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The miR-491-3p/mTORC2/FOXO1 regulatory loop modulates chemo-sensitivity in human tongue cancer.

Zheng G, Jia X, Peng C, Deng Y, Yin J, Zhang Z, Li N, Deng M, Liu X, Liu H, Lu M, Wang C, Gu Y, He Z - Oncotarget (2015)

Bottom Line: We found that miR-491-3p directly targeted mTORC2 component Rictor and inhibited mTORC2 activity, which was increased in resistant TC cells with high p-Akt(Ser473), p-SGK1(Ser422) and p-FOXO1(Thr24) levels.As a feedback loop, mTORC2 downregulated miR-491-3p expression by inactivating FOXO1, which otherwise would transcriptionally induce miR-491-3p expression.Levels of miR-491-3 and Rictor or mTORC2 activity negatively correlated in TC tissues.

View Article: PubMed Central - PubMed

Affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Guangzhou 510095, Guangdong, China.

ABSTRACT
We found that levels of miR-491-3p were decreased in multidrug-resistant tongue cancer (TC) cells. Induction of miR-491-3p expression sensitized TC cells to chemotherapy. In agreement, functional inhibition of miR-491-3p enhanced resistance of TC cells to chemotherapy. We found that miR-491-3p directly targeted mTORC2 component Rictor and inhibited mTORC2 activity, which was increased in resistant TC cells with high p-Akt(Ser473), p-SGK1(Ser422) and p-FOXO1(Thr24) levels. Inhibition of mTORC2 activity via either Rictor knockdown or mTOR inhibitor in turn sensitized TC cells to chemotherapy. In agreement, overexpression of Rictor increased the mTORC2 activity and induced resistance of TC cells to chemotherapy. As a feedback loop, mTORC2 downregulated miR-491-3p expression by inactivating FOXO1, which otherwise would transcriptionally induce miR-491-3p expression. Levels of miR-491-3 and Rictor or mTORC2 activity negatively correlated in TC tissues. Finally, low levels of miR-491-3p and highly expressed Rictor were associated with poor prognosis in tongue cancer patients. These data provide a rationale for targeted intervention on miR-491-3p/mTORC2 axis to enhance the efficacy of chemotherapy against tongue cancer.

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miR-491-3p repressed Rictor protein expression(A) Schematic of predicted miR-491-3p site in the 3′UTR of human Rictor mRNA, which broadly conserved among vertebrates. (B) The expression pattern of Rictor in mRNA levels in selected cell lines detected by qRT-PCR by normalizing to GAPDH as endogenous control and the expression level in Tca8113 was set as 1. (C) The expression pattern of Rictor on protein level and mTORC2 activity in selected cell lines detected by western blot. (D) Functional interference of miR-491-3p negatively regulated Rictor protein expression and Rictor activity determined with western blot. (E) and (F) Endogenous expression pattern and functional interference of miR-491-3p negatively associated with the activity of luciferase gene linked with the 3′UTR sequence of Rictor and a renilla luciferase reporter for normalization. Luciferase activities were measured at 48 hours after transfection and the data was obtained from three independent experiments. The mean of the results from Tca8113 cells transfected with pMir-Wt, and cells transfected with pMir-Wt and interference control were set as 100% respectively. *p < 0.01. **p < 0.001.
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Figure 3: miR-491-3p repressed Rictor protein expression(A) Schematic of predicted miR-491-3p site in the 3′UTR of human Rictor mRNA, which broadly conserved among vertebrates. (B) The expression pattern of Rictor in mRNA levels in selected cell lines detected by qRT-PCR by normalizing to GAPDH as endogenous control and the expression level in Tca8113 was set as 1. (C) The expression pattern of Rictor on protein level and mTORC2 activity in selected cell lines detected by western blot. (D) Functional interference of miR-491-3p negatively regulated Rictor protein expression and Rictor activity determined with western blot. (E) and (F) Endogenous expression pattern and functional interference of miR-491-3p negatively associated with the activity of luciferase gene linked with the 3′UTR sequence of Rictor and a renilla luciferase reporter for normalization. Luciferase activities were measured at 48 hours after transfection and the data was obtained from three independent experiments. The mean of the results from Tca8113 cells transfected with pMir-Wt, and cells transfected with pMir-Wt and interference control were set as 100% respectively. *p < 0.01. **p < 0.001.

