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miR-942 promotes cancer stem cell-like traits in esophageal squamous cell carcinoma through activation of Wnt/β-catenin signalling pathway.

Ge C, Wu S, Wang W, Liu Z, Zhang J, Wang Z, Li R, Zhang Z, Li Z, Dong S, Wang Y, Xue Y, Yang J, Tan Q, Wang Z, Song X - Oncotarget (2015)

Bottom Line: Here, we report that miR-942 is significantly upregulated in esophageal squamous cell carcinoma (ESCC), and miR-942 levels are associated with poor prognosis in ESCC patients.In addition, our results indicate that c-myc directly binds to the miR-942 promoter and promotes its expression.Taken together, our findings establish an oncogenic role of miR-942 in ESCC and indicate that miR-942 might be an effective therapeutic target for ESCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province), Kunming, Yunnan, People's Republic of China.

ABSTRACT
The Wnt/β-catenin signalling pathway is known to play a vital role in the maintenance of cancer stem cells (CSCs), which are reported to be the origin of malignant cancers, and result in poor prognosis of multiple kinds of cancer. Therefore, it is of great importance to illuminate the mechanism by which the Wnt/β-catenin pathway regulates the cancer stem cell-like traits in cancers. Here, we report that miR-942 is significantly upregulated in esophageal squamous cell carcinoma (ESCC), and miR-942 levels are associated with poor prognosis in ESCC patients. Overexpression of miR-942 promotes, whereas inhibition of miR-942 decreases, the tumor sphere formation, the CD90+ subpopulation cells and the expression of pluripotency associated markers. Moreover, in vivo assay shows that miR-942 overexpressing cells form larger tumors and display higher tumourigenesis. Furthermore, we demonstrate that miR-942 upregulates the Wnt/β-catenin signaling activity via directly targeting sFRP4, GSK3β and TLE1, which are multiple level negative regulators of the Wnt/β-catenin signaling cascade. In addition, our results indicate that c-myc directly binds to the miR-942 promoter and promotes its expression. Taken together, our findings establish an oncogenic role of miR-942 in ESCC and indicate that miR-942 might be an effective therapeutic target for ESCC.

No MeSH data available.


Related in: MedlinePlus

miR-942 directly targets sFRP4, GSK3β, and TLE1(A) Indicated cells transfected with TOP flash/FOP flash and Renilla pRL-TK plasmids were subjected to dual-luciferase assays 48 hours after transfection. Reporter activity detected was normalized by Renilla luciferase activity. (B) Predicted miR-942 target sequences in the 3′-UTRs of sFRP4, GSK3β and TLE1 and mutant containing three altered nucleotides in the seed sequence of miR-942 (miR-942-mu). (C) Western blotting analysis of sFRP4, GSK3β, TLE1 and nuclear β-catenin in the indicated cells. α-Tubulin served as the loading control. (D) Luciferase activity of targets 3′UTR in the indicated cells. (E) miRNP immunoprecipitation assay showed associations of miR-942 with sFRP4, GSK3β and TLE1. GAPDH and 5s rRNA served as a negative control. (F) Quantification of indicated tumoursphere cells with specific siRNA transfection. (G) Luciferase activity of TOP flash/FOP flash in the indicated cells. Each bar represents the mean ± SD of three independent experiments. * P < 0.05.
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Figure 5: miR-942 directly targets sFRP4, GSK3β, and TLE1(A) Indicated cells transfected with TOP flash/FOP flash and Renilla pRL-TK plasmids were subjected to dual-luciferase assays 48 hours after transfection. Reporter activity detected was normalized by Renilla luciferase activity. (B) Predicted miR-942 target sequences in the 3′-UTRs of sFRP4, GSK3β and TLE1 and mutant containing three altered nucleotides in the seed sequence of miR-942 (miR-942-mu). (C) Western blotting analysis of sFRP4, GSK3β, TLE1 and nuclear β-catenin in the indicated cells. α-Tubulin served as the loading control. (D) Luciferase activity of targets 3′UTR in the indicated cells. (E) miRNP immunoprecipitation assay showed associations of miR-942 with sFRP4, GSK3β and TLE1. GAPDH and 5s rRNA served as a negative control. (F) Quantification of indicated tumoursphere cells with specific siRNA transfection. (G) Luciferase activity of TOP flash/FOP flash in the indicated cells. Each bar represents the mean ± SD of three independent experiments. * P < 0.05.

