Figure 2
Figure 2. CCNT2 is a target gene of both miR-29a and miR-142-3p, and CCNT2 plays a role in inhibiting PMA-induced monocytic differentiation in THP-1 and HL-60 cells. (A) The nucleotide sequences of miR-29a, miR-142-3p, and their complementary sequences in CCNT2 mRNA. The predicted miR-29a and miR-142-3p binding sites in the CCNT2 3′-UTR and the mutated nucleotides (gray boxes) of the 3′-UTR site are shown. CCNT2_WT is a reporter construct containing the entire wild-type 3′-UTR sequence of CCNT2. CCNT2_29aM, CCNT2_142-3pM, and CCNT2_DM are reporter constructs containing mutations in the miR-29a binding site, the miR-142-3p binding site mutation, and both sites. (B) Luciferase activity in 293T cells cotransfected with one of the 4 reporter constructs and either one of the miRNA (miR-29a or miR-142-3p) mimics or scramble control, as indicated. The reporter constructs with perfect complementary sites for miR-29a and miR-142-3p (p29a_site and p142-3p site, respectively) were used as positive controls. The activities were calculated as a ratio of firefly to renilla luciferase activity, and are expressed as mean ± SD of 3 separate experiments. *P < .05 (Student t test). **P < .01 (Student t test). (C) Luciferase activity in 293T cells cotransfected with either the wild-type or double mutant, and both miR-29a and miR-142-3p mimics simultaneously or scramble control. (D) Western blot analysis of CCNT2 expression in 293T and myeloid cell lines THP-1, HL-60, and NB4 transfected with miR-29a mimic, miR-142-3p mimic, or scramble control. Densitometric values normalized on the basis of GAPDH expression are indicated below the corresponding lanes and shown as fold changes. N.D indicates a densitometric value cannot be given because the band is almost undetectable. (E) Western blot analysis of CCNT2 expression in monocytic cell line THP-1 transfected with an inhibitor for miR-29a (anti-29a), an inhibitor for miR-142-3p (anti–142-3p), or an inhibitor control. (F) Western blot analysis showing expression of the 2 CCNT2 isoforms during PMA-induced monocytic differentiation in THP-1 and HL-60 cells; GAPDH was detected for checking equal protein loading. (G) Western blot analysis showing the expression of CCNT2 in HL-60 and THP-1 cells that were transfected with CCNT2 siRNA (si_CCNT2) or siRNA control (si_control) and subsequently treated with PMA for 72 hours. (H) Flow cytometric analysis of CD11b in THP-1 cells transfected with si_CCNT2 or si_control. Numbers in the graphs represent the percentages of positively stained after 72 hours of differentiation (the right peak) compared with untreated cells (the left peak). A representative experiment of 3 is presented. (I) Cell proliferation assay of THP-1 cells transfected with si_CCNT2 or si_control using cell counting kit 8. The cells were cultured in medium with or without PMA. (J) Western blots showing pRb and Rb protein levels in THP-1 cells after transfection with si_CCNT2 or si_control, and in HL-60 cells after transfection with miR-29a mimic, miR-142-3p mimic, or scramble control.

CCNT2 is a target gene of both miR-29a and miR-142-3p, and CCNT2 plays a role in inhibiting PMA-induced monocytic differentiation in THP-1 and HL-60 cells. (A) The nucleotide sequences of miR-29a, miR-142-3p, and their complementary sequences in CCNT2 mRNA. The predicted miR-29a and miR-142-3p binding sites in the CCNT2 3′-UTR and the mutated nucleotides (gray boxes) of the 3′-UTR site are shown. CCNT2_WT is a reporter construct containing the entire wild-type 3′-UTR sequence of CCNT2. CCNT2_29aM, CCNT2_142-3pM, and CCNT2_DM are reporter constructs containing mutations in the miR-29a binding site, the miR-142-3p binding site mutation, and both sites. (B) Luciferase activity in 293T cells cotransfected with one of the 4 reporter constructs and either one of the miRNA (miR-29a or miR-142-3p) mimics or scramble control, as indicated. The reporter constructs with perfect complementary sites for miR-29a and miR-142-3p (p29a_site and p142-3p site, respectively) were used as positive controls. The activities were calculated as a ratio of firefly to renilla luciferase activity, and are expressed as mean ± SD of 3 separate experiments. *P < .05 (Student t test). **P < .01 (Student t test). (C) Luciferase activity in 293T cells cotransfected with either the wild-type or double mutant, and both miR-29a and miR-142-3p mimics simultaneously or scramble control. (D) Western blot analysis of CCNT2 expression in 293T and myeloid cell lines THP-1, HL-60, and NB4 transfected with miR-29a mimic, miR-142-3p mimic, or scramble control. Densitometric values normalized on the basis of GAPDH expression are indicated below the corresponding lanes and shown as fold changes. N.D indicates a densitometric value cannot be given because the band is almost undetectable. (E) Western blot analysis of CCNT2 expression in monocytic cell line THP-1 transfected with an inhibitor for miR-29a (anti-29a), an inhibitor for miR-142-3p (anti–142-3p), or an inhibitor control. (F) Western blot analysis showing expression of the 2 CCNT2 isoforms during PMA-induced monocytic differentiation in THP-1 and HL-60 cells; GAPDH was detected for checking equal protein loading. (G) Western blot analysis showing the expression of CCNT2 in HL-60 and THP-1 cells that were transfected with CCNT2 siRNA (si_CCNT2) or siRNA control (si_control) and subsequently treated with PMA for 72 hours. (H) Flow cytometric analysis of CD11b in THP-1 cells transfected with si_CCNT2 or si_control. Numbers in the graphs represent the percentages of positively stained after 72 hours of differentiation (the right peak) compared with untreated cells (the left peak). A representative experiment of 3 is presented. (I) Cell proliferation assay of THP-1 cells transfected with si_CCNT2 or si_control using cell counting kit 8. The cells were cultured in medium with or without PMA. (J) Western blots showing pRb and Rb protein levels in THP-1 cells after transfection with si_CCNT2 or si_control, and in HL-60 cells after transfection with miR-29a mimic, miR-142-3p mimic, or scramble control.

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