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MicroRNA-26a promotes anoikis in human hepatocellular carcinoma cells by targeting alpha5 integrin.

Zhang X, Cheng SL, Bian K, Wang L, Zhang X, Yan B, Jia LT, Zhao J, Gammoh N, Yang AG, Zhang R - Oncotarget (2015)

Bottom Line: Previous studies demonstrate that microRNA-26a (miR-26a) is an important tumor suppressor that inhibits the proliferation and invasion of HCC cells by targeting multiple oncogenic proteins.However, whether miR-26a can also influence anoikis has not been well established.Here, we discovered that miR-26a promotes anoikis of HCC cells both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, the Fourth Military Medical University, Xi'an, Shaanxi, China.

ABSTRACT
Metastasis is the major reason for the death of patients suffering from malignant diseases such as human hepatocellular carcinoma (HCC). Among the complex metastatic process, resistance to anoikis is one of the most important steps. Previous studies demonstrate that microRNA-26a (miR-26a) is an important tumor suppressor that inhibits the proliferation and invasion of HCC cells by targeting multiple oncogenic proteins. However, whether miR-26a can also influence anoikis has not been well established. Here, we discovered that miR-26a promotes anoikis of HCC cells both in vitro and in vivo. With a combinational analysis of bioinformatics and public clinical databases, we predicted that alpha5 integrin (ITGA5), an integrin family member, is a putative target of miR-26a. Furthermore, we provide experimental evidence to confirm that ITGA5 is a bona fide target of miR-26a. Through gain- and loss-of-function studies, we demonstrate that ITGA5 is a functional target of miR-26a-induced anoikis in HCC cells. Collectively, our findings reveal that miR-26a is a novel player during anoikis and a potential therapeutic target for the treatment of metastatic HCC.

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Inactivation of Akt is an essential downstream signal in ITGA5 suppression induced tumor cell anoikis(A) Western blot analysis was performed to determine the phosphorylation of Akt(Ser473) in BEL-7404 cells after over-expression of miR-26a or knockdown of ITGA5. (B) Western blot analysis was performed to determine the phosphorylation of ERK1/2(Thr202/Tyr204) in BEL-7404 cells after over-expression of miR-26a or knockdown of ITGA5. (C) The anoikis activity of BEL-7404 cells treated with DMSO or Akt inhibitor (15 μM) was evaluated by Annexin-V/PI staining. (D) The anoikis activity of BEL-7404 cells treated with DMSO or ERK inhibitor (5 μM) was evaluated by Annexin-V/PI staining. In &D, cells (1×106) were cultured for 48 h in poly-HEMA pre-coated plates before evaluation. ***, P < 0.001. Error bars, s.d. ns, no significance. Luc, luciferase.
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Figure 6: Inactivation of Akt is an essential downstream signal in ITGA5 suppression induced tumor cell anoikis(A) Western blot analysis was performed to determine the phosphorylation of Akt(Ser473) in BEL-7404 cells after over-expression of miR-26a or knockdown of ITGA5. (B) Western blot analysis was performed to determine the phosphorylation of ERK1/2(Thr202/Tyr204) in BEL-7404 cells after over-expression of miR-26a or knockdown of ITGA5. (C) The anoikis activity of BEL-7404 cells treated with DMSO or Akt inhibitor (15 μM) was evaluated by Annexin-V/PI staining. (D) The anoikis activity of BEL-7404 cells treated with DMSO or ERK inhibitor (5 μM) was evaluated by Annexin-V/PI staining. In &D, cells (1×106) were cultured for 48 h in poly-HEMA pre-coated plates before evaluation. ***, P < 0.001. Error bars, s.d. ns, no significance. Luc, luciferase.

Mentions: Our study suggests that miR-26a induces anoikis of HCC cells negatively regulating ITGA5. However, the downstream molecular events of how anoikis is regulated are still elusive. Previous data show that Akt and mitogen-activated protein kinase (MAPK, also known as EKR1/2) signaling networks play important roles in the malignant behavior of tumor cells [33, 34]. Therefore, we tested whether miR-26a-ITGA5 effects on anoikis acts through the Akt and ERK1/2 pathways. As shown in Figure 6A-B, the upregulation of miR-26a and downregulation of ITGA5 resulted in reduced phosphorylation of Akt and ERK1/2, whereas the total Akt and ERK levels were not affected, indicating both Akt and ERK1/2 signaling pathways could be the downstream of miR-26-ITGA5 axis. Intriguingly, pharmacological inhibition of the Akt pathway, but not the ERK pathway, resulted in a significant increase of anoikis in tumor cells (Figure 6C-D). This suggests that the ERK1/2 pathway is dispensable during anoikis of HCC cells in our system. Collectively, these findings demonstrated that Akt inactivation is an essential downstream signaling pathway during ITGA5 suppression-induced tumor cell anoikis.


