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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Necl-2/CADM1 is down-regulated by the promoter hypermethylation and/or the loss of heterozygosity at chromosome 11q23.2 in many types of cancers and serves as a tumor suppressor by interacting in cis with ErbB3 and suppressing the ligand-induced ErbB2/ErbB3 signaling for cell movement and death. However, the incidence of these epigenetic and genetic abnormalities of Necl-2 is 30–60% in these cancers. We investigated here other mechanisms that down-regulate Necl-2. miR-214, that is frequently up-regulated in a variety of cancers, targeted the 3′UTR of the Necl-2 mRNA directly, suppressed the translation of Necl-2 and enhanced the ligand-induced ErbB2/ErbB3 signaling in human colon cancer Caco-2 cells. Hypoxia reduced the Necl-2 protein level in a manner independent of miR-214 or hypoxia-inducible factor-1α in Caco-2 cells. These results indicate that miR-214 and hypoxia are novel regulators that down-regulate Necl-2 and enhance ErbB2/ErbB3 signaling.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Nectin-like molecule-2 (Necl-2), also termed as IGSF4, RA175, SgIGSF, TSLC1, SynCAM1 or CADM1 (Gomyo et al. 1999; Kuramochi et al. 2001; Urase et al. 2001; Wakayama et al. 2001; Biederer et al. 2002), is an immunoglobulin (Ig)-like cell adhesion molecule and is a member of the Necl family consisting of five members, Necl-1, Necl-2, Necl-3, Necl-4 and Necl-5 (Takai et al. 2008b). This family comprises a superfamily with the nectin family, which consists of four members, nectin-1, -2, -3 and -4 (Takai et al. 2008b). All members of these families have similar domain structures: one extracellular region with three Ig-like domains, one transmembrane segment and one cytoplasmic region. Necl-2 is widely expressed in various tissues and organs, although it is undetectable in fibroblast cells, such as NIH3T3, Swiss3T3 and L cells (Takai et al. 2008a). In epithelial cells of the gall bladder, Necl-2 is localized at the basolateral plasma membrane, but not at specialized cell–cell junctions, such as tight junctions, adherens junctions and desmosomes (Shingai et al. 2003). Necl-2 regulates many cellular functions, such as cell polarization, proliferation, differentiation, survival and sorting (Takai et al. 2008a). It is also involved in immunological responses, synapse formation, spermatogenesis and epithelial–mesenchymal transition (EMT) (Takai et al. 2008a). Pathologically, Necl-2 serves as a tumor suppressor in many types of cancers (Murakami 2005).

Necl-2 interacts in trans with other members of the nectin and Necl families, such as Necl-1, nectin-3 and class-I-restricted T-cell-associated molecule, through its extracellular region (Masuda et al. 2002; Shingai et al. 2003; Galibert et al. 2005). The juxtamembrane region of the cytoplasmic region of Necl-2 contains a band 4.1-binding motif and binds DAL-1 (Yageta et al. 2002). The cytoplasmic region has the PDZ-binding motif at its C-terminal region and binds Pals2, Dlg3/MPP3, CASK and PTPN13 (Takai et al. 2008b; Kawano et al. 2009). In addition, we previously showed that Necl-2 interacts in cis with ErbB3 and integrin α6β4 through its extracellular region (Kawano et al. 2009; Mizutani et al. 2011).

ErbB3 is a member of the ErbB receptor tyrosine kinase family that consists of four members, ErbB1/EGFR, ErbB2/Neu, ErbB3 and ErbB4. Amplification and/or mutation of the ErbB2 and ErbB3 genes are observed in many types of cancers, including lung and breast cancers (Schraml et al. 1999; Yarden & Sliwkowski 2001; Lahiry et al. 2010), and causes enhanced signaling for cell movement and survival, eventually resulting in tumorigenesis, invasion and metastasis. Necl-2 inhibits the heregulin (HRG)-induced, ErbB2-catalyzed tyrosine phosphorylation of ErbB3 and subsequent activation of Rac and Akt, resulting in the inhibition of cell movement and death (Kawano et al. 2009). PTPN13, a protein tyrosine phosphatase known to be a tumor suppressor (Abaan & Toretsky 2008), binds to the cytoplasmic region of Necl-2 and mediates the inhibitory role of Necl-2 in suppressing the ErbB2/ErbB3 signaling (Kawano et al. 2009). Necl-2 thus serves as a tumor suppressor through the suppression of the ErbB2/ErbB3 signaling.

