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SUMMARY: Keloid scar is a unique benign fibroproliferative tumor of the human skin. Previously, it was reported that early growth response 1 (EGR1), a transcription factor, promotes keloid fibrosis; however, the mechanism by which EGR1 modulates keloid formation was not elaborated. In this research, the specific function and the microRNA (miRNA) regulatory network of EGR1 in keloids was examined. Keloid fibroblasts (KFs) were transfected with EGR1-small interfering RNA (siEGR1), EGR1-overexpression plasmid (pcDNA3.1-EGR1), and microRNA (miR-183-5p)-mimics to regulate the expression of EGR1 and miR-183-5p. The study employed dual-luciferase reporter assays to explore the targeting regulation of miR-183-5p on EGR1. Additionally, Western blotting, flow cytometry, qRT-PCR, cell count kit-8 (CCK-8), transwell, and wound healing assays, and RNA sequencing were conducted. EGR1 was upregulated in KFs, and EGR1 silencing diminished proliferation, fibrosis, migration, invasion, and apoptosis of cells. In KFs, the expression of miR- 183-5p was reduced, leading to the inhibition of cell proliferation, migration, and invasion. Conversely, it enhanced apoptosis. By targeting EGR1, miR-183-5p partially counteracted the impact of EGR1 on migration, invasion, and fibrosis in KFs. The findings imply that miR-183-5p suppresses keloid formation by targeting EGR1. As a result, EGR1 holds promise as a potential therapeutic target for preventing and treating keloids.
La cicatriz queloide es un tumor fibroproliferativo benigno único de la piel humana. Anteriormente, se informó que la respuesta de crecimiento temprano 1 (EGR1), un factor de transcripción, promueve la fibrosis queloide; sin embargo, no se explicó el mecanismo por el cual EGR1 modula la formación de queloides. En esta investigación, se examinó la función específica y la red reguladora de microARN (miARN) de EGR1 en queloides. Se transfectaron fibroblastos queloides (KF) con ARN de interferencia pequeño de EGR1 (siEGR1), plásmido de sobreexpresión de EGR1 (pcDNA3.1-EGR1) y miméticos de microARN (miR-183-5p) para regular la expresión de EGR1 y miR-183. -5p. El estudio empleó ensayos de indicador de luciferasa dual para explorar la regulación dirigida de miR-183-5p en EGR1. Además, se realizaron pruebas de transferencia Western, citometría de flujo, qRT-PCR, kit de recuento celular-8 (CCK-8), transwell y curación de heridas, y secuenciación de ARN. EGR1 estaba regulado positivamente en KF, y el silenciamiento de EGR1 disminuyó la proliferación, fibrosis, migración, invasión y apoptosis de las células. En KF, la expresión de miR- 183-5p se redujo, lo que llevó a la inhibición de la proliferación, migración e invasión celular. Por el contrario, mejoró la apoptosis. Al apuntar a EGR1, miR-183-5p contrarrestó parcialmente el impacto de EGR1 en la migración, invasión y fibrosis en KF. Los hallazgos implican que miR-183-5p suprime la formación de queloides al apuntar a EGR1. Como resultado, EGR1 es prometedor como objetivo terapéutico potencial para prevenir y tratar los queloides.
