We conclude from this study that integrin expression correlates with specific patterns of miRNA expression and that β4 integrin status affects the expression of specific families of miRNAs. Manipulation of β4 expression in two breast cancer cell lines provided in vitro
model systems for analysis, while a collection of invasive breast carcinoma specimens established an in vivo
link to the cell line data. The novel qNPA array technology identified two miRNA families, miR-25/32/92abc/363/363-3p/367 and miR-99ab/100, as undergoing repression in the presence of β4 across all systems. An analysis of published Affymetrix GeneChip data (Chen et al., 2009
) identified 54 common putative targets of these two miRNA families within β4-regulated genes. Many of these identified genes are established mediators of cell adhesion, cell motility, and signal transduction. Statistical analysis established that this population is enriched in genes involved in cell migration. These data reveal previously unrecognized β4 targets, which could contribute to the ability of β4 to promote carcinoma progression. Finally, gene set enrichment analysis detected an enrichment in predicted targets of several miRNA families, including miR-92ab, within β4-regulated genes, substantiating the physiological relevance of our findings with respect to the effect of β4 on the expression of distinct miRNA families.
Although the fields of integrin and miRNA biology have been extensively linked to cancer initiation and progression, the connection between these two disciplines has remained elusive. Our novel observation that a specific integrin correlates with miRNA expression has implications for development and disease, especially tumorigenesis. Along these lines, tyrosine kinase receptors, such as EGFR, have also been shown to regulate miRNA expression (Avraham et al., 2010
). Our data support the hypothesis that cells utilize this small class of RNAs to respond to external cues in their microenvironment, employing surface receptors like integrins as intermediates in the delivery of key information. An interesting observation that emerged from the results of the miRNA microarray analysis involves the predominantly repressive effect of β4 on global miRNA expression. This is consistent with published data describing global downregulation of miRNA expression in cancers (Gaur et al., 2007
; Lu et al., 2005
). Differential expression of the endogenous miRNA processing machinery represents a potential explanation for the repressive patterns of miRNA expression that we observed, as recent reports have highlighted the importance of miRNA processing genes in the regulation of miRNA biogenesis and function (Cheng et al., 2009
; Van der Auwera et al., 2010
). We examined the expression of dicer, drosha, ago1, ago2, and trpb2 mRNAs between the β4 and mock transfectants using Affymetrix GeneChip data but observed no change that could account for the downregulated pattern of miRNA expression (data not shown).
Our observation that family members miR-92a and miR-92b are consistently downregulated in the presence β4 in our arrays is interesting considering the defined role of miR-92a as an “oncomir” (Olive et al., 2010
). miR-92a belongs to the miR-17-92 cluster, a group of six miRNAs generated from a single polycistronic transcript that includes miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a. This cluster confers potent oncogenic potential and is overexpressed in a variety of cancers, often the result of genomic amplification (Olive et al., 2010
). These findings are seemingly at odds with our observation that miR-92a inversely correlates with the expression of β4, an integrin with a well-established role in potentiating carcinoma cell migration, invasion, and survival. Recent data, however, have identified a role for miRNAs from this family as tumor suppressors (O'Donnell et al., 2005
), highlighting the importance of cellular and molecular context in determining the role of specific miRNAs in tumorigenesis. Interestingly, an analysis of the arrays failed to identify consistent downregulation of other members from this miRNA cluster with the exception of miR-19b, which was repressed in two of the three arrays (data not shown). miR-92b, despite sharing the same seed sequence and common putative mRNA targets with miR-92a, is transcribed from an independent genomic locus and is less well characterized from a functional standpoint. Its intergenic location near the THBS3 gene, which is known to share a common promoter with MTX1, prompted us to examine both thrombospondin 3 and metataxin 1 mRNA expression using our Affymetrix GeneChip data from the MDA-MB-435/β4 cells. Conveniently, miR-92b was downregulated in this particular miRNA array; however, no detectable changes were observed in the expression of either thrombospondin 3 or metataxin 1 mRNA levels in this system (data not shown). This finding, along with the paucity of other downregulated miRNAs from the miR-17–92 cluster, suggest changes in miR-92a and miR-92b expression are not mediated at a transcriptional level, rather the presence of this integrin likely affects the stability of these previously transcribed miRNAs. Our hypothesis is intriguing in light of recent data linking miRNA decay to changes in cell adhesion (Kim et al., 2011
), as well as the general notion that global miRNA expression is typically downregulated in cancer (Gaur et al., 2007
; Lu et al., 2005
The role of miR-99a, miR-99b, and miR-100, the other miRNA family identified by our array, in tumorigenesis appears to be controversial. However, downregulation of members of this miRNA family has been linked to breast carcinoma, hepatocellular carcinoma, prostate carcinoma, nasopharyngeal carcinoma, oral carcinomas, hepatoblastoma, and ovarian carcinoma (Cairo et al., 2010
; Henson et al., 2009
; Lobert et al., 2011
; Nam et al., 2008
; Petrelli et al., 2012
; Shi et al., 2010
; Sun et al., 2011
; Wong et al., 2008
). All three miRNAs are transcribed from independent genomic loci with clustered miRNAs. miR-99a is co-transcribed with let-7c, miR-99b is co-transcribed with let-7e and miR-125a, and miR-100 is an intergenic miRNA co-transcribed with let-7a. Again using the Affymetrix GeneChip data from the MDA-MB-435/β4 cells, we detected no change in the expression of genes surrounding the miR-100 cluster despite downregulation of miR-100 in this system (data not shown). However, we noted that all of the other co-transcribed clustered miRNAs were repressed across arrays (). In fact, let-7a, let-7c, and let-7e belong to the let-7/98/4458/4500 miRNA family and miR-125a belongs to the miR-125a-5p/125b-5p/351/670/4319 miRNA family, both of which we identified to be downregulated by β4 in two of the three arrays (). Unlike miR-92a and miR-92b, these observations suggest a complex transcriptional mechanism that induces repression of miRNAs known to be genomically and functionally linked. This observation provides compelling evidence that the relationship between β4 and the expression patterns of these miRNAs is biologically driven and highly conserved. Furthermore, this observation diminishes our negative finding that the population of β4-regulated mRNAs does not contain an enrichment for miR-99ab/100 targets.
Our observations that miR-92ab and miR-99ab/100 both target β4-regulated genes involved in cell motility and signal transduction suggests a novel miRNA-mediated mechanism by which β4 promotes carcinoma cell migration and invasion. Moreover, these data contribute to our understanding of β4 function in the context of signal transduction, implying that this integrin not only activates signaling cascades through phosphorylation events but it also may alter the expression of key molecules involved in these complex processes by regulating miRNAs. Future studies aimed at exploring the mechanism of regulation of miR-25/32/92abc/363/363-3p/36 and miR-99ab/100 miRNA families in the presence of β4, as well as the role of putative targets in mediating cell motility downstream of this integrin, will provide further insight into the role of β4 function in promoting carcinoma progression.