Mentions: As Wnt/β-catenin signalling pathways is the major molecular pathway controlling CSCs and promotes tumour progression [27], we hypothesis miR-942 might regulate Wnt/β-catenin signalling. As expected, miR-942 overexpression markedly increased the luciferase activity of the TOP flash/FOP flash reporter. Conversely, transfection of antagomir-942 reduced the luciferase activity, and mutant miR-942 had no effect (Fig. 5A), indicating that miR-942 activates Wnt/β-catenin signalling. Using publicly available algorithms (TargetScan6.2 and miRanda), we found that sFRP4, GSK3β, and TLE1, the multiple negative regulators of Wnt/β-catenin pathway, might be the potential targets of miR-942 (Fig. 5B). Western blot analysis revealed that the expressions of sFRP4, GSK3β, and TLE1 was drastically decreased in miR-942-transduced cells but pronouncedly elevated in antagomir-942 cells, compared with their corresponding control cells (Fig. 5C). In addition, results of the luciferase reporter activities, which linked to the 3′UTRs of sFRP4, GSK3β, and TLE1, indicated that miR-942 overexpression significantly repressed, whereas inhibition of endogenous miR-942 increased, the luciferase activity of 3′UTRs, and ectopically expressing mutant miR-942 had no inhibitory effect on the 3′UTRs luciferase activity (Fig. 5D). Importantly, results of the miRNP immunoprecipitation assay showed that miR-942 selectively associated with sFRP4, GSK3β, and TLE1, but not GAPDH or 5s rRNA (Fig. 5E). Hence, our results indicate that sFRP4, GSK3β, and TLE1 are the bona fide targets of miR-942.


miR-942 promotes cancer stem cell-like traits in esophageal squamous cell carcinoma through activation of Wnt/β-catenin signalling pathway.

Ge C, Wu S, Wang W, Liu Z, Zhang J, Wang Z, Li R, Zhang Z, Li Z, Dong S, Wang Y, Xue Y, Yang J, Tan Q, Wang Z, Song X - Oncotarget (2015)

miR-942 directly targets sFRP4, GSK3β, and TLE1(A) Indicated cells transfected with TOP flash/FOP flash and Renilla pRL-TK plasmids were subjected to dual-luciferase assays 48 hours after transfection. Reporter activity detected was normalized by Renilla luciferase activity. (B) Predicted miR-942 target sequences in the 3′-UTRs of sFRP4, GSK3β and TLE1 and mutant containing three altered nucleotides in the seed sequence of miR-942 (miR-942-mu). (C) Western blotting analysis of sFRP4, GSK3β, TLE1 and nuclear β-catenin in the indicated cells. α-Tubulin served as the loading control. (D) Luciferase activity of targets 3′UTR in the indicated cells. (E) miRNP immunoprecipitation assay showed associations of miR-942 with sFRP4, GSK3β and TLE1. GAPDH and 5s rRNA served as a negative control. (F) Quantification of indicated tumoursphere cells with specific siRNA transfection. (G) Luciferase activity of TOP flash/FOP flash in the indicated cells. Each bar represents the mean ± SD of three independent experiments. * P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4484432&req=5

