GRIM19 is involved in WT1 expression and epithelial-to-mesenchymal transition in adenomyotic lesions

The epithelial-to-mesenchymal transition may play a role in adenomyosis. GRIM19 expression is downregulated in adenomyotic lesions, and the effects of this downregulation in adenomyosis remain relatively unclear. We aimed to explore whether aberrant GRIM19 expression is associated with the epithelial-to-mesenchymal transition in adenomyosis. In this study, expression of both GRIM19 and WT1 was low, and epithelial-to-mesenchymal transition, which included significant changes in CDH1, CDH2 and KRT8 expression, occurred in adenomyotic lesions, as confirmed by western blotting and quantitative real-time polymerase chain reaction. We provided novel insights into WT1 expression in adenomyosis, revealing that WT1 expression was increased in the endometrial glands of adenomyotic lesions by immunohistochemistry. In vitro, knockdown of GRIM19 expression by small-interfering RNA promoted the proliferation, migration and invasion of Ishikawa cells, as measured by Cell Counting Kit-8, wound healing assay and Transwell assays. Western blotting and quantitative real-time polymerase chain reaction confirmed that WT1 expression increased and epithelial-to-mesenchymal transition was induced, including upregulation of CDH2 and downregulation of CDH1 and KRT8 after transfecting the GRIM19 small interfering RNA to Ishikawa cells. Furthermore, WT1 expression was upregulated and epithelial-to-mesenchymal transition was observed, including downregulation of CDH1 and KRT8 in GRIM19 gene-knockdown mice. Upregulation of WT1 expression in the endometrial glands of GRIM19 knockdown mice was also verified by immunohistochemistry. Taken together, these results reveal that low expression of GRIM19 in adenomyosis may upregulate WT1 expression and induce epithelial-to-mesenchymal transition in the endometria, providing new insights into the pathogenesis of adenomyosis.

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Long Noncoding RNA Tug1 Promotes Angiotensin II–Induced Renal Fibrosis by Binding to Mineralocorticoid Receptor and Negatively Regulating MicroR-29b-3p

Hypertension ◽

10.1161/hypertensionaha.120.16395 ◽

2021 ◽

Author(s):

Juhong Zhang ◽

Yuqing Zhang ◽

Jing Gao ◽

Meihui Wang ◽

Xiaoting Li ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Long Noncoding Rna ◽

Mineralocorticoid Receptor ◽

Renal Fibrosis ◽

Noncoding Rna ◽

Epithelial To Mesenchymal Transition ◽

Chain Reaction ◽

Mesenchymal Transition ◽

Polymerase Chain

Inappropriately activation of renin-angiotensin-aldosterone system induced renal fibrosis is characterized by partial epithelial-to-mesenchymal transition. Previously, we have indicated that miR-29b-3p in inhibiting partial epithelial-to-mesenchymal transition by negatively regulating extracellular matrix gene expression in the kidney. Despite the critical role of miR-29b-3p in fibrosis, the molecular mechanisms by which miR-29b-3p is regulated under the condition of profibrotic stimuli are largely unknown. Our aim is to search for the long noncoding RNA that mediated sponge regulatory on miR-29b-3p, and whether the long noncoding RNA could be activated by renin-angiotensin-aldosterone system and its consequent effects on renal fibrosis. Bioinformatics analysis predicted that Tug1 might directly bound to miR-29b-3p and function as a competing endogenous RNA. Dual-luciferase reporter assay, fluorescence in situ hybridization, and real-time polymerase chain reaction were performed to indicate that Tug1 interact with miR-29b-3p in a sequence-specific manner. Decreased Tug1 led to an increase in extracellular matrix measured by Western blot, and this effect was enhanced by miR-29b-3p measured by real-time polymerase chain reaction and Western blot, suggesting cross-regulation between the RNAs. Bioinformatics analysis predicted that mineralocorticoid receptor might bind to long noncoding RNA Tug1 . Notably, mineralocorticoid receptor antagonism reduced Tug1 expression in the presence or absence of angiotensin II or aldosterone measured by real-time polymerase chain reaction, and RNA immunoprecipitation assays confirmed that the mineralocorticoid receptor directly bound to Tug1 . Finally, we confirmed that Tug1 expression was enhanced in fibrotic compared with nonfibrotic human renal biopsy samples using RNA-in situ hybridization. Our findings provide novel insights into the molecular mechanism of Ang II–induced renal fibrosis and identify the Tug1 –miR-29b-3p axis as an important target of MR activation.

