Genome-wide Characterization of miR-34a Induced Changes in Protein and mRNA Expression by a Combined Pulsed SILAC and Microarray Analysis

Abstract

The gene encoding the miR-34a microRNA is a transcriptional target of the p53 tumor suppressor protein and subject to epigenetic inactivation in colorectal cancer and numerous other tumor types. Here, we combined pulsed SILAC (pSILAC) and microarray analyses to identify miR34a-induced changes in protein and mRNA expression. pSILAC allowed to quantify the de novo protein synthesis of 1206 proteins after activation of a conditional miR-34a allele in a colorectal cancer cell line. ϳ19% of the detected proteins were differentially regulated, with 113 proteins being down-and 115 up-regulated. The proteins with a miR-34a seed-matching-sequence in the 3-untranslated region (UTR) of the corresponding mRNA showed a clear bias toward translational repression. Proteins involved in DNA replication, e.g. the MCM proteins, and cell proliferation, were over-represented among indirectly down-regulated proteins lacking a miR-34a seedmatch. The decrease in de novo protein synthesis of direct miR-34a targets correlated with reduced levels of the corresponding mRNA in most cases, indicating an interdependence of both types of regulation. In addition, 43 mRNAs encoding proteins not detected by pSILAC were down-regulated after miR-34a expression and contained miR-34a seed-matches. The direct regulation of selected miR-34a target-mRNAs was confirmed using reporter assays. Via down-regulation of the proteins encoded by these mRNAs miR-34a presumably inhibits glycolysis (LDHA), WNT-signaling (LEF1), invasion/migration (AXL) and lipid metabolism (ACSL1, ACSL4). Furthermore, miR34a may activate p53 by inhibiting its acetylation (MTA2, HDAC1) and degradation (YY1). In summary, miR-34a presumably participates in multiple tumor suppressive pathways by directly and indirectly suppressing the expression of numerous, critical proteins. Molecular & Cellular

miR-34 and SNAIL form a double-negative feedback loop to regulate epithelial-mesenchymal transitions

“…Results represent the average ± SD (n=3). downregulation of SNAIL by miR-34, further indirect inhibition of SNAIL functions may be mediated by the downregulation of HDAC1 by miR-34a, which we have recently described, 14 since HDAC1 functions as a co-repressor of SNAIL. 26 Interestingly, pancreatic acinar cells with deletion of p53, which, therefore, presumably also express low levels of miR-34a and miR-34b/c, express high levels of SNAIL, SLUG, TWIST and ZEB1/2, show increased stemness markers and undergo EMT upon subculture.…”

“…Recently, we identified additional miR-34a targets, such as AXL, LDH1, MTA2 and LEF1, using a combined proteomic and transcriptomic approach. 14 MiRNAs regulate their targets via association of a ~7 nucleotide stretch, the so-called seed sequence, located in their 5' portion with a complementary sequence in the 3'-UTR of the target mRNA. 15,16 Additional base pairing may occur via nucleotides in the middle and 3' portion of the miRNA.…”

Reciprocal regulation between microRNAs and epigenetic machinery in colorectal cancer

“…Furthermore, miR-34a is a transcriptional target of p53, suggesting a positive feedback loop between p53 and miR-34a through SIRT1-dependent regulation (103). Notably, another study reported that HDAC1 also exhibits a robust decrease in protein levels following the induction of miR-34a in CRC cells (104). In a previous study evaluating the role of miRNAs in the antitumour action of calcitriol, miRNA expression profiles were examined in colon cancer cells treated with calcitriol (105).…”

Gene Expression Profile and Signaling Pathways in MCF-7 Breast Cancer Cells Mediated by Acyl-Coa Synthetase 4 Overexpression

“…Current views on cancer cell mutations hold that there are two different types: driver mutations, which are behind cancer growth because they give tumor cells a growth advantage, and passenger mutations, which are just along for the ride. It has been suggested that ACSL4 overexpression is led by a driver mutation [18,34]; however, as it is capable per se of changing cancer cell phenotypes, ACSL4 overexpression may be thought of as a backseat driver factor which generates changes in gene expression and signaling pathways toward a highly aggressive phenotype, and which acts in addition to passenger mutations when the driver gene is mutated. In summary, this study derives an ACSL4 overexpression gene and functional proteomic signature which might reveal important information about novel mediators of breast cancer cell aggressiveness.…”