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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

SOX2 is a functional target of miR-371-5p(A) Expression of EMT related markers and target genes of Wnt/β-catenin signaling in cells treated with Zip-371-5p or Zip-371-5p/shSOX2 by Western blot. Expression levels were normalized to Tubulin. (B) Immunofluorescence images of E-cadherin, Vimentin expression and nuclear translocation of β-catenin in SW480/NC, SW480/Zip-371-5p and SW480/Zip-371-5p/shSOX2 cells. Red scale bars represent 10 μm, whereas yellow scale bars represent 5 μm. (C) Expression of OCT4, SOX2 and CD133 in SW480 cells treated with Zip-371-5p or Zip-371-5p/shSOX2 by qRT-PCR. The relative expression levels in NC cells were normalized to 1. (D) HCT116/NC, HCT116/Zip-371-5p and HCT116/Zip-371-5p/shSOX2 cells (1×106) were injected in the subcutaneous tissue of nude mice (n = 5). The weight of subcutaneous tumors was measured. (E) HCT116/NC, HCT116/Zip-371-5p and HCT116/Zip-371-5p/shSOX2 cells (2×106) were injected into the tail vein of nude mice (n = 5) for 2 months. Yellow arrows in top panels point at lung metastatic nodules. Scale bars in bottom panels represent 100 μm. The number of lung metastatic nodules per mouse was counted under the microscope. * P < 0.05, ** P < 0.01. Data represent the mean ± SD.
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Figure 5: SOX2 is a functional target of miR-371-5p(A) Expression of EMT related markers and target genes of Wnt/β-catenin signaling in cells treated with Zip-371-5p or Zip-371-5p/shSOX2 by Western blot. Expression levels were normalized to Tubulin. (B) Immunofluorescence images of E-cadherin, Vimentin expression and nuclear translocation of β-catenin in SW480/NC, SW480/Zip-371-5p and SW480/Zip-371-5p/shSOX2 cells. Red scale bars represent 10 μm, whereas yellow scale bars represent 5 μm. (C) Expression of OCT4, SOX2 and CD133 in SW480 cells treated with Zip-371-5p or Zip-371-5p/shSOX2 by qRT-PCR. The relative expression levels in NC cells were normalized to 1. (D) HCT116/NC, HCT116/Zip-371-5p and HCT116/Zip-371-5p/shSOX2 cells (1×106) were injected in the subcutaneous tissue of nude mice (n = 5). The weight of subcutaneous tumors was measured. (E) HCT116/NC, HCT116/Zip-371-5p and HCT116/Zip-371-5p/shSOX2 cells (2×106) were injected into the tail vein of nude mice (n = 5) for 2 months. Yellow arrows in top panels point at lung metastatic nodules. Scale bars in bottom panels represent 100 μm. The number of lung metastatic nodules per mouse was counted under the microscope. * P < 0.05, ** P < 0.01. Data represent the mean ± SD.

Mentions: Transcriptional regulator SOX2 was identified as an oncogene in many cancers, including colon cancer. It plays an important role in cancer stem cell, EMT and metastasis of colon cancer [21-23]. To determine whether cancer cell phenotypes associated with miR-371-5p expression could be reversed via restoration of SOX2, we transfected miR-371-5p-depleting cells with shRNAs toward SOX2 in HCT116 and SW480 cells (Supplementary Figure 5B). In miR-371-5p-depleting cells, depletion of SOX2 reversed, at least partially, miR-371-5p knockdown-imposed proliferation and invasion (Supplementary Figure 5C). SOX2 rescued miR-371-5p's dependent MET morphogical changes (Supplementary Figure 5D). Reintroduction of SOX2 shRNAs in miR-371-5p depleting cells led to down-regulations of Vimentin and Slug, up-regulation of E-cadherin (Figure 5A and 5B), cytoplasmic translocation of β-catenin (Figure 5B), TCF/LEF transcriptional inactivation (Supplementary Figure 5E) and decreased expression of target genes CyclinD1, C-myc and DKK1 (Figure 5A). Moreover, depletion of SOX2 abolished the promoting effect of miR-371-5p knockdown on stemness, as shown by decreased expression of stem cell pluripotency factors OCT4, SOX2 and stem cell marker CD133 (Figure 5C) and decreased sphere-forming capacity (Supplementary Figure 5F). We also evaluated whether SOX2 rescued miR-371-5p's effects on tumor growth and metastasis in vivo. Depletion of SOX2 reversed, at least in part, tumor growth and lung metastases resulting from knockdown of miR-371-5p (Figure 5D and 5E). These results reveal that miR-371-5p is a crucial and unexpected switch for EMT, stemness and metastasis of CRC via repression of SOX2.


