Limits...
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.

Show MeSH

Related in: MedlinePlus

AKT2 knockdown phenocopies the tumor-suppressive effect of let-7b/g in gastric cancer cells. (A) Western blot of AKT2, p-AKT (S473) and pS6 after siAKT2 transfection in AGS, NCI-N87 and MKN45 cells. (B) AKT2 knockdown suppressed cell proliferation (**, P < 0.001). 6 wells were measured for each group to get SDs. (C) AKT2 knockdown decreased monolayer colony formation in AGS, NCI-N87 and MKN45 cells (**, P < 0.001). The experiments was repeated in 3 wells to get SDs. (D) siAKT2 inhibited cell invasion of gastric cancer cells (**, P < 0.001). The invaded cells in 3 random vision fields were counted for SDs achieving. (E) siAKT2 suppressed the formation of xenografts in nude mice compared with siScramble group (*, P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: AKT2 knockdown phenocopies the tumor-suppressive effect of let-7b/g in gastric cancer cells. (A) Western blot of AKT2, p-AKT (S473) and pS6 after siAKT2 transfection in AGS, NCI-N87 and MKN45 cells. (B) AKT2 knockdown suppressed cell proliferation (**, P < 0.001). 6 wells were measured for each group to get SDs. (C) AKT2 knockdown decreased monolayer colony formation in AGS, NCI-N87 and MKN45 cells (**, P < 0.001). The experiments was repeated in 3 wells to get SDs. (D) siAKT2 inhibited cell invasion of gastric cancer cells (**, P < 0.001). The invaded cells in 3 random vision fields were counted for SDs achieving. (E) siAKT2 suppressed the formation of xenografts in nude mice compared with siScramble group (*, P < 0.05).

Mentions: We have demonstrated that let-7b/g targeted AKT2 and exerted tumor suppressor function in gastric cancer. siRNA-mediated AKT2 knockdown was therefore performed to investigate if AKT2 downregulation phenocopied the tumor-suppressive effect of let-7b/g. We transfected siAKT2 into gastric cancer cell lines AGS, NCI-N87 and MKN45. Successful AKT2 knockdown was confirmed by Western blot analysis. p-AKT (S473) and pS6 protein showed decreased expression upon siAKT2 transfection (Figure 5A). AKT2 knockdown suppressed AGS, NCI-N87 and MKN45 cell proliferation in a 5-day MTT assays (P < 0.001, Figure 5B). AKT2 knockdown also decreased monolayer colony formation in gastric cancer cells to a significant level (Figure 5C). Moreover, siAKT2 inhibited cell invasion of gastric cancer cells (P < 0.001, Figure 5D). The effect of siAKT2 on tumor growth in vivo was also investigated. The siAKT2-MKN45 and vector control cells were injected into nude mice subcutaneously. The tumor growth in siAKT2 groups was significantly decreased compared with the vector control after 22 days (P < 0.05, Figure 5E).


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)

AKT2 knockdown phenocopies the tumor-suppressive effect of let-7b/g in gastric cancer cells. (A) Western blot of AKT2, p-AKT (S473) and pS6 after siAKT2 transfection in AGS, NCI-N87 and MKN45 cells. (B) AKT2 knockdown suppressed cell proliferation (**, P < 0.001). 6 wells were measured for each group to get SDs. (C) AKT2 knockdown decreased monolayer colony formation in AGS, NCI-N87 and MKN45 cells (**, P < 0.001). The experiments was repeated in 3 wells to get SDs. (D) siAKT2 inhibited cell invasion of gastric cancer cells (**, P < 0.001). The invaded cells in 3 random vision fields were counted for SDs achieving. (E) siAKT2 suppressed the formation of xenografts in nude mice compared with siScramble group (*, P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: AKT2 knockdown phenocopies the tumor-suppressive effect of let-7b/g in gastric cancer cells. (A) Western blot of AKT2, p-AKT (S473) and pS6 after siAKT2 transfection in AGS, NCI-N87 and MKN45 cells. (B) AKT2 knockdown suppressed cell proliferation (**, P < 0.001). 6 wells were measured for each group to get SDs. (C) AKT2 knockdown decreased monolayer colony formation in AGS, NCI-N87 and MKN45 cells (**, P < 0.001). The experiments was repeated in 3 wells to get SDs. (D) siAKT2 inhibited cell invasion of gastric cancer cells (**, P < 0.001). The invaded cells in 3 random vision fields were counted for SDs achieving. (E) siAKT2 suppressed the formation of xenografts in nude mice compared with siScramble group (*, P < 0.05).
Mentions: We have demonstrated that let-7b/g targeted AKT2 and exerted tumor suppressor function in gastric cancer. siRNA-mediated AKT2 knockdown was therefore performed to investigate if AKT2 downregulation phenocopied the tumor-suppressive effect of let-7b/g. We transfected siAKT2 into gastric cancer cell lines AGS, NCI-N87 and MKN45. Successful AKT2 knockdown was confirmed by Western blot analysis. p-AKT (S473) and pS6 protein showed decreased expression upon siAKT2 transfection (Figure 5A). AKT2 knockdown suppressed AGS, NCI-N87 and MKN45 cell proliferation in a 5-day MTT assays (P < 0.001, Figure 5B). AKT2 knockdown also decreased monolayer colony formation in gastric cancer cells to a significant level (Figure 5C). Moreover, siAKT2 inhibited cell invasion of gastric cancer cells (P < 0.001, Figure 5D). The effect of siAKT2 on tumor growth in vivo was also investigated. The siAKT2-MKN45 and vector control cells were injected into nude mice subcutaneously. The tumor growth in siAKT2 groups was significantly decreased compared with the vector control after 22 days (P < 0.05, Figure 5E).

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