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.     

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.     

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.     

Long non-coding RNA LINC01116 accelerates the progression of keloid formation by regulating miR-203/SMAD5 axis

BackgroundEmerging evidence reveals the importance of long non-coding RNAs (lncRNAs) in the development and progression of keloid formation. However, the roles and molecular mechanism of lncRNA LINC01116 in the progression of keloid formation remain largely unknown.MethodsThe expression levels of LINC01116, microRNA-203 (miR-203) and SMAD family member 5 (SMAD5) were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell proliferation, migration and invasion were detected by Cell counting Kit-8 (CCK-8) assay and transwell assay. Flow cytometry and western blot assay were used to examine cell apoptosis and extracellular matrix (ECM) production. The interaction between miR-203 and LINC01116 or SMAD5 was predicted by bioinformatics analysis and verified by dual-luciferase reporter and RNA Immunoprecipitation (RIP) and RNA pull-down assays.ResultsLINC01116 and SMAD5 were upregulated while miR-203 was downregulated in keloid tissues and keloid fibroblasts. LINC01116 knockdown suppressed the proliferation, migration, invasion, and ECM production but induced apoptosis in keloid fibroblasts through enhancing miR-203 and inhibiting SMAD5. Moreover, SMAD5 was identified as a direct target of miR-203 and miR-203 could directly bind to LINC01116. Besides, LINC01116 regulated SMAD5 expression by targeting miR-203.ConclusionDownregulation of LINC01116 inhibited the progression of keloid formation by regulating miR-203/SMAD5 axis, which might provide a novel target for keloid therapy.     

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.     

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。     

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。     

长链非编码RNA人去泛素化酶含有卵巢肿瘤结构域的6B反义RNA 1通过微小RNA-122-5p促进肝癌细胞增殖、侵袭和迁移的研究

目的探讨长链非编码RNA(lncRNA)人去泛素化酶含有卵巢肿瘤结构域的6B反义RNA 1(OTUD6B-AS1)通过微小RNA(microRNA,miR)-122-5p对肝癌细胞增殖、侵袭和迁移的影响。方法反转录-聚合酶链反应(RT-PCR)检测2010年1月至2013年1月河南科技大学第一附属医院手术切除的98例肝癌组织标本及其配对的癌旁组织、肝癌细胞系(Hhu7、Hep3B、HCCLM3、MHCC97H)和人正常肝细胞(LO2)中OTUD6B-AS1的相对表达量。用脂质体2000(Lipofectamine^TM2000)分别将sh-NC(sh-NC组)和sh-OTUD6B-AS1(sh-OTUD6B-AS1组)转染入Hep3B细胞。克隆形成实验和细胞计数试剂盒(CCK-8)法检测细胞增殖;Transwell检测细胞侵袭;细胞划痕实验检测细胞迁移。用TargetScan预测OTUD6B-AS1与miR-122-5p的互补结合位点,随后用双荧光素酶报告基因实验确定两者的靶向关系。为了进一步验证两者调控关系,将Hep3B细胞分别分为sh-NC+miR-NC组、sh-OTUD6B-AS1+miR-NC组、sh-OTUD6B-AS1+anti-miR-122-5p组,比较3组细胞增殖、侵袭和迁移。两两比较采用t检验;多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验;生存分析采用Log-rank检验。结果肝癌组织中OTUD6B-AS1相对表达量为2.42±0.96,明显高于癌旁组织(0.92±0.39,t=14.331,P<0.05),差异有统计学意义。肝癌细胞系Hhu7(2.84±0.23)、Hep3B(3.80±0.68)、HCCLM3(1.89±0.25)和MHCC97H(2.01±0.71)的OTUD6B-AS1相对表达量明显高于正常肝细胞LO2(1.04±0.23,F=51.827,P<0.05),差异有统计学意义。sh-OTUD6B-AS1组细胞增殖、侵袭和迁移能力均低于sh-NC组(t=5.556、2.454,P值均<0.05),差异均有统计学意义。双荧光素酶报告基因实验结果证实OTUD6B-AS1可以靶向调控miR-122-5p。sh-NC+miR-NC组和sh-OTUD6B-AS1+anti-miR-122-5p组增殖、侵袭和迁移能力明显高于sh-OTUD6B-AS1+miR-NC组(F=111.908、0.301,P值均<0.05),差异均有统计学意义。结论OTUD6B-AS1可能通过负调控miR-122-5p促进肝癌细胞的增殖、侵袭和迁移。     

