- Authors: Wang S. et al.
- Year: 2021
- Journal: Front Genet 12 615340
- Applications: in vitro / DNA, siRNA / INTERFERin, jetOPTIMUS
- Cell type: HT22
cDNA of GAS5 isoform 210 was cloned into pcDNA3.1(+) (Invitrogen) OE vector. pcDNA3.1(+) empty vector was used as a negative control. Cells were seeded in 12-well culture plates and transfected with an OE vector when cells had reached 60 to 80% confluences. Then, cells were transfected with 2-μg GAS5-iso210 pcDNA3.1(+) or empty pcDNA3.1(+) using jetOPTIMUS reagent (Polyplus, 117-01, France) at 1 of 500 final dilutions for 48 h. Small interfering RNA (siRNA) was used to knock down the expression of GAS5 non-coding RNA in HT22 cells and were synthesized by GenePharma. Cells were seeded in 12-well culture plates and transfected with siRNA when cells had reached 30 to 40% confluences. Then, cells were transfected with 10-nM GAS5 siRNA or NC siRNA using INTERFERin reagent (Polyplus, 409-10, France) at 1 of 250 final dilutions for 48 h.
Increasing studies show that long non-coding RNAs (lncRNAs) play essential roles in various fundamental biological processes. Long non-coding RNA growth arrest-specific transcript 5 (GAS5) showed differential expressions between young and old mouse brains in our previous RNA-Seq data, suggesting its potential role in senescence and brain aging. Examination using quantitative reverse transcription-polymerase chain reaction revealed that GAS5 had a significantly higher expression level in the old mouse brain hippocampus region than the young one. Cellular fractionation using hippocampus-derived HT22 cell line confirmed its nucleoplasm and cytoplasm subcellular localization. Overexpression or knockdown of GAS5 in HT22 cell line revealed that GAS5 inhibits cell cycle progression and promotes cell apoptosis. RNA-Seq analysis of GAS5-knockdown HT22 cells identified differentially expressed genes related to cell proliferation (e.g., DNA replication and nucleosome assembly biological processes). RNA pull-down assay using mouse brain hippocampus tissues showed that potential GAS5 interacting proteins could be enriched into several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and some of them are involved in senescence-associated diseases such as Parkinson's and Alzheimer's diseases. These results contribute to understand better the underlying functional network of GAS5 and its interacting proteins in senescence at brain tissue and brain-derived cell line levels. Our study may also provide a reference for developing diagnostic and clinic biomarkers of GAS5 in senescence and brain aging.