Limits...
MicroRNA-135b, a HSF1 target, promotes tumor invasion and metastasis by regulating RECK and EVI5 in hepatocellular carcinoma.

Li Y, Xu D, Bao C, Zhang Y, Chen D, Zhao F, Ding J, Liang L, Wang Q, Liu L, Li J, Yao M, Huang S, He X - Oncotarget (2015)

Bottom Line: MicroRNAs (miRNAs) often localize to chromosomal fragile sites and are associated with cancer.In this study, we screened for the aberrant and functional miRNAs in the regions of copy number alterations (CNAs) in hepatocellular carcinoma (HCC), and found that miR-135b was frequently amplified and upregulated in HCC tissues.The newly identified HSF1/miR-135b/RECK&EVI5 axis provides novel insight into the mechanisms of HCC metastasis, which may facilitate the development of new therapeutics against HCC.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
MicroRNAs (miRNAs) often localize to chromosomal fragile sites and are associated with cancer. In this study, we screened for the aberrant and functional miRNAs in the regions of copy number alterations (CNAs) in hepatocellular carcinoma (HCC), and found that miR-135b was frequently amplified and upregulated in HCC tissues. The expression level of miR-135b was inversely correlated with the occurrence of tumor capsules. In addition, miR-135b promoted HCC cell migration and invasion in vitro and metastasis in vivo. The reversion-inducing-cysteine-rich protein with kazal motifs (RECK) and ecotropic viral integration site 5 (EVI5) were identified as the direct and functional targets of miR-135b in HCC. Furthermore, we observed that heat shock transcription factor 1 (HSF1) directly activated miR-135b expression, consequently enhancing HCC cell motility and invasiveness. The newly identified HSF1/miR-135b/RECK&EVI5 axis provides novel insight into the mechanisms of HCC metastasis, which may facilitate the development of new therapeutics against HCC.

Show MeSH

Related in: MedlinePlus

MiR-135b is regulated by heat shock transcription factor 1 (HSF1)(A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. DNA-protein complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering RNA (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P < 0.05; **P < 0.01 (Student's t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4385861&req=5

Figure 5: MiR-135b is regulated by heat shock transcription factor 1 (HSF1)(A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. DNA-protein complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering RNA (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P < 0.05; **P < 0.01 (Student's t test).

Mentions: MiR-135b expression is upregulated in 54.2% of HCC tissues, while only 30.2% of miR-135b genomic regions are amplified. Thus, additional mechanisms besides gene amplification are likely responsible for the dysregulation of miR-135b in HCC. An enrichment of the H3K27Ac histone mark, often found near active regulatory elements, was observed in the 3-kb region upstream of pre-miR-135b (Figure 5A). A series of luciferase reporter constructs containing fragments from this region (from −3000 to +1 bp) were cloned into the pGL3-basic vector (Figure 5A), and luciferase activity was measured after transfection of these constructs into SMMC-7721 cells. The highest activity was associated with the −2000 to −1500 bp fragment (Figure 5B), indicating that it contained regulatory elements critical for the transcription of miR-135b. Next, we used TFSEARCH[16] to predict the transcription factor binding sites in this region. We found this region contained two binding sites for HSF1 (Figure 5C). Chromatin immunoprecipitation (ChIP) assays revealed that HSF1 could bind to this region at binding site 2 in HCC cells (Figure 5D). We also found deletion of binding site 1 does not affect the reporter activities, but deletion of binding site 2 significantly reduces the activities (Supplementary Figures S9A and B). Moreover, both EMSA (Figure 5E) and antibody-supershift assays (Figure 5F) showed an interaction between HSF1 and the binding site 2 in vitro. When co-transfection of the luciferase reporter containing the binding site 2 with HSF1 expressing vector or siRNA against HSF1, we found HSF1 overexpression increased the reporter activities (Figure 5G), and inhibition of HSF1 decreased its activities (Figure 5H). Consistent with this result, we observed the level of miR-135b expression was significantly upregulated by ectopic expression of HSF1 (Figure 5I), and downregulated by silencing of HSF1 expression (Figure 5J). Taken together, these findings demonstrate that HSF1 directly regulates miR-135b expression by binding to its upstream region.


