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let-7b/g silencing activates AKT signaling to promote gastric carcinogenesis.

Kang W, Tong JH, Lung RW, Dong Y, Yang W, Pan Y, Lau KM, Yu J, Cheng AS, To KF - J Transl Med (2014)

Bottom Line: Functional analyses including MTT proliferation, monolayer colony formation, cell invasion assays and in vivo study were performed in both ectopic expression and knockdown approaches. let-7b/g was found down-regulated in gastric cancer and its downregulation was associated with poor survival and correlated with lymph node metastasis. let-7b/g inhibited AKT2 expression by directly binding to its 3'UTR, reduced p-AKT (S473) activation and suppressed expression of the downstream effector pS6.Short interfering RNA (siRNA) mediated knockdown of AKT2 phenocopied the tumor-suppressive effects of let-7b/g.Moreover, AKT2 re-expression partly abrogated the growth-inhibitory effect of let-7b/g.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomical and Cellular Pathology, State Key Laboratory in Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, SAR, People's Republic of China. kfto@cuhk.edu.hk.

ABSTRACT

Background: Aberrant AKT activation contributes to gastric cancer cell survival and chemotherapy resistance, however its regulation is poorly understood. microRNAs have been established to be important regulators in gastric carcinogenesis. Here, we showed the functional role and putative target of let-7b and let-7g (let-7b/g) in gastric carcinogenesis.

Methods: The expression of let-7b/g in gastric cancer cell lines and primary tumors were evaluated by miRNA qRT-PCR. The putative target gene of let-7b/g was explored by TargetScan followed by further validation. Functional analyses including MTT proliferation, monolayer colony formation, cell invasion assays and in vivo study were performed in both ectopic expression and knockdown approaches.

Results: let-7b/g was found down-regulated in gastric cancer and its downregulation was associated with poor survival and correlated with lymph node metastasis. let-7b/g inhibited AKT2 expression by directly binding to its 3'UTR, reduced p-AKT (S473) activation and suppressed expression of the downstream effector pS6. AKT2 mRNA expression showed negative correlation with the expression of let-7b/g in primary tumors. Short interfering RNA (siRNA) mediated knockdown of AKT2 phenocopied the tumor-suppressive effects of let-7b/g. Moreover, AKT2 re-expression partly abrogated the growth-inhibitory effect of let-7b/g.

Conclusion: In conclusion, our findings reveal decreased let-7b/g contributes to aberrant AKT activation in gastric tumorigenesis and provide a potential therapeutic strategy for gastric cancer.

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Related in: MedlinePlus

let-7b/g targets AKT2 in gastric cancer. (A) The binding sites of let-7b/g on the AKT2 3′UTR as predicted by TargetScan (www.targetscan.org). (B) AKT2 mRNA expression was down-regulated upon ectopic let-7b/g expression (*, P < 0.05; **, P < 0.001). (C) Western blot analysis of AKT2, p-AKT (S473) and pS6 upon let-7b/g ectopic expression in AGS, NCI-N87 and MKN45 cells. (D) Relative luciferase activity in the constructs containing wild-type binding site or mutated binding site of AKT2 3′UTR after let-7b/g transfection (Wild type, the construct containing the complementary sequence of seed region; Mutation, the binding site was mutated; *, P < 0.05).
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Fig3: let-7b/g targets AKT2 in gastric cancer. (A) The binding sites of let-7b/g on the AKT2 3′UTR as predicted by TargetScan (www.targetscan.org). (B) AKT2 mRNA expression was down-regulated upon ectopic let-7b/g expression (*, P < 0.05; **, P < 0.001). (C) Western blot analysis of AKT2, p-AKT (S473) and pS6 upon let-7b/g ectopic expression in AGS, NCI-N87 and MKN45 cells. (D) Relative luciferase activity in the constructs containing wild-type binding site or mutated binding site of AKT2 3′UTR after let-7b/g transfection (Wild type, the construct containing the complementary sequence of seed region; Mutation, the binding site was mutated; *, P < 0.05).

Mentions: AKT2 was predicted to be a putative target of let-7b/g by TargetScan (www.targetscan.org). The putative binding sites of let-7b/g within the 3′UTR of AKT2 were shown in Figure 3A. We transfected let-7b/g into gastric cancer cells and decreased AKT2 mRNA expression level was observed in AGS, NCI-N87 and MKN45 cells (P < 0.05; left bar chart of Figure 3B). However, ectopic expression of let-7b/g did not change the AKT1 mRNA expression (right bar chart of Figure 3B), indicating let-7b/g only exerts its inhibitory effect on AKT2 but not on AKT1. AKT2 protein showed a decrease expression after ectopic let-7b/g expression (Figure 3C), suggesting that let-7b/g triggered a silencing effect on the endogenous AKT2 expression. Meanwhile, the phosphorylated AKT (S473) and its direct downstream effector of AKT-mTOR pathway, pS6, showed decreased expression upon let-7b/g overexpression. A luciferase reporter assay was performed to test whether AKT2 is a direct target of let-7b/g. As shown in Figure 3D, let-7b/g decreased the relative luciferase activity of constructs encompassing AKT2 3′UTR binding sites in NCI-N87 cells (P < 0.05). Meanwhile, let-7b/g did not suppress the luciferase activity in the constructs containing the mutated binding site. The results in this part revealed that let-7b/g specifically recognized the binding sites in AKT2 3′UTR and directly repressed AKT2 expression through mRNA degradation and protein synthesis inhibition.Figure 4


let-7b/g silencing activates AKT signaling to promote gastric carcinogenesis.