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Young Adult , Early Growth Response Protein 1 , Fibroblasts , Keloid/genetics , Keloid/pathology , Wound Healing , Transfection , Down-Regulation , Cell Movement , Blotting, Western , Sequence Analysis, RNA , Apoptosis , MicroRNAs/physiology , Cell Proliferation , Real-Time Polymerase Chain ReactionABSTRACT
Objective:To observe the expressions of miR-183 and retinal dehydrogenase 11 (RDH11) in exosomes derived from bone marrow mesenchymal stem cells (BMSC), and to preliminarily explore their targeting relationship and their effects on retinal pigment epithelial (RPE) cells.Methods:BMSC from C57BL/6 (C57) mice were isolated and cultured, and BMSC-derived exosomes were identified. BMSC were divided into blank group, simulation blank control group (mimic-NC group), miR-183 simulation group (miR-183-mimic group). C57 mice and retinal degeneration 10 (rd10) mouse RPE cells were cultured with reference to literature methods. RPE cells from rd10 mice were transfected with BMSC exosomes and co-cultured and divided into control group, exosome group, mimic-NC-exosome group (mimic-NC-exo group), miR-183-mimic-exosome group (miR-183-mimic-exo group). The relative expression levels of miR-183, RDH11 mRNA and protein in C57 mice, rd10 mice and RPE cells in each group were detected by real-time quantitative polymerase chain reaction and western blotting. The targeting relationship between miR-183 and RDH11 was analyzed by bioinformatics website and dual luciferase reporter. Cell counting kit 8 was used to detect the effect of miR-183 on BMSC exosomes on RPE cell proliferation; in situ labeling end labeling method was used to detect RPE cells apoptosis. One-way ANOVA was used to compare multiple groups.Results:Compared with C57 mouse RPE cells, the relative expression of miR-183 in rd10 mouse RPE cells was down-regulated, and the relative expression of RDH11 mRNA was up-regulated, and the differences were statistically significant ( t=5.230, 8.548; P=0.006, 0.001). Compared with the blank group and the mimic-NC group, the relative expression of miR-183 mRNA in the exosomes of the miR-183-mimics group was significantly increased ( F=60.130, P <0.05 ). After 24 h of co-culture, exosomes entered RPE cells. Compared with the mimic-NC-exo group, the relative expression of miR-183 mRNA in RPE cells in the miR-183-mimic-exo group was significantly increased, the proliferation ability was enhanced ( t=7.311, P=0.002), and the number of apoptotic cells was decreased ( F=10.949, P=0.012), and the differences were statistically significant ( t=4.571, P=0.002). Bioinformatics website and dual-luciferase report confirmed that miR-183 has a targeting relationship with RDH11. Compared with the mimic-NC group, the relative expression of RDH11 mRNA and protein in the exosomes of the miR-183-mimic group was decreased, and the difference was statistically significant ( t=5.361, 6.591; P=0.006, 0.003). After co-culture, compared with the control group, there was no significant difference in the relative expression of RDH11 mRNA and protein in RPE cells in the exosome group ( t=0.169, 1.134; P=0.874, 0.320); The relative expressions of RDH11 mRNA and protein in RPE cells in -183-mimic-exo group were decreased, and the difference was statistically significant ( t=5.554, 5.546; P=0.005, 0.005). Conclusion:Up-regulation of BMSC-derived exosomal miR-183 promote the proliferation of RPE cells in vitro by targeting the expression of RDH11 and reduce the number of apoptosis.
