Mimic miRNA Overexpression Plasmids

The miRNA expression plasmids were generated by PCR amplification of nucleotides 91,350,335–91,350,756 of chromosome 13 for miR-17, 91,350,951–91,351,273 of chromosome 13 for miR-20a, 134,169,622–134,170,000 of X chromosome for miR-20b, 134,169,722–134,170,440 of X chromosome for miR-106a and nucleotides 100,093,902–100,094,174 of chromosome 7 for miR-106b from human genomic DNA and by insertion into the pSG5 vector (Agilent technologies, Ratingen, Germany). Overexpression of the corresponding miRNAs after transfection in HEK293T and LNCaP cells was verified by qRT-PCR (S1 Fig). The nucleotides 2005–3089 of the CCND1 mRNA (accession number: NM_053056.2) containing a part of the corresponding 3’UTR were amplified via PCR using specific primers from human genomic DNA and inserted into pMIR-RNL-TK reporter vector which is described elsewhere [20 (link)]. The mutagenesis of the predicted target site seed sequences of pMIR-RNL-TK reporter constructs were performed with QuickChange Site Directed Mutagenesis Kit (Stratagene, Heidelberg, Germany), following the instructions of the manufacturer's manual. The primer sequences used for cloning and site directed mutagenesis are shown in S1 Table.