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Silencing of miRNA-148a by hypermethylation activates the integrin-mediated signaling pathway in nasopharyngeal carcinoma.

Li HP, Huang HY, Lai YR, Huang JX, Chang KP, Hsueh C, Chang YS - Oncotarget (2014)

Bottom Line: For this study, we found that miR-148a is downregulated through hypermethylation in NPC biopsies and NPC cell lines compared with adjacent normal and NP cells respectively.The ectopic expression of miR-148a inhibits cell migration in NPC cells through the suppression of integrin-mediated signaling by targeting VAV2, WASL and ROCK1.Furthermore, immunohistochemical staining and Western blotting analysis revealed that the 3 oncogenic targets of miR-148a were overexpressed in NPC biopsies, suggesting that the inactivation of miR-148a caused by DNA methylation promotes NPC progression.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan, Republic of China (ROC). Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan, Republic of China (ROC). Department of Microbiology and Immunology, School of Medicine, Chang Gung University, Taoyuan, Taiwan, Republic of China (ROC). Contributed equally to this work.

ABSTRACT
MicroRNAs (miRNAs) play a pivotal role in carcinogenesis by suppressing oncogenes or tumor suppressor genes. Various studies have identified numerous miRNAs and their diverse targets; however, the consequences of dysregulated miRNAs in nasopharyngeal carcinoma (NPC) remain unclear. For this study, we found that miR-148a is downregulated through hypermethylation in NPC biopsies and NPC cell lines compared with adjacent normal and NP cells respectively. Promoter assays demonstrated that upstream stimulatory factor 1 (USF1) is a crucial transcription factor that activates miR-148a promoter activity. EMSA assays confirmed that purified USF1 binds better toward the unmethylated than the methylated CG-containing USF1 consensus probe. The ectopic expression of miR-148a inhibits cell migration in NPC cells through the suppression of integrin-mediated signaling by targeting VAV2, WASL and ROCK1. Biochemical and functional assays provided supporting evidence that these 3 genes are the downstream targets of miR-148a in NPC cells. Furthermore, immunohistochemical staining and Western blotting analysis revealed that the 3 oncogenic targets of miR-148a were overexpressed in NPC biopsies, suggesting that the inactivation of miR-148a caused by DNA methylation promotes NPC progression. Overall, our findings revealed that miR-148a can act as tumor suppressor miRNA and serve as a biomarker as well as a therapeutic target for NPC.

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miR-148a inhibits integrin signaling pathway by targeting ITGA11, ITGB8, VAV2, and WASLThe target proteins were knockdown by transfecting shROCK1, shVAV2, and shWASL in both TW02 and TW06 cells. (a) Cell migration and (b) wound-healing assays were measured in TW02 and TW06 cells. (c) Wound-healing and (d) transwell cell migration assays were performed after the TW02 cells were cotransfected with miRNA vector or miR-148a; and cDNA vector control or target gene expressing clones (ITGA11, ITGB8, VAV2, and WASL) as indicicated. The p values between 2 groups are indicated. (e) IHC analysis was performed to determine the expression levels of endogenous ROCK1, VAV2, and WASL in 21 paired NPC tissues. Only 6 paired NPC tissues are shown. (f) The correlation between miR148a RNA expression and the protein expression of 3 target genes in 5 paired NPC tissues were indicated. Protein expression fold change in each NPC tumor was normalized with the actin and non-tumor (T/N) (left panel). The expression fold change of miR-148a (T/N) quantitated using qRT-PCR and the fold change of the endogenous protein expression of target genes (T/N) in A-E NPC tissues were listed in a table (right panel). (g) The correlation of relative miR-148a levels in T/N (X-axis) and the endogenous protein levels of ROCK1, VAV2, and WASL in T/N (Y-axis) in 5 paired NPC tissues were indicated. (r: correlation coefficient)
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Figure 5: miR-148a inhibits integrin signaling pathway by targeting ITGA11, ITGB8, VAV2, and WASLThe target proteins were knockdown by transfecting shROCK1, shVAV2, and shWASL in both TW02 and TW06 cells. (a) Cell migration and (b) wound-healing assays were measured in TW02 and TW06 cells. (c) Wound-healing and (d) transwell cell migration assays were performed after the TW02 cells were cotransfected with miRNA vector or miR-148a; and cDNA vector control or target gene expressing clones (ITGA11, ITGB8, VAV2, and WASL) as indicicated. The p values between 2 groups are indicated. (e) IHC analysis was performed to determine the expression levels of endogenous ROCK1, VAV2, and WASL in 21 paired NPC tissues. Only 6 paired NPC tissues are shown. (f) The correlation between miR148a RNA expression and the protein expression of 3 target genes in 5 paired NPC tissues were indicated. Protein expression fold change in each NPC tumor was normalized with the actin and non-tumor (T/N) (left panel). The expression fold change of miR-148a (T/N) quantitated using qRT-PCR and the fold change of the endogenous protein expression of target genes (T/N) in A-E NPC tissues were listed in a table (right panel). (g) The correlation of relative miR-148a levels in T/N (X-axis) and the endogenous protein levels of ROCK1, VAV2, and WASL in T/N (Y-axis) in 5 paired NPC tissues were indicated. (r: correlation coefficient)