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@#［摘要］ 目的：探讨miR-183 对脑胶质瘤细胞放射敏感性的影响。方法：2020 年10 月至2021 年6 月，收集秦皇岛市第一 医院40 例脑胶质瘤组织标本，对T98G 细胞进行梯度剂量X 射线（0、2、4、6 Gy）照射。采用qPCR 检测miR-183、细胞质多腺 苷酸化元件结合蛋白1（cytoplasmic polyadenylation element-binding protein 1，CPEB1）在脑胶质瘤组织、T98G 细胞和经X 射线 照射的T98G 细胞中的表达量。将miR-183 inhibitor 转染T98G 细胞后下调miR-183 表达，经6 Gy X 射线垂直照射，CCK-8 法、 流式细胞术和WB 法，分别检测T98G 细胞增殖能力、细胞凋亡率及BAX 和Bcl2 蛋白表达量。Targetscan 软件预测和双荧光 素酶报告基因实验检测miR-183 与CPEB1 的靶向关系。下调CPEB1 表达后，经6 Gy X 射线照射，分别用CCK-8 法、流式细胞 术和WB 法检测T98G 细胞增殖能力、细胞凋亡率及BAX 和Bcl2 蛋白表达量。将pcDNA-CPEB1 或CPEB1 siRNA 质粒转染 T98G细胞，分别下调或过表达CPEB1 后，检测miR-183 通过CPEB1 对T98G细胞放射敏感性的影响。结果：脑胶质瘤组织和 细胞中miR-183 呈高表达，CPEB1 mRNA 呈低表达。T98G 细胞中miR-183 的表达量随着X 射线放射剂量增加而降低 （P<0.05），CPEB1 表达量随着X 射线放射剂量增加而升高（P<0.05）。6 Gy X 射线照射T98G 细胞后，下调miR-183 可降低细 胞增殖能力、增加细胞凋亡率，而过表达miR-183 则起到相反作用（P<0.05）。miR-183 靶向CPEB1 mRNA 且负调控CPEB1 表 达。下调CPEB1 表达后，经6 Gy X 射线照射可显著提高T98G 细胞增殖能力（P<0.05）、降低细胞凋亡率（P<0.05），miR-183 可 逆转CPEB1 过表达对细胞T98G 放射敏感性的促进作用（P<0.05）。结论：下调miR-183 的表达能够负调控CPEB1，从而增强 脑胶质瘤细胞的放射敏感性。
ABSTRACT
Intracellular trafficking is a field that has been intensively studied for years and yet there remains much to be learned. Part ofthe reason that there is so much obscurity remaining in this field is due to all the pathways and the stages that define cellulartrafficking. One of the major steps in cellular trafficking is fusion. Fusion is defined as the terminal step that occurs when acargo-laden vesicle arrives at the proper destination. There are two types of fusion within a cell: homotypic and heterotypicfusion. Homotypic fusion occurs when the two membranes merging together are of the same type such as vacuole tovacuole fusion. Heterotypic fusion occurs when the two membranes at play are of different types such as when anendosomal membrane fuses with a Golgi membrane. In this review, we will focus on all the protein components – Rabs,Golgins, Multisubunit tethers, GTPases, protein phosphatases and SNAREs – that have been known to function in both ofthese types of fusion. We hope to develop a model of how all of these constituents function together to achieve membranefusion. Membrane fusion is a biological process absolutely necessary for proper intracellular trafficking. Due to the degreeof importance multiple proteins are required for it to be properly carried through. Whether we are talking about heterotypicor homotypic fusion, any defects in the fusion machinery can result in disease states such as Parkinson’s and Alzheimer’sdisease. Although much research has significantly expanded our knowledge of fusion, there is still much more to belearned.
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PURPOSE: Hepatocellular carcinoma (HCC) is the most common malignant tumor of liver cells. Researchers have reported that cancer susceptibility candidate 2 (CASC2), a long non-coding RNA, is down-regulated in various cancers, including HCC. Our study aimed to investigate the molecular mechanism(s) of CASC2 in HCC. MATERIALS AND METHODS: Real-time quantitative PCR (RT-qPCR) was used to analyze the expression of CASC2 and miR-183 in HCC tissues and cells. The viability of HCC SMMC-7721 and Huh-7 cells was detected through MTT assay. Colony formation assay was performed to assess the colony formation ability of HCC cells. The migration and invasion abilities of HCC cells were evaluated by Transwell assay. Western blot was conducted to examine levels of key Wnt/β-catenin signaling pathway factors, C-myc, cyclinD, survivin, and β-catenin. The interaction between CASC2 and miR-183 was affirmed by bioinformatics analysis and luciferase reporter assay. RESULTS: CASC2 was down-regulated in HCC tissues and cell lines, while miR-183 was up-regulated. The expression of miR-183 was negatively correlated with CASC2 expression in HCC tissues. Overexpression of CASC2 inhibited cell viability, colony formation, migration, and invasion in HCC cells, as well as Wnt/β-catenin signaling pathway activity. miR-183 was a downstream target of CASC2 and negatively regulated by CASC2. Introduction of miR-183 rescued CASC2-induced suppressive effects on HCC cell viability, colony formation, migration, and invasion and Wnt/β-catenin signaling. CONCLUSION: CASC2 inhibited cell viability and the colony formation, migration, and invasion abilities of HCC cells by directly downregulating miR-183 through inactivation of the Wnt/β-catenin signaling pathway.
