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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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Related in: MedlinePlus

ITGA5 is a bona fide target gene of mir-26a(A) Gene Ontology classification of miR-26a's potential target genes predicted by integrating the results of two algorithms (Targetscan and PicTar). (B) The correlation of ITGA5 mRNA and miR-26a in human hepatocellular cancer tissues. The Pearson product-moment correlation coefficient and significance levels are indicated. Expression levels of ITGA5 and miR-26a were from two correlative normalized GEO datasets (GSE14520 & GSE6857). (C) The correlation between miR-26a expression and the levels of ITGA5 in different human HCC cell lines and normal hepatocyte-derived cell line (L-02). The miRNA expression was evaluated by qRT-PCR and protein abundance was evaluated by western blot. (D) Schematic diagram of the miR-26a target site of human and other representative mammal ITGA5 3′UTRs. (E) The wild-type 3′UTR of ITGA5 and mutant 3′UTR sequences that abolished binding. (F) A reporter vector containing the WT(wild-type) or MUT(mutant) ITGA5 3′UTR was transfected, along with miR-NC or miR-26a, into HEK-293 cells. Luciferase activity was measured in three independent experiments after 48 hours of transfection and normalized to Renilla luciferase activity. (G) Western blot analysis was performed to determine ITGA5 protein expression post infection with Lv-Luc or Lv-miR-26a. **, P < 0.01. NC, negative control. ns, no significance. Luc, luciferase.
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Figure 3: ITGA5 is a bona fide target gene of mir-26a(A) Gene Ontology classification of miR-26a's potential target genes predicted by integrating the results of two algorithms (Targetscan and PicTar). (B) The correlation of ITGA5 mRNA and miR-26a in human hepatocellular cancer tissues. The Pearson product-moment correlation coefficient and significance levels are indicated. Expression levels of ITGA5 and miR-26a were from two correlative normalized GEO datasets (GSE14520 & GSE6857). (C) The correlation between miR-26a expression and the levels of ITGA5 in different human HCC cell lines and normal hepatocyte-derived cell line (L-02). The miRNA expression was evaluated by qRT-PCR and protein abundance was evaluated by western blot. (D) Schematic diagram of the miR-26a target site of human and other representative mammal ITGA5 3′UTRs. (E) The wild-type 3′UTR of ITGA5 and mutant 3′UTR sequences that abolished binding. (F) A reporter vector containing the WT(wild-type) or MUT(mutant) ITGA5 3′UTR was transfected, along with miR-NC or miR-26a, into HEK-293 cells. Luciferase activity was measured in three independent experiments after 48 hours of transfection and normalized to Renilla luciferase activity. (G) Western blot analysis was performed to determine ITGA5 protein expression post infection with Lv-Luc or Lv-miR-26a. **, P < 0.01. NC, negative control. ns, no significance. Luc, luciferase.

Mentions: To identify the effectors of miR-26a-induced anoikis, we used combined analyses of TargetScan (http://www.targetscan.org/) and PicTar (http://pictar.mdc-berlin.de/) databases to predict the putative target genes of miR-26a (Figure 3A). Using DAVID Bioinformatics Resources (http://david.abcc.ncifcrf.gov/), gene ontology (GO) analysis revealed that the candidate genes were functionally enriched in several biological processes (Figure 3A). We focused on genes related to the focal adhesion pathway because of its close relationship with anoikis as reported previously [30]. The putative target genes in the focal adhesion pathway were COL1A2, COL5A1, PDGFRA, ITGA5 and ITGA6 (Figure S1). Since a previous study has demonstrated that the most target genes of miRNAs are regulated in the mRNA level [31], to further narrow down the candidates, we performed in silico studies. Analyses of two normalized GEO datasets (GSE14520 & GSE6857) show that only ITGA5 mRNA levels were inversely correlated with miR-26a (Figure 3B, Figure S2). Furthermore, we examined the expression levels of miR-26a and ITGA5 in a panel of human HCC cell lines. qRT-PCR and Western blotting analyses revealed a significant (P < 0.05) inverse correlation between miR-26a and ITGA5 expression levels (Figure 3C, Figure 1A and Figure S3). The seed sequence of miR-26a is complementary to the 3′UTR of ITGA5 and is highly conserved in six different species (Figure 3D). These findings indicate that ITGA5 is a potential target of miR-26a.