Mentions: The integrin signaling cascade, which regulates cell motility and migration, is frequently activated in cancer cells [26]. To investigate whether miR-148a inhibits migration via the targeting integrin pathway, we performed genes loss- and gain-of-function experiments on miR-148a target genes in the presence or absence of miR-148a in NPC cells. The knockdown of endogenous VAV2, ROCK1, and WASL alone triggered by individual shRNAs reduced cell migration by approximately 50% (Fig. 5a) as well as wound-healing ability (Fig. 5b) in TW02 cells, suggesting that these genes are involved in promoting cell migration. Next, we transfected miR-148a to knockdown its collective downstream target genes, and concurrently reintroduced an individual target gene sequentially to counteract the repressive effect of miR-148a in NPC cells. After 24 h of post-transfection, we performed transwell and wound-healing assays. The results showed that the cotransfection of miR-148a with each target gene (ITGA11, ITGB8, VAV2, and WASL) could promote cell migration (by approximately 20%) and wound-healing abilities (Figs. 5c-5d). These results revealed that, despite the expression of exogenous miR-148a, which inhibited the expression of hundreds of target genes, the reintroduction of an miR-148a target gene could restore the cell migration ability, suggesting that ITGA11, ITGB8, VAV2, and WASL are specific targets of miR-148a.


Silencing of miRNA-148a by hypermethylation activates the integrin-mediated signaling pathway in nasopharyngeal carcinoma.

Li HP, Huang HY, Lai YR, Huang JX, Chang KP, Hsueh C, Chang YS - Oncotarget (2014)

