Luciferase reporter constructs were generated by introducing the ANG-1 3′-UTR, carrying a putative miR-204/211 binding site, into the vector pGL3 control (Promega, Madison, WI, USA). The 3′-UTR sequence was amplified by PCR using the primers ANG-1-UTR-Sense (5′-CGGGGTACCGCGCAATGTCAGAAGCGATTATG-3′) and ANG-1-UTR-Antisense (5′-GGAAGATCTGTAGTTTGAAGCACAGCAAGC-3′) to introduce KpnI and BglII restriction sites (underlined). The PCR product was cloned into pGL3 control and the resultant plasmid (pGL3C-WT) was used as a template to produce the mutant plasmid pGL3C-MUT. Site-directed mutagenesis of the miR-204/211 binding site was performed using the Quik-Change Site-Directed Mutagenesis kit (Stratagene, Heidelberg, Germany). Specifically, the bases AAA, complementary to UUU in the seed sequence (UUUCCCUU) of miR-204/211, were mutated to TTT; the three base pairs were mutated without introducing or removing other nucleotides in the binding site. All plasmid DNA was purified using the QIAfilter plasmid kit (Qiagen, Hilden, Germany) and confirmed to have the correct sequence by sequencing (Takara, Dalian, China). miR-NC (empty vector), miR-204 (miRBase accession number: MI0000247) and miR-211 (miRBase accession number: MI0000708) overexpressing retroviral vector carrying were constructed as described by Huang et al (19 (link),20 (link)).