scholarly journals Hyperbranched Cationic Glycogen Derivative-Mediated IκBα Gene Silencing Regulates the Uveoscleral Outflow Pathway in Rats

2020 ◽  
Vol 2020 ◽  
pp. 1-17
Author(s):  
Rui Zeng ◽  
Jinmiao Li ◽  
Haijun Gong ◽  
Jiahao Luo ◽  
Zijing Li ◽  
...  
Keyword(s):  
Protein Expression ◽  
Gene Silencing ◽  
Nuclear Translocation ◽  
Gene Transfection ◽  
Gelatin Zymography ◽  
Ciliary Muscle ◽  
Uveoscleral Outflow ◽  
Mrna And Protein Expression ◽  
Outflow Pathway ◽  
Sirna Group

The role of the IκB/NF-κB signaling pathway in the uveoscleral outflow pathway was investigated with IκBα gene silencing mediated by the 3-(dimethylamino)-1-propylamine-conjugated glycogen (DMAPA-Glyp) derivative. The IκBα-siRNA-loaded DMAPA-Glyp complex was transfected into the ciliary muscles of rats by intracameral injection (labeled as the DMAPA-Glyp+siRNA group). The Lipofectamine™ 2000 (Lipo)/siRNA complex and the naked siRNA were set as the controls. The mRNA and protein expression of IκBα, NF-κBp65, and MMP-2 were analyzed by real-time PCR, western blotting, and in situ gelatin zymography. Nuclear translocation of NF-κBp65 was analyzed by immunofluorescence. Rat intraocular pressure (IOP) was monitored pre- and postinjection. Gene transfection efficiency and toxicity of the DMAPA-Glyp derivative were also evaluated. After RNA interference (RNAi), IκBα mRNA and protein expression were significantly inhibited. NF-κBp65 mRNA and protein expression showed no significant differences. Nevertheless, nuclear translocation of NF-κBp65 occurred in the DMAPA-Glyp+siRNA group. Both mRNA expression and activity of MMP-2 increased, with the largest increase in the DMAPA-Glyp+siRNA group. IOP in the DMAPA-Glyp+siRNA group fell to the lowest level on day 3 after RNAi. The levels of Cy3-siRNA in the ciliary muscle of the DMAPA-Glyp+siRNA group did not significantly decrease over time. At 7 and 14 d after RNAi, no significant pathological damage was detectable in the eyes injected with the DMAPA-Glyp derivative or the DMAPA-Glyp/siRNA complex. Taken together, our results suggest that downregulation of IκBα expression in the ciliary muscle plays a crucial role in reducing the IOP values of rats. IκBα may become a new molecular target for lowering IOP in glaucoma. The DMAPA-Glyp derivative is safe and feasible as an effective siRNA vector in rat eyes.

scholarly journals Effects of PKM2 Gene Silencing on the Proliferation and Apoptosis of Colorectal Cancer LS-147T and SW620 Cells

2017 ◽  
Vol 42 (5) ◽  
pp. 1769-1778 ◽  
Author(s):  
Ran Ao ◽  
Lin Guan ◽  
Ying Wang ◽  
Jia-Ni Wang
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Gene Silencing ◽  
Cell Counting ◽  
Qrt Pcr ◽  
Empty Plasmid ◽  
Proliferation And Apoptosis ◽  
Mrna And Protein Expression ◽  
Sw620 Cells

Background/Aims: This paper aims to explore the effects of pyruvate kinase (PK) M2 gene silencing on the proliferation and apoptosis of colorectal cancer (CRC) LS-147T and SW620 cells. Methods: CRC LS-147T and SW620 cells highly expressing PKM2 were randomly selected by quantitative real-time polymerase chain reaction (qRT-PCR) and then assigned into the blank (no transfection), PKM2-shRNA (transfection with shRNA) and empty plasmid (transfection with empty plasmid) groups. Immunofluorescence was applied to detect PKM2 protein expression. qRT-PCR and Western blotting were conducted to assess mRNA and protein expression of PKM2, p53 and p21. The cell counting kit-8 (CCK-8) assay was used to assess cell proliferation. Flow cytometry was used to assess the cell cycle and apoptosis rate, and a senescence-associated β-galactosidase staining kit was used to assess cell senescence. Results: PKM2 exhibited high mRNA expression among CRC LS-147T and SW620 cells with remarkable protein expression noted in the cytoplasm and nucleus. The PKM2-shRNA group exhibited reduced PKM2 mRNA and protein expression, whereas p53 and p21 expression was increased compared with the blank and empty plasmid groups. Cell proliferation in PKM2-shRNA cells decreased significantly compared with the blank group and empty plasmid groups. The PKM2-shRNA group exhibited more cells in the G1 phase and fewer cells in the G2/M phase compared with the blank and empty plasmid groups. In addition, the PKM2-shRNA group exhibited significantly increased apoptosis rates and β-galactosidase activity compared with the blank and empty plasmid groups. Conclusion: Our study demonstrates that PKM2 gene silencing suppresses proliferation and promotes apoptosis in LS-147T and SW620 cells.