miR-148a inhibits integrin signaling pathway by targeting ITGA11, ITGB8, VAV2, and WASLThe target proteins were knockdown by transfecting shROCK1, shVAV2, and shWASL in both TW02 and TW06 cells. (a) Cell migration and (b) wound-healing assays were measured in TW02 and TW06 cells. (c) Wound-healing and (d) transwell cell migration assays were performed after the TW02 cells were cotransfected with miRNA vector or miR-148a; and cDNA vector control or target gene expressing clones (ITGA11, ITGB8, VAV2, and WASL) as indicicated. The p values between 2 groups are indicated. (e) IHC analysis was performed to determine the expression levels of endogenous ROCK1, VAV2, and WASL in 21 paired NPC tissues. Only 6 paired NPC tissues are shown. (f) The correlation between miR148a RNA expression and the protein expression of 3 target genes in 5 paired NPC tissues were indicated. Protein expression fold change in each NPC tumor was normalized with the actin and non-tumor (T/N) (left panel). The expression fold change of miR-148a (T/N) quantitated using qRT-PCR and the fold change of the endogenous protein expression of target genes (T/N) in A-E NPC tissues were listed in a table (right panel). (g) The correlation of relative miR-148a levels in T/N (X-axis) and the endogenous protein levels of ROCK1, VAV2, and WASL in T/N (Y-axis) in 5 paired NPC tissues were indicated. (r: correlation coefficient)
© Copyright Policy - open-access
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Figure 5: miR-148a inhibits integrin signaling pathway by targeting ITGA11, ITGB8, VAV2, and WASLThe target proteins were knockdown by transfecting shROCK1, shVAV2, and shWASL in both TW02 and TW06 cells. (a) Cell migration and (b) wound-healing assays were measured in TW02 and TW06 cells. (c) Wound-healing and (d) transwell cell migration assays were performed after the TW02 cells were cotransfected with miRNA vector or miR-148a; and cDNA vector control or target gene expressing clones (ITGA11, ITGB8, VAV2, and WASL) as indicicated. The p values between 2 groups are indicated. (e) IHC analysis was performed to determine the expression levels of endogenous ROCK1, VAV2, and WASL in 21 paired NPC tissues. Only 6 paired NPC tissues are shown. (f) The correlation between miR148a RNA expression and the protein expression of 3 target genes in 5 paired NPC tissues were indicated. Protein expression fold change in each NPC tumor was normalized with the actin and non-tumor (T/N) (left panel). The expression fold change of miR-148a (T/N) quantitated using qRT-PCR and the fold change of the endogenous protein expression of target genes (T/N) in A-E NPC tissues were listed in a table (right panel). (g) The correlation of relative miR-148a levels in T/N (X-axis) and the endogenous protein levels of ROCK1, VAV2, and WASL in T/N (Y-axis) in 5 paired NPC tissues were indicated. (r: correlation coefficient)
Mentions: The integrin signaling cascade, which regulates cell motility and migration, is frequently activated in cancer cells [26]. To investigate whether miR-148a inhibits migration via the targeting integrin pathway, we performed genes loss- and gain-of-function experiments on miR-148a target genes in the presence or absence of miR-148a in NPC cells. The knockdown of endogenous VAV2, ROCK1, and WASL alone triggered by individual shRNAs reduced cell migration by approximately 50% (Fig. 5a) as well as wound-healing ability (Fig. 5b) in TW02 cells, suggesting that these genes are involved in promoting cell migration. Next, we transfected miR-148a to knockdown its collective downstream target genes, and concurrently reintroduced an individual target gene sequentially to counteract the repressive effect of miR-148a in NPC cells. After 24 h of post-transfection, we performed transwell and wound-healing assays. The results showed that the cotransfection of miR-148a with each target gene (ITGA11, ITGB8, VAV2, and WASL) could promote cell migration (by approximately 20%) and wound-healing abilities (Figs. 5c-5d). These results revealed that, despite the expression of exogenous miR-148a, which inhibited the expression of hundreds of target genes, the reintroduction of an miR-148a target gene could restore the cell migration ability, suggesting that ITGA11, ITGB8, VAV2, and WASL are specific targets of miR-148a.

Bottom Line: For this study, we found that miR-148a is downregulated through hypermethylation in NPC biopsies and NPC cell lines compared with adjacent normal and NP cells respectively.The ectopic expression of miR-148a inhibits cell migration in NPC cells through the suppression of integrin-mediated signaling by targeting VAV2, WASL and ROCK1.Furthermore, immunohistochemical staining and Western blotting analysis revealed that the 3 oncogenic targets of miR-148a were overexpressed in NPC biopsies, suggesting that the inactivation of miR-148a caused by DNA methylation promotes NPC progression.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan, Republic of China (ROC). Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan, Republic of China (ROC). Department of Microbiology and Immunology, School of Medicine, Chang Gung University, Taoyuan, Taiwan, Republic of China (ROC). Contributed equally to this work.

ABSTRACT
MicroRNAs (miRNAs) play a pivotal role in carcinogenesis by suppressing oncogenes or tumor suppressor genes. Various studies have identified numerous miRNAs and their diverse targets; however, the consequences of dysregulated miRNAs in nasopharyngeal carcinoma (NPC) remain unclear. For this study, we found that miR-148a is downregulated through hypermethylation in NPC biopsies and NPC cell lines compared with adjacent normal and NP cells respectively. Promoter assays demonstrated that upstream stimulatory factor 1 (USF1) is a crucial transcription factor that activates miR-148a promoter activity. EMSA assays confirmed that purified USF1 binds better toward the unmethylated than the methylated CG-containing USF1 consensus probe. The ectopic expression of miR-148a inhibits cell migration in NPC cells through the suppression of integrin-mediated signaling by targeting VAV2, WASL and ROCK1. Biochemical and functional assays provided supporting evidence that these 3 genes are the downstream targets of miR-148a in NPC cells. Furthermore, immunohistochemical staining and Western blotting analysis revealed that the 3 oncogenic targets of miR-148a were overexpressed in NPC biopsies, suggesting that the inactivation of miR-148a caused by DNA methylation promotes NPC progression. Overall, our findings revealed that miR-148a can act as tumor suppressor miRNA and serve as a biomarker as well as a therapeutic target for NPC.

Show MeSH
Related in: MedlinePlus