Integrin α6β4 is abundantly expressed in normal epithelial cells and forms hemidesmosomes, one of the cell–extracellular matrix junctions (Wilhelmsen et al. 2006). Hemidesmosomes play roles in keeping epithelial cell morphology and inhibiting cell movement. In many types of cancer cells, integrin α6β4 is up-regulated and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and a facilitation of their invasion (Mercurio et al. 2001). Necl-2 inhibits its disassembly from hemidesmosomes and thereby serves as a tumor suppressor (Mizutani et al. 2011).

Necl-2 is down-regulated by the hypermethylation of the Necl-2 gene promoter and/or the loss of heterozygosity (LOH) at chromosome 11q23.2 in many types of cancers, such as lung, liver, breast and pancreas cancers (Gomyo et al. 1999; Kuramochi et al. 2001; Murakami 2005). However, the incidence of these epigenetic and genetic abnormalities of Necl-2 is approximately 30%–60% in these cancers and other mechanisms for the down-regulation of Necl-2 are presumed to be present. We attempted here to investigate novel mechanisms for the down-regulation of Necl-2, focusing on micro-RNAs (miRNAs) and hypoxia, because the 3′untranslated region (UTR) of the Necl-2 mRNA was predicted to be targeted by multiple miRNAs (Lewis et al. 2005), and hypoxia is known to drive EMT and enhances cell movement, invasion and metastasis of cancer cells (Nieto 2011).

miRNAs are short noncoding RNAs that regulate protein expression from targeted genes by pairing complementary sequences in the 3′UTR (Bartel 2009). miRNAs regulate various cellular processes, such as differentiation, proliferation, apoptosis and angiogenesis. In addition, miRNAs have oncogenic or tumor suppressive roles, and alterations in miRNAs and other short or long noncoding RNA are involved in initiation, progression and metastasis of human cancers. Among miRNAs, miR-214 is up-regulated in various human malignancies, including pancreatic, gastric, breast and ovarian cancers and malignant melanoma (Volinia et al. 2006; Sempere et al. 2007; Yang et al. 2008a; Shimono et al. 2009; Ueda et al. 2010; Yin et al. 2010; Zhang et al. 2010; Penna et al. 2011). Hypoxia regulates the protein level and activity of hypoxia-inducible factor-1α (HIF-1α), which up-regulates expression of the proteins that induce EMT, such as Twist1 and Zeb1 (Yang & Wu 2008). We found here that miR-214 and hypoxia are novel regulators that down-regulate Necl-2 and enhance the ErbB2/ErbB3 signaling.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Direct targeting of Necl-2 by miR-214

We first examined whether miR-214 was able to directly bind to and regulate the activity of the 3′UTR of the Necl-2 mRNA because Necl-2 is predicted by TargetScan to have a miR-214 targeted site (Lewis et al. 2005). The 3′UTR of Necl-2, which contains a predicted target site for miR-214, was cloned into the pGL3-MC vector downstream of a luciferase minigene (pGL3-Necl-2-3′UTR wild type) (Fig. 1A). In addition, two nucleotides within the predicted miR-214 target site were mutated in the pGL3-Necl-2-3′UTR-mutant vector (Fig. 1A). Human embryonic kidney (HEK) 293 cells that do not express miR-214 were cotransfected with the pGL3-Necl-2-3′UTR wild-type vector, its mutant vector, or the control luciferase vector, together with pRL-TK Renilla luciferase vector and miRNA precursors. Cotransfection of the miR-214 precursor suppressed luciferase activity of the pGL3-Necl-2-3′UTR wild-type vector by 40%, indicating that miR-214 suppressed its translation through the Necl-2 3′UTR (Fig. 1B). Mutation of the predicted miR-214 target site in the Necl-2 3′UTR abrogated the repressive ability of miR-214, demonstrating the specificity of the target sequence for Necl-2 (Fig. 1B).

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Figure 1. Targeting of Necl-2 by miR-214. (A) Schematic representation of the pGL3 luciferase expression plasmid. The sequence of the Necl-2 3′UTR containing the predicted miR-214 targeting site was cloned downstream of a luciferase minigene. Two nucleotides complementary to the seed sequence (nucleotides 2–7 of miRNA) for miR-214 were mutated in the Necl-2 mutant plasmid. The number indicates the position of the nucleotides in the reference wild-type sequence of Necl-2 (NM_001098517). (B) Activity of luciferase gene linked to the 3′UTR of the Necl-2 mRNA. The mean of the results from the cells transfected with control precursor was set at 1.0. The data are mean and standard deviation (SD) of three separate transfections (n = 3, *P = 0.01). n.s., not significant (P > 0.05).

