Figure 5
Figure 5. The target genes of miR-128b and miR-221. (A) Positions of the predicted (target scan) miR-128b target sequences are marked (X) in the 3′ UTRs of human MLL and AF4 mRNAs. The solid arrows represent the position of primers used for cloning the 3′UTR segments that contains the predicted miR-128b. (B) Expression of the Lenilla luciferase reporter is significantly reduced when MLL-UTR-128b and AF4-UTR-128b reporter vectors, containing the miR-128b binding sites of the 3′-UTR of MLL and AF4, respectively, are cotransfected together with miR-128b. This reduction is not observed when control or miR-128bmut expression vectors are used. The Renilla/firefly luciferase ratio is calculated and normalized against the control (MDH) (n = 3). **P < .05. (C) Relative expression levels of MLL-AF4, AF4-MLL, AF4-MLL+MLL, and MLL-AF4+AF4 mRNAs are normalized to U1A expression levels using quantitative PCR in RS4;11 cells, which are overexpressing miR-128b, control vector, or miR-221 (n = 3). **P < .05. (D) Western blot analyses for N320 (MLL N-terminal protein), MLL-AF4, and C180 (MLL C-terminal protein) from total protein extracts of RS4;11 cells, which are overexpressing miR-128b, control vector, or miR-221. The relative intensities of each band, N320 (top in top panel), MLL-AF4 (bottom in top panel), or C180 (middle panel), were normalized to the GAPDH loading control using Multigage software and are indicated below each lane. (Bottom panels) Relative expression levels of HOXa9 mRNAs, normalized to U1A expression levels, using quantitative PCR in SEM cells (left) and RS4;11 (right) cells, which are overexpressing miR-128b, let7b, or miR-221 (n = 3). **P < .05. (E) Positions of the predicted (target scan) miR-221 target sequences are marked (X) in the 3′ UTRs of human CDKN1B mRNA. The solid arrows represent the position of primers used for cloning the 3′UTR segments that contains the predicted miR-221 binding sites. (F) Expression of the Lenilla luciferase reporter activity is significantly reduced when the CDKN1B-UTR-221 reporter vector containing the miR-221 binding sites of the 3′-UTR of CDKN1B is cotransfected together with miR-221. This reduction is not observed when the control and miR-221mut reporter vectors are used (n = 3). **P < .05. (G) Relative expression levels of CDKN1B mRNA is normalized to U1A mRNA expression levels using quantitative PCR in RS4;11 cells, which are overexpressing either miR-221 or a control vector. (H) Western blot analyses for p27 from total protein extracts of RS4;11 cells, which are overexpressing miR-221, or control vector. The relative intensities of each band were normalized to the β-actin loading control using Multigage software Version 2.2 (Fuji) and are indicated below each lane. (I) Cell-cycle analyses for SEM cells, which are overexpressing miR-221, miR-128b, let7b, or control vector (MDH). G1, S, and G2 populations are calculated using FlowJo software Version 7 (TreeStar). A representative experiment is shown (n = 3).

The target genes of miR-128b and miR-221. (A) Positions of the predicted (target scan) miR-128b target sequences are marked (X) in the 3′ UTRs of human MLL and AF4 mRNAs. The solid arrows represent the position of primers used for cloning the 3′UTR segments that contains the predicted miR-128b. (B) Expression of the Lenilla luciferase reporter is significantly reduced when MLL-UTR-128b and AF4-UTR-128b reporter vectors, containing the miR-128b binding sites of the 3′-UTR of MLL and AF4, respectively, are cotransfected together with miR-128b. This reduction is not observed when control or miR-128bmut expression vectors are used. The Renilla/firefly luciferase ratio is calculated and normalized against the control (MDH) (n = 3). **P < .05. (C) Relative expression levels of MLL-AF4, AF4-MLL, AF4-MLL+MLL, and MLL-AF4+AF4 mRNAs are normalized to U1A expression levels using quantitative PCR in RS4;11 cells, which are overexpressing miR-128b, control vector, or miR-221 (n = 3). **P < .05. (D) Western blot analyses for N320 (MLL N-terminal protein), MLL-AF4, and C180 (MLL C-terminal protein) from total protein extracts of RS4;11 cells, which are overexpressing miR-128b, control vector, or miR-221. The relative intensities of each band, N320 (top in top panel), MLL-AF4 (bottom in top panel), or C180 (middle panel), were normalized to the GAPDH loading control using Multigage software and are indicated below each lane. (Bottom panels) Relative expression levels of HOXa9 mRNAs, normalized to U1A expression levels, using quantitative PCR in SEM cells (left) and RS4;11 (right) cells, which are overexpressing miR-128b, let7b, or miR-221 (n = 3). **P < .05. (E) Positions of the predicted (target scan) miR-221 target sequences are marked (X) in the 3′ UTRs of human CDKN1B mRNA. The solid arrows represent the position of primers used for cloning the 3′UTR segments that contains the predicted miR-221 binding sites. (F) Expression of the Lenilla luciferase reporter activity is significantly reduced when the CDKN1B-UTR-221 reporter vector containing the miR-221 binding sites of the 3′-UTR of CDKN1B is cotransfected together with miR-221. This reduction is not observed when the control and miR-221mut reporter vectors are used (n = 3). **P < .05. (G) Relative expression levels of CDKN1B mRNA is normalized to U1A mRNA expression levels using quantitative PCR in RS4;11 cells, which are overexpressing either miR-221 or a control vector. (H) Western blot analyses for p27 from total protein extracts of RS4;11 cells, which are overexpressing miR-221, or control vector. The relative intensities of each band were normalized to the β-actin loading control using Multigage software Version 2.2 (Fuji) and are indicated below each lane. (I) Cell-cycle analyses for SEM cells, which are overexpressing miR-221, miR-128b, let7b, or control vector (MDH). G1, S, and G2 populations are calculated using FlowJo software Version 7 (TreeStar). A representative experiment is shown (n = 3).

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