A full length of the human PLAU 3′-untranslated region (932 bp) and partial length of HIC2 3′-UTR (1131 bp, 1862-2992 from a 4719-bp full length) with the miR-193a-3p targeting sequence were cloned at the downstream of the firefly luciferase gene in pGL3 (Invitrogen) to construct pGL3-luc-PLAU and pGL3-luc-HIC2, respectively. All the constructs were confirmed by restriction digestion.
Cells were seeded into 96-well plates at around 1 × 104 cells per well and transfected with a mixture of 50 ng pGL3-luc-PLAU or pGL3-luc-HIC2, 5 ng Renilla plus 5 pmol mimic or scramble control (NC) nucleotides, with the riboFECT CP transfection reagents according to the manufacturer's instruction. Both firefly and Renilla luciferase activities were measured 18 h after transfection by the Dual-Luciferase Reporter Assay System (Promega) using a Promega GloMax 20/20 luminometer. The relative firefly luciferase activities were normalized with the Renilla luciferase activities as a for transfection efficiency.
The pathway luciferase reporter constructs (Supplementary Figure S2A ): (1) the negative control construct: the firefly luciferase gene is under the control of the minimal promoter. (2) the pathway reporter construct: a tandem repeat of the cognate consensus motif that is recognized by each master transcription factor for the corresponding pathway was placed at the upstream of the minimal promoter in the construct 1. (3) the positive control construct: the firefly luciferase gene is under the control of CMV promoter and (4) the internal control construct. The firefly luciferase gene in construct 3 was replaced with the Renilla luciferase gene. The analysis was carried out according to the manufacturer's instruction (Qiagen).42 (link) Briefly, the cells were transfected in triplet with each firefly luciferase reporter construct in combination with the Renilla luciferase construct using ribo FECT CP transfection reagent, and both luciferase activities in cell extracts at 24 h after transfection were measured by a Promega Dual-Luciferase Reporter assay (Promega) using a Promega GloMax 20/20 luminometer.43 Firefly luciferase activities from each set were normalized to the activity of Renilla luciferase to control the inter-transfection bias. The relative luciferase activities (luciferase unit) of the pathway reporter over the negative control in the transfected cells were calculated as a measurement of the pathway activity.
Chemotherapeutics: All the chemotherapeutic drugs used are of the clinic grade44 (link), 45 (link) (NCI Dictionary of Cancer Terms,http://www.cancer.gov/dictionary ), Pi: Pirarubicin hydrochloride (Wanle, Shenzhen, China); Pa: Paclitaxel (Shuanglu, Beijing, China); Ad: Adriamycin (Haizheng, Zhejiang, China); EH: Epirubicin hydrochloride (Haizheng, Zhejiang, China).
Chemoresistance profiling (IC50 measurements): Cells in the logarithmic phase of growth were seeded in triplicate in 96-well plates at the density of 0.5 × 104/well and treated with four-fold serially diluted drugs for 72 h. Cell survival was then measured by a thiazolyl blue tetrazolium bromide (MTT, 490 nm reading)-based cell proliferation assay.46 (link) Both the linear regression parameters and the IC50 (the concentration of drug required for 50% of cells to be killed) with the no-drug control as the reference were calculated. The relative chemoresistance was presented as the fold for each of the cell line over the lowest IC50.
Cells were seeded into 96-well plates at around 1 × 104 cells per well and transfected with a mixture of 50 ng pGL3-luc-PLAU or pGL3-luc-HIC2, 5 ng Renilla plus 5 pmol mimic or scramble control (NC) nucleotides, with the riboFECT CP transfection reagents according to the manufacturer's instruction. Both firefly and Renilla luciferase activities were measured 18 h after transfection by the Dual-Luciferase Reporter Assay System (Promega) using a Promega GloMax 20/20 luminometer. The relative firefly luciferase activities were normalized with the Renilla luciferase activities as a for transfection efficiency.
The pathway luciferase reporter constructs (
Chemotherapeutics: All the chemotherapeutic drugs used are of the clinic grade44 (link), 45 (link) (NCI Dictionary of Cancer Terms,
Chemoresistance profiling (IC50 measurements): Cells in the logarithmic phase of growth were seeded in triplicate in 96-well plates at the density of 0.5 × 104/well and treated with four-fold serially diluted drugs for 72 h. Cell survival was then measured by a thiazolyl blue tetrazolium bromide (MTT, 490 nm reading)-based cell proliferation assay.46 (link) Both the linear regression parameters and the IC50 (the concentration of drug required for 50% of cells to be killed) with the no-drug control as the reference were calculated. The relative chemoresistance was presented as the fold for each of the cell line over the lowest IC50.
