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microRNA-182 targets special AT-rich sequence-binding protein 2 to promote colorectal cancer proliferation and metastasis.

Yang MH, Yu J, Jiang DM, Li WL, Wang S, Ding YQ - J Transl Med (2014)

Bottom Line: We found that miR-182 expression is increased in CRC cells that originated from metastatic foci and human primary CRC tissues with lymph node metastases.Further research showed that miR-182 could directly target the 3'untranslated region (3'UTR) of SATB2 mRNA and subsequently repress both the mRNA and protein expressions of SATB2, which we identified in previous studies as a CRC metastasis-associated protein.Restoring SATB2 expression could reverse the effects of miR-182 on CRC cell proliferation and migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. shuangw@126.com.

ABSTRACT

Background: Increasing evidence has revealed that microRNAs (miRNA) played a pivotal role in regulating cancer cell proliferation and metastasis. The deregulation of miR-182 has been identified in colorectal cancer (CRC). However, the role and mechanism of miR-182 in CRC have not been completely understood yet.

Methods: The expression levels of miR-182 in CRC tissues and CRC cell lines were examined by performing stem-loop quantitative RT-PCR. The stable over-expression miR-182 cell lines and control cell lines were constructed by lentivirus infection. Subsequently, CCK-8 assay, plate colony formation assay, cell migration, invasion assay and experimental animal models were performed to detect the biological functions of miR-182 in vitro and in vivo. A luciferase reporter assay was conducted to confirm target associations. Western blot and immunohistochemical analysis were performed to examine the expression changes of molecular markers that are regulated by miR-182.

Results: We found that miR-182 expression is increased in CRC cells that originated from metastatic foci and human primary CRC tissues with lymph node metastases. The ectopic expression of miR-182 enhanced cell proliferation, invasion, and migration in vitro. Stable overexpression of miR-182 also facilitated tumor growth and metastasis in vivo too. Further research showed that miR-182 could directly target the 3'untranslated region (3'UTR) of SATB2 mRNA and subsequently repress both the mRNA and protein expressions of SATB2, which we identified in previous studies as a CRC metastasis-associated protein. Restoring SATB2 expression could reverse the effects of miR-182 on CRC cell proliferation and migration. Investigations of possible mechanisms underlying these behaviors induced by miR-182 revealed that miR-182 induced epithelial-mesenchymal transition (EMT) by modulating the expression of key cellular molecules in EMT.

Conclusions: Our results illustrated that the up-regulation of miR-182 played a pivotal role in CRC tumorigenesis and metastasis, which suggesting a potential implication of miR-182 in the molecular therapy for CRC.

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SATB2 was a direct target of miR-182 in CRC cells. (A) Schematic illustration of the SATB2 3’UTR- containing reporter constructs. The mutant binding site was underlined. wt denotes wild type; mt denotes mutant. (B) Luciferase reporter assays in SW480 cells with cotransfection of wt or mt 3’UTR and miRNA as indicated. (C, D) Levels of SATB2 mRNA and protein after miR-182 ectopic over-expression in CRC cells were examined by real-time RT-PCR (C) and western blot (D). The immunosignal was quantified using Quantity One Software and relative protein abundance was determined by normalizing with β-actin. (E) A statistically significant inverse correlation was reported between miR-182 and SATB2 mRNA levels in CRC tissues (Spearman's correlation analysis, r = -0.7007; p = 0.001, n = 18).
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Figure 5: SATB2 was a direct target of miR-182 in CRC cells. (A) Schematic illustration of the SATB2 3’UTR- containing reporter constructs. The mutant binding site was underlined. wt denotes wild type; mt denotes mutant. (B) Luciferase reporter assays in SW480 cells with cotransfection of wt or mt 3’UTR and miRNA as indicated. (C, D) Levels of SATB2 mRNA and protein after miR-182 ectopic over-expression in CRC cells were examined by real-time RT-PCR (C) and western blot (D). The immunosignal was quantified using Quantity One Software and relative protein abundance was determined by normalizing with β-actin. (E) A statistically significant inverse correlation was reported between miR-182 and SATB2 mRNA levels in CRC tissues (Spearman's correlation analysis, r = -0.7007; p = 0.001, n = 18).

Mentions: To explore the mechanism that facilitates the effects on proliferation and migration induced by miR-182, we analyzed the putative miR-182 targets by a bioinformatic screen that was developed using three algorithms: TargetScan, PicTar, and miRanda. We focused on SATB2 from which orginated 3’UTR containing a binding site of miR-182. The 3’ UTR sequence of SATB2 (wt 3’UTR) or the mutant sequence (mt 3’UTR) was cloned into a luciferase reporter vector (Figure 5A). A luciferase reporter assay was carried out to determine whether miR-182 can directly regulate the expression of SATB2 in CRC SW480 cells. The results indicated a significant decrease in luciferase activity that was induced by miR-182 over-expression when compared with the empty vector control (p < 0.001, Figure 5B). Compared with anti-miR-control, the transfection with anti-miR-182 in SW480 cells led to a significant increase in luciferase activity (p = 0.009, Figure 5B). However, the activity of mt 3UTR vector was unaffected with a simultaneous transfection with miR-182 (p > 0.05, Figure 5B).


microRNA-182 targets special AT-rich sequence-binding protein 2 to promote colorectal cancer proliferation and metastasis.