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)

SOX2 is a functional target of miR-371-5p(A) Expression of EMT related markers and target genes of Wnt/β-catenin signaling in cells treated with Zip-371-5p or Zip-371-5p/shSOX2 by Western blot. Expression levels were normalized to Tubulin. (B) Immunofluorescence images of E-cadherin, Vimentin expression and nuclear translocation of β-catenin in SW480/NC, SW480/Zip-371-5p and SW480/Zip-371-5p/shSOX2 cells. Red scale bars represent 10 μm, whereas yellow scale bars represent 5 μm. (C) Expression of OCT4, SOX2 and CD133 in SW480 cells treated with Zip-371-5p or Zip-371-5p/shSOX2 by qRT-PCR. The relative expression levels in NC cells were normalized to 1. (D) HCT116/NC, HCT116/Zip-371-5p and HCT116/Zip-371-5p/shSOX2 cells (1×106) were injected in the subcutaneous tissue of nude mice (n = 5). The weight of subcutaneous tumors was measured. (E) HCT116/NC, HCT116/Zip-371-5p and HCT116/Zip-371-5p/shSOX2 cells (2×106) were injected into the tail vein of nude mice (n = 5) for 2 months. Yellow arrows in top panels point at lung metastatic nodules. Scale bars in bottom panels represent 100 μm. The number of lung metastatic nodules per mouse was counted under the microscope. * P < 0.05, ** P < 0.01. Data represent the mean ± SD.
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Related In: Results  -  Collection

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Figure 5: SOX2 is a functional target of miR-371-5p(A) Expression of EMT related markers and target genes of Wnt/β-catenin signaling in cells treated with Zip-371-5p or Zip-371-5p/shSOX2 by Western blot. Expression levels were normalized to Tubulin. (B) Immunofluorescence images of E-cadherin, Vimentin expression and nuclear translocation of β-catenin in SW480/NC, SW480/Zip-371-5p and SW480/Zip-371-5p/shSOX2 cells. Red scale bars represent 10 μm, whereas yellow scale bars represent 5 μm. (C) Expression of OCT4, SOX2 and CD133 in SW480 cells treated with Zip-371-5p or Zip-371-5p/shSOX2 by qRT-PCR. The relative expression levels in NC cells were normalized to 1. (D) HCT116/NC, HCT116/Zip-371-5p and HCT116/Zip-371-5p/shSOX2 cells (1×106) were injected in the subcutaneous tissue of nude mice (n = 5). The weight of subcutaneous tumors was measured. (E) HCT116/NC, HCT116/Zip-371-5p and HCT116/Zip-371-5p/shSOX2 cells (2×106) were injected into the tail vein of nude mice (n = 5) for 2 months. Yellow arrows in top panels point at lung metastatic nodules. Scale bars in bottom panels represent 100 μm. The number of lung metastatic nodules per mouse was counted under the microscope. * P < 0.05, ** P < 0.01. Data represent the mean ± SD.
Mentions: Transcriptional regulator SOX2 was identified as an oncogene in many cancers, including colon cancer. It plays an important role in cancer stem cell, EMT and metastasis of colon cancer [21-23]. To determine whether cancer cell phenotypes associated with miR-371-5p expression could be reversed via restoration of SOX2, we transfected miR-371-5p-depleting cells with shRNAs toward SOX2 in HCT116 and SW480 cells (Supplementary Figure 5B). In miR-371-5p-depleting cells, depletion of SOX2 reversed, at least partially, miR-371-5p knockdown-imposed proliferation and invasion (Supplementary Figure 5C). SOX2 rescued miR-371-5p's dependent MET morphogical changes (Supplementary Figure 5D). Reintroduction of SOX2 shRNAs in miR-371-5p depleting cells led to down-regulations of Vimentin and Slug, up-regulation of E-cadherin (Figure 5A and 5B), cytoplasmic translocation of β-catenin (Figure 5B), TCF/LEF transcriptional inactivation (Supplementary Figure 5E) and decreased expression of target genes CyclinD1, C-myc and DKK1 (Figure 5A). Moreover, depletion of SOX2 abolished the promoting effect of miR-371-5p knockdown on stemness, as shown by decreased expression of stem cell pluripotency factors OCT4, SOX2 and stem cell marker CD133 (Figure 5C) and decreased sphere-forming capacity (Supplementary Figure 5F). We also evaluated whether SOX2 rescued miR-371-5p's effects on tumor growth and metastasis in vivo. Depletion of SOX2 reversed, at least in part, tumor growth and lung metastases resulting from knockdown of miR-371-5p (Figure 5D and 5E). These results reveal that miR-371-5p is a crucial and unexpected switch for EMT, stemness and metastasis of CRC via repression of SOX2.

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