dependent kinase 6 (CDK6) expression. The biological functions of keloid fibroblasts were determined by MTT assay, flow cytometry, transwell assay and wound healing assay. Western blot analysis was used to measure the protein levels of extracellular matrix (ECM) markers and CDK6. The interaction between miR-331-3p and circ-PDE7B or CDK6 was confirmed by dual-luciferase reporter assay and RIP assay. Circ-PDE7B was found to be upregulated in keloid tissues and fibroblasts. Downregulation of circ-PDE7B could suppress the proliferation, invasion, migration, ECM accumulation and accelerate the apoptosis of keloid fibroblasts. Circ-PDE7B could serve as a sponge of miR-331-3p, and the regulation of silenced circ-PDE7B on the biological functions of keloid fibroblasts could be abolished by miR-331-3p inhibitor. Additionally, CDK6 was a target of miR-331-3p, and its overexpression could reverse the negative regulation of miR-331-3p on the biological functions of keloid fibroblasts. Circ-PDE7B sponged miR-331-3p to positively regulate CDK6 expression. Taken together, circ-PDE7B promoted the proliferation, invasion, migration and ECM accumulation of keloid fibroblasts by regulating the miR-331-3p/CDK6 axis, suggesting that circ-PDE7B might be a potential target for keloid treatment.     

Circ_0008450 regulates keloid-derived fibroblast proliferation,migration, invasion and apoptosis with increased IGFBP5 through sponging miR-1224-5p

BackgroundKeloids (KD) are benign fibroproliferative tumors and circular RNAs (circRNAs) may participate in KD progression. At present, whether circ_0008450 regulates keloid-derived fibroblast phenotypes remains unclear. This study aimed to explore the functions of circ_0008450 in keloid (KD)-derived fibroblast phenotypes and the underlying mechanism.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) or western blot assay was performed to determine the expression of circ_0008450, miR-1224-5p, insulin like growth factor binding protein 5 (IGFBP5) and extracellular matrix (ECM)-related markers. 5-Ethynyl-2′-deoxyuridine (EdU) assay was conducted to assess cell proliferation ability. Flow cytometry analysis was used to analyze cell cycle and cell apoptosis. Scratch assay and transwell assay were utilized to examine cell migration and invasion. Mechanism assays were executed to verify the relations of circ_0008450, miR-1224-5p and IGFBP5.ResultsCirc_0008450 was highly expressed in KD tissues and KD-derived fibroblasts. Circ_0008450 silencing inhibited KD-derived fibroblast proliferation, cell cycle, and motility and promoted apoptosis. The effect of circ_0008450 knockdown on KD-derived fibroblast processes was ameliorated by miR-1224-5p downregulation. IGFBP5 was a target gene of miR-1224-5p. IGFBP5 upregulation abated miR-1224-5p-mediated effects on KD-derived fibroblast processes.ConclusionCirc_0008450 promoted KD-derived fibroblast proliferation, migration, and invasion and repressed apoptosis via sponging miR-1224-5p and elevating IGFBP5.     