MicroRNA-26a promotes anoikis in human hepatocellular carcinoma cells by targeting alpha5 integrin.

Zhang X, Cheng SL, Bian K, Wang L, Zhang X, Yan B, Jia LT, Zhao J, Gammoh N, Yang AG, Zhang R - Oncotarget (2015)

Inactivation of Akt is an essential downstream signal in ITGA5 suppression induced tumor cell anoikis(A) Western blot analysis was performed to determine the phosphorylation of Akt(Ser473) in BEL-7404 cells after over-expression of miR-26a or knockdown of ITGA5. (B) Western blot analysis was performed to determine the phosphorylation of ERK1/2(Thr202/Tyr204) in BEL-7404 cells after over-expression of miR-26a or knockdown of ITGA5. (C) The anoikis activity of BEL-7404 cells treated with DMSO or Akt inhibitor (15 μM) was evaluated by Annexin-V/PI staining. (D) The anoikis activity of BEL-7404 cells treated with DMSO or ERK inhibitor (5 μM) was evaluated by Annexin-V/PI staining. In &D, cells (1×106) were cultured for 48 h in poly-HEMA pre-coated plates before evaluation. ***, P < 0.001. Error bars, s.d. ns, no significance. Luc, luciferase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385851&req=5

Figure 6: Inactivation of Akt is an essential downstream signal in ITGA5 suppression induced tumor cell anoikis(A) Western blot analysis was performed to determine the phosphorylation of Akt(Ser473) in BEL-7404 cells after over-expression of miR-26a or knockdown of ITGA5. (B) Western blot analysis was performed to determine the phosphorylation of ERK1/2(Thr202/Tyr204) in BEL-7404 cells after over-expression of miR-26a or knockdown of ITGA5. (C) The anoikis activity of BEL-7404 cells treated with DMSO or Akt inhibitor (15 μM) was evaluated by Annexin-V/PI staining. (D) The anoikis activity of BEL-7404 cells treated with DMSO or ERK inhibitor (5 μM) was evaluated by Annexin-V/PI staining. In &D, cells (1×106) were cultured for 48 h in poly-HEMA pre-coated plates before evaluation. ***, P < 0.001. Error bars, s.d. ns, no significance. Luc, luciferase.
Mentions: Our study suggests that miR-26a induces anoikis of HCC cells negatively regulating ITGA5. However, the downstream molecular events of how anoikis is regulated are still elusive. Previous data show that Akt and mitogen-activated protein kinase (MAPK, also known as EKR1/2) signaling networks play important roles in the malignant behavior of tumor cells [33, 34]. Therefore, we tested whether miR-26a-ITGA5 effects on anoikis acts through the Akt and ERK1/2 pathways. As shown in Figure 6A-B, the upregulation of miR-26a and downregulation of ITGA5 resulted in reduced phosphorylation of Akt and ERK1/2, whereas the total Akt and ERK levels were not affected, indicating both Akt and ERK1/2 signaling pathways could be the downstream of miR-26-ITGA5 axis. Intriguingly, pharmacological inhibition of the Akt pathway, but not the ERK pathway, resulted in a significant increase of anoikis in tumor cells (Figure 6C-D). This suggests that the ERK1/2 pathway is dispensable during anoikis of HCC cells in our system. Collectively, these findings demonstrated that Akt inactivation is an essential downstream signaling pathway during ITGA5 suppression-induced tumor cell anoikis.

Bottom Line: Previous studies demonstrate that microRNA-26a (miR-26a) is an important tumor suppressor that inhibits the proliferation and invasion of HCC cells by targeting multiple oncogenic proteins.However, whether miR-26a can also influence anoikis has not been well established.Here, we discovered that miR-26a promotes anoikis of HCC cells both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, the Fourth Military Medical University, Xi'an, Shaanxi, China.

ABSTRACT
Metastasis is the major reason for the death of patients suffering from malignant diseases such as human hepatocellular carcinoma (HCC). Among the complex metastatic process, resistance to anoikis is one of the most important steps. Previous studies demonstrate that microRNA-26a (miR-26a) is an important tumor suppressor that inhibits the proliferation and invasion of HCC cells by targeting multiple oncogenic proteins. However, whether miR-26a can also influence anoikis has not been well established. Here, we discovered that miR-26a promotes anoikis of HCC cells both in vitro and in vivo. With a combinational analysis of bioinformatics and public clinical databases, we predicted that alpha5 integrin (ITGA5), an integrin family member, is a putative target of miR-26a. Furthermore, we provide experimental evidence to confirm that ITGA5 is a bona fide target of miR-26a. Through gain- and loss-of-function studies, we demonstrate that ITGA5 is a functional target of miR-26a-induced anoikis in HCC cells. Collectively, our findings reveal that miR-26a is a novel player during anoikis and a potential therapeutic target for the treatment of metastatic HCC.

Show MeSH
Related in: MedlinePlus