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The SOX17/miR-371-5p/SOX2 axis inhibits EMT, stem cell properties and metastasis in colorectal cancer.

Li Y, Lv Z, He G, Wang J, Zhang X, Lu G, Ren X, Wang F, Zhu X, Ding Y, Liao W, Ding Y, Liang L - Oncotarget (2015)

Bottom Line: In the study, miR-371-5p was obviously down-regulated in primary CRC tissues compared with matched adjacent normal mucosa and correlated significantly with differentiation, tumor size, lymphatic and liver metastases.It also suppressed EMT by regulating Wnt/β-catenin signaling and strongly decreased the CRC stemness phenotypes.A positive relationship between SOX17 and miR-371-5p expression and a negative one between miR-371-5p and SOX2 expression were observed in CRC cell lines and tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, People's Republic of China.

ABSTRACT
Cancer stem cells (CSCs) and EMT-type cells, which share molecular characteristics with CSCs, have been believed to play critical roles in tumor metastasis. Although much progress has been garnered in elucidating the molecular pathways that trigger EMT, stemness and metastasis, a number of key mechanistic gaps remain elusive. In the study, miR-371-5p was obviously down-regulated in primary CRC tissues compared with matched adjacent normal mucosa and correlated significantly with differentiation, tumor size, lymphatic and liver metastases. MiR-371-5p could attenuate proliferation, invasion in vitro and metastasis in vivo in CRC cells. It also suppressed EMT by regulating Wnt/β-catenin signaling and strongly decreased the CRC stemness phenotypes. Moreover, demethylation of SOX17 induced miR-371-5p expression and consequently suppressed its direct target SOX2 in CRC cells. MiR-371-5p was necessary for SOX17 mediated cancer-related traits and SOX2 was a functional target of miR-371-5p. A positive relationship between SOX17 and miR-371-5p expression and a negative one between miR-371-5p and SOX2 expression were observed in CRC cell lines and tissues. In conclusion, we identified miR-371-5p as an important "oncosuppressor" in CRC progression and elucidated a novel mechanism of the SOX17/miR-371-5p/SOX2 axis in the regulation of EMT, stemness and metastasis, which may be a potential therapeutic target.

No MeSH data available.


Related in: MedlinePlus

SOX17 transcriptionally regulates miR-371-5p in CRC cells and is sufficient to suppress EMT by regulating miR-371-5p(A) Luciferase activity of miR-371-5p-promoter-luc construct after transfection of SOX17 plasmid in HEK293 and SW480 cells. (B) ChIP assay in HCT116 and SW480 cells. PCR was performed with primers specific for 3 regions in miR-371-5p promoter (R1, R2 and R3), which include 7 putative SOX17 binding sites. Input was used as a positive control, whereas IgG was a negative one. (C) Expression of miR-371-5p in SOX17 depleting HCT116 and SW480 cells by qRT-PCR. The relative expression levels of miR-371-5p in NC cells were normalized to 1. (D) Expression of EMT related markers and target genes of Wnt/β-catenin signaling in cells treated with shSOX17 or shSOX17/miR-371-5p by Western blot. Expression levels were normalized to Tubulin. (E) Immunofluorescence images of E-cadherin, Vimentin expression and nuclear translocation of β-catenin in SW480/NC, SW480/shSOX17 and SW480/shSOX17/miR-371-5p cells. Red scale bars represent 10 μm, whereas yellow scale bars represent 5 μm. * P < 0.05, ** P < 0.01. Data represent the mean ± SD.
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Figure 3: SOX17 transcriptionally regulates miR-371-5p in CRC cells and is sufficient to suppress EMT by regulating miR-371-5p(A) Luciferase activity of miR-371-5p-promoter-luc construct after transfection of SOX17 plasmid in HEK293 and SW480 cells. (B) ChIP assay in HCT116 and SW480 cells. PCR was performed with primers specific for 3 regions in miR-371-5p promoter (R1, R2 and R3), which include 7 putative SOX17 binding sites. Input was used as a positive control, whereas IgG was a negative one. (C) Expression of miR-371-5p in SOX17 depleting HCT116 and SW480 cells by qRT-PCR. The relative expression levels of miR-371-5p in NC cells were normalized to 1. (D) Expression of EMT related markers and target genes of Wnt/β-catenin signaling in cells treated with shSOX17 or shSOX17/miR-371-5p by Western blot. Expression levels were normalized to Tubulin. (E) Immunofluorescence images of E-cadherin, Vimentin expression and nuclear translocation of β-catenin in SW480/NC, SW480/shSOX17 and SW480/shSOX17/miR-371-5p cells. Red scale bars represent 10 μm, whereas yellow scale bars represent 5 μm. * P < 0.05, ** P < 0.01. Data represent the mean ± SD.