miR-483-5p通过下调CDK15促进肾上腺皮质癌增殖和侵袭

目的探讨miR-483-5p在肾上腺皮质癌中的作用及其可能的作用机制。方法荧光定量PCR法检测miR-483-5p和CDK15在肾上腺皮质癌组织和细胞系中的表达,CCK-8增殖试验测定miR-483-5p对细胞增殖的影响,Transwell法检测ACC细胞侵袭性的变化。荧光素酶试验和挽救实验验证miR-483-5p与CDK15相关的分子机制。结果miR-483-5p在肾上腺皮质癌组织中高表达(2.36±1.02 vs 1.09±0.43),CDK15在肾上腺皮质癌组织中低表达(0.57±0.26 vs 1.06±0.32)。荧光素酶检测证实CDK15是miR-483-5p的直接靶点。过表达miR-483-5p可通过下调CDK15的表达促进ACC细胞的增殖(24 h:0.26±0.03 vs 0.23±0.04,48 h:0.56±0.05 vs 0.41±0.03,72 h:0.73±0.04 vs 0.59±0.03)和侵袭能力(95.78±4.66 vs 23.89±2.52)。结论miR-483-5p可通过下调CDK15的表达促进肾上腺皮质癌的发生发展,其可作为肾上腺皮质癌的潜在生物标志物及治疗肾上腺皮质癌的新的作用靶点。     

miR-622在胃癌细胞中的表达及其功能的研究

目的 研究miR-622在胃癌细胞中的表达,探讨miR-622在胃癌中的生物学功能.方法 采用实时荧光定量PCR检测miR-622在胃癌细胞中的表达,以胃癌细胞SGC-7901和NCI-N87为模型瞬时转染miR-622寡核苷酸前体或抑制体,通过克隆形成、侵袭和划痕实验验证其在胃癌细胞中的功能.结果 荧光定量PCR实验结果表明:miR-622在胃癌细胞SGC-7901中相对表达量为1.29±0.58,而在NCI-N87细胞中相对表达量为10.96±1.02.在SGC-7901细胞中转染了miR-622 寡核苷酸前体,克隆形成率为76％.划痕试验中愈合能力为(11±7)μm,胃癌细胞侵袭能力为(732±3)个,与对照组相比差异均有统计学意义(均P＜0.05).结论 miR-622能够促进胃癌细胞克隆形成、迁移和侵袭.     

环状RNA FBXW7在三阴乳腺癌中的表达及其临床意义

背景与目的：环状RNA（circRNA）是参与多种癌症进程的重要调节因子。据报道,环状RNA FBXW7（circFBXW7）在胶质瘤中通过编码一种新蛋白发挥抑癌作用。然而,其在三阴性乳腺癌（TNBC）中的功能和机制尚不明确。本研究探讨了circFBXW7在TNBC中的作用及其临床意义。 方法：收集240例TNBC患者的新鲜癌组织及临床资料,通过qRT-PCR检测癌组织中circFBXW7的表达,用Kaplan-Meier法分析TNBC患者circFBXW7表达水平与预后的关系,并用Cox回归方法分析TNBC患者的预后因素。用CCK8实验、Transwell实验、qRT-PCR观察TNBC细胞株MDA-MB-231和HCC1806过表达circFBXW7后增殖与迁移、侵袭能力以及miR-197-3p表达的变化,在上述两种细胞中,敲除circFBXW7并转染miR-197-3p抑制剂后,用Transwell实验与克隆形成实验分析增殖与侵袭能力的变化。 结果：240例TNBC患者癌组织标本中circFBXW7的平均表达水平为1.043±0.268,以1.043为临界值分组生存分析显示,circFBXW7低表达患者的总生存期和无病生存期明显短于circFBXW7高表达患者（均P<0.05）;circFBXW7表达以及组织学分级、TNM分期为TNBC患者预后的独立影响因素（均P<0.05）。细胞实验显示,过表达circFBXW7后,MDA-MB-231和HCC1806细胞的增殖、迁移与侵袭能力以及miR-197-3p表达均明显降低（均P<0.05）;共转染miR-197-3p抑制剂可逆转由于敲除circFBXW7所致的MDA-MB-231和HCC1806细胞的增殖与侵袭能力的增强（均P<0.05）。 结论：circFBXW7的表达在TNBC中发挥抑癌作用,机制可能与其竞争性吸附miR-197-3p,从而抑制TNBC细胞的增殖与侵袭有关,FBXW7可能是一种新型TNBC预后生物标志物及潜在治疗靶标。     