scholarly journals The effect of Sep15 gene silencing on the expression of crystall oid protein in human lens epithelial cells

2020 ◽  
Vol 185 ◽  
pp. 03005
Author(s):  
Meng Wang
Keyword(s):  
Endoplasmic Reticulum ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Gene Silencing ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Lens Protein ◽  
Expression Levels ◽  
Human Lens Epithelial Cells ◽  
Mrna And Protein Expression

Objective: To investigate the effect of Sep15 gene silencing on lens protein expression in human lens epithelial cells. Method: Human lens epithelial cell (HLEC) SRA01/04 was used as the study object, Using MTT method, RT-PCRs, Small molecule RNA interference technology and protein immunoblotting method, The survival rate of hLE cells stimulated by different concentrations of bFGF and Tm (tunicamycin, an endoplasmic reticulum stress inducing reagent), the changes of the mRNA and protein expression levels of intracellular beta-crystallin and GRP78 proteins, and the changes of Sep15 protein expression levels in hLE cells were determined, respectively, to evaluate the effect of Sep15 gene silencing on the expression of lens protein in human lens epithelial cells. Result: First, The survival rate of hLE cells induced by endoplasmic reticulum stress inducing reagents tunicamycin (Tm) and bFGF was investigated by MTT method. The results showed that a certain concentration of Tm and bFGF could inhibit or lead to the death of hLE cells; Secondly, The effects of Tm on the levels of mRNA and protein expression of the endoplasmic reticulum stress marker protein GRP78 and the effects of bFGF on the levels of alpha-, were detected by real-time fluorescence quantitative PCR and protein immunoblotting, respectively, Effects of beta-crystallin mRNA and protein expression levels, To determine the optimal concentration and time of Tm and bFGF on hLE cells, The results showed that 40 ng/mL of bFGF-treated cells for 48 h was the optimal reaction condition for bFGF to stimulate the differentiation of lens epithelial cells. Third, The co-action of Tm and bFGF on hLE cells was detected by real-time fluorescence quantitative PCR and protein immunoblotting. For Intracellular GRP78, α-, β- crystallin, Effects of mRNA and protein expression levels, The results showed that the occurrence of ER stress could upregulate its expression. Fourth, The changes of Sep15 protein expression level in hLE cells after Sep15 gene silencing and the effects of Tm and bFGF on Sep15 gene silencing in cells were detected by Western blotting. The results showed that the addition of Tm and bFGF basically did not affect the silencing effect of Sep15 gene. Fifth, The expression of GRP78 and β- crystallin in hLE cells after silencing of Sep15 gene was detected by Western blot. The results showed that Sep15 may be involved in the process of protecting the differentiation of lens epithelial cells. Conclusion: Sep15 gene silencing has an inhibitory effect on lens protein expression in human lens epithelial cells, and strengthening its clinical research is of positive significance for improving the clinical treatment and prevention of cataract diseases.

scholarly journals Interleukin-12-mediated expression of matrix metalloproteinases in human periodontal ligament fibroblasts involves in NF-κB activation

2017 ◽  
Vol 37 (6) ◽  
Author(s):  
Li Miao ◽  
Shujun Zhan ◽  
Jiyan Liu
Keyword(s):  
Matrix Metalloproteinases ◽  
Protein Expression ◽  
Periodontal Ligament ◽  
Nuclear Translocation ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Interleukin 12 ◽  
Periodontal Ligament Fibroblasts ◽  
Protein Levels ◽  
Mrna And Protein Expression ◽  
Human Periodontal Ligament Fibroblasts

Interleukin-12 (IL-12) is a proinflammatory cytokine, and its increased level correlates with the severity of periodontitis. However, its role in the pathogenesis of tooth periapical lesions is controversial and has not been completely clarified. The present study aimed to investigate whether IL-12 affects the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human periodontal ligament fibroblasts (hPDLFs). After treatment with IL-12 for different times, real-time PCR and Western blotting were used to determine the mRNA and protein levels of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, and TIMP-2, respectively. ELISA was applied to measure MMPs and TIMPs secretion production. The results indicated that IL-12 significantly increased the mRNA and protein expression levels of MMP-1, MMP-3, and MMP-13, but down-regulated MMP-2 and MMP-9 mRNA and protein expression in the hPDLFs. Furthermore, IL-12 (10 ng/ml) enhanced the secreted protein production of MMP-1, MMP-3, and MMP-13, and conversely lowered MMP-2 and MMP-9 secretion levels. However, IL-12 treatment did not exert a significant effect on the mRNA and protein levels of TIMP-1 and TIMP-2 and their secreted production. Additionally, IL-12 increased the phosphorylated levels of IκBα and nuclear factor-κB P65 (NF-κB P65), and promoted NF-κB P65 subunit nuclear translocation. Pretreatment with NF-κB inhibitor not only attenuated IL-12-induced IκBα and NF-κB P65 phosphorylation and inhibited NF-κB P65 subunit into nucleus, but also antagonized IL-12-mediated MMP-1, MMP-2, MMP-3, MMP-9, and MMP-13 expression in the hPDLFs. These findings indicate that NF-κB-dependent activation is possibly indispensable for IL-12-mediated MMP expression in hPDLFs.