Mentions: To explore whether miR-371-5p expression levels was associated with the promoter hypermethylation, we treated CRC cells with methyltransferase inhibitor 5′AZC or Genistein, and found that 5′AZC or Genistein treatment induced the increased expression of miR-371-5p (Supplementary Figure 3A). However, no CpG islands were found in the 1kb region directly upstream of miR-371-5p (promoter) (Supplementary Figure 3B). We then predicted the possible transcription factor binding sites in the promoter of miR-371-5p by using Consite (http://consite.genereg.net/) and TFsearch (http://www.cbrc.jp/research/db/TFSEARCH.html) databases. The possible binding motifs of SOX17 were found in the promoter of miR-371-5p in both databases. It was observed that SOX17 effectively stimulated the luciferase activity of miR-371-5p promoter in HEK293 and SW480 cells (Figure 3A). ChIP results also showed that SOX17 could directly bind the region of R2 (−777~−361bp) and R3 (−376~−86bp) in the promoter of miR-371-5p (Figure 3B). Moreover, knockdown of SOX17 led to decreased expression of miR-371-5p in HCT116 and SW480 cells (Supplementary Figure 3C and Figure 3C). Interestingly, we also found that Genistein treatment in CRC cells induced the increased expression of SOX17 (Supplementary Figure 3D). In CRC, SOX17 silence was found to be due to promoter hypermethylation and contribute to aberrant activation of Wnt signaling [20]. Therefore, the above results indicate that demethylation of SOX17 in CRC can positively regulated miR-371-5p expression.


The SOX17/miR-371-5p/SOX2 axis inhibits EMT, stem cell properties and metastasis in colorectal cancer.

Li Y, Lv Z, He G, Wang J, Zhang X, Lu G, Ren X, Wang F, Zhu X, Ding Y, Liao W, Ding Y, Liang L - Oncotarget (2015)

