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Cited by 91

The DOI matches its Crossref record; 43 of the 43 references it cites were found in a registry or an index.

MiR-132 Suppresses the Migration and Invasion of Lung Cancer Cells via Targeting the EMT Regulator ZEB2

You, Jiacong, Li, Yang, Fang, Nianzhen, Liu, Bin, Zu, Lingling, Chang, Rui, Li, Xuebing, Zhou, Qinghua·2014·PLoS ONE
DOI 10.1371/journal.pone.0091827PMC PMC3953608
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Abstract

MicroRNAs (miRNAs) are small, non-coding RNAs which can function as oncogenes or tumor suppressor genes in human cancers. Emerging evidence reveals that deregulation of miRNAs contributes to the human non-small cell lung cancer (NSCLC). In the present study, we demonstrated that the expression levels of miR-132 were dramatically decreased in examined NSCLC cell lines and clinical NSCLC tissue samples. Then, we found that introduction of miR-132 significantly suppressed the migration and invasion of lung cancer cells in vitro, suggesting that miR-132 may be a novel tumor suppressor. Further studies indicated that the EMT-related transcription factor ZEB2 was one direct target genes of miR-132, evidenced by the direct binding of miR-132 with the 3' untranslated region (3' UTR) of ZEB2. Further, miR-132 could decrease the expression of ZEB2 at the levels of mRNA and protein. Notably, the EMT marker E-cadherin or vimentin, a downstream of ZEB2, was also down-regulated or up-regulated upon miR-132 treatment. Additionally, over-expressing or silencing ZEB2 was able to elevate or inhibit the migration and invasion of lung cancer cells, parallel to the effect of miR-132 on the lung cancer cells. Meanwhile, knockdown of ZEB2 reversed the enhanced migration and invasion mediated by anti-miR-132. These results indicate that miR-132 suppresses the migration and invasion of NSCLC cells through targeting ZEB2 involving the EMT process. Thus, our finding provides new insight into the mechanism of NSCLC progression. Therapeutically, miR-132 may serve as a potential target in the treatment of human lung cancer.