Subject(s)
Blotting, Western , Carcinoma, Hepatocellular , Cell Line , Cell Survival , Computational Biology , Liver , Luciferases , Polymerase Chain Reaction , RNA, Long NoncodingABSTRACT
Objective To detect the expression and significance of serum miRNA-183 and TK1 in patients with colorectal cancer, and the mechanism thereof. Methods Fifty-two serum samples of colorectal cancer patients and paired health serum samples were collected. The expression of miRNA-183 was detected by real-time quantitative PCR, and TK1 was detected by Western blot enhanced chemiluminescence assay. The correlation between miRNA-183 and TK1 and their relations with the clinicpathologic characteristics were analyzed. Results The serum miRNA-183 expression was significantly higher in colorectal cancer group than that in normal control group (P<0.01). The expression of serum miRNA-183 was significantly higher in stage Ⅲ-Ⅳ group than that in stage Ⅰ-Ⅱ group (P < 0.01). There was more significant increase in serum miRNA-183 in lymphatic metastasis group than that without lymphatic metastasis group (P < 0.01). Receiver operating characteristic (ROC) curve showed that of there was a diagnostic value for serum miRNA-183 in colorectal cancer, with an optimal value of 1.15. The diagnostic sensitivity and specificity were 78.8%and 67.3%, and the positive predictive value and negative predictive value were 78.9%and 70.2%. The serum TK1 expression was also higher in colorectal cancer group compared with that of normal control group (P<0.01). And the TK1 expression was also higher in stageⅢ-Ⅳgroup than that in stageⅠ-Ⅱgroup (P<0.01). Furthermore, miRNA-183 expression was positively correlated with TK1 expression (rs=0.692, P<0.01). Conclusion The serum expression levels of miR-183 and TK1 may act as tumor markers for early colorectal cancer diagnosis, and also can be used to predict the malignant degree and prognosis of colorectal cancer.
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Objective To study the expression and clinical significance of miR-183-5p, TβRⅠ and TβRⅡ in esophageal squamous cell carcinoma (ESCC). Methods The mRNA and protein expression of miR-183-5p, TβRⅠ and TβRⅡ were examined in ESCC cell lines ECA-109, TE-1, normal esophageal epithelial cells, tumor tissues and tumor-free tissues from 72 ESCC patients. Their clinical significance and the relationship between miR-183-5p and the latter two were analyzed. The effects of miR-183-5p on the expression of TβRⅠand TβRⅡ in ECA-109 cells and the cell functions of ECA-109 were also investigated. Results Compared with the normal esophageal epithelia cells, ESCC cell lines TE-1 and ECA-109 were statistically characterized by a high expression of miR-183-5p (all P<0.05) and low expression of TβRⅠand TβRⅡ(all P<0.05). The expression of miR-183-5p in ESCC tissues was higher than that in adjacent normal tissues, while the expressions of TβRⅠ and TβRⅡ were lower (all P< 0.05). The expression of miR-183-5p was closely related to sex, tumor differentiation, tumor staging, distant metastasis, lymphatic metastasis, and tumor location (all P<0.05). TβRⅠlevel was associated with sex, lymph node metastasis and tumor size (all P<0.05). Experimental data showed the negative correlation between the expression of miR-183-5p and TβRⅠin ESCC tissues (r= -0.521, P< 0.05). Over expression of miR-183-5p significantly inhibited the expression of TβRⅠ in ECA-109 cells (P< 0.05) and promoted the growth, invasion and metastasis of ECA-109 cells (P< 0.05). Low expression of miR-183-5p significantly promoted the expression of TβRⅠ in ECA-109 cells (P< 0.05), and suppressed the growth, invasion and metastasis of ECA-109 cells (P< 0.05). There was no significant change in the expression of TβRⅡ in the transfection experiments. Conclusion MiR-183-5p is closely related to the abnormal expression of TβRⅠ, which may exert an important role in the progression of lymphatic metastasis.