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We then examined the effect(s) of miR-214 on the Necl-2 protein level in human colon cancer Caco-2 cells by Western blotting. The immunoreactive bands of Necl-2 were observed at approximately 90 kDa with several additional bands in the control-precursor-transfected cells (Fig. 2). The multiple immunoreactive bands of Necl-2 were present because of the differences of posttranslational modifications (A. Minami, Y. Shimono, K. Mizutani, K. Nobutani, K. Momose, T. Azuma and Y. Takai, unpublished data). The intensities of the bands were reduced by 40% in the miR-214-precursor-transfected cells (Fig. 2). The reduction in Necl-2 protein level by miR-214 was confirmed using human lung cancer A549 cells (K. Momose, A. Minami, Y. Shimono, K. Mizutani, K. Nobutani, T. Azuma and Y. Takai, unpublished data). These results indicate that miR-214 targets the 3′UTR of the Necl-2 mRNA and reduces its protein level.

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Figure 2. Down-regulation of the Necl-2 protein level by miR-214. Caco-2 cells were transfected with the indicated miRNA precursors and cultured for 72 h. The cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting using the anti-Necl-2 polyclonal antibody (pAb) and the anti-actin monoclonal antibody (mAb). The intensities of the Necl-2 protein bands were evaluated using imagej software. The mean of the intensities of the Necl-2 protein bands from the cells transfected with control precursor was set at 1.0. The reduction in the Necl-2 protein level in the miR-214-precursor-transfected cells was statistically significant (n = 6, *P = 0.006).

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Enhancement of HRG-induced ErbB2/ErbB3 signaling by miR-214

We examined the effect of miR-214 on the HRG-induced ErbB2/ErbB3 signaling by analyzing the phosphorylation of ErbB3 in Caco-2 cells. We previously showed that this cell line expresses ErbB2, ErbB3 and Necl-2 and that Necl-2 inhibits the HRG-induced ErbB2/ErbB3 signaling (Kawano et al. 2009). Caco-2 cells were serum starved and stimulated with HRG. The intensities of the immunoreactive bands of ErbB3, in which the tyrosine 1289 was phosphorylated, were increased in response to HRG in both miR-214-expressing and miR-control-expressing Caco-2 cells, but the intensities were much higher in miR-214-expressing Caco-2 cells than in miR-control-expressing Caco-2 cells (Fig. 3). These results indicate that miR-214 enhances the HRG-induced ErbB2/ErbB3 signaling at least partly through the reduction in the Necl-2 protein level.

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Figure 3. Enhancement of the HRG-induced ErbB2/ErbB3 signaling by miR-214. Caco-2 cells were transfected with control or miR-214 precursors and cultured for 72 h. The cells were serum starved for 20 h and were stimulated by 20 ng/mL HRG for the indicated periods of time. The lysates were subjected to SDS-PAGE, followed by Western blotting using the anti-Necl-2, anti-phospho-ErbB3 and anti-ErbB3 pAbs and the anti-actin mAb.

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Reduction in the Necl-2 protein level by hypoxia

We next analyzed the Necl-2 protein level under hypoxic conditions because hypoxia and induction of a transcription factor HIF-1α are involved in the progression of cancer (Wilson & Hay 2011). Hypoxia increased the HIF-1α protein level in Caco-2 cells and reduced the Necl-2 protein level by 25% at 48 h of hypoxia (Fig. 4A).

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Figure 4. Reduction in the Necl-2 protein level under hypoxic conditions. (A) Caco-2 cells were cultured under hypoxic conditions for 72 h. The lysates were subjected to SDS-PAGE, followed by Western blotting using the anti-Necl-2 and anti-HIF-1α pAbs and the anti-actin mAb. The intensities of the Necl-2 protein bands were evaluated using imagej software. The mean of the intensities of the Necl-2 protein bands from the cells under normoxic conditions was set at 1.0. The reduction in the Necl-2 protein level under hypoxic conditions was statistically significant (n = 5, *P = 0.002). (B) The amount of mRNA was not significantly changed in Caco-2 cells by hypoxia (n = 3). The mean of the amounts of the Necl-2 mRNA from the cells under normoxic conditions was set at 1.0. n.s., not significant (P > 0.05).

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We then analyzed the effect of hypoxia on transcription of the Necl-2 mRNA in Caco-2 cells. Amount of the Necl-2 mRNA was not significantly changed under hypoxic conditions (Fig. 4B). Expression of various miRNAs is enhanced or suppressed under hypoxic conditions (Galanis et al. 2008). We therefore analyzed whether hypoxia was able to affect miR-214 expression to down-regulate the Necl-2 protein level posttranscriptionally. In Caco-2 cells, miR-214 was faintly expressed under normoxic conditions and was not increased by hypoxia as estimated by real-time PCR analysis (K. Momose, A. Minami, Y. Shimono, K. Mizutani, K. Nobutani, T. Azuma and Y. Takai, unpublished data).