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Real-time quantification of antibody–short interfering RNA conjugate in serum by antigen capture reverse transcription–polymerase chain reaction

Analytical Biochemistry ◽

10.1016/j.ab.2012.08.018 ◽

2012 ◽

Vol 430 (2) ◽

pp. 171-178 ◽

Cited By ~ 8

Author(s):

Martha Tan ◽

Jean-Michel Vernes ◽

Joyce Chan ◽

Trinna L. Cuellar ◽

Aarati Asundi ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Reverse Transcription ◽

Short Interfering Rna ◽

Chain Reaction ◽

Antigen Capture ◽

Polymerase Chain ◽

Interfering Rna

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Long non-coding RNA TIRY promotes tumor metastasis by enhancing epithelial-to-mesenchymal transition in oral cancer

Experimental Biology and Medicine ◽

10.1177/1535370220903673 ◽

2020 ◽

Vol 245 (7) ◽

pp. 585-596 ◽

Cited By ~ 4

Author(s):

Nuo Jin ◽

Nianqiang Jin ◽

Wenhuan Bu ◽

Xing Li ◽

Lili Liu ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Epithelial To Mesenchymal Transition ◽

Tumor Biology ◽

Progression Free Survival ◽

Cancer Associated Fibroblasts ◽

Chain Reaction ◽

Free Survival ◽

Mesenchymal Transition ◽

Polymerase Chain

Long non-coding RNAs (lncRNAs) modulate a variety of cancerous biological processes, including the promotion of tumorigenicity in tumor parenchymal cells. However, there is a lack of studies assessing the regulation of lncRNAs in cancer-associated fibroblasts. In the present study, a novel lncRNA, TIRY, was found to act as a miRNA sponge and to downregulate miR-14 expression in oral squamous cell carcinoma (OSCC). Fluorescence in situ hybridization assay was used to evaluate TIRY expression in OSCC tissues. Survival analysis in a prospective cohort revealed a correlation between high TIRY expression and short progression-free survival. Subsequently, TIRY expression in cancer-associated fibroblasts and primary fibroblasts from adjacent normal (para-carcinoma) tissues was assessed using quantitative reverse transcription polymerase chain reaction. TIRY overexpression in cancer-associated fibroblasts isolated from OSCC tissues was induced by overexpressing the TIRY plasmid, and candidate microRNA expressions were assessed using quantitative real-time polymerase chain reaction. Moreover, the expression of proteins related to epithelial-to-mesenchymal transition (EMT) was determined; the proliferation, metastasis, and invasion of cancer cells co-cultured with TIRY-overexpressing cancer-associated fibroblasts were determined. We found significantly decreased miR-14 expression in cancer-associated fibroblast-derived exosomes and increased expression of EMT markers including transcription factors (Snail and FOXC2) and cellular scaffolding proteins (α-SMA, β-catenin, and FSP1). TIRY overexpression in cancer-associated fibroblasts activated the Wnt/β-catenin signaling pathway and promoted the invasion and metastasis of OSCC cells through miR-14 sponging based on cancer-associated exosome secretion. Our findings provide a novel molecular mechanism underlying the role of TIRY in cancer-associated fibroblasts in tumor biology; moreover, TIRY is a potential therapeutic target in OSCC. Impact statement This study demonstrated the novel lncRNA, TIRY, enhances epithelial-to-mesenchymal transition in cancer-associated fibroblasts and promotes the metastasis of tumor via miR-14 sponging in oral squamous cell carcinoma, and thus provide a novel molecular mechanism underlying the role of TIRY in CAFs in tumor biology and a potential target in OSCC. Further, the data showed that TIRY expression was negatively correlated with miR-14 transcription levels and was associated with poor prognosis in OSCC specimens. Therefore, TIRY may be a potential prognostic biomarker of overall survival and progression-free survival in OSCC. Moreover, TIRY adds to the understanding of regulatory mechanisms involved in CAFs and epithelial cancer cells in OSCC and may provide novel insights for further understanding tumor biology.

