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Downregulation of miR-432 activates Wnt/β-catenin signaling and promotes human hepatocellular carcinoma proliferation.

Jiang N, Chen WJ, Zhang JW, Xu C, Zeng XC, Zhang T, Li Y, Wang GY - Oncotarget (2015)

Bottom Line: Sustained cell growth and proliferation, one of the hallmarks of cancer, is considered to responsible for cancer-related deaths by disorganizing the balance of growth promotion and growth limitation.Elucidating the molecular mechanism of this abnormality in hepatocellular carcinoma carcinogenesis may improve diagnostic and therapeutic strategies for this malignancy.Furthermore, miR-432 directly targeted and suppressed multiple regulators of the Wnt/β-catenin signaling cascade, including LRP6, TRIM29 and Pygo2, which subsequently deactivated Wnt/β-catenin signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatic Surgery, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, China.

ABSTRACT
Sustained cell growth and proliferation, one of the hallmarks of cancer, is considered to responsible for cancer-related deaths by disorganizing the balance of growth promotion and growth limitation. Aberrant activation of the Wnt/β-catenin signaling pathway leads to cell proliferation, growth and survival, and promotes the development of various human tumors, including hepatocellular carcinoma. Elucidating the molecular mechanism of this abnormality in hepatocellular carcinoma carcinogenesis may improve diagnostic and therapeutic strategies for this malignancy. Herein, we report that the expression of miR-432 was markedly downregulated in hepatocellular carcinoma cell lines and tissues, and upregulation of miR-432 inhibited, whereas downregulation of miR-432 enhanced the proliferation and tumorigenicity of hepatocellular carcinoma cells both in vitro and in vivo. Furthermore, miR-432 directly targeted and suppressed multiple regulators of the Wnt/β-catenin signaling cascade, including LRP6, TRIM29 and Pygo2, which subsequently deactivated Wnt/β-catenin signaling pathway. Finally, miR-432 expression was inversely correlated with three targets in clinical hepatocellular carcinoma samples. These results demonstrated that miR-432 functions as a tumor-suppressive miRNA by suppressing Wnt/β-catenin signaling activation and may represent a therapeutic target for hepatocellular carcinoma.

No MeSH data available.


Related in: MedlinePlus

LRP6, TRIM29 and Pygo2 are direct targets of miR-432(A). Indicated cells transfected with TOPflash or FOPflash 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). Altered nuclear translocation of β-catenin in response to deregulated miR-432 expression. Nuclear fractions of indicated cells were analyzed by western blotting; p84 was used as the loading control. (C). Predicted miR-432 target sequences in the 3′UTRs of LRP6, TRIM29 and Pygo2. The miR-432 mutant (miR-432-mut) contained three altered nucleotides in the seed sequence. (D). Western blotting analysis of LRP6, TRIM29 and Pygo2 expression in the indicated HCC cells. α-Tubulin served as loading control. (E). Luciferase assay of pGL3- LRP6-3′UTR, pGL3- TRIM29-3′UTR and pGL3- Pygo2-3′UTR reporter cotransfected with miR-432, miR-432-inhibitor and miR-432-mut oligonucleotides in HCC cells. (F). Luciferase assay of TCF/LEF transcriptional activity in indicated cells transfected with specific siRNA. Error bars represent the mean ± SD from of three independent experiments. *P < 0.05.
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Figure 5: LRP6, TRIM29 and Pygo2 are direct targets of miR-432(A). Indicated cells transfected with TOPflash or FOPflash 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). Altered nuclear translocation of β-catenin in response to deregulated miR-432 expression. Nuclear fractions of indicated cells were analyzed by western blotting; p84 was used as the loading control. (C). Predicted miR-432 target sequences in the 3′UTRs of LRP6, TRIM29 and Pygo2. The miR-432 mutant (miR-432-mut) contained three altered nucleotides in the seed sequence. (D). Western blotting analysis of LRP6, TRIM29 and Pygo2 expression in the indicated HCC cells. α-Tubulin served as loading control. (E). Luciferase assay of pGL3- LRP6-3′UTR, pGL3- TRIM29-3′UTR and pGL3- Pygo2-3′UTR reporter cotransfected with miR-432, miR-432-inhibitor and miR-432-mut oligonucleotides in HCC cells. (F). Luciferase assay of TCF/LEF transcriptional activity in indicated cells transfected with specific siRNA. Error bars represent the mean ± SD from of three independent experiments. *P < 0.05.

Mentions: The wnt/β-catenin pathway is an important regulator of tumor initiation and progression, and palys an important role in cell proliferation. Therefore, we detect the effect of miR-432 in regulating the wnt/β-catenin signaling. As shown in Figure 5A, miR-432 overexpression markedly decreased the luciferase activity of TOPflash or FOPflash reporter; conversely, transfection of miR-432 inhibitor increased the luciferase activity of TOPflash or FOPflash reporter, compare with vector or negative control, respectively. Furthermore, cellular fractionation showed that miR-432 overexpression inhibit nuclear accumulation of β-catenin (Figure 5B), indicating that miR-432 deactivates Wnt/β-catenin pathway by inhibiting β-catenin nuclear accumulation.


