Pgl vector

The promoter fragments of CRP (+3 to −157 bp, +3 to −550 bp, +3 to −1000 bp, or +3 to −1500 bp), SAP (−1 to −1000 bp), or KLHL5 (−1 to −1500 bp) were cloned into pGL2.0, pGL3.0, or pGL4.10 vector (Promega; catalog numbers: E1541, E1751, or E6651). Enhancer fragments were inserted upstream the promoter fragments. Hep3B cells (cell bank of Chinese Academy of Sciences) were cotransfected with 1.5 μg of pGL vector and 0.075 μg of phRL-TK (Promega; catalog number: E6241) using X-tremeGENE 9 DNA transfection reagent (Roche; catalog number: 06365787001) for 16 h. Cells were then treated with or without IL-6 (10 ng/ml) and IL-1β (1 ng/ml) for 24 h followed by measurement of luciferase activities with Dual-Luciferase Reporter Assay System (Promega; catalog number: E1960) by a Synergy HTX Multi-Mode Microplate Reader (BioTek). Activities of firefly luciferase driven by inserted promoters were normalized with activities of cotransfected Renilla luciferase.

The predicted binding sites between miR-202 and K-Ras were obtained using TargetScan 7.0 (http://www.targetscan.org/vert_70/) and miRanda (v3.3a; http://www.microrna.org) online softwares. The full-length 3′-UTR of the wild-type (wt) human K-Ras gene was amplified by PCR (2X Phanta^® Master Mix; Vazyme Biotech Co., Ltd.) using human cDNA isolated from ectopic endometrium tissues as the template using the following primers: Forward, 5′-CCG GGT ACC ATG ACT GAA TAT AAA CTT GTG G-3′ and reverse, 5′-CCG CTC GAG TTA CAT AAT TAC ACA CTT TGT C-3′. Thermocycling conditions consisted of an initial incubation at 95°C for 5 min, followed by 30 cycles of 94°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec, followed by a final 10-min extension step at 72°C. The resultant amplicon was then cloned into the KpnI and XhoI sites of the pGL vector (Promega Corporation). The mutant (mt)-K-Ras vector was also constructed. This vector contains mutations in the major miR-202 binding site made using the GeneArt™ Site-Directed Mutagenesis Plus System according to the manufacturer's protocols (Thermo Fisher Scientific, Inc.). Successful plasmid construction was confirmed by Sanger sequencing by Sangon Biotech Co., Ltd.