Journal: Developmental cell

Article Title: MIR205HG is a Long Noncoding RNA that Regulates Growth Hormone and Prolactin Production in the Anterior Pituitary

doi: 10.1016/j.devcel.2019.03.012

Figure Lengend Snippet: MIR205HG regulates Pit1/Zbtb20 transcriptional activity independent of miR-205. A. Luciferase reporter assays were performed using the prolactin promoter cloned upstream of luciferase. Transient transfection assays were performed in HEK293T cells with plasmids containing the rat prolactin-luciferase reporter construct with/without expression constructs for rat Pit1, Zbtb20, and rat MIR205HG, the latter added in a dose-dependent manner. Data are representative of two independent experiments with p values calculated using student t-tests. B. The luciferase reporter assays were performed as in A. using the growth hormone promoter cloned upstream of luciferase. Data are representative of two independent experiments with p values calculated using student t-tests. C. MMQ cells were incubated with a control inhibitor or one specifically targeting miR-205 (antagomir). The levels of miR-205 were determined 48 hrs post antagomir addition using quantitative miRNA-RT-PCR. Primers specific for rat Pit1, Zbtb20, prolactin, and growth hormone were used for regular qRT-PCR. Significance was determined by t-tests with differences that were not significant listed as ns. D. HEK293T cells were transfected with either wild-type or the knock-in mutation in miR-205 in the full-length MIR205HG construct. These cells were also transfected with rat Pit1 and rat Zbtb20. Forty-eight hrs post-transfection, the cells were UV-cross-linked, lysed, and control IgG or anti-FLAG IgG immunoprecipitations undertaken. The precipitates were washed, proteinase K treatment, and RNA isolated. The RNA was used for qRT-PCR reactions with probes for human gapdh, murine MIR205HG targeting exon 1 (Probe 1, primers 1403/1406), exon 2b (Probe 2, primers 945/946) and exon 2c (Probe 3, primers 1194/1195), the latter detecting transcripts distal to miR-205. Results are obtained from two independent experiments, performed with triplicate samples. Statistical significance was determined by Student t-tests and is shown.

Article Snippet: Human embryonic kidney cells HEK293T (ATCC CRL-3216) and a rat lactotrope cell line MMQ (ATCC CRL-10609) were obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Activity Assay, Luciferase, Clone Assay, Transfection, Construct, Expressing, Incubation, Control, miRNA RT, Quantitative RT-PCR, Knock-In, Mutagenesis, Isolation