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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

miR-371-5p is sufficient to suppress stem cell properties and metastasis of CRC cells(A) Expression of OCT4, SOX2 and CD133 in SW480 cells treated with Zip-371-5p by qRT-PCR. The relative expression levels in NC cells were normalized to 1. (B) Lovo/mock and Lovo/miR-371-5p cells (1×106), and HCT116/NC and HCT116/Zip-371-5p cells (1×106) were injected in the hindlimbs of nude mice (n = 5). The weight of subcutaneous tumors was measured. (C) Local invasion of subcutaneous tumors in HCT116/NC group or HCT116/Zip-371-5p group by HE staining. Scale bars represent 50 μm. (D) Intestinal and hepatic metastatic nodules after subcutaneous tumors of HCT116/NC and HCT116/Zip-371-5p were transplanted in the mesentery at the tail end of cecum (n = 5) for six weeks. Yellow arrows in top panels point at metastatic nodules. Scale bars in bottom panels represent 50 μm. The number of intestinal or hepatic metastatic nodules per mouse was counted under the microscope. (E) HCT116/NC and HCT116/Zip-371-5p cells (2×106) were injected in the tail vein of nude mice (n = 5) for 2 months. Yellow arrows point at lung metastatic nodules. Scale bars 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 2: miR-371-5p is sufficient to suppress stem cell properties and metastasis of CRC cells(A) Expression of OCT4, SOX2 and CD133 in SW480 cells treated with Zip-371-5p by qRT-PCR. The relative expression levels in NC cells were normalized to 1. (B) Lovo/mock and Lovo/miR-371-5p cells (1×106), and HCT116/NC and HCT116/Zip-371-5p cells (1×106) were injected in the hindlimbs of nude mice (n = 5). The weight of subcutaneous tumors was measured. (C) Local invasion of subcutaneous tumors in HCT116/NC group or HCT116/Zip-371-5p group by HE staining. Scale bars represent 50 μm. (D) Intestinal and hepatic metastatic nodules after subcutaneous tumors of HCT116/NC and HCT116/Zip-371-5p were transplanted in the mesentery at the tail end of cecum (n = 5) for six weeks. Yellow arrows in top panels point at metastatic nodules. Scale bars in bottom panels represent 50 μm. The number of intestinal or hepatic metastatic nodules per mouse was counted under the microscope. (E) HCT116/NC and HCT116/Zip-371-5p cells (2×106) were injected in the tail vein of nude mice (n = 5) for 2 months. Yellow arrows point at lung metastatic nodules. Scale bars 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: The EMT is known to be a central mechanism responsible for invasiveness and metastasis of breast cancer and is also associated with normal and malignant mammary stem cell function [17]. Since the microRNA-371-373 cluster is thought to be involved in stem cell pluripotency [18, 19], we speculated that miR-371-5p could also induce stemness. Substantially, miR-371-5p knockdown resulted in up-regulations of stem cell pluripotency factors OCT4 and SOX2 and stem cell marker CD133 (Figure 2A). Over-expression of miR-371-5p decreased the ability of cells to develop into spheres, and vice versa (Supplementary Figure 2E). Because of its effects on in vitro traits associated with high-grade malignancy, we asked whether miR-371-5p could inhibit tumor growth and metastasis in vivo. MiR-371-5p over-expressing cells or miR-371-5p silencing cells were injected into the subcutaneous site of mice, respectively. Noticeably, over-expression of miR-371-5p inhibited primary tumor growth, while silence of miR-371-5p showed the opposite effect (Figure 2B). Unexpectedly, control cell primary tumors were well encapsulated and noninvasive, while miR-371-5p depleting tumors displayed evidence of local invasion (Figure 2C). Accordingly, we used orthotopic transplant to evaluate metastasis in nude mice. Orthotopic transplant of miR-371-5p depleting tumors into nude mice gave rise to metastatic nodules similar in size to control tumors, and showed a significant increase in the number of spontaneous intestinal and liver metastases (Figure 2D). Next, we also examined whether miR-371-5p only limits the ability of tumor cells to disseminate from the primary site, or if it also affects the late stages of metastasis, for example, colonization. We inoculated miR-371-5p depleting cells or control cells into the circulation of nude mice through the tail vein. Knockdown of miR-371-5p in HCT116 cells strikingly enhanced their capacity to seed lung metastases (Figure 2E). MiR-371-5p depleting metastatic tumors in intestine, liver or lung tended to infiltrate the adjacent tissues compared to control groups (Figure 2D and 2E). The above results indicate that miR-371-5p inhibited stemness and metastasis of CRC cells.