Bone sialoprotein mRNA and protein expression in human multiple myeloma cell lines and patients

2000 ◽  
Vol 111 (4) ◽  
pp. 1118-1121 ◽  
Author(s):  
A. Bellahcene ◽  
I. Van Riet ◽  
C. de Greef ◽  
N. Antoine ◽  
M. F. Young ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Expression ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Bone Sialoprotein ◽  
Multiple Myeloma Cell ◽  
Human Multiple Myeloma ◽  
Mrna And Protein Expression

Changes in mRNA and Protein Expression of Antioxidant Enzymes in Leukocytes of Children with Bronchial Asthma after Interval Hypoxic Training

2012 ◽  
Vol 3 (2) ◽  
pp. 125-134
Author(s):  
Klaudia V. Nesvitaylova ◽  
Olga A. Gonchar ◽  
Tatyana I. Drevitskaya ◽  
Ludmila P. Arabskaya ◽  
Mikhail M. Steshenko ◽  
...  
Keyword(s):  
Antioxidant Enzymes ◽  
Protein Expression ◽  
Bronchial Asthma ◽  
Hypoxic Training ◽  
Mrna And Protein Expression

Surgical resection of the sclera is a non-penetrating operation for the treatment of patients with primary open-angle and secondary glaucoma

GlaucomaNews ◽  
2020 ◽  
pp. 62-65
Author(s):  
А.Y. Kazantseva ◽  
◽  
O.A. Rumyantseva ◽  
Keyword(s):  
Surgical Resection ◽  
Primary Open Angle Glaucoma ◽  
Open Angle Glaucoma ◽  
Secondary Glaucoma ◽  
Ophthalmological Examination ◽  
Open Angle ◽  
Visual Functions ◽  
Dynamic Observation ◽  
Uveoscleral Outflow ◽  
Outflow Pathway

Purpose. To evaluate the effectiveness of surgical resection of the sclera in patients with primary open-angle and secondary glaucoma. Materials and methods. The study included 84 patients with POAG and SG stages III-IV and decompensated IOP level (not higher than 32 mm Hg). In order to normalize the increased ophthalmotonus, a non - penetrating operation was performed-surgical resection of the sclera (SRS). The patients underwent complex ophthalmological examination and dynamic observation. Result. In the studied groups of patients after surgical treatment there was a decrease in elevated IOP levels by 33.42%, an improvement in the coefficient of ease of outflow and a weakening of the hypotensive regime. Stabilization of visual functions was observed in all patients. Summary. The proposed new SRS technique provides a smooth decrease in IOP, preservation of visual functions and is not accompanied by intra-and postoperative complications. Key words: primary open-angle glaucoma, surgical resection of sclera, secondary glaucoma, uveoscleral outflow pathway (USPO), intraocular pressure, EO coefficient .

Icariin Stimulates hFOB 1.19 Osteoblast Proliferation and Differentiation via OPG/RANKL Mediated by the Estrogen Receptor

2020 ◽  
Vol 22 (1) ◽  
pp. 168-175 ◽  
Author(s):  
Lin-Jun Sun ◽  
Chong Li ◽  
Xiang-hao Wen ◽  
Lu Guo ◽  
Zi-Fen Guo ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Estrogen Receptor ◽  
Protein Expression ◽  
Chinese Herb ◽  
Human Osteoblast ◽  
Osteoblast Cells ◽  
Expression Ratio ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Mrna And Protein Expression

Background:: Icariin (ICA), one of the main effective components isolated from the traditional Chinese herb Epimedium brevicornu Maxim., has been reported to possess extensive pharmacological actions, including enhanced sexual function, immune regulation, anti-inflammation, and antiosteoporosis. Methods:: Our study was designed to investigate the effect of ICA on cell proliferation and differentiation and the molecular mechanism of OPG/RANKL mediated by the Estrogen Receptor (ER) in hFOB1.19 human osteoblast cells. Results:: The experimental results show that ICA can stimulate cell proliferation and increase the activity of Alkaline Phosphatase (ALP), Osteocalcin (BGP) and I Collagen (Col I) and a number of calcified nodules. Furthermore, the mRNA and protein expression of OPG and RANKL and the OPG/ RANKL mRNA and protein expression ratios were upregulated by ICA. The above-mentioned results indicated that the optimal concentration of ICA for stimulating osteogenesis was 50ng/mL. Subsequent mechanistic studies comparing 50ng/mL ICA with an estrogen receptor antagonist demonstrated that the effect of the upregulated expression is connected with the estrogen receptor. In conclusion, ICA can regulate bone formation by promoting cell proliferation and differentiation and upregulating the OPG/RANKL expression ratio by the ER in hFOB1.19 human osteoblast cells.

Sign in / Sign up

Export Citation Format

Share Document