3171 Epub [Extra Quality]
Sekeres MA, Gore SD, Stablein DM, DiFronzo N, Abel GA, DeZern AE, Troy JD, Rollison DE, Thomas JW, Waclawiw MA, Liu JJ, Al Baghdadi T, Walter MJ, Bejar R, Gorak EJ, Starczynowski DT, Foran JM, Cerhan JR, Moscinski LC, Komrokji RS, Deeg HJ, Epling-Burnette PK. The National MDS Natural History Study: design of an integrated data and sample biorepository to promote research studies in myelodysplastic syndromes. Leuk Lymphoma. 2019 Dec;60(13):3161-3171. doi: 10.1080/10428194.2019.1616186. Epub 2019 May 21. PMID: 31111762. =top&needAccess=true
To the best of our knowledge, the present study wasthe first to investigate the role of OTUD6B-AS1 in CRC, and toexamine whether OTUD6B-AS1 could affect the proliferation, invasionand migration of CRC cells by regulating miR-3171.
The OTUD6B-AS1 overexpression plasmid(Oe-OTUD6B-AS1; 1 µg) and empty vector (Oe-NC; 1 µg) were obtainedfrom Shanghai GenePharma Co., Ltd. miR-3171 mimics (40 nM; cat. no.miR10015046-1-5) and corresponding scrambled mimic negative control(mimic-NC; 40 nM; cat. no. miR1N0000001-1-5) were purchased fromGuangzhou RiboBio Co., Ltd. Cells (1106 cells/well) weretransfected with the aforementioned oligonucleotides usingLipofectamine 3000 (Invitrogen; Thermo FisherScientific, Inc.) according to the manufacturer's protocols at 37Cfor 48 h. At 48 h after transfection, HCT116 cells were harvestedfor further experiments, and successful transfection was verifiedusing reverse transcription-quantitative PCR (RT-qPCR).
Potential target genes of lncRNA OTUD6B-AS1 werepredicted using an online bioinformatics software Starbase 2.0( ) (21). HCT116 cells were reseeded into24-well plates and cultured for 24 h. The fragments of OTUD6B-AS1containing predicted wild-type (WT) and mutant (MUT) miR-3171binding sequences were amplified by Shanghai GenePharma Co., Ltd.,and inserted into the luciferase reporter gene of the pmirGLOvector (Promega Corporation) to produce the reporter plasmidsOTUD6B-AS1-WT and OTUD6B-AS1-MUT, respectively. Subsequently, thecells (1104 cells/well) were co-transfected with plasmids andmiR-3171 mimic (40 nM; cat. no. miR10015046-1-5; Guangzhou RiboBioCo., Ltd.) or mimic-NC vector (40 nM; cat. no. miR1N0000001-1-5;Guangzhou RiboBio Co., Ltd.) using Lipofectamine 2000(Invitrogen; Thermo Fisher Scientific, Inc.) for 24 h. At 48 hafter transfection, the culture medium was removed and the cellswere rinsed twice with PBS. The HCT116 cells were lysed to obtaincell lysates, which were swirled for 10 min and centrifuged at12,000 g for 10 min at 4C, and the supernatant was transferredto a new Eppendorf tube. According to the instructions of thedual-luciferase reporter assay kit (Beijing Solarbio Science &Technology Co., Ltd.), the fluorescence value was used as aninternal reference, and the fluorescence value was detected usingan enzyme marker. The results were normalized to Renillaluciferase activity.
The binding site between OTUD6B-AS1 and miR-3171 waspredicted using Starbase 2.0 (Fig.4A). Subsequently, RT-qPCR was utilized to detect theexpression levels of miR-3171 in CRC cell lines and HIEC cells. Itwas identified that miR-3171 expression was markedly upregulated inCRC cell lines compared with in the HIEC cell line, particularly inHCT116 cells (Fig. 4B). The miR-3171level was markedly elevated following transfection with miR-3171mimic (Fig. 4C). The dual-luciferasereporter assay demonstrated the binding of miR-3171 and OTUD6B-AS1,since the miR-3171 mimic + OTUD6B-AS1 WT group exhibited lowerluciferase activity compared with the mimic-NC + OTUD6B-AS1 WTgroup (Fig. 4D). As expected, theresults of the RT-qPCR assay (Fig.4E) indicated that miR-3171 expression was markedly reducedfollowing OTUD6B-AS1 overexpression compared with that in the OE-NCgroup. These results suggested that miR-3171 is a direct target ofOTUD6B-AS1.