长链非编码RNA LINC00313与miR-342-3p/膜联蛋白A2轴在肝细胞癌细胞中的作用与机制

背景与目的 肝细胞癌（HCC）是造成癌症相关性死亡的常见原因之一，研究表明长链非编码RNA（lncRNA）调控微小RNA（miRNA）的表达，进而通过抑制靶mRNA翻译或促进mRNA降解来参与肿瘤发生及进展过程。LINC00313作为一种具有致癌活性的lncRNA参与肿瘤发生及进展过程；膜联蛋白A2（ANXA2）在包括HCC的多种恶性肿瘤中表达上调，促进恶性表型的发生，并可能受上游miR-342-3p的调控。因此，本研究探讨LINC00313、miR-342-3p、ANXA2在HCC细胞中的表达及其相互关系。方法 用qRT-PCR与Western blot检测人肝实质细胞及HCC细胞系（Li-7、HuH-7、Hep3B2.1-7）中LINC00313、miR-342-3p及ANXA2表达。将体外培养的Li-7细胞分为空白对照组（无处理）、LINC00313 siRNA组（转染LINC00313 siRNA）、miR-342-3p模拟物组（转染miR-342-3p模拟物）、共转染阴性对照组（转染阴性siRNA序列与阴性miRNA序列）、共转染组（转染LINC00313 siRNA及miR-342-3p抑制物），用qRT-PCR与Western blot检测各组细胞LINC00313、miR-342-3p及ANXA2表达；MTT实验及平板集落形成实验检测各组细胞增殖；进行TUNEL染色检测各组细胞凋亡；Transwell侵袭及Western blot分别检测各组细胞侵袭数目及上皮-间充质转化（EMT）相关蛋白波形蛋白（vimentin）、E-钙黏蛋白（E-cadherin）表达；免疫荧光染色检测各组细胞Bcl-2关联X蛋白（Bax）/B淋巴细胞瘤-2（Bcl-2）；双荧光素酶报告实验分析Li-7细胞中LINC00313对miR-342-3p、miR-342-3p对ANXA2的靶向调控。建立皮下裸鼠异种移植瘤模型，验证LINC00313沉默对Li-7细胞体内生长的影响。结果 与人肝实质细胞比较，Li-7、HuH-7、Hep3B2.1-7细胞的LINC00313、ANXA2 mRNA及蛋白表达均明显升高，而miR-342-3p表达均明显降低（均P<0.05）。与对照组比较，LINC00313 siRNA组、miR-342-3p模拟物组细胞ANXA2 mRNA及蛋白表达、增殖率、集落生成率、侵袭细胞数目、vimentin蛋白表达均明显降低（P<0.05），miR-342-3p表达、凋亡率、E-cadherin蛋白表达、Bax/Bcl-2比值均明显升高（均P<0.05）；与LINC00313 siRNA组比较，共转染组细胞ANXA2 mRNA及蛋白表达、增殖率、集落生成率、侵袭细胞数目、vimentin蛋白表达均明显升高，而miR-342-3p表达、凋亡率、E-cadherin蛋白表达、Bax/Bcl-2比值均明显降低（均P<0.05）。Li-7细胞中，LINC00313可靶向下调miR-342-3p表达，miR-342-3p可靶向下调其ANXA2表达（均P<0.05）。体内实验结果显示，与无处理的Li-7细胞移植瘤比较，LINC00313敲低的Li-7细胞移植瘤的体积与质量均明显降低，肿瘤组织中LINC00313、ANXA2 mRNA及蛋白表达均明显降低，而miR-342-3p表达明显升高（均P<0.05）。结论 LINC00313在HCC细胞中的表达上调，LINC00313可能通过抑制miR-342-3p而增加后者靶基因ANXA2的表达，进而促进HCC细胞的恶性表型。     

CircSLC8A1 targets miR-181a-5p/HIF1AN pathway to inhibit the growth,migration and extracellular matrix deposition of human keloid fibroblasts

BackgroundCircular RNAs (circRNAs) are identified as important regulators in human diseases, including keloid. The purpose of this study is to reveal the role and molecular mechanism of circSLC8A1 in keloid formation.MethodsExpression of circSLC8A1, microRNA (miR)-181a-5p, and hypoxia inducible factor 1 alpha inhibitor (HIF1AN) were detected by quantitative real-time PCR. Protein expression of extracellular matrix (ECM) deposition markers and HIF1AN was detected by western blot analysis. Furthermore, the interaction between miR-181a-5p and circSLC8A1 or HIF1AN was confirmed by dual-luciferase reporter assay, RIP assay and RNA pull-down assay.ResultsExpression of circSLC8A1 was downregulated in keloid tissues and HKFs. Overexpression of circSLC8A1 suppressed HKFs proliferation, migration, ECM deposition, and promoted apoptosis. MiR-181a-5p is targeted by circSLC8A1, and its mimic reversed the effect of circSLC8A1 on the biological function of HKFs. HIF1AN was a target of miR-181a-5p, and it was positively regulated by circSLC8A1. Knockdown of HIF1AN also reversed the negatively regulation of circSLC8A1 on the biological functions of HKFs.ConclusionOur data showed that circSLC8A1 regulates the miR-181a-5p/HIF1AN axis to restrain HKFs biological functions, confirming that circSLC8A1 might serve as a novel therapeutic target for keloids.     