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Figures

Figure 1 MiR-132 is frequently down-regulated in highly metastatic lung cancer cells and tissue specimens. (A) The relative mRNA levels of miR-132 were detected by qRT-PCR and normalized against an endogenous control (U6 RNA) in several lung cancer cell lines with distinct metastatic ability. Data are reported as mean ±SD for three independent experiments (**P<0.01, Student’s t- test). (B) qRT-PCR analysis of miR-132 expression in 45 pairs of primary NSCLC tissues and their corresponding lymph node metastases. MiR-132 expression in those two types of tissues was compared by way of Wilcoxon signed-rank test (***P<0.001, Student’s t- test).
Figure 1 MiR-132 is frequently down-regulated in highly metastatic lung cancer cells and tissue specimens. (A) The relative mRNA levels of miR-132 were detected by qRT-PCR and normalized against an endogenous control (U6 RNA) in several lung cancer cell lines with distinct metastatic ability. Data are reported as mean ±SD for three independent experiments (**P<0.01, Student’s t- test). (B) qRT-PCR analysis of miR-132 expression in 45 pairs of primary NSCLC tissues and their corresponding lymph node metastases. MiR-132 expression in those two types of tissues was compared by way of Wilcoxon signed-rank test (***P<0.001, Student’s t- test).CC BY · PubMed Central
Figure 2 MiR-132 is able to inhibit the migration and invasion of NSCLC cells in vitro. (A) The wound healing or was used to detected the migration ability of L9981 and A549 cells, respectively. (B, C) Boyden chamber assay was employed to examine the invasion ability of 95D, L9981, and A549 cells, respectively. The results were from three independent experiments (*P<0.05, **P<0.01, Student’s t- test). The migratory cell number in each group was normalized to the control. Cells were transfected with pcDNA3.1 (NC) or pcDNA3.1-miR-132 (miR-132) constructs, and miR-132 inhibitor (anti-miR-132) or inhibitor control (anti-miR-NC). The migratory A549 cells in lower chambers from one experiment were shown in B.
Figure 2 MiR-132 is able to inhibit the migration and invasion of NSCLC cells in vitro. (A) The wound healing or was used to detected the migration ability of L9981 and A549 cells, respectively. (B, C) Boyden chamber assay was employed to examine the invasion ability of 95D, L9981, and A549 cells, respectively. The results were from three independent experiments (*P<0.05, **P<0.01, Student’s t- test). The migratory cell number in each group was normalized to the control. Cells were transfected with pcDNA3.1 (NC) or pcDNA3.1-miR-132 (miR-132) constructs, and miR-132 inhibitor (anti-miR-132) or inhibitor control (anti-miR-NC). The migratory A549 cells in lower chambers from one experiment were shown in B.CC BY · PubMed Central
Figure 3 MiR-132 directly inhibits the expression of ZEB2 through its 3′UTR and regulates the EMT of NSCLC cells. (A) The miR-132 binding site predicted in the 3′UTR of ZEB2 mRNA. (B) Mutant was generated at the seed region of ZEB2 3′ UTR as indicated by the underline. A 3′ UTR fragment of ZEB2 mRNA containing wild-type or mutant of the miR-132 binding sequence was cloned into the downstream of the luciferase gene in pMIR vector. (C) L9981 cells were transfected with pMIR reporter vectors containing either wild-type or mutant ZEB2 3′UTR (indicated as pMIR-ZEB2-3′ UTR-wt and pMIR-ZEB2-3′ UTR-mut) with either pcDNA3.1 (indicated as NC) or pcDNA3.1-miR-132 vector (indicated as miR-132). Luciferase activity was determined 48 h after transfection. (D) ZEB2 mRNA was detected by qRT-PCR in cell lines transfected with pcDNA3.1 (indicated as NC) or pcDNA3.1-miR-132 vector (indicated as miR-132). (E, F) The protein levels of ZEB2, E-cadherin or vimentin was examined by Western blot in cells transfected with different plasmids. Figure F shows the relative gray values of each band (normalized to β-actin). Protein bands from three independent Western blot assays were quantified using Quantity One software (Bio-Rad, USA). Data are reported as mean ±SD (**P<0.01, Student’s t- test).
Figure 3 MiR-132 directly inhibits the expression of ZEB2 through its 3′UTR and regulates the EMT of NSCLC cells. (A) The miR-132 binding site predicted in the 3′UTR of ZEB2 mRNA. (B) Mutant was generated at the seed region of ZEB2 3′ UTR as indicated by the underline. A 3′ UTR fragment of ZEB2 mRNA containing wild-type or mutant of the miR-132 binding sequence was cloned into the downstream of the luciferase gene in pMIR vector. (C) L9981 cells were transfected with pMIR reporter vectors containing either wild-type or mutant ZEB2 3′UTR (indicated as pMIR-ZEB2-3′ UTR-wt and pMIR-ZEB2-3′ UTR-mut) with either pcDNA3.1 (indicated as NC) or pcDNA3.1-miR-132 vector (indicated as miR-132). Luciferase activity was determined 48 h after transfection. (D) ZEB2 mRNA was detected by qRT-PCR in cell lines transfected with pcDNA3.1 (indicated as NC) or pcDNA3.1-miR-132 vector (indicated as miR-132). (E, F) The protein levels of ZEB2, E-cadherin or vimentin was examined by Western blot in cells transfected with different plasmids. Figure F shows the relative gray values of each band (normalized to β-actin). Protein bands from three independent Western blot assays were quantified using Quantity One software (Bio-Rad, USA). Data are reported as mean ±SD (**P<0.01, Student’s t- test).CC BY · PubMed Central
Figure 4 ZEB2 contributes to miR-132- suppressed migration and invasion of NSCLC cells. (A) The expression of ZEB2 was examined by Western blot in NSCLC cells. (B) The relative mRNA levels of ZEB2 were detected in 45 paired NSCLC primary tumor tissues and their lymph node metastasis counterparts. ZEB2 expression in those tissues was compared by way of Wilcoxon signed-rank test (***P<0.001, Student’s t- test). (C) Inverse correlation between miR-132 and ZEB2 expression in NSCLC tissues. ZEB2 expression was analyzed by qRT-PCR and normalized to GAPDH. The miR-132 expression was detected by qRT-PCR analysis and normalized to U6 expression. Statistical analysis was performed using Pearson’s correlation coefficient (r = −0.68, ***P<0.001). (D) The cell invasion was detected by Boyden chamber assay in NL9980 or 95C cells transfected with pCMV or pCMV-ZEB2 vectors, respectively. (E, F) The effect of ZEB2 knockdown on the cell migration or invasion was assessed by wound healing or Boyden chamber assay, respectively (**P<0.01, Student’s t- test). Additionally, the silencing efficiency of ZEB2 by siRNA was examined by Western blot. (G, H) The wound healing or Boyden chamber assay was used to detect the migration or invasion ability of NL9980 cells with different treatments, respectively (*P<0.05, **P<0.01, Student’s t- test). Additionally, the silencing efficiency of ZEB2 by siRNA was examined by Western blot.
Figure 4 ZEB2 contributes to miR-132- suppressed migration and invasion of NSCLC cells. (A) The expression of ZEB2 was examined by Western blot in NSCLC cells. (B) The relative mRNA levels of ZEB2 were detected in 45 paired NSCLC primary tumor tissues and their lymph node metastasis counterparts. ZEB2 expression in those tissues was compared by way of Wilcoxon signed-rank test (***P<0.001, Student’s t- test). (C) Inverse correlation between miR-132 and ZEB2 expression in NSCLC tissues. ZEB2 expression was analyzed by qRT-PCR and normalized to GAPDH. The miR-132 expression was detected by qRT-PCR analysis and normalized to U6 expression. Statistical analysis was performed using Pearson’s correlation coefficient (r = −0.68, ***P<0.001). (D) The cell invasion was detected by Boyden chamber assay in NL9980 or 95C cells transfected with pCMV or pCMV-ZEB2 vectors, respectively. (E, F) The effect of ZEB2 knockdown on the cell migration or invasion was assessed by wound healing or Boyden chamber assay, respectively (**P<0.01, Student’s t- test). Additionally, the silencing efficiency of ZEB2 by siRNA was examined by Western blot. (G, H) The wound healing or Boyden chamber assay was used to detect the migration or invasion ability of NL9980 cells with different treatments, respectively (*P<0.05, **P<0.01, Student’s t- test). Additionally, the silencing efficiency of ZEB2 by siRNA was examined by Western blot.CC BY · PubMed Central

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Metadata

Authors
You, J., Li, Y., Fang, N., Liu, B., Zu, L., Chang, R., Li, X., & Zhou, Q.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-03-13
Volume
9
Issue
3
Pages
e91827
ISSN
1932-6203
DOI
10.1371/journal.pone.0091827
PMC
PMC3953608
Cited by
91
References
43
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)

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You, J., Li, Y., Fang, N., Liu, B., Zu, L., Chang, R., Li, X., & Zhou, Q. (2014). MiR-132 Suppresses the Migration and Invasion of Lung Cancer Cells via Targeting the EMT Regulator ZEB2. PLoS ONE. https://doi.org/10.1371/journal.pone.0091827

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