HIF-1α is involved in diverse biological processes not only as a transcription factor but also as a transcriptional coactivator and a director of proteasomal degradation (Greer et al. 2012). However, the Necl-2 protein level was unaffected in Caco-2 cells transfected with the small interfering RNA (siRNA) against HIF-1α (Fig. 5). Taken together, these results indicate that hypoxia down-regulates Necl-2 in a manner independent of miR-214, the Necl-2 mRNA transcription and HIF-1α.

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Figure 5. No requirement of HIF-1α for the reduction in the Necl-2 protein level under hypoxic conditions. Caco-2 cells transfected with an siRNA for HIF-1α or a control siRNA were cultured under hypoxic conditions for 72 h. The lysates were subjected to SDS-PAGE, followed by Western blotting using the anti-HIF-1α and anti-Necl-2 pAbs and the anti-actin mAb.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

It is established that expression of Necl-2 is down-regulated in cancer cells because of the hypermethylation of the Necl-2 gene promoter and/or the LOH at chromosome 11q23.2 (Murakami 2005). In addition to these two mechanisms, we showed here two novel mechanisms in which miR-214 and hypoxia were the regulators that down-regulate Necl-2 (Fig. 6).

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Figure 6. Four mechanisms for the down-regulation of Necl-2. Schematic representation of the mechanisms for the down-regulation of Necl-2. Two novel mechanisms identified in this study are presented in the black boxes with white letters.

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miRNAs target multiple mRNAs simultaneously and regulate gene translation. Recently, two miRNAs, miR-10b and miR-216a, were shown to target the 3′UTR of the Necl-2 mRNA and to be involved in the progression of hepatocellular carcinoma (Chen et al. 2012; Li et al. 2012). In this study, we found that miR-214 directly bound to the target site within the 3′UTR of the Necl-2 mRNA and reduced its protein level. We recently found that miR-199 that was clustered with miR-214 at the intron of Dnm3os gene, indirectly reduced the Necl-2 protein level (A. Minami, Y. Shimono, K. Mizutani, K. Nobutani, K. Momose, T. Azuma and Y. Takai, unpublished data). We are currently analyzing the mode of action for the suppression of Necl-2 by miR-199. In addition, transcription of the miRNA cluster for miR-199 and miR-214 is regulated by a transcription factor TWIST, one of the important EMT inducers (Yin et al. 2010). In accordance with its high expression in tumor-initiating cells and highly metastatic tumors, expression of miR-214 in cancer cells induces cell migration, invasion and chemoresistance (Yang et al. 2008a; Yin et al. 2010; Penna et al. 2011). miR-214 targets and down-regulates PTEN, resulting in the activation of the Akt pathway for cell survival and cisplatin resistance in human ovarian cancer (Yang et al. 2008a). High expression of miR-214 is associated with progression of gastric cancer and melanoma (Ueda et al. 2010; Penna et al. 2011). miR-214 targets and down-regulates p53, indirectly induces the expression of Nanog and enhances chemoresistance of ovarian cancer cells (Xu et al. 2012). Taken together, miR-214 and miR-199 may cooperatively reduce the Necl-2 protein level and regulates cell invasion, metastasis and EMT during physiological and pathological processes, such as development and tumor progression.

We further found here that the Necl-2 protein level was reduced under hypoxic conditions in Caco-2 cells. Hypoxia actively contributes to disease progression and/or tissue recovery through the promotion of processes including angiogenesis, inflammation, metabolism and development. Hypoxia modulates expression of membrane proteins, such as E-cadherin, N-cadherin and chemokine (C-X-C motif) receptor 4 (CXCR4), at least partly through its induction of EMT. Down-regulation of E-cadherin under hypoxic conditions is mediated by TWIST that is directly induced by HIF-1α (Yang et al. 2008b). Expression of CXCR4 is dependent on HIF-1α and is negatively regulated by von Hippel–Lindau tumor suppressor protein that down-regulates HIF-1α (Schioppa et al. 2003; Staller et al. 2003). Although an increased expression of many hypoxia-induced proteins is mediated by HIF-1α and nuclear factor-κB (NFκB) (Semenza 1998; Taylor & Cummins 2009), it is not well characterized yet which factor or protein complex mediates the hypoxia-induced reduction in protein expression. In addition, hypoxia affects expression of a number of miRNAs, including miR-210 that is a direct transcriptional target of HIF-1α (Galanis et al. 2008). We found that miR-199 or miR-214 was not up-regulated in Caco-2 cells under hypoxic conditions (K. Momose, A. Minami, Y. Shimono, K. Mizutani, K. Nobutani, T. Azuma and Y. Takai, unpublished data), indicating that the Necl-2 protein level is not posttranslationally regulated by these miRNAs under hypoxic conditions. In addition, the expression of NFκB and IκB was not modulated by miR-214 under hypoxic conditions (K. Momose, A. Minami, Y. Shimono, K. Mizutani, N. Nobutani, T. Azuma and Y. Takai, unpublished data). We speculate that reduction in the Necl-2 protein level under hypoxic conditions is mediated by (i) enhancement of the Necl-2 protein degradation and/or (ii) targeting of the 3′UTR of the Necl-2 mRNA by other hypoxia-induced miRNAs.