MicroRNA-135b, a HSF1 target, promotes tumor invasion and metastasis by regulating RECK and EVI5 in hepatocellular carcinoma.

Li Y, Xu D, Bao C, Zhang Y, Chen D, Zhao F, Ding J, Liang L, Wang Q, Liu L, Li J, Yao M, Huang S, He X - Oncotarget (2015)

MiR-135b is regulated by heat shock transcription factor 1 (HSF1)(A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. DNA-protein complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering RNA (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P < 0.05; **P < 0.01 (Student's t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385861&req=5

Figure 5: MiR-135b is regulated by heat shock transcription factor 1 (HSF1)(A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. DNA-protein complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering RNA (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P < 0.05; **P < 0.01 (Student's t test).
Mentions: MiR-135b expression is upregulated in 54.2% of HCC tissues, while only 30.2% of miR-135b genomic regions are amplified. Thus, additional mechanisms besides gene amplification are likely responsible for the dysregulation of miR-135b in HCC. An enrichment of the H3K27Ac histone mark, often found near active regulatory elements, was observed in the 3-kb region upstream of pre-miR-135b (Figure 5A). A series of luciferase reporter constructs containing fragments from this region (from −3000 to +1 bp) were cloned into the pGL3-basic vector (Figure 5A), and luciferase activity was measured after transfection of these constructs into SMMC-7721 cells. The highest activity was associated with the −2000 to −1500 bp fragment (Figure 5B), indicating that it contained regulatory elements critical for the transcription of miR-135b. Next, we used TFSEARCH[16] to predict the transcription factor binding sites in this region. We found this region contained two binding sites for HSF1 (Figure 5C). Chromatin immunoprecipitation (ChIP) assays revealed that HSF1 could bind to this region at binding site 2 in HCC cells (Figure 5D). We also found deletion of binding site 1 does not affect the reporter activities, but deletion of binding site 2 significantly reduces the activities (Supplementary Figures S9A and B). Moreover, both EMSA (Figure 5E) and antibody-supershift assays (Figure 5F) showed an interaction between HSF1 and the binding site 2 in vitro. When co-transfection of the luciferase reporter containing the binding site 2 with HSF1 expressing vector or siRNA against HSF1, we found HSF1 overexpression increased the reporter activities (Figure 5G), and inhibition of HSF1 decreased its activities (Figure 5H). Consistent with this result, we observed the level of miR-135b expression was significantly upregulated by ectopic expression of HSF1 (Figure 5I), and downregulated by silencing of HSF1 expression (Figure 5J). Taken together, these findings demonstrate that HSF1 directly regulates miR-135b expression by binding to its upstream region.

Bottom Line: MicroRNAs (miRNAs) often localize to chromosomal fragile sites and are associated with cancer.In this study, we screened for the aberrant and functional miRNAs in the regions of copy number alterations (CNAs) in hepatocellular carcinoma (HCC), and found that miR-135b was frequently amplified and upregulated in HCC tissues.The newly identified HSF1/miR-135b/RECK&EVI5 axis provides novel insight into the mechanisms of HCC metastasis, which may facilitate the development of new therapeutics against HCC.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
MicroRNAs (miRNAs) often localize to chromosomal fragile sites and are associated with cancer. In this study, we screened for the aberrant and functional miRNAs in the regions of copy number alterations (CNAs) in hepatocellular carcinoma (HCC), and found that miR-135b was frequently amplified and upregulated in HCC tissues. The expression level of miR-135b was inversely correlated with the occurrence of tumor capsules. In addition, miR-135b promoted HCC cell migration and invasion in vitro and metastasis in vivo. The reversion-inducing-cysteine-rich protein with kazal motifs (RECK) and ecotropic viral integration site 5 (EVI5) were identified as the direct and functional targets of miR-135b in HCC. Furthermore, we observed that heat shock transcription factor 1 (HSF1) directly activated miR-135b expression, consequently enhancing HCC cell motility and invasiveness. The newly identified HSF1/miR-135b/RECK&EVI5 axis provides novel insight into the mechanisms of HCC metastasis, which may facilitate the development of new therapeutics against HCC.

Show MeSH
Related in: MedlinePlus