MicroRNA-200c通过TGF-β/Smad通路抑制人瘢痕疙瘩成纤维细胞增殖和胶原合成

目的:探讨microRNA-200c(miR-200c)对人瘢痕疙瘩成纤维细胞增殖及胶原合成的影响,并阐明其涉及的TGF-β/Smad通路机制。方法:将miR-200c mimics用oligofectami脂质体转染经TGF-β1诱导的人瘢痕疙瘩成纤维细胞,Cell Counting Kit-8(CCK-8)法测细胞增殖变化;3H-脯氨酸掺入法测胶原蛋白水平的变化。相关蛋白表达变化和TGF-β1分泌表达变化分别用Western blot和ELISA法检测。结果:miR-200c明显抑制经TGF-β1诱导的人瘢痕疙瘩纤维细胞的增殖和胶原合成;miR-200c能明显减低磷酸化Smad2和Smad3的蛋白表达水平及抑制博莱霉素诱导的TGF-β1分泌。结论:miR-200c能明显抑制人瘢痕疙瘩成纤维细胞增殖及胶原合成,其机制可能与抑制TGF-β/Smad通路相关。     

Circ_0000069 contributes to the growth,metastasis and glutamine metabolism in renal cell carcinoma (RCC) via regulating miR-125a-5p-dependent SLC1A5 expression

BackgroundCircular RNAs (circRNAs) have emerged as critical mediators in various cancers, including renal cell carcinoma (RCC). In the present research, the functions of circ_0000069 in RCC were explored.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) assay, western blot assay and immunohistochemistry (IHC) assay were performed for the expression of circ_0000069, microRNA-125a-5p (miR-125a-5p) and solute carrier family 1 member 5 (SLC1A5). Cell Counting Kit-8 (CCK-8) assay and 5′-ethynyl-2′-deoxyuridine (EdU) assay were performed for cell proliferation. Flow cytometry assay was manipulated for cell apoptosis. Transwell assay and wound-healing assay were utilized for cell invasion and migration. Glutamine metabolism level was evaluated by examining glutamine consumption, α-ketoglutarate production and glutamate production. Dual-luciferase reporter assay was used to analyze the relationships of circ_0000069, miR-125a-5p and SLC1A5. Murine xenograft model assay was conducted to analyze the function of circ_0000069 in vivo.ResultsCirc_0000069 level was abnormally upregulated in RCC tissues and cells. Knockdown of circ_0000069 inhibited the proliferation, invasion, migration and glutamine metabolism and promoted the apoptosis in RCC cells in vitro and restrained tumor growth in vivo. Circ_0000069 served as the sponge for miR-125a-5p. MiR-125a-5p inhibition ameliorated the effects of circ_0000069 knockdown on RCC cell malignant behaviors. SLC1A5 was identified as the target gene of miR-125a-5p. Moreover, miR-125a-5p overexpression repressed the progression of RCC cells, while SLC1A5 elevation abrogated the effect.ConclusionCirc_0000069 knockdown inhibited the carcinogenesis of RCC by regulating miR-125a-5p/SLC1A5 axis.     

Long non-coding RNA H19 promotes the proliferation,migration and invasion while inhibits apoptosis of hypertrophic scarring fibroblasts by targeting miR-3187-3p/GAB1 axis

BackgroundIt had been reported that long non-coding RNA (lncRNA) H19 was associated with the proliferation of fibroblasts. However, the regulatory mechanism of H19 remains unclear. Thus, the study was designed to explore the underlying mechanism of H19 in the process of Hypertrophic scarring (HS).MethodsThe expression levels of H19, miR-3187-3p, and growth factor receptor binding 2-associated binding protein 1 (GAB1) in HS tissues and HS fibroblasts were measured by real-time quantitative polymerase chain reaction (RT-qPCR) assay. The biological behaviors of HS fibroblasts, such as cell proliferation, apoptosis, migration, and invasion were assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT), colony formation, flow cytometry, and transwell assays, respectively. The protein expression level was quantified by western blot assay. The interaction association between miR-3187-3p and H19 or GAB1 was predicted by Starbase database analysis and confirmed by dual-luciferase reporter assay, respectively.ResultsH19 was significantly increased in HS tissues and HS fibroblasts. Loss-of-functional experiments revealed that knockdown of H19 inhibited the development of HS. Moreover, silencing of H19 impeded the proliferation, migration, and invasion, while enhanced apoptosis of HS fibroblasts by increasing miR-3187-3p expression. In addition, overexpression of GAB1 could abolish miR-3187-3p overexpression-induced effects on cell proliferation, apoptosis, migration, and invasion of HS fibroblasts. Mechanistically, H19 could act as a sponge of miR-3187-3p to upregulate the expression of GAB1 in HS fibroblasts.ConclusionCollectively, our results revealed that H19 promoted the proliferation, migration, and invasion, while impeded apoptosis of HS fibroblasts by targeting miR-3187-3p/GAB1 axis.     

