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SOX4 interacts with EZH2 and HDAC3 to suppress microRNA-31 in invasive esophageal cancer cells.

Koumangoye RB, Andl T, Taubenslag KJ, Zilberman ST, Taylor CJ, Loomans HA, Andl CD - Mol. Cancer (2015)

Bottom Line: We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3'-UTR.Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues.Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, 2213 Garland Ave. 10445 MRB IV, Nashville, TN, 37232-6840, USA. rainelli.koumangoye@vanderbilt.edu.

ABSTRACT

Background: Tumor metastasis is responsible for 90% of cancer-related deaths. Recently, a strong link between microRNA dysregulation and human cancers has been established. However, the molecular mechanisms through which microRNAs regulate metastasis and cancer progression remain unclear.

Methods: We analyzed the reciprocal expression regulation of miR-31 and SOX4 in esophageal squamous and adenocarcinoma cell lines by qRT-PCR and Western blotting using overexpression and shRNA knock-down approaches. Furthermore, methylation studies were used to assess epigenetic regulation of expression. Functionally, we determined the cellular consequences using migration and invasion assays, as well as proliferation assays. Immunoprecipitation and ChIP were used to identify complex formation of SOX4 and co-repressor components.

Results: Here, we report that SOX4 promotes esophageal tumor cell proliferation and invasion by silencing miR-31 via activation and stabilization of a co-repressor complex with EZH2 and HDAC3. We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3'-UTR. Additionally, miR-31 regulates EZH2 and HDAC3 indirectly. SOX4, EZH2 and HDAC3 levels inversely correlate with miR-31 expression in ESCC cell lines. Ectopic expression of miR-31 in ESCC and EAC cell lines leads to down regulation of SOX4, EZH2 and HDAC3. Conversely, pharmacologic and genetic inhibition of SOX4 and EZH2 restore miR-31 expression. We show that SOX4, EZH2 and HDAC3 form a co-repressor complex that binds to the miR-31 promoter, repressing miR-31 through an epigenetic mark by H3K27me3 and by histone acetylation. Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues.

Conclusions: Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas.

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Ectopic expression of miR-31 suppresses migration and invasion of ESCC and EAC cell lines. TE8 and FLO1 cells were transfected with pre-miR-31 containing vector (grey bars) or empty vector control (black bars). (A) Overexpression of miR-31 was verified by qRT-PCR. Pre-mature miR-31 expression was normalized to GAPDH and mature miR-31 expression was normalized to RNU6. (B) Cell migration was measured 24 hours post-transfection using Boyden chambers. (C) Cell invasion was measured 24 hours post-transfection using Matrigel-coated Boyden chambers. (D) Cell viability was measured using the WST-1 assay. (E) Cell growth was evaluated by colony formation assay and measured on the Oxford Optronix Gelcount. Results are means ± SD from at least three biological replicates.
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Fig2: Ectopic expression of miR-31 suppresses migration and invasion of ESCC and EAC cell lines. TE8 and FLO1 cells were transfected with pre-miR-31 containing vector (grey bars) or empty vector control (black bars). (A) Overexpression of miR-31 was verified by qRT-PCR. Pre-mature miR-31 expression was normalized to GAPDH and mature miR-31 expression was normalized to RNU6. (B) Cell migration was measured 24 hours post-transfection using Boyden chambers. (C) Cell invasion was measured 24 hours post-transfection using Matrigel-coated Boyden chambers. (D) Cell viability was measured using the WST-1 assay. (E) Cell growth was evaluated by colony formation assay and measured on the Oxford Optronix Gelcount. Results are means ± SD from at least three biological replicates.

Mentions: To examine the functional contribution of miR-31 in aggressive esophageal cancer, we transfected TE8 and FLO1 cells with vectors containing the precursor of miR-31 or an empty vector control. Ectopic expression of precursor and mature miR-31 in the respective cell lines was tested by quantitative RT-PCR (Figure 2A). In Boyden chamber migration and invasion assays, precursor miR-31 transfection significantly decreased cell migration and invasion in TE8 and FLO1 cells (Figure 2B, C, respectively). miR-31 expression had no significant effect on proliferation in TE8 cells and did not alter the number of colonies in colony formation assays (Figure 2D, E). In FLO1 cells, however, miR-31 suppressed proliferation and colony formation (Figure 2D, E, respectively), indicating miR-31 regulates esophageal carcinoma cell growth in some cell lines. These data suggest that miR-31 suppresses esophageal cancer cell motility and invasiveness, but cell growth depending on the cellular context.Figure 2


SOX4 interacts with EZH2 and HDAC3 to suppress microRNA-31 in invasive esophageal cancer cells.

