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

2012 ◽  
Vol 102 (4) ◽  
pp. 299-308 ◽  
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)

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

Reproduction ◽  
2021 ◽  
Author(s):  
Chen Geng ◽  
Hao-ran Liu ◽  
Yue Zhao ◽  
Yang Yang ◽  
Lan Chao
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Western Blotting ◽  
Small Interfering Rna ◽  
Epithelial To Mesenchymal Transition ◽  
Chain Reaction ◽  
Mesenchymal Transition ◽  
Ishikawa Cells ◽  
Polymerase Chain ◽  
Interfering Rna

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.

scholarly journals The knockdown of c-myc expression by RNAi inhibits cell proliferation in human colon cancer HT-29 cells in vitro and in vivo

2009 ◽  
Vol 14 (2) ◽  
Author(s):  
Xiao Zhang ◽  
Yin-Lin Ge ◽  
Run-Hua Tian
Keyword(s):  
Colon Cancer ◽  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Human Colon ◽  
Chain Reaction ◽  
Human Colon Cancer ◽  
Polymerase Chain ◽  
Ht 29

AbstractWe investigated the effects of RNA interference-mediated silencing of the c-myc gene on celluar proliferation and apoptosis in human colon cancer HT-29 cells in vitro and in vivo. A small interfering RNA (siRNA) targeting c-myc was designed, the DNA template was synthesized, and the siRNA was obtained by in vitro transcription. After siRNA transfection into HT-29 and human neuroblastoma IMR-32 cells with Lipofectamine 2000™, the proliferation of the HT-29 and IMR-32 cells was assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and Hoechst 33258 staining was used to observe cell apoptosis. Following gene transfer to HT-29 cells, the expression of c-myc mRNA was examined via reverse transcription polymerase chain reaction, and the level of the protein via Western blot assay. Growth curves were constructed and in vivo experiments were performed on nude mice to assess the effects of c-myc silencing on tumor growth. The c-myc expression in the tumor tissue was measured by reverse transcription polymerase chain reaction and subsequently by immunohistochemistry. Our paper demonstrates that the delivery of siRNA directed against c-myc not only efficiently down-regulated the expression of c-myc, inhibited the proliferation of HT-29 cells and induced apoptosis in vitro, but also suppressed the growth of colon cancer cells in vivo.

A TaqMan® Reverse Transcription Polymerase Chain Reaction (RT-PCR) In Vitro Potency Assay for Plasmid-based Vaccine Products

2008 ◽  
Vol 40 (1) ◽  
pp. 47-57 ◽  
Author(s):  
Rohit Mahajan ◽  
Beth Feher ◽  
Basil Jones ◽  
Doug Jones ◽  
Lana Marjerison ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Potency Assay ◽  
Polymerase Chain

A Microinjection Method for Infecting the Planthopper Sogatella furcifera (Hemiptera: Delphacidae) with the Southern Rice Black-Streaked Dwarf Virus

2019 ◽  
Vol 112 (4) ◽  
pp. 1541-1545
Author(s):  
Kui Hu ◽  
Lin Qiu ◽  
Yurong Zhang ◽  
Yu Du ◽  
Hualiang He ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Rice Plant ◽  
Dwarf Virus ◽  
Economic Damage ◽  
Sogatella Furcifera ◽  
Chain Reaction ◽  
Rice Plants ◽  
Polymerase Chain

Abstract The southern rice black-streaked dwarf virus (SRBSDV) causes significant economic damage to rice crops. This virus is transmitted to rice plants by the planthopper Sogatella furcifera (Horváth) in a persistent, circular, and propagative manner. Researchers currently lack suitable methods for assaying the activity of SRBSDV in vitro and preserving the virus all year. We used a microinjection method to directly inject SRBSDV extracts into the hemocoel of S. furcifera nymphs. SRBSDV was subsequently detected by Reverse Transcription-Polymerase Chain Reaction in more than 56.7% of the insects after 5 d and 60% of healthy rice plants fed by these insects also became SRBSDV infected. Moreover, injecting planthopper with an extract of SRBSDV-infected rice plant that had been frozen at −80°C for 220 d caused 63.3% to become viruliferous. These results indicate that SRBSDV can be successfully transmitted to S. furcifera by microinjection, and that extracts of SRBSDV-infected rice plants frozen at −80°C for 220 d still contain sufficient active SRBSDV to infect S. furcifera. We provide a novel way to preserve SRBSDV all year by injecting S. furcifera with the SRBSDV extract.

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