Decreased expression of inhibitory SMAD6 and SMAD7 in keloid scarring.

Keloids are benign skin tumours occurring during wound healing in genetically predisposed patients. They are characterised by an abnormal deposition of extracellular matrix components, in particular collagen. There is evidence that transforming growth factor-beta (TGFbeta) is involved in keloid formation. SMAD proteins play a crucial role in TGFbeta signaling and in terminating the TGFbeta signal by a negative feedback loop through SMAD6 and 7. It is unclear how TGFbeta signaling is connected to the pathogenesis of keloids. Therefore, we investigated the expression of SMAD mRNA and proteins in keloids, in normal skin and in normal scars. Dermal fibroblasts were obtained from punch-biopsies of keloids, normal scars and normal skin. Cells were stimulated with TGFbeta1 and the expression of SMAD2, 3, 4, 6 and 7 mRNA was analysed by real time RT-PCR. Protein expression was determined by Western blot analysis. Our data demonstrate a decreased mRNA expression of the inhibitory SMAD6 and 7 in keloid fibroblasts as compared to normal scar (p<0.01) and normal skin fibroblasts (p<0.05). SMAD3 mRNA was found to be lower in keloids (p<0.01) and in normal scar fibroblasts (p<0.001) compared to normal skin fibroblasts. Our data showed for the first time a decreased expression of the inhibitory SMAD6 and SMAD7 in keloid fibroblasts. This could explain why TGFbeta signaling is not terminated in keloids leading to overexpression of extracellularmatrix in keloids. These data support a possible role of SMAD6 and 7 in the pathogenesis of keloids.     

长链非编码RNA 909靶向调控miR-194-5p/DACH1轴对胰腺癌细胞增殖、迁移和侵袭的影响

背景与目的:长链非编码RNA 909(LINC00909)是一个新发现的长度约为2 kb的长链非编码RNA(lncRNA),在胶质瘤和白血病中被报道发挥癌基因的作用.然而LINC00909在胰腺癌中的作用鲜有报道.本研究旨探讨LINC00909在胰腺癌中的表达及其对患者预后的影响,以及LINC00909与其调控网络对胰...     

miR-21对瘢痕疙瘩成纤维细胞胶原分泌及细胞增殖的影响

目的 探讨mi R-21(micro RNA-21)在瘢痕疙瘩中的表达及其生物学作用,为瘢痕疙瘩的防治提供新的思路。方法 收集临床患者正常皮肤及瘢痕疙瘩标本,部分进行组织学检测;部分进行体外细胞培养,Real-time PCR检测体外培养的正常皮肤来源和瘢痕疙瘩来源的成纤维细胞中,mi R-21、Smad7(mi R-21靶基因)、Col 1 A1、Col 3 A1(纤维化相关基因)的表达;应用mi RNA-21的模拟物和抑制剂,以Western-Blot和Real-time PCR分别检测Col 1 A1、Col 3 A1及Smad7的蛋白和核酸水平表达变化,结晶紫实验检测细胞增殖能力的变化。结果 瘢痕疙瘩标本组织学检测,其胶原的含量明显高于正常皮肤;瘢痕疙瘩成纤维细胞中mi R-21、Col 1 A1、Col 3 A1表达高于正常皮肤组织,Smad7表达低于正常皮肤组织;正常皮肤来源成纤维细胞转染mi R-21模拟物后,Smad7表达降低,纤维化相关基因的表达、细胞增殖能力增加;瘢痕疙瘩组织来源成纤维细胞转染mi R-21抑制剂后,Smad7表达增加,纤维化相关基因的表达、细胞增殖能力降低。结论 瘢痕疙瘩中mi R-21表达升高,促进了细胞外基质中纤维化相关胶原基因的表达,促进成纤维细胞的体外增殖能力,可能是导致瘢痕疙瘩形成的重要原因。     