To determine whether OTUD6B-AS1 can exert itseffects on proliferation, invasion and migration of HCT116 cells bytargeting miR-3171, a series of functional experiments wasperformed. As shown in Fig. 5A-C,co-transfection of OTUD6B-AS1 overexpression plasmid and miR-3171mimics reversed the inhibitory effects of OTUD6B-AS1 overexpressionalone on the proliferation, colony formation abilities and the Ki67expression of HCT116 cells. Furthermore, the addition of miR-3171mimics attenuated the effects of OTUD6B-AS1 overexpression on theinvasive and migratory abilities of HCT116 cells (Fig. 6A-D). Additionally, the expressionlevels of migration-associated proteins were elevated when theHCT116 cells with overexpression of OTUD6B-AS1 were co-transfectedwith miR-3171 mimic (Fig. 6E). Theseresults indicated that OTUD6B-AS1 inhibited the proliferation,invasion and migration of HCT116 cells by targeting miR-3171.
In conclusion, to the best of our knowledge, thepresent study was the first to investigate the role of OTUD6B-AS1in CRC cells, and to reveal that lncRNA OTUD6B-AS1 overexpressioninhibited the proliferation, invasion and migration of HCT116cells, at least partially, by targeting miR-3171. Therefore,OTUD6B-AS1 may serve as a potential novel biomarker and target forthe diagnosis and treatment of CRC. However, the use of only oneCRC cell line in the cell function experiments and mechanismexperiments, and lack of data obtained from clinical samples andinvestigations in animal models were limitations of the presentstudy. Therefore, a comprehensive analysis is required in thefuture. Additionally, the use of only one TCGA dataset toinvestigate OTUD6B-AS1 expression is another limitation of thepresent study, and future studies should improve upon this and theother limitations.
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There were 142 miRNAs with differing expression in the CSB compared to the PB of AF patients; among them, 6 miRNAs were upregulated and 8 were downregulated significantly, especially the miR-1266 increased by 5.55-, 8.41- and 4.70-fold, and miR-3171 decreased 0.43-, 0.47- and 0.32-fold in the ParoAF, PersAF and PermAF groups, respectively (Fig 2, Table 4).
The chip data of five candidate microRNAs (miR-1266, miR-4279, miR-892a, miR-3149, and miR-3171) were validated by using real-time PCR. The expression levels of these five miRNAs confirmed the previously observed significant upregulation or downregulation (Fig 4).
Compared to the peripheral blood of normal subjects, the levels of miR-3171 were lower in the CSB and PB of AF patients, indicating that it is mainly secreted by tissues other than the myocardium, and it may participate in the regulation of AF through coronary circulation. The continuous decrease of miR-3171 in patients with paroxysmal, persistent and permanent AF makes it possible for it to become a biomarker of this disease.
The differential expression of miRNAs in coronary circulation in Three Types AF Patients in our study indicated that: 1) the expression of miRNAs in the CSB may better reflect the real metabolism, expression and regulation status of miRNAs in patients with AF; 2) the levels of miRNA changes may reflect the severity of AF clinical and pathophysiological advance; and 3) the miR-3171, miR-892a and miR-3149, variably expressed from the early to the end stage of AF in PB, may be used as biomarkers for earlier diagnosis of AF; the miR-1266, miR-4279, and miR-4666a-3p, obviously increased in the CSB of AF patients, may serve as potential intervening targets for AF in the future.
Kunimi, H.; Miwa, Y.; Inoue, H.; Tsubota, K.; Kurihara, T. A Novel HIF Inhibitor Halofuginone Prevents Neurodegeneration in a Murine Model of Retinal Ischemia-Reperfusion. Int. J. Mol. Sci. 2019, 20, 3171.
Kunimi H, Miwa Y, Inoue H, Tsubota K, Kurihara T. A Novel HIF Inhibitor Halofuginone Prevents Neurodegeneration in a Murine Model of Retinal Ischemia-Reperfusion. International Journal of Molecular Sciences. 2019; 20(13):3171.
Kunimi, Hiromitsu, Yukihiro Miwa, Hiroyoshi Inoue, Kazuo Tsubota, and Toshihide Kurihara. 2019. "A Novel HIF Inhibitor Halofuginone Prevents Neurodegeneration in a Murine Model of Retinal Ischemia-Reperfusion" International Journal of Molecular Sciences 20, no. 13: 3171.
The Republic of Plato is one of the classic gateway texts into the study and practice of philosophy, and it is just the sort of book that has been able to arrest and redirect lives. How it has been able to do this, and whether or not it will be able to do this in your own case, is something you can only discover for yourself. The present guidebook aims to help a person get fairly deep, fairly quickly, into the project. It divides the dialogue into 96 sections and provides commentary on each section as well as questions for reflection and exploration. It is organized with a table of contents and is stitched together with a system of navigating bookmarks. Links to external sites such as the Perseus Classical Library are used throughout. This book is suitable for college courses or independent study.
Wheeler PG, Ng BG, Sanford L, Sutton VR, Bartholomew DW, Pastore MT, Bamshad MJ, Kircher M, Buckingham KJ, Nickerson DA, Shendure J, Freeze HH. SRD5A3-CDG: expanding the phenotype of a congenital disorder of glycosylation with emphasis on adult-onset features. Am J Med Genet A 2016;170:3165-3171. 041b061a72