Figure 5: miR-942 directly targets sFRP4, GSK3β, and TLE1(A) Indicated cells transfected with TOP flash/FOP flash and Renilla pRL-TK plasmids were subjected to dual-luciferase assays 48 hours after transfection. Reporter activity detected was normalized by Renilla luciferase activity. (B) Predicted miR-942 target sequences in the 3′-UTRs of sFRP4, GSK3β and TLE1 and mutant containing three altered nucleotides in the seed sequence of miR-942 (miR-942-mu). (C) Western blotting analysis of sFRP4, GSK3β, TLE1 and nuclear β-catenin in the indicated cells. α-Tubulin served as the loading control. (D) Luciferase activity of targets 3′UTR in the indicated cells. (E) miRNP immunoprecipitation assay showed associations of miR-942 with sFRP4, GSK3β and TLE1. GAPDH and 5s rRNA served as a negative control. (F) Quantification of indicated tumoursphere cells with specific siRNA transfection. (G) Luciferase activity of TOP flash/FOP flash in the indicated cells. Each bar represents the mean ± SD of three independent experiments. * P < 0.05.
Mentions: As Wnt/β-catenin signalling pathways is the major molecular pathway controlling CSCs and promotes tumour progression [27], we hypothesis miR-942 might regulate Wnt/β-catenin signalling. As expected, miR-942 overexpression markedly increased the luciferase activity of the TOP flash/FOP flash reporter. Conversely, transfection of antagomir-942 reduced the luciferase activity, and mutant miR-942 had no effect (Fig. 5A), indicating that miR-942 activates Wnt/β-catenin signalling. Using publicly available algorithms (TargetScan6.2 and miRanda), we found that sFRP4, GSK3β, and TLE1, the multiple negative regulators of Wnt/β-catenin pathway, might be the potential targets of miR-942 (Fig. 5B). Western blot analysis revealed that the expressions of sFRP4, GSK3β, and TLE1 was drastically decreased in miR-942-transduced cells but pronouncedly elevated in antagomir-942 cells, compared with their corresponding control cells (Fig. 5C). In addition, results of the luciferase reporter activities, which linked to the 3′UTRs of sFRP4, GSK3β, and TLE1, indicated that miR-942 overexpression significantly repressed, whereas inhibition of endogenous miR-942 increased, the luciferase activity of 3′UTRs, and ectopically expressing mutant miR-942 had no inhibitory effect on the 3′UTRs luciferase activity (Fig. 5D). Importantly, results of the miRNP immunoprecipitation assay showed that miR-942 selectively associated with sFRP4, GSK3β, and TLE1, but not GAPDH or 5s rRNA (Fig. 5E). Hence, our results indicate that sFRP4, GSK3β, and TLE1 are the bona fide targets of miR-942.

Bottom Line: Here, we report that miR-942 is significantly upregulated in esophageal squamous cell carcinoma (ESCC), and miR-942 levels are associated with poor prognosis in ESCC patients.In addition, our results indicate that c-myc directly binds to the miR-942 promoter and promotes its expression.Taken together, our findings establish an oncogenic role of miR-942 in ESCC and indicate that miR-942 might be an effective therapeutic target for ESCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province), Kunming, Yunnan, People's Republic of China.

ABSTRACT
The Wnt/β-catenin signalling pathway is known to play a vital role in the maintenance of cancer stem cells (CSCs), which are reported to be the origin of malignant cancers, and result in poor prognosis of multiple kinds of cancer. Therefore, it is of great importance to illuminate the mechanism by which the Wnt/β-catenin pathway regulates the cancer stem cell-like traits in cancers. Here, we report that miR-942 is significantly upregulated in esophageal squamous cell carcinoma (ESCC), and miR-942 levels are associated with poor prognosis in ESCC patients. Overexpression of miR-942 promotes, whereas inhibition of miR-942 decreases, the tumor sphere formation, the CD90+ subpopulation cells and the expression of pluripotency associated markers. Moreover, in vivo assay shows that miR-942 overexpressing cells form larger tumors and display higher tumourigenesis. Furthermore, we demonstrate that miR-942 upregulates the Wnt/β-catenin signaling activity via directly targeting sFRP4, GSK3β and TLE1, which are multiple level negative regulators of the Wnt/β-catenin signaling cascade. In addition, our results indicate that c-myc directly binds to the miR-942 promoter and promotes its expression. Taken together, our findings establish an oncogenic role of miR-942 in ESCC and indicate that miR-942 might be an effective therapeutic target for ESCC.

No MeSH data available.


Related in: MedlinePlus