miR-195-5p通过靶向ROCK1抑制肾癌细胞迁移、侵袭和上皮-间质转化

目的探讨miR-195-5p对肾癌细胞迁移、侵袭和上皮-间质转化的影响。方法通过转染miR-195-5p mimics或inhibitors分别过表达或抑制肾癌细胞中miR-195-5p的表达,转染靶向Rho相关螺旋蛋白激酶1(ROCK1)的小干扰RNA敲低肾癌细胞中ROCK1的表达量,利用细胞划痕实验和Transwell小室实验分别检测肾癌细胞的迁移和侵袭能力。通过双荧光素酶报告实验验证miR-195-5p对ROCK1的靶向调控作用,利用免疫印迹试验检测ROCK1及上皮-间质转化相关蛋白的表达水平。结果过表达miR-195-5p可显著抑制肾癌细胞的迁移、侵袭和上皮-间质转化,而抑制miR-195-5p的表达可明显促进肾癌细胞的迁移、侵袭和上皮-间质转化(P<0.05)。miR-195-5p可通过靶向ROCK1调控其在肾癌细胞中表达。敲低ROCK1后可部分抵消miR-195-5p inhibitors对肾癌细胞迁移、侵袭和上皮-间质转化的影响。结论miR-195-5p可通过靶向ROCK1抑制肾癌细胞的迁移、侵袭和上皮-间质转化。     

miR-542-3p对肝癌细胞恶性生物学行为的影响及机制探讨

目的探讨microRNA-542-3p（miR-542-3p）在人肝癌细胞系及组织中的表达水平及其对肝癌细胞增殖、侵袭转移能力的影响。方法采用RT-PCR检测miR-542-3p在肝癌细胞系（HCCLM3、Hep3B、Huh7、SMMC-7721、MHCC-97H、MHCC-97 L）以及人正常肝细胞LO2中的表达;同时采用RT-PCR检测肝癌及对应癌旁组织中miR-542-3p的表达。分别以转染miR-542-3p mimics的MHCC-97H和HCCLM3细胞作为过表达组,以转染空载体的MHCC-97H和HCCLM3细胞作为阴性对照组;采用CCK-8法检测各组细胞增殖能力,采用Transwell法检测各组细胞体外侵袭能力,Western blotting检测各组细胞上皮间质转化（epithelial mesenchymal transition,EMT）相关蛋白的表达,免疫荧光法检测miR-542-3p对肝癌细胞EMT的影响。结果miR-542-3p在肝癌细胞系HCCLM3（0.221±0.034）、Hep3B（0.764±0.059）、Huh7（0.561±0.029）、SMMC-7721（0.688±0.049）、MHCC-97H（0.162±0.031）、MHCC-97L（0.473±0.041）中的表达均低于在人正常肝细胞LO2（1.0）的表达水平（P＜0.05）;在肝癌组织表达低于癌旁组织[（0.208±0.064）vs（0.746±0.093）,P＜0.05]。MHCC-97H和HCCLM3过表达组细胞增殖、侵袭转移能力显著低于阴性对照组,EMT相关蛋白荧光明显低于阴性对照组（P＜0.05）。结论miR-542-3p在肝癌组织及肝癌细胞中低表达,miR-542-3p的过表达可有效抑制肝癌细胞恶性生物学行为,该过程可能与EMT相关通路有关。miR-542-3p有望成为肝癌生物治疗的潜在靶点。     