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)

ITGA5 is a bona fide target gene of mir-26a(A) Gene Ontology classification of miR-26a's potential target genes predicted by integrating the results of two algorithms (Targetscan and PicTar). (B) The correlation of ITGA5 mRNA and miR-26a in human hepatocellular cancer tissues. The Pearson product-moment correlation coefficient and significance levels are indicated. Expression levels of ITGA5 and miR-26a were from two correlative normalized GEO datasets (GSE14520 & GSE6857). (C) The correlation between miR-26a expression and the levels of ITGA5 in different human HCC cell lines and normal hepatocyte-derived cell line (L-02). The miRNA expression was evaluated by qRT-PCR and protein abundance was evaluated by western blot. (D) Schematic diagram of the miR-26a target site of human and other representative mammal ITGA5 3′UTRs. (E) The wild-type 3′UTR of ITGA5 and mutant 3′UTR sequences that abolished binding. (F) A reporter vector containing the WT(wild-type) or MUT(mutant) ITGA5 3′UTR was transfected, along with miR-NC or miR-26a, into HEK-293 cells. Luciferase activity was measured in three independent experiments after 48 hours of transfection and normalized to Renilla luciferase activity. (G) Western blot analysis was performed to determine ITGA5 protein expression post infection with Lv-Luc or Lv-miR-26a. **, P < 0.01. NC, negative control. ns, no significance. Luc, luciferase.
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Figure 3: ITGA5 is a bona fide target gene of mir-26a(A) Gene Ontology classification of miR-26a's potential target genes predicted by integrating the results of two algorithms (Targetscan and PicTar). (B) The correlation of ITGA5 mRNA and miR-26a in human hepatocellular cancer tissues. The Pearson product-moment correlation coefficient and significance levels are indicated. Expression levels of ITGA5 and miR-26a were from two correlative normalized GEO datasets (GSE14520 & GSE6857). (C) The correlation between miR-26a expression and the levels of ITGA5 in different human HCC cell lines and normal hepatocyte-derived cell line (L-02). The miRNA expression was evaluated by qRT-PCR and protein abundance was evaluated by western blot. (D) Schematic diagram of the miR-26a target site of human and other representative mammal ITGA5 3′UTRs. (E) The wild-type 3′UTR of ITGA5 and mutant 3′UTR sequences that abolished binding. (F) A reporter vector containing the WT(wild-type) or MUT(mutant) ITGA5 3′UTR was transfected, along with miR-NC or miR-26a, into HEK-293 cells. Luciferase activity was measured in three independent experiments after 48 hours of transfection and normalized to Renilla luciferase activity. (G) Western blot analysis was performed to determine ITGA5 protein expression post infection with Lv-Luc or Lv-miR-26a. **, P < 0.01. NC, negative control. ns, no significance. Luc, luciferase.
Mentions: To identify the effectors of miR-26a-induced anoikis, we used combined analyses of TargetScan (http://www.targetscan.org/) and PicTar (http://pictar.mdc-berlin.de/) databases to predict the putative target genes of miR-26a (Figure 3A). Using DAVID Bioinformatics Resources (http://david.abcc.ncifcrf.gov/), gene ontology (GO) analysis revealed that the candidate genes were functionally enriched in several biological processes (Figure 3A). We focused on genes related to the focal adhesion pathway because of its close relationship with anoikis as reported previously [30]. The putative target genes in the focal adhesion pathway were COL1A2, COL5A1, PDGFRA, ITGA5 and ITGA6 (Figure S1). Since a previous study has demonstrated that the most target genes of miRNAs are regulated in the mRNA level [31], to further narrow down the candidates, we performed in silico studies. Analyses of two normalized GEO datasets (GSE14520 & GSE6857) show that only ITGA5 mRNA levels were inversely correlated with miR-26a (Figure 3B, Figure S2). Furthermore, we examined the expression levels of miR-26a and ITGA5 in a panel of human HCC cell lines. qRT-PCR and Western blotting analyses revealed a significant (P < 0.05) inverse correlation between miR-26a and ITGA5 expression levels (Figure 3C, Figure 1A and Figure S3). The seed sequence of miR-26a is complementary to the 3′UTR of ITGA5 and is highly conserved in six different species (Figure 3D). These findings indicate that ITGA5 is a potential target of miR-26a.

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