Necl-2 is involved in a variety of cellular functions as described above and plays roles in a wide range of events from normal development to tumorigenesis. Therefore, it is speculated that multiple regulatory mechanisms for the Necl-2 protein level have developed. Epigenetic regulation, such as promoter hypermethylation for stable and inheritable down-regulation of Necl-2, may be suitable for the regulation in normal development and tumorigenesis. However, environmental regulation, such as hypoxia, is potentially reversible and transient and may be suitable for the regulation of cellular events, such as EMT and cell movement. The regulation by miRNAs may be suitable for both stable and transient regulations of a variety of cellular and biological processes in concert with other miRNA-target proteins not only in normal development but also in tumorigenesis. Considering that up-regulation of miR-214 and hypoxia are highly associated with cancer progression, the two novel mechanisms described here will be involved in the down-regulation of Necl-2 in a variety of human cancers, cooperatively or independently of promoter hypermethylation and LOH.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Cell culture and hypoxic culture

Caco-2 cells were purchased from the European Collection of Cell Cultures. Caco-2 and HEK 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin and cultured at 5% CO2 at 37 °C. In addition, 0.1 mm nonessential amino acids were added in the culture medium for Caco-2 cells. For hypoxic culture, cells were incubated in the multigas incubator (APM-30D; Astec) at 1% O2 at 37 °C.

Plasmid vectors

Total RNA was extracted from Caco-2 cells and reverse transcribed using Superscript III (Invitrogen) according to the manufacturer's protocol. The partial 3′UTR sequences of the putative miR-214 target genes were amplified by PCR using the cDNA as a template and cloned at the 3′ of the luciferase gene of the firefly luciferase reporter pGL3-MC vector (Shimono et al. 2009). The following primers with restriction enzyme sites were used for cloning of the 3′UTR sequence of Necl-2: Necl-2 3′UTR forward, 5′-GCGGGCCCCCTTTCTTTTCCCCCTTTGTGCAC-3′; reverse, 5′-GCACTAGTCTATGAAATCTAAGCTGTGCTGGTTC-3′. Mutation of the putative miR-214 target sequences within the 3′UTR was generated using a site-specific mutagenesis method. The primers used for mutagenesis were as follows: Necl-2 mutant forward, 5′-CTCCCATTTTGGAATTTGCGTGGTGGGATTCCTTAG-3′; reverse, 5′-GGAAAAGAGTCTAAGGAATCCCACCACGCAAATTCC-3′.

miRNA/siRNA transfection

The miR-214 and control precursors were purchased from Applied Biosystems. Stealth RNAi duplex against human HIF-1α and stealth RNAi negative control duplex were purchased from Invitrogen. The sequence of the stealth RNAi duplex selected for the knockdown of HIF-1α was 5′-GGGAUUAACUCAGUUUGAACUAACU-3′. HEK 293 cells or Caco-2 cells were plated in six-well plates, reverse transfected with 10 nm pre-miRNA precursor or 20 nm siRNA using Lipofectamine RNAiMAX reagent (Invitrogen), according to the manufacturer's instructions, and cultured for 48 h.

Dual-luciferase reporter assay

HEK293 cells were plated in 48-well plates precoated with collagen I (Iwaki) at 80% confluency and cultured for 24 h. The cells were cotransfected with 360 ng of the pGL3-MC firefly luciferase constructs with the wild-type or mutated 3′UTR of the Necl-2 mRNA and 40 ng of pRL-TK Renilla luciferase plasmid (Promega), together with 35 nm miR-214 precursor or negative control precursor using Lipofectamine 2000 (Invitrogen). The lysates were collected 48 h after the transfection, and the activities of firefly luciferase and Renilla luciferase were measured using the Dual-Luciferase Reporter System (Promega) and normalized to Renilla luciferase activity. The mean of the results from the cells transfected with control precursor was set at 1.0. All experiments were carried out in triplicate. The results were statistically analyzed using the Student's t-test.