Circular RNA circ_0114876 regulates osteoarthritis through upregulating ADAM10 via targeting miR-1227-3p

BackgroundOsteoarthritis (OA) was a chronic degenerative joint disease. The dysregulation of circular RNAs (circRNAs) has been identified in OA progression. However, the function and regulation mechanism of circ_0114876 in OA remains largely unknown.MethodFirstly, we used LPS-treated C28/I2 cells as a cellular model of OA. Quantificational real-time polymerase chain reaction (qRT-PCR) was used to determine the expression levels of circ_0114876, miRNA-1227-3p, and ADAM10 in OA chondrocytes. Cell Counting Kit-8 (CCK8), 5-ethynyl-20-deoxyuridine (EdU) incorporation assays, flow cytometry, Enzyme-linked immunosorbent assay (ELISA) kit, and western blot were applied to confirm cell proliferation, apoptosis, inflammation, and extracellular matrix.of circ_0114876 in vitro. The interaction between circ_0114876 and its downstream target (miR-1227-3p) and mRNA target ADAM metallopeptidase domain 10 (ADAM10), was evaluated by luciferase assay and RNA immunoprecipitation (RIP) assay.ResultCirc_0114876 and ADAM10 were upregulated and miR-1227-3p was decreased in OA tissues and LPS-treated chondrocytes. Low expression of circ_0114876 promoted proliferation and inhibited apoptosis, inflammation, and extracellular matrix of the LPS-treated chondrocytes. Mechanistically, circ_0114876 functioned in human chondrocytes through targeting miR-1227-3p and ADAM10. Furthermore, miRNA-1227-3p inhibitor reversed the effect of circ_0114876 knockdown on the OA chondrocytes, and ADAM10 overexpression reversed the effect of miR-1227-3p mimic on the OA chondrocytes.ConclusionCirc_0114876 was increased in OA tissues and cells. Circ_0114876 facilitated the progression in the LPS-induced OA cell model via regulating the miR-1227-3p/ADAM10 axis. This study would provide a potentially effective therapeutic strategy for OA progression.     

Notch signaling pathway in keloid disease: Enhanced fibroblast activity in a Jagged‐1 peptide‐dependent manner in lesional vs. extralesional fibroblasts

Keloid disease (KD) is a fibroproliferative disorder of unknown etiopathogenesis with ill‐defined treatment. There is increasing evidence to suggest that aberrant Notch signaling may contribute directly to skin pathogenesis and altered expression of Notch receptors identified in KD. Therefore, the aim of this study was to investigate the Notch signaling pathway in KD compared to normal skin (NS). In this study, we employed in vitro primary cell culture models to elucidate the role of Notch signaling in 44 tissue samples from patients with KD split into keloid and extralesional (EL) samples (internal control) from the same patients, and six NS tissue samples (external control). We show the presence of a significant (p < 0.05) up‐regulation of Notch receptors and ligand Jagged‐1 (JAG‐1) in KD compared to EL and NS tissue samples. Cell spreading, attachment, and proliferation were significantly (p < 0.05) reduced in JAG‐1 antisense‐treated primary dermal fibroblasts isolated from KD and treated with γ‐secretase inhibitor (blocks proteolytic cleavage and activation of Notch), evaluated by real‐time cell analyzer (RTCA) on a microelectronic sensory array. In contrast, extralesional skin fibroblasts (ELF) treated with recombinant human JAG‐1 (rh‐JAG‐1) peptide showed significant (p < 0.05) enhancement of cell spreading, attachment, and proliferation in RTCA. Activation/inhibition of JAG‐1 and Notch signaling significantly (p < 0.05) altered the behavior of primary keloid fibroblasts and ELF, in cell migration (using a scratch wound assay), invasion (using a 3D invasion assay), and angiogenesis (in vitro coculture tube formation assay). In conclusion, this is the first study to demonstrate a potential role for the Notch signaling pathway in KD progression and that targeting this pathway may provide a novel strategy for treatment of KD.