%0 Journal Article
%T ΔNp63 regulates the expression of hyaluronic acid-related genes in breast cancer cells
%A Angelo Peschiaroli
%A Claudia Fierro
%A Elke Katrin Markert
%A Federica Giangrazi
%A Gerry Melino
%A Lucilla Bongiorno-Borbone
%A Mirco Compagnone
%A Veronica Gatti
%J Archive of "Oncogenesis".
%D 2018
%R 10.1038/s41389-018-0073-3
%X a HCC1954 and HCC1937 breast carcinoma cell lines were grown in RPMI-1640 medium (Gibco, Invitrogen); H1299 cells (non-smal cell lung cancer cell line) were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Invitrogen) supplemented with 10% v/v fetal bovine serum (FBS), 100？μg/mL penicillin and 100？μg/mL streptomycin (Gibco, Invitrogen). Cells were cultured at 37？°C with 5% CO2. Breast carcinoma cell lines were purchased from ATCC and routinely tested for mycoplasma contaminations. HAS3 and ΔNp63 mRNA levels were quantified by Real-Time PCR analysis (qRT-PCR) in the indicated basal-type breast tumor cell lines transfected with scrambled (SCR) or p63 siRNA (sip63) oligos. For siRNA oligos transfection cells were seeded at a density of 1.4？×？105 cells/well in a six-well plate and transfected with oligos using RNAimax (Invitrogen) according to manufacturer’s instructions. Smart pool siRNA oligos direct against p63, HAS3 mRNA, and non-relevant gene (scramble) were purchased by Dharmacon (Thermo Scientific). Cells were collected 48？h after transfection and lysates were subjected to qRT-PCR analysis. Total mRNA was isolated using the RNeasy mini kit (Qiagen, Duesseldorf, Gemrany) following the manufacturer’s recommendations. Total RNA was quantified using a NanoDrop Spectophotometer (Thermo Scientific, Delaware, USA) and used for cDNA synthesis using Superscript Reverse Transcriptase (Promega, Fithburg, WI, USA), according to the manufacturer’s protocol. cDNA was subsequently used for qRT-PCR. Each 25？μl reaction contained 2X SYBR-Green PCR Master Mix (Promega), 2？μl cDNA and the appropriate specific primers (0.5？μM). Amplification and fluorescence detection according to the manufacturer’s instructions was performed using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, France). The expression of each gene was defined from threshold cycle (Ct), and the relative expression levels were calculated using the 2-ΔΔCt method. The primers used for qRT-PCR are the following: human ΔNp63 for 5′-GAAGAAAGGACAGCAGCATTG-3′; rev 5′-GGGACTGGTGGACGAGGAG-3′; human HAS3 for 5′-TGTGCATTGCCGCATACC-3′; rev 5′-CCGAGCGCAGGCACTT-3′; human GAPDH for 5′-AGCCACATCGCTCAGACA-3′; rev 5′-GCCCAATACGACCAAATC-3′; human Actin for 5′-GTTGCTATCCAGGCTGTG-3′; rev 5′-AATGTCACGCACGATTTCCCG-3′; Bars represent the mean of three technical replicates (n？=？3, PCR runs)？±？SD and are representative of three independent experiments (n？=？3 biological replicates). No data were excluded form the analysis. *p- value？<？0.05. b HYAL-1 mRNA levels were measured by qRT-PCR in HCC1937 cells
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107578/