Yang MH, Yu J, Jiang DM, Li WL, Wang S, Ding YQ - J Transl Med (2014)

SATB2 was a direct target of miR-182 in CRC cells. (A) Schematic illustration of the SATB2 3’UTR- containing reporter constructs. The mutant binding site was underlined. wt denotes wild type; mt denotes mutant. (B) Luciferase reporter assays in SW480 cells with cotransfection of wt or mt 3’UTR and miRNA as indicated. (C, D) Levels of SATB2 mRNA and protein after miR-182 ectopic over-expression in CRC cells were examined by real-time RT-PCR (C) and western blot (D). The immunosignal was quantified using Quantity One Software and relative protein abundance was determined by normalizing with β-actin. (E) A statistically significant inverse correlation was reported between miR-182 and SATB2 mRNA levels in CRC tissues (Spearman's correlation analysis, r = -0.7007; p = 0.001, n = 18).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4020308&req=5

Figure 5: SATB2 was a direct target of miR-182 in CRC cells. (A) Schematic illustration of the SATB2 3’UTR- containing reporter constructs. The mutant binding site was underlined. wt denotes wild type; mt denotes mutant. (B) Luciferase reporter assays in SW480 cells with cotransfection of wt or mt 3’UTR and miRNA as indicated. (C, D) Levels of SATB2 mRNA and protein after miR-182 ectopic over-expression in CRC cells were examined by real-time RT-PCR (C) and western blot (D). The immunosignal was quantified using Quantity One Software and relative protein abundance was determined by normalizing with β-actin. (E) A statistically significant inverse correlation was reported between miR-182 and SATB2 mRNA levels in CRC tissues (Spearman's correlation analysis, r = -0.7007; p = 0.001, n = 18).
Mentions: To explore the mechanism that facilitates the effects on proliferation and migration induced by miR-182, we analyzed the putative miR-182 targets by a bioinformatic screen that was developed using three algorithms: TargetScan, PicTar, and miRanda. We focused on SATB2 from which orginated 3’UTR containing a binding site of miR-182. The 3’ UTR sequence of SATB2 (wt 3’UTR) or the mutant sequence (mt 3’UTR) was cloned into a luciferase reporter vector (Figure 5A). A luciferase reporter assay was carried out to determine whether miR-182 can directly regulate the expression of SATB2 in CRC SW480 cells. The results indicated a significant decrease in luciferase activity that was induced by miR-182 over-expression when compared with the empty vector control (p < 0.001, Figure 5B). Compared with anti-miR-control, the transfection with anti-miR-182 in SW480 cells led to a significant increase in luciferase activity (p = 0.009, Figure 5B). However, the activity of mt 3UTR vector was unaffected with a simultaneous transfection with miR-182 (p > 0.05, Figure 5B).

Bottom Line: We found that miR-182 expression is increased in CRC cells that originated from metastatic foci and human primary CRC tissues with lymph node metastases.Further research showed that miR-182 could directly target the 3'untranslated region (3'UTR) of SATB2 mRNA and subsequently repress both the mRNA and protein expressions of SATB2, which we identified in previous studies as a CRC metastasis-associated protein.Restoring SATB2 expression could reverse the effects of miR-182 on CRC cell proliferation and migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. shuangw@126.com.

ABSTRACT

Background: Increasing evidence has revealed that microRNAs (miRNA) played a pivotal role in regulating cancer cell proliferation and metastasis. The deregulation of miR-182 has been identified in colorectal cancer (CRC). However, the role and mechanism of miR-182 in CRC have not been completely understood yet.

Methods: The expression levels of miR-182 in CRC tissues and CRC cell lines were examined by performing stem-loop quantitative RT-PCR. The stable over-expression miR-182 cell lines and control cell lines were constructed by lentivirus infection. Subsequently, CCK-8 assay, plate colony formation assay, cell migration, invasion assay and experimental animal models were performed to detect the biological functions of miR-182 in vitro and in vivo. A luciferase reporter assay was conducted to confirm target associations. Western blot and immunohistochemical analysis were performed to examine the expression changes of molecular markers that are regulated by miR-182.

Results: We found that miR-182 expression is increased in CRC cells that originated from metastatic foci and human primary CRC tissues with lymph node metastases. The ectopic expression of miR-182 enhanced cell proliferation, invasion, and migration in vitro. Stable overexpression of miR-182 also facilitated tumor growth and metastasis in vivo too. Further research showed that miR-182 could directly target the 3'untranslated region (3'UTR) of SATB2 mRNA and subsequently repress both the mRNA and protein expressions of SATB2, which we identified in previous studies as a CRC metastasis-associated protein. Restoring SATB2 expression could reverse the effects of miR-182 on CRC cell proliferation and migration. Investigations of possible mechanisms underlying these behaviors induced by miR-182 revealed that miR-182 induced epithelial-mesenchymal transition (EMT) by modulating the expression of key cellular molecules in EMT.

Conclusions: Our results illustrated that the up-regulation of miR-182 played a pivotal role in CRC tumorigenesis and metastasis, which suggesting a potential implication of miR-182 in the molecular therapy for CRC.

Show MeSH
Related in: MedlinePlus