title: MicroRNA-363 and GATA-1 are regulated by HIF-1α in K562 cells under hypoxia.
abstract: The aim of the present study was to investigate regulatory relationships among hypoxia-inducible factor-1α (HIF-1α), microRNA and erythroid transcription factors. K562 cells were transfected with HIF-1α knockout or with overexpression lentivirus of plasmid (MOI 10). The cells were divided into 3 groups: the negative control, overexpressing and interference groups. The cells were cultured under normoxia and hypoxia. Expression of miR-17*, miR-363 and miR-574-5p in the three groups was determined by quantitative PCR. Expression levels of erythroid transcription factor mRNAs such as GATA-1/GATA-2 and nuclear factor‑erythroid 2 (NF-E2) were measured using RT-qPCR while the protein expression was studied using western blot analysis. Under normoxia or hypoxia, the levels of miR-17*, miR-363 and miR‑574-5p in the overexpression group were higher than those in the other groups. Differences were statistically significant (P<0.05). Under hypoxia, the level of miR-363 in the interference group was less than that in the negative control group and difference was statistically significant (P<0.05). The level of GATA-1 mRNA in the overexpression group was higher than that in the negative control group, however, in the interference group the level was lower than that in the overexpression group under both normoxic and hypoxic conditions. The level of GATA-2 mRNA in the interference group was higher than that in other two groups under normoxic or hypoxic conditions. The NF-E2 mRNA was reversely related to GATA-2. The levels of HIF-1α, GATA-1 and NF-E2 mRNAs in the negative control under hypoxia were higher than those of normoxia. The level of HIF-1α mRNA in the overexpression group in hypoxia was lower than that in normoxia, while the GATA-1 and GATA-2 mRNA showed a reverse association. The levels of HIF-1α and GATA-2 mRNA in the interference group under hypoxia were higher compared to those of normoxia. Differences were statistically significant (P<0.05). Western blot results suggested that GATA-1, GATA-2 and NF-E2 protein expression correlated with changes in their respective mRNA transcription levels. The results therefore suggested that GATA-l and miR-363 were involved in the regulation of hematopoiesis via the HIF-1α pathway in K562 cells under hypoxic condition. The hsa-miR-17* and hsa-miR‑574-5p were not entirely dependent on HIF-1α, suggesting possible complex regulatory mechanisms involved in hypoxia.
authors.affiliation: Qinghai University, Qinghai Provincial People's Hospital, Xining, Qinghai 810000, P.R. China.
authors.forename: Youbang
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Qinghai Provincial People's Hospital, Xining, Qinghai 810007, P.R. China.
authors.forename: Wenqian
authors.initials: W
authors.lastname: Li
authors.affiliation: Qinghai Provincial People's Hospital, Xining, Qinghai 810007, P.R. China.
authors.forename: Jianming
authors.initials: J
authors.lastname: Feng
authors.affiliation: Qinghai High Altitude Medical Research Institute, Xining, Qinghai 810000, P.R. China.
authors.forename: Tianyi
authors.initials: T
authors.lastname: Wu
authors.affiliation: Qinghai Provincial People's Hospital, Xining, Qinghai 810007, P.R. China.
authors.forename: Jianping
authors.initials: J
authors.lastname: Li
mesh_terms: D050982:GATA1 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015973:Gene Expression Regulation, Leukemic; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D000860:Hypoxia / Q000235:genetics* / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D020014:K562 Cells; D016086:Lentivirus / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D009928:Organ Specificity / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D050982:GATA1 Transcription Factor; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C574861:MIRN363 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger