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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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Functions of ITGA5 in HCC(A) Western blot analysis was performed to determine ITGA5 protein expression post-infection with Lv-shGFP or Lv-shITGA5. (B, C) The anoikis activity of BEL-7404(left) and FHCC-98(right) cells treated with Lv-shGFP or Lv-shITGA5 was evaluated by caspase-3 activity(B) and Annexin-V/PI staining(C). Cells (1×106) were cultured for 48 h in poly-HEMA pre-coated plates before evaluation. (D) BEL-7404 Gluc stably ITGA5 knockdown cells and control cells (1×106) were injected into nude mice (n=5) through tail vein. Gluc activity analysis was performed to determine anoikis in different HCC cell lines in vivo. (E) Comparisons of ITGA5 expression levels in paired tumor and non-tumor samples from 213 HCC patients, with the use of Student's t test; dashed line is shown at the mean of non-tumor group. Expression levels of ITGA5 were from a normalized GEO dataset (GSE14520). (F) Relative ITGA5 levels assessed by Deep Sequencing in HCC tissues stratified according to the stages of HCC. (G) Kaplan-Meier graph representing the probabilities of overall survival in HCC patients stratified according to the expression levels of ITGA5. (H) The correlation of ITGA5 and fibronection 1 (FN1) mRNA in human HCC tissues. The Pearson product-moment correlation coefficient and significance levels are indicated. In, expression levels of ITGA5 and FN1 were downloaded from the TCGA Liver hepatocellular carcinoma (LIHC) mRNA dataset. **, P < 0.01. *, P < 0.05. Error bars, s.d. (experiments depicted in B, C performed in triplicate).
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Figure 4: Functions of ITGA5 in HCC(A) Western blot analysis was performed to determine ITGA5 protein expression post-infection with Lv-shGFP or Lv-shITGA5. (B, C) The anoikis activity of BEL-7404(left) and FHCC-98(right) cells treated with Lv-shGFP or Lv-shITGA5 was evaluated by caspase-3 activity(B) and Annexin-V/PI staining(C). Cells (1×106) were cultured for 48 h in poly-HEMA pre-coated plates before evaluation. (D) BEL-7404 Gluc stably ITGA5 knockdown cells and control cells (1×106) were injected into nude mice (n=5) through tail vein. Gluc activity analysis was performed to determine anoikis in different HCC cell lines in vivo. (E) Comparisons of ITGA5 expression levels in paired tumor and non-tumor samples from 213 HCC patients, with the use of Student's t test; dashed line is shown at the mean of non-tumor group. Expression levels of ITGA5 were from a normalized GEO dataset (GSE14520). (F) Relative ITGA5 levels assessed by Deep Sequencing in HCC tissues stratified according to the stages of HCC. (G) Kaplan-Meier graph representing the probabilities of overall survival in HCC patients stratified according to the expression levels of ITGA5. (H) The correlation of ITGA5 and fibronection 1 (FN1) mRNA in human HCC tissues. The Pearson product-moment correlation coefficient and significance levels are indicated. In, expression levels of ITGA5 and FN1 were downloaded from the TCGA Liver hepatocellular carcinoma (LIHC) mRNA dataset. **, P < 0.01. *, P < 0.05. Error bars, s.d. (experiments depicted in B, C performed in triplicate).

Mentions: ITGA5 is a member of the integrin family mediating cell-to-cell adhesion and can drive migration in tumor cells [32]. To investigate the role of ITGA5 on anoikis in HCC cells, we stably silenced endogenous ITGA5 using a lentivirus delivery system. ITGA5 expression was reduced by more than 90% (Figure 4A). Analysis of the cell detachment assay shows that knockdown of ITGA5 significantly increased anoikis of HCC cells in vitro (Figure 4B-C). Enhanced anoikis sensitivity by ITGA5 silencing mimicked the phenotype induced by overexpression of miR-26a in HCC cells. Furthermore, in our in vivo assay, silencing endogenous ITGA5 dramatically promoted tumor cell anoikis upon tail vein injection in nude mice (Figure 4D). Our results suggest that reduction of ITGA5 levels has similar effects on the HCC cells to miR-26a overexpression, further confirming that ITGA5 may act as a downstream functional mediator of miR-26a during 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)

Functions of ITGA5 in HCC(A) Western blot analysis was performed to determine ITGA5 protein expression post-infection with Lv-shGFP or Lv-shITGA5. (B, C) The anoikis activity of BEL-7404(left) and FHCC-98(right) cells treated with Lv-shGFP or Lv-shITGA5 was evaluated by caspase-3 activity(B) and Annexin-V/PI staining(C). Cells (1×106) were cultured for 48 h in poly-HEMA pre-coated plates before evaluation. (D) BEL-7404 Gluc stably ITGA5 knockdown cells and control cells (1×106) were injected into nude mice (n=5) through tail vein. Gluc activity analysis was performed to determine anoikis in different HCC cell lines in vivo. (E) Comparisons of ITGA5 expression levels in paired tumor and non-tumor samples from 213 HCC patients, with the use of Student's t test; dashed line is shown at the mean of non-tumor group. Expression levels of ITGA5 were from a normalized GEO dataset (GSE14520). (F) Relative ITGA5 levels assessed by Deep Sequencing in HCC tissues stratified according to the stages of HCC. (G) Kaplan-Meier graph representing the probabilities of overall survival in HCC patients stratified according to the expression levels of ITGA5. (H) The correlation of ITGA5 and fibronection 1 (FN1) mRNA in human HCC tissues. The Pearson product-moment correlation coefficient and significance levels are indicated. In, expression levels of ITGA5 and FN1 were downloaded from the TCGA Liver hepatocellular carcinoma (LIHC) mRNA dataset. **, P < 0.01. *, P < 0.05. Error bars, s.d. (experiments depicted in B, C performed in triplicate).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Functions of ITGA5 in HCC(A) Western blot analysis was performed to determine ITGA5 protein expression post-infection with Lv-shGFP or Lv-shITGA5. (B, C) The anoikis activity of BEL-7404(left) and FHCC-98(right) cells treated with Lv-shGFP or Lv-shITGA5 was evaluated by caspase-3 activity(B) and Annexin-V/PI staining(C). Cells (1×106) were cultured for 48 h in poly-HEMA pre-coated plates before evaluation. (D) BEL-7404 Gluc stably ITGA5 knockdown cells and control cells (1×106) were injected into nude mice (n=5) through tail vein. Gluc activity analysis was performed to determine anoikis in different HCC cell lines in vivo. (E) Comparisons of ITGA5 expression levels in paired tumor and non-tumor samples from 213 HCC patients, with the use of Student's t test; dashed line is shown at the mean of non-tumor group. Expression levels of ITGA5 were from a normalized GEO dataset (GSE14520). (F) Relative ITGA5 levels assessed by Deep Sequencing in HCC tissues stratified according to the stages of HCC. (G) Kaplan-Meier graph representing the probabilities of overall survival in HCC patients stratified according to the expression levels of ITGA5. (H) The correlation of ITGA5 and fibronection 1 (FN1) mRNA in human HCC tissues. The Pearson product-moment correlation coefficient and significance levels are indicated. In, expression levels of ITGA5 and FN1 were downloaded from the TCGA Liver hepatocellular carcinoma (LIHC) mRNA dataset. **, P < 0.01. *, P < 0.05. Error bars, s.d. (experiments depicted in B, C performed in triplicate).
Mentions: ITGA5 is a member of the integrin family mediating cell-to-cell adhesion and can drive migration in tumor cells [32]. To investigate the role of ITGA5 on anoikis in HCC cells, we stably silenced endogenous ITGA5 using a lentivirus delivery system. ITGA5 expression was reduced by more than 90% (Figure 4A). Analysis of the cell detachment assay shows that knockdown of ITGA5 significantly increased anoikis of HCC cells in vitro (Figure 4B-C). Enhanced anoikis sensitivity by ITGA5 silencing mimicked the phenotype induced by overexpression of miR-26a in HCC cells. Furthermore, in our in vivo assay, silencing endogenous ITGA5 dramatically promoted tumor cell anoikis upon tail vein injection in nude mice (Figure 4D). Our results suggest that reduction of ITGA5 levels has similar effects on the HCC cells to miR-26a overexpression, further confirming that ITGA5 may act as a downstream functional mediator of miR-26a during 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