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)

miR-371-5p is sufficient to suppress stem cell properties and metastasis of CRC cells(A) Expression of OCT4, SOX2 and CD133 in SW480 cells treated with Zip-371-5p by qRT-PCR. The relative expression levels in NC cells were normalized to 1. (B) Lovo/mock and Lovo/miR-371-5p cells (1×106), and HCT116/NC and HCT116/Zip-371-5p cells (1×106) were injected in the hindlimbs of nude mice (n = 5). The weight of subcutaneous tumors was measured. (C) Local invasion of subcutaneous tumors in HCT116/NC group or HCT116/Zip-371-5p group by HE staining. Scale bars represent 50 μm. (D) Intestinal and hepatic metastatic nodules after subcutaneous tumors of HCT116/NC and HCT116/Zip-371-5p were transplanted in the mesentery at the tail end of cecum (n = 5) for six weeks. Yellow arrows in top panels point at metastatic nodules. Scale bars in bottom panels represent 50 μm. The number of intestinal or hepatic metastatic nodules per mouse was counted under the microscope. (E) HCT116/NC and HCT116/Zip-371-5p cells (2×106) were injected in the tail vein of nude mice (n = 5) for 2 months. Yellow arrows point at lung metastatic nodules. Scale bars 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 2: miR-371-5p is sufficient to suppress stem cell properties and metastasis of CRC cells(A) Expression of OCT4, SOX2 and CD133 in SW480 cells treated with Zip-371-5p by qRT-PCR. The relative expression levels in NC cells were normalized to 1. (B) Lovo/mock and Lovo/miR-371-5p cells (1×106), and HCT116/NC and HCT116/Zip-371-5p cells (1×106) were injected in the hindlimbs of nude mice (n = 5). The weight of subcutaneous tumors was measured. (C) Local invasion of subcutaneous tumors in HCT116/NC group or HCT116/Zip-371-5p group by HE staining. Scale bars represent 50 μm. (D) Intestinal and hepatic metastatic nodules after subcutaneous tumors of HCT116/NC and HCT116/Zip-371-5p were transplanted in the mesentery at the tail end of cecum (n = 5) for six weeks. Yellow arrows in top panels point at metastatic nodules. Scale bars in bottom panels represent 50 μm. The number of intestinal or hepatic metastatic nodules per mouse was counted under the microscope. (E) HCT116/NC and HCT116/Zip-371-5p cells (2×106) were injected in the tail vein of nude mice (n = 5) for 2 months. Yellow arrows point at lung metastatic nodules. Scale bars 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: The EMT is known to be a central mechanism responsible for invasiveness and metastasis of breast cancer and is also associated with normal and malignant mammary stem cell function [17]. Since the microRNA-371-373 cluster is thought to be involved in stem cell pluripotency [18, 19], we speculated that miR-371-5p could also induce stemness. Substantially, miR-371-5p knockdown resulted in up-regulations of stem cell pluripotency factors OCT4 and SOX2 and stem cell marker CD133 (Figure 2A). Over-expression of miR-371-5p decreased the ability of cells to develop into spheres, and vice versa (Supplementary Figure 2E). Because of its effects on in vitro traits associated with high-grade malignancy, we asked whether miR-371-5p could inhibit tumor growth and metastasis in vivo. MiR-371-5p over-expressing cells or miR-371-5p silencing cells were injected into the subcutaneous site of mice, respectively. Noticeably, over-expression of miR-371-5p inhibited primary tumor growth, while silence of miR-371-5p showed the opposite effect (Figure 2B). Unexpectedly, control cell primary tumors were well encapsulated and noninvasive, while miR-371-5p depleting tumors displayed evidence of local invasion (Figure 2C). Accordingly, we used orthotopic transplant to evaluate metastasis in nude mice. Orthotopic transplant of miR-371-5p depleting tumors into nude mice gave rise to metastatic nodules similar in size to control tumors, and showed a significant increase in the number of spontaneous intestinal and liver metastases (Figure 2D). Next, we also examined whether miR-371-5p only limits the ability of tumor cells to disseminate from the primary site, or if it also affects the late stages of metastasis, for example, colonization. We inoculated miR-371-5p depleting cells or control cells into the circulation of nude mice through the tail vein. Knockdown of miR-371-5p in HCT116 cells strikingly enhanced their capacity to seed lung metastases (Figure 2E). MiR-371-5p depleting metastatic tumors in intestine, liver or lung tended to infiltrate the adjacent tissues compared to control groups (Figure 2D and 2E). The above results indicate that miR-371-5p inhibited stemness and metastasis of CRC cells.

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