Mentions: We next used miRNA database TargetScan (http://www.targetscan.org) to predict potential targets of miR-491-3p. The mTORC2 component Rictor with a conserved binding site of miR-491-3p was selected for further identification (Figure 3A). There was no significant difference of the Rictor mRNA level in selected cell lines (Figure 3B). However, the Rictor protein level in Tca8113/PYM cells was much higher than that in Tca8113, SCC-25 and CAL-27 cell lines (Figure 3C). Notably, transfection with miR-491-3p mimics significantly downregulated Rictor protein level in Tca8113/PYM cells, and the miR-491-3p inhibitor clearly upregulated Rictor protein level in Tca8113, SCC-25 and CAL-27 cell lines (Figure 3D). To assess whether Rictor is a direct target of miR-491-3p, a luciferase reporter vector containing the putative Rictor 3′UTR target site for miR-491-3p (pMir-Wt, as wildtype version) or a mutant version with a deletion of 7bp in the seed sequence was constructed (pMir-Mut). As shown, in Tca8113, SCC-25 and CAL-27 cell lines, the luciferase activities from mutant version were higher than that from wildtype version. In contrast the luciferase activity from wildtype version in Tca8113/PYM cells was higher than that in Tca8113, SCC-25 and CAL-27 cells (Figure 3E), suggesting that endogenous miR-491-3p inhibits Rictor expression by binding to the seed sequence in the 3′UTR of Rictor mRNA. Moreover, miR-491-3p mimics significantly repressed the luciferase activity of the vector with wild-type Rictor 3′UTR in Tca8113/PYM cells, but the mutant version abrogated the repressive ability of miR-491-3p (Figure 3F). Inversely, miR-491-3p inhibitor increased the luciferase activities from the wildtype version, and the mutant version abrogated the facilitative effect of miR-491-3p inhibitor (Figure 3F). These results strongly demonstrated the specificity of miR-491-3p targeting Rictor mRNA.


The miR-491-3p/mTORC2/FOXO1 regulatory loop modulates chemo-sensitivity in human tongue cancer.

Zheng G, Jia X, Peng C, Deng Y, Yin J, Zhang Z, Li N, Deng M, Liu X, Liu H, Lu M, Wang C, Gu Y, He Z - Oncotarget (2015)

miR-491-3p repressed Rictor protein expression(A) Schematic of predicted miR-491-3p site in the 3′UTR of human Rictor mRNA, which broadly conserved among vertebrates. (B) The expression pattern of Rictor in mRNA levels in selected cell lines detected by qRT-PCR by normalizing to GAPDH as endogenous control and the expression level in Tca8113 was set as 1. (C) The expression pattern of Rictor on protein level and mTORC2 activity in selected cell lines detected by western blot. (D) Functional interference of miR-491-3p negatively regulated Rictor protein expression and Rictor activity determined with western blot. (E) and (F) Endogenous expression pattern and functional interference of miR-491-3p negatively associated with the activity of luciferase gene linked with the 3′UTR sequence of Rictor and a renilla luciferase reporter for normalization. Luciferase activities were measured at 48 hours after transfection and the data was obtained from three independent experiments. The mean of the results from Tca8113 cells transfected with pMir-Wt, and cells transfected with pMir-Wt and interference control were set as 100% respectively. *p < 0.01. **p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4466660&req=5