MicroRNA-7对HCCC9810细胞增殖、迁移和侵袭的研究

目的探讨MicroRNA-7(miR-7)对于肝内胆管癌细胞HCCC9810增殖、迁移、侵袭能力的影响。方法利用实时定量PCR对于肝内胆管癌病人的癌组织和癌旁组织,以及正常肝内胆管上皮细胞和肝内胆管癌细胞HCCC9810中miR-7表达水平的验证;培养人肝内胆管癌细胞HCCC9810,将其分为研究组(miR-7 mimics)和阴性对照组(miR-7 NC),分别进行miR-模拟物以及miR-阴性序列的转染,效率验证后,通过CCK-8实验,EDU实验,克隆形成实验,以及划痕和Transwell实验分别验证两组细胞的增殖、迁移和侵袭的能力。结果肝内胆管癌病人中癌组织miR-7的表达水平低于癌旁组织,肿瘤细胞HCCC9810 miR-7表达水平低于正常胆管上皮细胞;细胞转染效率验证显示,miR-7 mimics组HCCC9810细胞中miR-7的相对表达水平远高于miR-7 NC组,差异具有统计学意义(P<0.05);与miR-7NC相比,miR-7 mimics组细胞的增殖、迁移以及侵袭能力被明显抑制,差异均有统计学意义(均P<0.05)。结论miR-7在肝内胆管癌中表达水平下调,而且过表达miR-7能够抑制肿瘤细胞的增殖、迁移以及侵袭过程。     

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。     

Identifying miRNA modules associated with progression of keloids through weighted gene co-expression network analysis and experimental validation in vitro

Keloid is a type of skin fibroproliferative disease, characterized by excessive deposition of collagen in the extracellular matrix, myofibroblast activation and invasive growth to the surrounding normal skin tissue. However, the specific pathogenesis of keloids is not yet fully understood and existing treatment strategies are unsatisfied. It is therefore urgent to explore new biomarkers associated with its progression for keloids. In this study, the microarray dataset GSE113620 was downloaded from the Gene Expression Omnibus (GEO) database to screen out the differential expression of miRNAs (DEMs). The DEMs with large variance were applied to construct a weighted gene co-expression network to identify miRNA modules that are closely relevant to keloid progression. It is worth noting that miR-424-3p in the blue module (r = 0.98, p = 1e-18) is considered to be the ultimate target most relevant to keloid progression through co-expressed network analysis. Subsequently, the results of molecular biology experiments determine that miR-424-3p targeting Smad7 significantly enhanced the ability of cell proliferation, migration and collagen secretion after transfection with miR-424-3p mimic, while the apoptosis rate was significantly reduced. On the contrary, the miR-424-3p inhibitor performs the exact opposite function.     

miR-26b的表达及其与Foxf2相互作用在乳腺癌细胞生物学行为中的作用

目的:探讨miR-26b在乳腺癌细胞中的表达及其对乳腺癌细胞生物学行为的的影响。方法:比较正常乳腺细胞系MCF-10A及乳腺癌细胞系MCF-7中miR-26b的表达差异。以无处理的MCF-7细胞为空白对照,分别检测MCF-7细胞转染miR-26b模拟物(miR-26b组)、空质粒(阴性对照组)后的miR-26b表达与增殖、迁移、侵袭能力,以及Foxf2的mRNA与蛋白表达的变化。用双荧光素酶报告系统检测miR-26b对MCF-7细胞中Foxf2转录活性的影响。结果:miR-26b在MCF-7细胞中的表达水平明显低于MCF-10A细胞(P0.05)。与空白对照组和阴性对照组比较,miR-26b组miR-26b mRNA表达水平明显升高、细胞增殖迁移、侵袭能力明显降低,Foxf2的mRNA和蛋白表达量均明显下调(P0.05)。转染miR-26b模拟物后,MCF-7细胞中Foxf2-3'UTR的转录活性明显抑制(P0.05)。结论:miR-26b在乳腺癌细胞中表达降低、增加其表达能抑制乳腺癌细胞的恶性生物学行为,机制可能与其下调Foxf2的表达有关。     