Koumangoye RB, Andl T, Taubenslag KJ, Zilberman ST, Taylor CJ, Loomans HA, Andl CD - Mol. Cancer (2015)

Ectopic expression of miR-31 suppresses migration and invasion of ESCC and EAC cell lines. TE8 and FLO1 cells were transfected with pre-miR-31 containing vector (grey bars) or empty vector control (black bars). (A) Overexpression of miR-31 was verified by qRT-PCR. Pre-mature miR-31 expression was normalized to GAPDH and mature miR-31 expression was normalized to RNU6. (B) Cell migration was measured 24 hours post-transfection using Boyden chambers. (C) Cell invasion was measured 24 hours post-transfection using Matrigel-coated Boyden chambers. (D) Cell viability was measured using the WST-1 assay. (E) Cell growth was evaluated by colony formation assay and measured on the Oxford Optronix Gelcount. Results are means ± SD from at least three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374188&req=5

Fig2: Ectopic expression of miR-31 suppresses migration and invasion of ESCC and EAC cell lines. TE8 and FLO1 cells were transfected with pre-miR-31 containing vector (grey bars) or empty vector control (black bars). (A) Overexpression of miR-31 was verified by qRT-PCR. Pre-mature miR-31 expression was normalized to GAPDH and mature miR-31 expression was normalized to RNU6. (B) Cell migration was measured 24 hours post-transfection using Boyden chambers. (C) Cell invasion was measured 24 hours post-transfection using Matrigel-coated Boyden chambers. (D) Cell viability was measured using the WST-1 assay. (E) Cell growth was evaluated by colony formation assay and measured on the Oxford Optronix Gelcount. Results are means ± SD from at least three biological replicates.
Mentions: To examine the functional contribution of miR-31 in aggressive esophageal cancer, we transfected TE8 and FLO1 cells with vectors containing the precursor of miR-31 or an empty vector control. Ectopic expression of precursor and mature miR-31 in the respective cell lines was tested by quantitative RT-PCR (Figure 2A). In Boyden chamber migration and invasion assays, precursor miR-31 transfection significantly decreased cell migration and invasion in TE8 and FLO1 cells (Figure 2B, C, respectively). miR-31 expression had no significant effect on proliferation in TE8 cells and did not alter the number of colonies in colony formation assays (Figure 2D, E). In FLO1 cells, however, miR-31 suppressed proliferation and colony formation (Figure 2D, E, respectively), indicating miR-31 regulates esophageal carcinoma cell growth in some cell lines. These data suggest that miR-31 suppresses esophageal cancer cell motility and invasiveness, but cell growth depending on the cellular context.Figure 2

Bottom Line: We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3'-UTR.Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues.Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, 2213 Garland Ave. 10445 MRB IV, Nashville, TN, 37232-6840, USA. rainelli.koumangoye@vanderbilt.edu.

ABSTRACT

Background: Tumor metastasis is responsible for 90% of cancer-related deaths. Recently, a strong link between microRNA dysregulation and human cancers has been established. However, the molecular mechanisms through which microRNAs regulate metastasis and cancer progression remain unclear.

Methods: We analyzed the reciprocal expression regulation of miR-31 and SOX4 in esophageal squamous and adenocarcinoma cell lines by qRT-PCR and Western blotting using overexpression and shRNA knock-down approaches. Furthermore, methylation studies were used to assess epigenetic regulation of expression. Functionally, we determined the cellular consequences using migration and invasion assays, as well as proliferation assays. Immunoprecipitation and ChIP were used to identify complex formation of SOX4 and co-repressor components.

Results: Here, we report that SOX4 promotes esophageal tumor cell proliferation and invasion by silencing miR-31 via activation and stabilization of a co-repressor complex with EZH2 and HDAC3. We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3'-UTR. Additionally, miR-31 regulates EZH2 and HDAC3 indirectly. SOX4, EZH2 and HDAC3 levels inversely correlate with miR-31 expression in ESCC cell lines. Ectopic expression of miR-31 in ESCC and EAC cell lines leads to down regulation of SOX4, EZH2 and HDAC3. Conversely, pharmacologic and genetic inhibition of SOX4 and EZH2 restore miR-31 expression. We show that SOX4, EZH2 and HDAC3 form a co-repressor complex that binds to the miR-31 promoter, repressing miR-31 through an epigenetic mark by H3K27me3 and by histone acetylation. Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues.

Conclusions: Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas.

Show MeSH
Related in: MedlinePlus