Real-time PCR assay

Total RNA was isolated from cells using TRIzol Reagent (Invitrogen) according to the manufacturer's instruction. Abundance of mRNAs in Caco-2 cells transfected with miRNA precursors was measured by qRT-PCR method using a SYBR GREEN master mix (Applied Biosystems) and Thermal Cycler Dice (TaKaRa). The results were statistically analyzed using the Student's t-test.

Antibodies

A rabbit anti-Necl-2 pAb was described previously (Kawano et al. 2009). The following rabbit pAbs were purchased from commercial sources: anti-ErbB3 pAb (Santa Cruz Biotechnology), anti-phospho-ErbB3 Tyr1289 pAb (Cell Signaling) and anti-HIF-1α pAb (Novus Biologicals). An anti-actin mAb was purchased from Millipore. A horseradish peroxidase–conjugated secondary Ab was purchased from GE Healthcare.

Western blotting

Samples were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride filters (Millipore). After being blocked with 2% skim milk in 0.05% Tween 20/Tris-buffered saline, filters were incubated with 1 : 1000 diluted anti-Necl-2 pAb or 1 : 2000 diluted anti-actin mAb. Then, 1 : 1000 diluted peroxidase-conjugated anti-rabbit or mouse IgG Ab was added and developed using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The intensities of the immunoreactive bands were evaluated using the imagej software. The results were statistically analyzed using the Student's t-test.

