ABSTRACT
PURPOSE: Circular RNAs (circRNAs) play essential roles in the progression of human tumors, including retinoblastoma (RB). In this study, we aimed to explore the functions and potential mechanisms of circ_0000527 in RB. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR), Western blot assay and immunohistochemistry (IHC) assay were conducted to determine the levels of circ_0000527, microRNA-1236-3p (miR-1236-3p) and SMAD family member 2 (SMAD2). RNase R assay and actinomycin D assay were conducted to analyze the characteristic of circ_0000527. Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, and colony formation assay were performed for cell proliferation ability. Wound healing assay and transwell assay were applied to assess cell migration and invasion. Tube formation assay was utilized for angiogenesis ability. Flow cytometry analysis was adopted to analyze cell apoptosis. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to analyze the relationships among circ_0000527, miR-1236-3p, and SMAD2. Murine xenograft model assay was conducted for the role of circ_0000527 in vivo. RESULTS: Circ_0000527 was overexpressed in RB patients and related to advanced TNM stages, optic nerve invasion and choroidal invasion. Circ_0000527 knockdown suppressed cell proliferation, migration, invasion and angiogenesis and promoted apoptosis in RB cells in vitro. Circ_0000527 sponged miR-1236-3p, which directly targeted SMAD2. MiR-1236-3p level was decreased in RB tissues and cells. MiR-1236-3p inhibition reversed circ_0000527 knockdown-mediated effects on RB cell malignant behaviors. Moreover, miR-1236-3p overexpression suppressed RB cell progression, with SMAD2 elevation abrogated the effect. Additionally, circ_0000527 knockdown restrained tumor formation in vivo. CONCLUSIONS: Circ_0000527/miR-1236-3p/SMAD2 axis played a positive role in the progression of RB.
Subject(s)
MicroRNAs , Retinal Neoplasms , Retinoblastoma , Animals , Cell Proliferation , Humans , Mice , MicroRNAs/genetics , Neovascularization, Pathologic , RNA, Circular/genetics , Retinal Neoplasms/genetics , Retinal Neoplasms/pathology , Retinoblastoma/genetics , Retinoblastoma/pathology , Smad2 Protein/genetics , Smad2 Protein/metabolismABSTRACT
AIM: To develop a new method to produce recombinant reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, in soluble format with low cost for the generation of induced pluripotent stem cells (iPSCs). METHODS: A short polypeptide sequence derived from the HIV trans-activator of transcription protein (TAT) and the nucleus localization signal (NLS) polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into pCold-SUMO vector which can extremely improve the solubility of recombinant proteins. Then these vector plasmids were transformed into E. coli BL21 (DE3) Chaperone competent cells for amplification. The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining. The recombinant proteins were purified by Ni-NTA resin and identified by Western blot. The transduction of these proteins into HEK 293T cells were evaluated by immunofluorescence staining. RESULTS: These four reprogramming proteins could be produced in soluble format in pCold-SUMO expression vector system with the assistance of chaperone proteins in bacteria. The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells. CONCLUSION: The results in the present study indicate the four important reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, can be produced in soluble format in bacteria with low cost. Our new method thus might be expected to greatly contribute to the future study of iPSCs.
ABSTRACT
MicroRNAs (miRNAs) regulate gene expression by targeted repression of transcription and translation, and are involved in carcinogenesis. In this study, we demonstrated that the expression of miR-454 was up-regulated in uveal melanoma tissues compared to normal tissues. Ectopic expression of miR-454 resulted in significant promotion of cell proliferation, colony formation, invasion and induction of cell cycle in uveal melanoma cells. Furthermore, we identified PTEN as a direct target of miR-454. Our data revealed that ectopic expression of PTEN restored the effects of miR-454 on cell proliferation and invasion in uveal melanoma cells. These findings support an oncogene role of miR-454 in development of uveal melanoma.
Subject(s)
Melanoma/genetics , Melanoma/metabolism , MicroRNAs/genetics , Oncogenes , PTEN Phosphohydrolase/genetics , Uveal Neoplasms/genetics , Uveal Neoplasms/metabolism , Base Sequence , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation , Humans , Melanoma/pathology , Neoplasm Invasiveness , PTEN Phosphohydrolase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/metabolism , Up-Regulation/genetics , Uveal Neoplasms/pathologyABSTRACT
MicroRNAs (miRNAs) are a group endogenous small non-coding RNAs that inhibit protein translation through binding to specific target mRNAs. Recent studies have demonstrated that miRNAs are implicated in the development of cancer. However, the role of miR-144 in uveal melanoma metastasis remains largely unknown. MiR-144 was downregulated in both uveal melanoma cells and tissues. Transfection of miR-144 mimic into uveal melanoma cells led to a decrease in cell growth and invasion. After identification of two putative miR-144 binding sites within the 3' UTR of the human c-Met mRNA, miR-144 was proved to inhibit the luciferase activity inMUM-2B cells with a luciferase reporter construct containing the binding sites. In addition, the expression of c-Met protein was inhibited by miR-144. Furthermore, c-Met-mediated cell proliferation and invasion were inhibited by restoration of miR-144 in uveal melanoma cells. In conclusion, miR-144 acts as a tumor suppressor in uveal melanoma, through inhibiting cell proliferation and migration. miR-144 might serve as a potential therapeutic target in uveal melanoma patients.
Subject(s)
Gene Expression Regulation, Neoplastic , Melanoma/genetics , MicroRNAs/genetics , Proto-Oncogene Proteins c-met/genetics , RNA, Messenger/genetics , Uveal Neoplasms/genetics , 3' Untranslated Regions , Base Sequence , Binding Sites , Cell Line, Tumor , Cell Movement , Cell Proliferation , Down-Regulation , Gene Expression , Humans , Melanoma/pathology , RNA Interference , Uveal Neoplasms/pathologyABSTRACT
The present study was designed to investigate the effect of vanadium in alloxan-induced diabetes and cataract in rats. Different doses of vanadium was administered once daily for 8 weeks to alloxan-induced diabetic rats. To know the mechanism of action of vanadium, lens malondialdehyde (MDA), protein carbonyl content, activity of superoxide dismutase (SOD), activities of aldose reductase (AR), and sorbitol levels were assayed, respectively. Supplementation of vanadium to alloxan-induced diabetic rats decreased the blood glucose levels due to hyperglycemia, inhibited the AR activity, and delayed cataract progression in a dose-dependent manner. The observed beneficial effects may be attributed to polyol pathway activation but not decreased oxidative stress. Overall, the results of this study demonstrate that vanadium could effectively reduce the alloxan-induced hyperglycemia and diabetic cataracts in rats.