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Resveratrol Relieves Hyperoxia-Induced Brain Injury in Neonatal Rats by Activating Sirt1

American Journal of Perinatology ◽

10.1055/s-0040-1710352 ◽

2020 ◽

Author(s):

Lan Kang ◽

Wenbin Dong ◽

Xiaobin Li ◽

Ying Ruan ◽

Rong Zhang

Keyword(s):

Polymerase Chain Reaction ◽

Brain Injury ◽

Real Time ◽

Western Blotting ◽

P53 Protein ◽

Tunel Staining ◽

Neonatal Rats ◽

Chain Reaction ◽

P53 Expression ◽

Polymerase Chain

Abstract Objective Neonatal rats with hyperoxia-induced brain injury were treated with resveratrol to investigate its protective effects through analyzing changes in reactive oxygen species (ROS), Sirt1, p53, and acetylated p53 levels. Study Design Neonatal rats were randomly divided into hyperoxia and resveratrol intervened groups. Rats in both groups were placed in a hyperoxia chamber for 7 days to induce hyperoxia-induced brain injury. The rats in the resveratrol intervened group were administered resveratrol 60 μg/g body weight daily, whereas those in the hyperoxia group were administered a dimethyl sulfoxide-based solvent. Brain tissues were collected, and hematoxylin and eosin (H&E) and TUNEL staining, ROS measurements, real time-polymerase chain reaction, and western blotting were performed. Results H&E and TUNEL staining revealed increased cell damage and apoptosis in brain tissue from hyperoxia-exposed animals compared with the findings in animals in the resveratrol intervened group. Real time-polymerase chain reaction and western blotting identified increases in Sirt1 expression and decreases in p53 expression in the resveratrol intervened group. In addition, acetylated p53 protein expression was lower in the intervened group than in the hyperoxia group. Conclusion Resveratrol alleviated brain apoptosis induced by hyperoxia in neonatal rats by upregulating Sirt1-mediated pathways, suggesting its potentially beneficial role in the treatment of brain injury induced by hyperoxia.

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Quantitation of full-size small interfering RNA by tailing with terminal deoxynucleotidyl transferase and reverse transcription-polymerase chain reaction analysis

Analytical Biochemistry ◽

10.1016/j.ab.2008.11.037 ◽

2009 ◽

Vol 385 (2) ◽

pp. 386-388 ◽

Cited By ~ 1

Author(s):

Kazunobu Futami ◽

Yasuhiro Furuichi

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Small Interfering Rna ◽

Polymerase Chain Reaction Analysis ◽

Terminal Deoxynucleotidyl Transferase ◽

Chain Reaction ◽

Full Size ◽

Polymerase Chain ◽

Interfering Rna

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Inhibition of Matrix Metalloproteinase 9 Expression in Rat Dermal Fibroblasts Using Small Interfering RNA

Journal of the American Podiatric Medical Association ◽

10.7547/1020299 ◽

2012 ◽

Vol 102 (4) ◽

pp. 299-308 ◽

Cited By ~ 5

Author(s):

Xiao-Ying Xie ◽

Chuan Yang ◽

Meng Ren ◽

Shao-Yun Hao ◽

Ping Zhu ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Small Interfering Rna ◽

Dermal Fibroblasts ◽

Diabetic Rat ◽

Rat Skin ◽

Chain Reaction ◽

Polymerase Chain ◽

Interfering Rna

Background: Matrix metalloproteinases (MMPs) degrade extracellular matrix components. Increased MMP-9 content in diabetic skin contributes to skin vulnerability and refractory foot ulcers. To identify ways to decrease MMP-9 levels in skin, inhibition of MMP-9 expression in dermal fibroblasts using small interfering RNA was investigated in vitro. Methods: A full-thickness wound was created on the midback of streptozotocin-induced diabetic rats; skin biopsies were performed 3 days later. Skin MMP-9 expression was observed by immunohistochemical analysis. Dermal fibroblasts from 1-day-old normal Sprague Dawley rats cultured with high glucose and homocysteine concentrations were transfected with small interfering RNA complexes. Cells were collected 30, 48, and 72 hours after transfection, and reverse transcription–polymerase chain reaction, Western blot analysis, and gelatin zymography for MMP-9 were performed. Results: Expression of MMP-9 was increased in diabetic rat skin, especially around wounds. After 30-, 48-, and 72-hour transfection with each MMP-9–specific small interfering RNA, reverse transcription–polymerase chain reaction showed markedly decreased MMP-9 messenger RNA expression, protein abundance, and activity. Of four MMP-9 small interfering RNAs, one sequence had a stable high inhibition rate (>70% at 30 and 48 hours after transfection). Conclusions: Expression of MMP-9 was increased in diabetic rat skin, especially around wounds, and was markedly inhibited after MMP-9 small interfering RNA transfection in vitro (P < .05). These findings may provide new treatments for diabetic skin wounds. (J Am Podiatr Med Assoc 102(4): 299–308, 2012)

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