Serum starvation and HRG stimulation

Caco-2 cells were transfected with the pre-miR precursors using Lipofectamine RNAiMAX reagent (Invitrogen), plated in six-well plates at a density of 2.5 × 104/cm2 and cultured for 48 h. The cells were starved of serum with DMEM containing 0.5% fatty acid-free bovine serum albumin (BSA) (Sigma) for 20 h, followed by stimulation with 20 ng/mL HRG (Sigma) in DMEM containing 0.5% fatty acid-free BSA. The cells were washed with ice-cold PBS twice and lysed with RIPA buffer (20 mm Tris-HCl, pH 7.5, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10% glycerol, 135 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, 50 mm NaF, 1 mm Na3VO4, 10 μg/mL leupeptin, 2 μg/mL aprotinin, and 10 μm phosphatase inhibitor cocktail 3 (Sigma)). The lysates were then boiled in the SDS sample buffer (60 mm Tris–HCl, pH 6.7, 3% SDS, 2% 2-mercaptoethanol and 5% glycerol) for 5 min and subjected to SDS-PAGE, followed by Western blotting using the indicated Abs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This work was supported by the Global COE Program ‘Global Center for Education and Research in Integrative Membrane Biology’ and the Targeted Proteins Research Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan, by Grants-in-Aid from the Japan Society for the Promotion of Science, by the Core Research for Evolutional Science and Technology from the Japanese Science and Technology Agency, and by the grants from the Naito Foundation, the Sagawa Foundation, the Yasuda Medical Foundation, and the Cell Science Research Foundation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • Abaan, O.D. & Toretsky, J.A. (2008) PTPL1: a large phosphatase with a split personality. Cancer Metastasis Rev. 27, 205214.
  • Bartel, D.P. (2009) MicroRNAs: target recognition and regulatory functions. Cell 136, 215233.
  • Biederer, T., Sara, Y., Mozhayeva, M., Atasoy, D., Liu, X., Kavalali, E.T. & Südhof, T.C. (2002) SynCAM, a synaptic adhesion molecule that drives synapse assembly. Science 297, 15251531.
  • Chen, P.J., Yeh, S.H., Liu, W.H., Lin, C.C., Huang, H.C., Chen, C.L., Chen, D.S. & Chen, P.J. (2012) Androgen pathway stimulates microRNA-216a transcription to suppress the tumor suppressor in lung cancer-1 gene in early hepatocarcinogenesis. Hepatology 56, 632643.
  • Galanis, A., Pappa, A., Giannakakis, A., Lanitis, E., Dangaj, D. & Sandaltzopoulos, R. (2008) Reactive oxygen species and HIF-1 signalling in cancer. Cancer Lett. 266, 1220.
  • Galibert, L., Diemer, G.S., Liu, Z., et al. (2005) Nectin-like protein 2 defines a subset of T-cell zone dendritic cells and is a ligand for class-I-restricted T-cell-associated molecule. J. Biol. Chem. 280, 2195521964.
  • Gomyo, H., Arai, Y., Tanigami, A., Murakami, Y., Hattori, M., Hosoda, F., Arai, K., Aikawa, Y., Tsuda, H., Hirohashi, S., Asakawa, S., Shimizu, N., Soeda, E., Sakaki, Y. & Ohki, M. (1999) A 2-Mb sequence-ready contig map and a novel immunoglobulin superfamily gene IGSF4 in the LOH region of chromosome 11q23.2. Genomics 62, 139146.
  • Greer, S.N., Metcalf, J.L., Wang, Y. & Ohh, M. (2012) The updated biology of hypoxia-inducible factor. EMBO J. 31, 24482460.
  • Kawano, S., Ikeda, W., Kishimoto, M., Ogita, H. & Takai, Y. (2009) Silencing of ErbB3/ErbB2 signaling by immunoglobulin-like Necl-2. J. Biol. Chem. 284, 2379323805.
  • Kuramochi, M., Fukuhara, H., Nobukuni, T., Kanbe, T., Maruyama, T., Ghosh, H.P., Pletcher, M., Isomura, M., Onizuka, M., Kitamura, T., Sekiya, T., Reeves, R.H. & Murakami, Y. (2001) TSLC1 is a tumor-suppressor gene in human non-small-cell lung cancer. Nat. Genet. 27, 427430.
  • Lahiry, P., Torkamani, A., Schork, N.J. & Hegele, R.A. (2010) Kinase mutations in human disease: interpreting genotype-phenotype relationships. Nat. Rev. Genet. 11, 6074.
  • Lewis, B.P., Burge, C.B. & Bartel, D.P. (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 1520.
  • Li, Q.J., Zhou, L., Yang, F., Wang, G.X., Zheng, H., Wang, D.S., He, Y. & Dou, K.F. (2012) MicroRNA-10b promotes migration and invasion through CADM1 in human hepatocellular carcinoma cells. Tumour Biol. 33, 14551465.
  • Masuda, M., Yageta, M., Fukuhara, H., Kuramochi, M., Maruyama, T., Nomoto, A. & Murakami, Y. (2002) The tumor suppressor protein TSLC1 is involved in cell-cell adhesion. J. Biol. Chem. 277, 3101431019.
  • Mercurio, A.M., Rabinovitz, I. & Shaw, L.M. (2001) The α6β4 integrin and epithelial cell migration. Curr. Opin. Cell Biol. 13, 541545.
  • Mizutani, K., Kawano, S., Minami, A., Waseda, M., Ikeda, W. & Takai, Y. (2011) Interaction of nectin-like molecule 2 with integrin α6β4 and inhibition of disassembly of integrin α6β4 from hemidesmosomes. J. Biol. Chem. 286, 3666736676.
  • Murakami, Y. (2005) Involvement of a cell adhesion molecule, TSLC1/IGSF4, in human oncogenesis. Cancer Sci. 96, 543552.
  • Nieto, M.A. (2011) The ins and outs of the epithelial to mesenchymal transition in health and disease. Annu. Rev. Cell Dev. Biol. 27, 347376.