SOX17 transcriptionally regulates miR-371-5p in CRC cells and is sufficient to suppress EMT by regulating miR-371-5p(A) Luciferase activity of miR-371-5p-promoter-luc construct after transfection of SOX17 plasmid in HEK293 and SW480 cells. (B) ChIP assay in HCT116 and SW480 cells. PCR was performed with primers specific for 3 regions in miR-371-5p promoter (R1, R2 and R3), which include 7 putative SOX17 binding sites. Input was used as a positive control, whereas IgG was a negative one. (C) Expression of miR-371-5p in SOX17 depleting HCT116 and SW480 cells by qRT-PCR. The relative expression levels of miR-371-5p in NC cells were normalized to 1. (D) Expression of EMT related markers and target genes of Wnt/β-catenin signaling in cells treated with shSOX17 or shSOX17/miR-371-5p by Western blot. Expression levels were normalized to Tubulin. (E) Immunofluorescence images of E-cadherin, Vimentin expression and nuclear translocation of β-catenin in SW480/NC, SW480/shSOX17 and SW480/shSOX17/miR-371-5p cells. Red scale bars represent 10 μm, whereas yellow scale bars represent 5 μm. * P < 0.05, ** P < 0.01. Data represent the mean ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: SOX17 transcriptionally regulates miR-371-5p in CRC cells and is sufficient to suppress EMT by regulating miR-371-5p(A) Luciferase activity of miR-371-5p-promoter-luc construct after transfection of SOX17 plasmid in HEK293 and SW480 cells. (B) ChIP assay in HCT116 and SW480 cells. PCR was performed with primers specific for 3 regions in miR-371-5p promoter (R1, R2 and R3), which include 7 putative SOX17 binding sites. Input was used as a positive control, whereas IgG was a negative one. (C) Expression of miR-371-5p in SOX17 depleting HCT116 and SW480 cells by qRT-PCR. The relative expression levels of miR-371-5p in NC cells were normalized to 1. (D) Expression of EMT related markers and target genes of Wnt/β-catenin signaling in cells treated with shSOX17 or shSOX17/miR-371-5p by Western blot. Expression levels were normalized to Tubulin. (E) Immunofluorescence images of E-cadherin, Vimentin expression and nuclear translocation of β-catenin in SW480/NC, SW480/shSOX17 and SW480/shSOX17/miR-371-5p cells. Red scale bars represent 10 μm, whereas yellow scale bars represent 5 μm. * P < 0.05, ** P < 0.01. Data represent the mean ± SD.
Mentions: To explore whether miR-371-5p expression levels was associated with the promoter hypermethylation, we treated CRC cells with methyltransferase inhibitor 5′AZC or Genistein, and found that 5′AZC or Genistein treatment induced the increased expression of miR-371-5p (Supplementary Figure 3A). However, no CpG islands were found in the 1kb region directly upstream of miR-371-5p (promoter) (Supplementary Figure 3B). We then predicted the possible transcription factor binding sites in the promoter of miR-371-5p by using Consite (http://consite.genereg.net/) and TFsearch (http://www.cbrc.jp/research/db/TFSEARCH.html) databases. The possible binding motifs of SOX17 were found in the promoter of miR-371-5p in both databases. It was observed that SOX17 effectively stimulated the luciferase activity of miR-371-5p promoter in HEK293 and SW480 cells (Figure 3A). ChIP results also showed that SOX17 could directly bind the region of R2 (−777~−361bp) and R3 (−376~−86bp) in the promoter of miR-371-5p (Figure 3B). Moreover, knockdown of SOX17 led to decreased expression of miR-371-5p in HCT116 and SW480 cells (Supplementary Figure 3C and Figure 3C). Interestingly, we also found that Genistein treatment in CRC cells induced the increased expression of SOX17 (Supplementary Figure 3D). In CRC, SOX17 silence was found to be due to promoter hypermethylation and contribute to aberrant activation of Wnt signaling [20]. Therefore, the above results indicate that demethylation of SOX17 in CRC can positively regulated miR-371-5p expression.

Bottom Line: In the study, miR-371-5p was obviously down-regulated in primary CRC tissues compared with matched adjacent normal mucosa and correlated significantly with differentiation, tumor size, lymphatic and liver metastases.It also suppressed EMT by regulating Wnt/β-catenin signaling and strongly decreased the CRC stemness phenotypes.A positive relationship between SOX17 and miR-371-5p expression and a negative one between miR-371-5p and SOX2 expression were observed in CRC cell lines and tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, People's Republic of China.

ABSTRACT
Cancer stem cells (CSCs) and EMT-type cells, which share molecular characteristics with CSCs, have been believed to play critical roles in tumor metastasis. Although much progress has been garnered in elucidating the molecular pathways that trigger EMT, stemness and metastasis, a number of key mechanistic gaps remain elusive. In the study, miR-371-5p was obviously down-regulated in primary CRC tissues compared with matched adjacent normal mucosa and correlated significantly with differentiation, tumor size, lymphatic and liver metastases. MiR-371-5p could attenuate proliferation, invasion in vitro and metastasis in vivo in CRC cells. It also suppressed EMT by regulating Wnt/β-catenin signaling and strongly decreased the CRC stemness phenotypes. Moreover, demethylation of SOX17 induced miR-371-5p expression and consequently suppressed its direct target SOX2 in CRC cells. MiR-371-5p was necessary for SOX17 mediated cancer-related traits and SOX2 was a functional target of miR-371-5p. A positive relationship between SOX17 and miR-371-5p expression and a negative one between miR-371-5p and SOX2 expression were observed in CRC cell lines and tissues. In conclusion, we identified miR-371-5p as an important "oncosuppressor" in CRC progression and elucidated a novel mechanism of the SOX17/miR-371-5p/SOX2 axis in the regulation of EMT, stemness and metastasis, which may be a potential therapeutic target.

No MeSH data available.


Related in: MedlinePlus