Kang W, Tong JH, Lung RW, Dong Y, Yang W, Pan Y, Lau KM, Yu J, Cheng AS, To KF - J Transl Med (2014)

let-7b/g targets AKT2 in gastric cancer. (A) The binding sites of let-7b/g on the AKT2 3′UTR as predicted by TargetScan (www.targetscan.org). (B) AKT2 mRNA expression was down-regulated upon ectopic let-7b/g expression (*, P < 0.05; **, P < 0.001). (C) Western blot analysis of AKT2, p-AKT (S473) and pS6 upon let-7b/g ectopic expression in AGS, NCI-N87 and MKN45 cells. (D) Relative luciferase activity in the constructs containing wild-type binding site or mutated binding site of AKT2 3′UTR after let-7b/g transfection (Wild type, the construct containing the complementary sequence of seed region; Mutation, the binding site was mutated; *, P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4196013&req=5

Fig3: let-7b/g targets AKT2 in gastric cancer. (A) The binding sites of let-7b/g on the AKT2 3′UTR as predicted by TargetScan (www.targetscan.org). (B) AKT2 mRNA expression was down-regulated upon ectopic let-7b/g expression (*, P < 0.05; **, P < 0.001). (C) Western blot analysis of AKT2, p-AKT (S473) and pS6 upon let-7b/g ectopic expression in AGS, NCI-N87 and MKN45 cells. (D) Relative luciferase activity in the constructs containing wild-type binding site or mutated binding site of AKT2 3′UTR after let-7b/g transfection (Wild type, the construct containing the complementary sequence of seed region; Mutation, the binding site was mutated; *, P < 0.05).
Mentions: AKT2 was predicted to be a putative target of let-7b/g by TargetScan (www.targetscan.org). The putative binding sites of let-7b/g within the 3′UTR of AKT2 were shown in Figure 3A. We transfected let-7b/g into gastric cancer cells and decreased AKT2 mRNA expression level was observed in AGS, NCI-N87 and MKN45 cells (P < 0.05; left bar chart of Figure 3B). However, ectopic expression of let-7b/g did not change the AKT1 mRNA expression (right bar chart of Figure 3B), indicating let-7b/g only exerts its inhibitory effect on AKT2 but not on AKT1. AKT2 protein showed a decrease expression after ectopic let-7b/g expression (Figure 3C), suggesting that let-7b/g triggered a silencing effect on the endogenous AKT2 expression. Meanwhile, the phosphorylated AKT (S473) and its direct downstream effector of AKT-mTOR pathway, pS6, showed decreased expression upon let-7b/g overexpression. A luciferase reporter assay was performed to test whether AKT2 is a direct target of let-7b/g. As shown in Figure 3D, let-7b/g decreased the relative luciferase activity of constructs encompassing AKT2 3′UTR binding sites in NCI-N87 cells (P < 0.05). Meanwhile, let-7b/g did not suppress the luciferase activity in the constructs containing the mutated binding site. The results in this part revealed that let-7b/g specifically recognized the binding sites in AKT2 3′UTR and directly repressed AKT2 expression through mRNA degradation and protein synthesis inhibition.Figure 4

Bottom Line: Functional analyses including MTT proliferation, monolayer colony formation, cell invasion assays and in vivo study were performed in both ectopic expression and knockdown approaches. let-7b/g was found down-regulated in gastric cancer and its downregulation was associated with poor survival and correlated with lymph node metastasis. let-7b/g inhibited AKT2 expression by directly binding to its 3'UTR, reduced p-AKT (S473) activation and suppressed expression of the downstream effector pS6.Short interfering RNA (siRNA) mediated knockdown of AKT2 phenocopied the tumor-suppressive effects of let-7b/g.Moreover, AKT2 re-expression partly abrogated the growth-inhibitory effect of let-7b/g.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomical and Cellular Pathology, State Key Laboratory in Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, SAR, People's Republic of China. kfto@cuhk.edu.hk.

ABSTRACT

Background: Aberrant AKT activation contributes to gastric cancer cell survival and chemotherapy resistance, however its regulation is poorly understood. microRNAs have been established to be important regulators in gastric carcinogenesis. Here, we showed the functional role and putative target of let-7b and let-7g (let-7b/g) in gastric carcinogenesis.

Methods: The expression of let-7b/g in gastric cancer cell lines and primary tumors were evaluated by miRNA qRT-PCR. The putative target gene of let-7b/g was explored by TargetScan followed by further validation. Functional analyses including MTT proliferation, monolayer colony formation, cell invasion assays and in vivo study were performed in both ectopic expression and knockdown approaches.

Results: let-7b/g was found down-regulated in gastric cancer and its downregulation was associated with poor survival and correlated with lymph node metastasis. let-7b/g inhibited AKT2 expression by directly binding to its 3'UTR, reduced p-AKT (S473) activation and suppressed expression of the downstream effector pS6. AKT2 mRNA expression showed negative correlation with the expression of let-7b/g in primary tumors. Short interfering RNA (siRNA) mediated knockdown of AKT2 phenocopied the tumor-suppressive effects of let-7b/g. Moreover, AKT2 re-expression partly abrogated the growth-inhibitory effect of let-7b/g.

Conclusion: In conclusion, our findings reveal decreased let-7b/g contributes to aberrant AKT activation in gastric tumorigenesis and provide a potential therapeutic strategy for gastric cancer.

Show MeSH
Related in: MedlinePlus