Figure 3: miR-491-3p repressed Rictor protein expression(A) Schematic of predicted miR-491-3p site in the 3′UTR of human Rictor mRNA, which broadly conserved among vertebrates. (B) The expression pattern of Rictor in mRNA levels in selected cell lines detected by qRT-PCR by normalizing to GAPDH as endogenous control and the expression level in Tca8113 was set as 1. (C) The expression pattern of Rictor on protein level and mTORC2 activity in selected cell lines detected by western blot. (D) Functional interference of miR-491-3p negatively regulated Rictor protein expression and Rictor activity determined with western blot. (E) and (F) Endogenous expression pattern and functional interference of miR-491-3p negatively associated with the activity of luciferase gene linked with the 3′UTR sequence of Rictor and a renilla luciferase reporter for normalization. Luciferase activities were measured at 48 hours after transfection and the data was obtained from three independent experiments. The mean of the results from Tca8113 cells transfected with pMir-Wt, and cells transfected with pMir-Wt and interference control were set as 100% respectively. *p < 0.01. **p < 0.001.
Mentions: We next used miRNA database TargetScan (http://www.targetscan.org) to predict potential targets of miR-491-3p. The mTORC2 component Rictor with a conserved binding site of miR-491-3p was selected for further identification (Figure 3A). There was no significant difference of the Rictor mRNA level in selected cell lines (Figure 3B). However, the Rictor protein level in Tca8113/PYM cells was much higher than that in Tca8113, SCC-25 and CAL-27 cell lines (Figure 3C). Notably, transfection with miR-491-3p mimics significantly downregulated Rictor protein level in Tca8113/PYM cells, and the miR-491-3p inhibitor clearly upregulated Rictor protein level in Tca8113, SCC-25 and CAL-27 cell lines (Figure 3D). To assess whether Rictor is a direct target of miR-491-3p, a luciferase reporter vector containing the putative Rictor 3′UTR target site for miR-491-3p (pMir-Wt, as wildtype version) or a mutant version with a deletion of 7bp in the seed sequence was constructed (pMir-Mut). As shown, in Tca8113, SCC-25 and CAL-27 cell lines, the luciferase activities from mutant version were higher than that from wildtype version. In contrast the luciferase activity from wildtype version in Tca8113/PYM cells was higher than that in Tca8113, SCC-25 and CAL-27 cells (Figure 3E), suggesting that endogenous miR-491-3p inhibits Rictor expression by binding to the seed sequence in the 3′UTR of Rictor mRNA. Moreover, miR-491-3p mimics significantly repressed the luciferase activity of the vector with wild-type Rictor 3′UTR in Tca8113/PYM cells, but the mutant version abrogated the repressive ability of miR-491-3p (Figure 3F). Inversely, miR-491-3p inhibitor increased the luciferase activities from the wildtype version, and the mutant version abrogated the facilitative effect of miR-491-3p inhibitor (Figure 3F). These results strongly demonstrated the specificity of miR-491-3p targeting Rictor mRNA.

Bottom Line: We found that miR-491-3p directly targeted mTORC2 component Rictor and inhibited mTORC2 activity, which was increased in resistant TC cells with high p-Akt(Ser473), p-SGK1(Ser422) and p-FOXO1(Thr24) levels.As a feedback loop, mTORC2 downregulated miR-491-3p expression by inactivating FOXO1, which otherwise would transcriptionally induce miR-491-3p expression.Levels of miR-491-3 and Rictor or mTORC2 activity negatively correlated in TC tissues.

View Article: PubMed Central - PubMed

Affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Guangzhou 510095, Guangdong, China.

ABSTRACT
We found that levels of miR-491-3p were decreased in multidrug-resistant tongue cancer (TC) cells. Induction of miR-491-3p expression sensitized TC cells to chemotherapy. In agreement, functional inhibition of miR-491-3p enhanced resistance of TC cells to chemotherapy. We found that miR-491-3p directly targeted mTORC2 component Rictor and inhibited mTORC2 activity, which was increased in resistant TC cells with high p-Akt(Ser473), p-SGK1(Ser422) and p-FOXO1(Thr24) levels. Inhibition of mTORC2 activity via either Rictor knockdown or mTOR inhibitor in turn sensitized TC cells to chemotherapy. In agreement, overexpression of Rictor increased the mTORC2 activity and induced resistance of TC cells to chemotherapy. As a feedback loop, mTORC2 downregulated miR-491-3p expression by inactivating FOXO1, which otherwise would transcriptionally induce miR-491-3p expression. Levels of miR-491-3 and Rictor or mTORC2 activity negatively correlated in TC tissues. Finally, low levels of miR-491-3p and highly expressed Rictor were associated with poor prognosis in tongue cancer patients. These data provide a rationale for targeted intervention on miR-491-3p/mTORC2 axis to enhance the efficacy of chemotherapy against tongue cancer.

Show MeSH
Related in: MedlinePlus