MiRNA-29a regulates the expression of numerous proteins and reduces the invasiveness and proliferation of human carcinoma cell lines
Introduction
MicroRNAs have emerged in recent years as key regulators in a broad spectrum of cellular functions – and dysfunctions – as evidenced by the proliferation of reports in the literature relating to their study. A large proportion of these have been in the field of cancer research with several focused on the invasive/metastatic process specifically.1
One of the challenges of studying miRNA function in the cellular environment is identifying potential protein targets. Many early studies started with the target gene, often well associated with some aspect of cancer, and then tried to identify miRNAs that might bind the transcript based on in silico predictions.2, 3 Conversely, in silico analysis has been used to predict the potential target genes based on the sequence of a particular miRNA.4 The rules being used to develop these algorithms are becoming more refined as experimental data builds, leading to improved predictive capacity of these software tools. More recently, high throughput proteomic and transcriptomic technologies have greatly increased the identification and understanding of bona fide miRNA targets in cell lines, tissue samples and in vivo.5
In this study we were interested in identifying downstream targets of miR-29a using such approaches. MiR-29a is found on chromosome 7q32.3 in tandem with miR-29b-1 and has been reported in studies focused on cancer biomarker identification6 as well as cancer-related phenotypes in vitro and in vivo. It has been shown to regulate the levels of Mcl-1 and subsequently increase TRAIL-mediated apoptosis in malignant cholangiocarcinoma cells.3 TCL-1, the oncogene implicated in progression of CLL, has been shown to be regulated, at least in part, by the action of miR-29a.7 In lung cancer cell lines miR-29a was shown to reverse aberrant methylation by down-regulating the expression of DNMT3A and 3B.4 Importantly, a study by Chang and colleagues demonstrated that miR-29a is a target for suppression by c-myc.8 Preliminary work in our laboratory identified miR-29a as being differentially expressed in an in vitro model of invasive lung cancer. In combination these findings point to an important role for this molecule in maintaining normal cellular function and behaviour and prompted us to further investigate other potential target proteins and pathways influenced by miR-29a in our model system.
Section snippets
Cell culture
Lung carcinoma cell lines, DLKP and DLKP-A9 were maintained in ATCC media supplemented with 5% FCS and incubated at 37 °C. DLKP is a poorly differentiated, squamous cell lung carcinoma with a non-invasive phenotype. DLKP-A is a highly invasive variant generated by chronic exposure of the parent cell line to the chemotherapeutic drug, Adriamycin. PANC-1 cell line is a highly invasive cell line derived from a pancreatic ductal carcinoma (ATCC) and maintained in DMEM supplemented with 5% (v/v) FCS
2D Difference gel electrophoresis (2D DIGE)
DLKP-A cells were transfected with pre-miR-29a, anti-miR-29a or their negative controls for 72 h and harvested. Lysates were minimally labelled with 200 pmol/50 μg of protein with either Cy3 (control) or Cy5 (test) for comparison on the same gel (dyes from GE Healthcare). A pool containing equal amounts of all samples was also prepared and labelled with Cy2 to be used as an internal standard on all gels to aid image matching and cross-gel statistical analysis. Three biological repeats of each
MiR-29a is down-regulated in invasive cancer cells
An initial bioarray-based study in our laboratory comparing microRNA expression in non- and highly invasive variants of the lung cancer cell line DLKP identified several differentially expressed miRNAs, one of which was miR-29a. Subsequent measurement by qRT-PCR confirmed that miR-29a expression in the invasive variant was less than 45% of that in the non-invasive cell line (Fig. 1).
MiR-29a decreases the invasive phenotype of cancer cells in vitro
Though ectopic expression of miR-29a has previously been shown to reduce the growth of lung cancer cells in vitro
Discussion
Several recent publications have established miR-29 family members as important molecules in suppressing tumourigenic phenotypes in cell line models. In vivo, their expression levels have been shown to positively correlate with favourable outcome in AML patients.6 Though several important target genes have been identified, typically by homology-based analysis of 3’UTR regions, it is expected that most miRNAs have the potential to influence the expression of many, if not hundreds of target
Conflict of interest statement
None declared.
Acknowledgements
This work was supported by the Higher Education Authority of Ireland via its PRTLI III funding scheme and by Science Foundation Ireland.
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2019, Molecular Aspects of MedicineCitation Excerpt :Moreover, a contemporary study by Zhao et al. has demonstrated that miR-124-5p expression in non-small cell lung carcinoma (NSCLC) inhibit the expression of glycolytic enzymes, GLUT1 and HK2, by targeting AKT1 and 2 (Protein Kinase B1 and 2). A recent study using 2D proteomic profiling approach has shown that the exogenous expression of miR-29a-3p in DLKP-A lung cancer cell lines downregulate the expression of key glycolytic enzymes Phosphoglucomutase (PGM1), Enolase (ENO1) and Phosphoglycerate kinase 1 (PGK1), and correlates well with the reduction in tumor proliferation and invasiveness (Muniyappa and Dowling, 2009). miR-320a, an oxidative stress-responsive microRNA was shown to regulate the glucose metabolism in lung cancer cells by controlling the expression of PFKm (Muscle type phosphofructokinase) (Tang and Lee, 2012).