Downregulation of miR-432 activates Wnt/β-catenin signaling and promotes human hepatocellular carcinoma proliferation.

Jiang N, Chen WJ, Zhang JW, Xu C, Zeng XC, Zhang T, Li Y, Wang GY - Oncotarget (2015)

LRP6, TRIM29 and Pygo2 are direct targets of miR-432(A). Indicated cells transfected with TOPflash or FOPflash 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). Altered nuclear translocation of β-catenin in response to deregulated miR-432 expression. Nuclear fractions of indicated cells were analyzed by western blotting; p84 was used as the loading control. (C). Predicted miR-432 target sequences in the 3′UTRs of LRP6, TRIM29 and Pygo2. The miR-432 mutant (miR-432-mut) contained three altered nucleotides in the seed sequence. (D). Western blotting analysis of LRP6, TRIM29 and Pygo2 expression in the indicated HCC cells. α-Tubulin served as loading control. (E). Luciferase assay of pGL3- LRP6-3′UTR, pGL3- TRIM29-3′UTR and pGL3- Pygo2-3′UTR reporter cotransfected with miR-432, miR-432-inhibitor and miR-432-mut oligonucleotides in HCC cells. (F). Luciferase assay of TCF/LEF transcriptional activity in indicated cells transfected with specific siRNA. Error bars represent the mean ± SD from of three independent experiments. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480722&req=5

Figure 5: LRP6, TRIM29 and Pygo2 are direct targets of miR-432(A). Indicated cells transfected with TOPflash or FOPflash 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). Altered nuclear translocation of β-catenin in response to deregulated miR-432 expression. Nuclear fractions of indicated cells were analyzed by western blotting; p84 was used as the loading control. (C). Predicted miR-432 target sequences in the 3′UTRs of LRP6, TRIM29 and Pygo2. The miR-432 mutant (miR-432-mut) contained three altered nucleotides in the seed sequence. (D). Western blotting analysis of LRP6, TRIM29 and Pygo2 expression in the indicated HCC cells. α-Tubulin served as loading control. (E). Luciferase assay of pGL3- LRP6-3′UTR, pGL3- TRIM29-3′UTR and pGL3- Pygo2-3′UTR reporter cotransfected with miR-432, miR-432-inhibitor and miR-432-mut oligonucleotides in HCC cells. (F). Luciferase assay of TCF/LEF transcriptional activity in indicated cells transfected with specific siRNA. Error bars represent the mean ± SD from of three independent experiments. *P < 0.05.
Mentions: The wnt/β-catenin pathway is an important regulator of tumor initiation and progression, and palys an important role in cell proliferation. Therefore, we detect the effect of miR-432 in regulating the wnt/β-catenin signaling. As shown in Figure 5A, miR-432 overexpression markedly decreased the luciferase activity of TOPflash or FOPflash reporter; conversely, transfection of miR-432 inhibitor increased the luciferase activity of TOPflash or FOPflash reporter, compare with vector or negative control, respectively. Furthermore, cellular fractionation showed that miR-432 overexpression inhibit nuclear accumulation of β-catenin (Figure 5B), indicating that miR-432 deactivates Wnt/β-catenin pathway by inhibiting β-catenin nuclear accumulation.

Bottom Line: Sustained cell growth and proliferation, one of the hallmarks of cancer, is considered to responsible for cancer-related deaths by disorganizing the balance of growth promotion and growth limitation.Elucidating the molecular mechanism of this abnormality in hepatocellular carcinoma carcinogenesis may improve diagnostic and therapeutic strategies for this malignancy.Furthermore, miR-432 directly targeted and suppressed multiple regulators of the Wnt/β-catenin signaling cascade, including LRP6, TRIM29 and Pygo2, which subsequently deactivated Wnt/β-catenin signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatic Surgery, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, China.

ABSTRACT
Sustained cell growth and proliferation, one of the hallmarks of cancer, is considered to responsible for cancer-related deaths by disorganizing the balance of growth promotion and growth limitation. Aberrant activation of the Wnt/β-catenin signaling pathway leads to cell proliferation, growth and survival, and promotes the development of various human tumors, including hepatocellular carcinoma. Elucidating the molecular mechanism of this abnormality in hepatocellular carcinoma carcinogenesis may improve diagnostic and therapeutic strategies for this malignancy. Herein, we report that the expression of miR-432 was markedly downregulated in hepatocellular carcinoma cell lines and tissues, and upregulation of miR-432 inhibited, whereas downregulation of miR-432 enhanced the proliferation and tumorigenicity of hepatocellular carcinoma cells both in vitro and in vivo. Furthermore, miR-432 directly targeted and suppressed multiple regulators of the Wnt/β-catenin signaling cascade, including LRP6, TRIM29 and Pygo2, which subsequently deactivated Wnt/β-catenin signaling pathway. Finally, miR-432 expression was inversely correlated with three targets in clinical hepatocellular carcinoma samples. These results demonstrated that miR-432 functions as a tumor-suppressive miRNA by suppressing Wnt/β-catenin signaling activation and may represent a therapeutic target for hepatocellular carcinoma.

No MeSH data available.


Related in: MedlinePlus