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miRNAs are essential factors of an extensively conserved post-transcriptional process controlling gene expression at mRNA level. Varoius biological processes such as growth and differentiation are regulated by miRNAs. Web of Science and PubMed databases were searched using the Endnote software for the publications about the role miRNA-183 family in inner ear: hair cell development and deafness published from 2000 to 2016. A triplet of these miRNAs particularly the miR-183 family is highly expressed in vertebrate hair cells, as with some of the peripheral neurosensory cells. Point mutations in one member of this family, miR-96, underlie DFNA50 autosomal deafness in humans and lead to abnormal hair cell development and survival in mice. In zebrafish, overexpression of the miR-183 family induces extra and ectopic hair cells, while knockdown decreases the number of hair cell. The miR-183 family (miR-183, miR-96 and miR-182) is expressed abundantly in some types of sensory cell in the eye, nose and inner ear. In the inner ear, mechanosensory hair cells have a robust expression level. Despite much similarity of these miRs sequences, small differences lead to distinct targeting of messenger RNAs targets. In the near future, miRNAs are likely to be explored as potential therapeutic agents to repair or regenerate hair cells, cell reprogramming and regenerative medicine applications in animal models because they can simultaneously down-regulate dozens or even hundreds of transcripts.
Subject(s)
Animals , Humans , Mice , Biological Phenomena , Cellular Reprogramming , Deafness , Ear, Inner , Gene Expression , Hair , Hearing Loss , MicroRNAs , Models, Animal , Nose , Point Mutation , Regenerative Medicine , RNA, Messenger , Triplets , Vertebrates , ZebrafishABSTRACT
BACKGROUND/AIMS: Intraductal papillary mucinous neoplasms (IPMN) are precursor lesions of fatal pancreatic cancer. Physiological function of microRNA is to regulate the stability and translation of mRNA. The aberrant microRNA expression is commonly observed in many cancers. The aim of this study was to analyze the expression pattern of microRNA in IPMN and evaluate the role of the microRNA. METHODS: Using two paraffin-embedded IPMN tissues, microRNA expression of normal tissue, IPMN adenoma and carcinoma were compared by cDNA-mediated annealing, selection, extension and ligation microarray assay. Using real time PCR, expression levels of aberrantly up-regulated microRNAs were assessed in another 20 IPMNs, four pancreatic cancer cell lines (Panc1, MiaPaCa-2, XPA-3, BxPC-3) and immortalized pancreatic ductal cell line (HPNE). Effect of suppressing highly over-expressed two microRNAs in pancreatic cancer cell lines with anti-microRNA inhibitors were evaluated using CCK-8 assay. RESULTS: Among aberrantly expressed 122 microRNAs in IPMN, miR-552, miR-25*, miR-183, miR-1300, miR-196a, miR-182*, and miR-30c-1* were consistently increased more than 3-fold. On average, miR-196a and miR-183 increased 10,824 folds and 26,519 folds in four pancreatic cancer cell lines compared with HPNE. These two microRNAs were also over-expressed in 20 IPMNs compared with HPNE. After applying anti-miRNA inhibitors, cell survival of four pancreatic cancer cell lines decreased by 24.5% with anti-miR-196a and by 14.2% with anti-miR-183 on average. CONCLUSIONS: Aberrant expression of 122 microRNAs was observed in IPMN. Two microRNAs, miR-196a and miR-183-increased in IPMN and pancreatic cancer cell lines compared with immortalized dancreatic ductal cell line. The inhibitions of these microRNAs repressed cell proliferation of pancreatic cancer cell lines.