长链非编码RNA ST8SIA6-AS1通过调节微小RNA-142-3p促进肝细胞癌的增殖和侵袭

目的探讨ST8SIA6-AS1靶向结合微小RNA(microRNA,miR)-142-3p,影响肝细胞癌的增殖和侵袭能力。方法用基因表达谱数据动态分析(GEPIA)验证ST8SIA6-AS1在肝细胞肝癌(HCC)和邻近正常组织中的差异表达。采用实时定量反转录聚合酶链反应(RT-qPCR)检测ST8SIA6-AS1和miR-142-3p在组织水平和细胞水平的表达量。采用细胞计数试剂盒(CCK-8)和集落形成实验检测人肝癌细胞Hep3B的增殖情况;采用迁移侵袭实验(Transwell法)检测肝癌Hep3B细胞迁移和侵袭情况。生物信息学、双荧光素酶报告实验验证ST8SIA6-AS1与miR-142-3P的靶向关系。组间采用t检验,多组间采用单因素方差分析进行比较,相关性分析采用Spearman秩检验。结果GEPIA结果显示ST8SIA6-AS1在肝癌组织中的表达明显高于正常组织。RT-qPCR结果显示ST8SIA6-AS1在HCC中的表达明显高于癌旁组织(0.778±0.363比1.951±0.575,t=11.370,P<0.05),差异有统计学意义。miR-142-3p在肝癌组织的表达量显著低于癌旁组织(0.881±0.374比0.371±0.164,t=9.395,P<0.05),差异有统计学意义。ST8SIA6-AS1在4种人HCC细胞株(HepG2、SMMC-7721、HCCLM3和Hep3B)的表达(3.84±0.27、5.81±0.60、4.47±0.55、5.94±0.69)明显高于正常肝细胞株L02(1.00±0.09,t=17.280,t=13.730,t=10.780,t=12.300,P<0.05),差异有统计学意义,ST8SIA6-AS1在敲低ST8SIA6-AS1的Hep3B细胞中的相对表达量低于对照组(0.26±0.04比1.00±0.01,t=9.423,P<0.05),差异有统计学意义。集落形成实验结果显示,敲低ST8SIA6-AS1的Hep3B细胞集落个数显著低于对照组[(32.60±6.70)个比(100.00±10.00)个,t=10.040,P<0.05],差异有统计学意义;CCK-8实验中敲低ST8SIA6-AS1的Hep3B细胞72 h吸光度(A)值低于对照组[(0.71±0.06)%比(1.14±0.09)%,t=6.886,P<0.05],差异有统计学意义。Transwell实验结果显示敲低ST8SIA6-AS1的Hep3B细胞垂直迁移率明显低于对照组[(53.38±6.49)%比(100.00±13.00)%,t=5.641,P<0.05],差异有统计学意义。敲低ST8SIA6-AS1的Hep3B细胞的侵袭率低于对照组[(44.98±8.42)%比(100.00±10.00)%,t=7.485,P<0.05],差异有统计学意义。双荧光素酶报告实验证实miR-142-3p过表达组明显低于对照组ST8SIA6-AS1-野生型(WT)细胞的荧光活性,ST8SIA6-AS1负向调控miR-142-3p的表达(0.42±0.05比1.00±0.09,t=9.757,P<0.05)。干扰ST8SIA6-AS1的表达可对肝癌细胞增殖、迁移、侵袭产生抑制作用,这种作用可通过沉默miR-142-3p发生逆转。结论ST8SIA6-AS1可通过竞争性结合miR-142-3p在肝癌中发挥致癌作用。     

miR-362-3p的表达及其靶基因与胆囊癌恶性特征的关系

目的:探讨miR-362-3p在胆囊癌中的表达及功能。方法:用qRT-PCR检测手术切除的44例胆囊癌患者手术标本中miR-362-3p的表达,并分析其表达与胆囊癌临床病理特征及预后的关系。miR-362-3p模拟物转染胆囊癌细胞后,分别用MTT法、细胞划痕试验及Transwell侵袭试验观察细胞增殖、迁移及侵袭的改变。通过生物信息学方法及双荧光素酶报告基因试验分析miR-362-3p的靶基因,并采用补救试验验证。结果:miR-362-3p在胆囊癌组织的表达明显低于相应癌旁组织(P0.05)。miR-362-3p的低表达与肿瘤TNM分期、淋巴结转移及远处转移明显有关(均P0.05)。低表达miR-362-3p患者总体生存率较高表达miR-362-3p患者明显降低(P0.05)。转染miR-362-3p模拟物后,胆囊癌细胞的增殖,迁移及侵袭能力明显减弱(均P0.05)。Nemo样激酶(NLK)被确定为miR-362-3p的潜在靶基因,转染NLK过表达载体后,miR-362-3p模拟物对胆囊癌细胞的上述作用被明显逆转(均P0.05)。结论:miR-362-3p在胆囊癌中表达下调,下调的miR-362-3p减少了对靶基因NLK的抑制,从而促进了胆囊癌细胞的增殖、迁移和侵袭。