  • Penna, E., Orso, F., Cimino, D., et al. (2011) microRNA-214 contributes to melanoma tumour progression through suppression of TFAP2C. EMBO J. 30, 19902007.
  • Schioppa, T., Uranchimeg, B., Saccani, A., Biswas, S.K., Doni, A., Rapisarda, A., Bernasconi, S., Saccani, S., Nebuloni, M., Vago, L., Mantovani, A., Melillo, G. & Sica, A. (2003) Regulation of the chemokine receptor CXCR4 by hypoxia. J. Exp. Med. 198, 13911402.
  • Schraml, P., Kononen, J., Bubendorf, L., Moch, H., Bissig, H., Nocito, A., Mihatsch, M.J., Kallioniemi, O.P. & Sauter, G. (1999) Tissue microarrays for gene amplification surveys in many different tumor types. Clin. Cancer Res. 5, 19661975.
  • Semenza, G.L. (1998) Hypoxia-inducible factor 1: master regulator of O2 homeostasis. Curr. Opin. Genet. Dev. 8, 588594.
  • Sempere, L.F., Christensen, M., Silahtaroglu, A., Bak, M., Heath, C.V., Schwartz, G., Wells, W., Kauppinen, S. & Cole, C.N. (2007) Altered MicroRNA expression confined to specific epithelial cell subpopulations in breast cancer. Cancer Res. 67, 1161211620.
  • Shimono, Y., Zabala, M., Cho, R.W., Lobo, N., Dalerba, P., Qian, D., Diehn, M., Liu, H., Panula, S.P., Chiao, E., Dirbas, F.M., Somlo, G., Pera, R.A., Lao, K. & Clarke, M.F. (2009) Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells. Cell 138, 592603.
  • Shingai, T., Ikeda, W., Kakunaga, S., Morimoto, K., Takekuni, K., Itoh, S., Satoh, K., Takeuchi, M., Imai, T., Monden, M. & Takai, Y. (2003) Implications of nectin-like molecule-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 in cell-cell adhesion and transmembrane protein localization in epithelial cells. J. Biol. Chem. 278, 3542135427.
  • Staller, P., Sulitkova, J., Lisztwan, J., Moch, H., Oakeley, E.J. & Krek, W. (2003) Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL. Nature 425, 307311.
  • Takai, Y., Ikeda, W., Ogita, H. & Rikitake, Y. (2008a) The immunoglobulin-like cell adhesion molecule nectin and its associated protein afadin. Annu. Rev. Cell Dev. Biol. 24, 309342.
  • Takai, Y., Miyoshi, J., Ikeda, W. & Ogita, H. (2008b) Nectins and nectin-like molecules: roles in contact inhibition of cell movement and proliferation. Nat. Rev. Mol. Cell Biol. 9, 603615.
  • Taylor, C.T. & Cummins, E.P. (2009) The role of NF-κB in hypoxia-induced gene expression. Ann. NY Acad. Sci. 1177, 178184.
  • Ueda, T., Volinia, S., Okumura, H., et al. (2010) Relation between microRNA expression and progression and prognosis of gastric cancer: a microRNA expression analysis. Lancet Oncol. 11, 136146.
  • Urase, K., Soyama, A., Fujita, E. & Momoi, T. (2001) Expression of RA175 mRNA, a new member of the immunoglobulin superfamily, in developing mouse brain. Neuroreport 12, 32173221.
  • Volinia, S., Calin, G.A., Liu, C.G., et al. (2006) A microRNA expression signature of human solid tumors defines cancer gene targets. Proc. Natl Acad. Sci. USA 103, 22572261.
  • Wakayama, T., Ohashi, K., Mizuno, K. & Iseki, S. (2001) Cloning and characterization of a novel mouse immunoglobulin superfamily gene expressed in early spermatogenic cells. Mol. Reprod. Dev. 60, 158164.
  • Wilhelmsen, K., Litjens, S.H. & Sonnenberg, A. (2006) Multiple functions of the integrin α6β4 in epidermal homeostasis and tumorigenesis. Mol. Cell. Biol. 26, 28772886.
  • Wilson, W.R. & Hay, M.P. (2011) Targeting hypoxia in cancer therapy. Nat. Rev. Cancer 11, 393410.
  • Xu, C.X., Xu, M., Tan, L., Yang, H., Permuth-Wey, J., Kruk, P.A., Wenham, R.M., Nicosia, S.V., Lancaster, J.M., Sellers, T.A. & Cheng, J.Q. (2012) MicroRNA miR-214 regulates ovarian cancer cell stemness by targeting p53/Nanog. J. Biol. Chem. 287, 34 97034 978.
  • Yageta, M., Kuramochi, M., Masuda, M., Fukami, T., Fukuhara, H., Maruyama, T., Shibuya, M. & Murakami, Y. (2002) Direct association of TSLC1 and DAL-1, two distinct tumor suppressor proteins in lung cancer. Cancer Res. 62, 51295133.
  • Yang, H., Kong, W., He, L., Zhao, J.J., O'Donnell, J.D., Wang, J., Wenham, R.M., Coppola, D., Kruk, P.A., Nicosia, S.V. & Cheng, J.Q. (2008a) MicroRNA expression profiling in human ovarian cancer: miR-214 induces cell survival and cisplatin resistance by targeting PTEN. Cancer Res. 68, 425433.
  • Yang, M.H. & Wu, K.J. (2008) TWIST activation by hypoxia inducible factor-1 (HIF-1): implications in metastasis and development. Cell Cycle 7, 20902096.
  • Yang, M.H., Wu, M.Z., Chiou, S.H., Chen, P.M., Chang, S.Y., Liu, C.J., Teng, S.C. & Wu, K.J. (2008b) Direct regulation of TWIST by HIF-1α promotes metastasis. Nat. Cell Biol. 10, 295305.
  • Yarden, Y. & Sliwkowski, M.X. (2001) Untangling the ErbB signalling network. Nat. Rev. Mol. Cell Biol. 2, 127137.
  • Yin, G., Chen, R., Alvero, A.B., Fu, H.H., Holmberg, J., Glackin, C., Rutherford, T. & Mor, G. (2010) TWISTing stemness, inflammation and proliferation of epithelial ovarian cancer cells through MIR199A2/214. Oncogene 29, 35453553.
  • Zhang, X.J., Ye, H., Zeng, C.W., He, B., Zhang, H. & Chen, Y.Q. (2010) Dysregulation of miR-15a and miR-214 in human pancreatic cancer. J. Hematol. Oncol. 3, 46.