Cyclin D1 siRNA (human) (sc-40489, Santa Cruz Biotechnology), Dicer siRNA (human) (sc-40489, Santa Cruz Biotechnology) and ESR1 siRNA (human) (sc-29305, Santa Cruz Biotechnology) were used for Knocking down [13 (link), 27 (link)]. Scrambled siRNA (sc-37007, Santa Cruz Biotechnology) was used as control. All small RNAs were transfected into MCF-7 or ZR75-1 cells by using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.
To construct the luciferase expression vector, the 3’-UTR containing the predicted miR-129-5p binding sites (wild-type, WT) and mutated 3’-UTR (Mut) of the human DICER1 gene were obtained by PCR, and inserted into the pGL3-Control. The recombinant plasmid was named pGL3-DICER1-WT and pGL3-DICER1-Mut, as previously did [27 (link), 35 (link)]. We also constructed the pGL3-ESR1-WT and pGL3-ESR1-Mut. The wildtype or mutant promoter regions of DICER1 was amplified and ligated into pGL3 reporter vector, and named as pGL3-DICER1p-Wt and pGL3-DICER1p-Mut. To identify the possible binding site between 3’-NUMB and Let-7b, a 62-bp fragment of NUMB 3’UTR was subcloned into the PmirGLO control vector to generate PmirGLO-NUMB-3’UTR-wt, Mutant construct of NUMB 3’UTR, named as PmirGLO-NUMB-3’UTR-mut, which carried a substitution of nucleotides in the core seed sequence of Let-7b.
To construct the luciferase expression vector, the 3’-UTR containing the predicted miR-129-5p binding sites (wild-type, WT) and mutated 3’-UTR (Mut) of the human DICER1 gene were obtained by PCR, and inserted into the pGL3-Control. The recombinant plasmid was named pGL3-DICER1-WT and pGL3-DICER1-Mut, as previously did [27 (link), 35 (link)]. We also constructed the pGL3-ESR1-WT and pGL3-ESR1-Mut. The wildtype or mutant promoter regions of DICER1 was amplified and ligated into pGL3 reporter vector, and named as pGL3-DICER1p-Wt and pGL3-DICER1p-Mut. To identify the possible binding site between 3’-NUMB and Let-7b, a 62-bp fragment of NUMB 3’UTR was subcloned into the PmirGLO control vector to generate PmirGLO-NUMB-3’UTR-wt, Mutant construct of NUMB 3’UTR, named as PmirGLO-NUMB-3’UTR-mut, which carried a substitution of nucleotides in the core seed sequence of Let-7b.
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