Global miRNA/proteomic analyses identify miRNAs at 14q32 and 3p21, which contribute to features of chronic iron-exposed fallopian tube epithelial cells

Abstract

Malignant transformation of fallopian tube secretory epithelial cells (FTSECs) is a key contributing event to the development of high-grade serous ovarian carcinoma (HGSOC). Our recent findings implicate oncogenic transformative events in chronic iron-exposed FTSECs, including increased expression of oncogenic mediators, increased telomerase transcripts, and increased growth/migratory potential. Herein, we extend these studies by implementing an integrated transcriptomic and mass spectrometry-based proteomics approach to identify global miRNA and protein alterations, for which we also investigate a subset of these targets to iron-induced functional alterations. Proteomic analysis identified > 4500 proteins, of which 243 targets were differentially expressed. Sixty-five differentially expressed miRNAs were identified, of which 35 were associated with the “top” proteomic molecules (> fourfold change) identified by Ingenuity Pathway Analysis. Twenty of these 35 miRNAs are at the 14q32 locus (encoding a cluster of 54 miRNAs) with potential to be regulated by DNA methylation and histone deacetylation. At 14q32, miR-432-5p and miR-127-3p were ~ 100-fold downregulated whereas miR-138-5p was 16-fold downregulated at 3p21 in chronic iron-exposed FTSECs. Combinatorial treatment with methyltransferase and deacetylation inhibitors reversed expression of these miRNAs, suggesting chronic iron exposure alters miRNA expression via epigenetic alterations. In addition, PAX8, an important target in HGSOC and a potential miRNA target (from IPA) was epigenetically deregulated in iron-exposed FTSECs. However, both PAX8 and ALDH1A2 (another IPA-predicted target) were experimentally identified to be independently regulated by these miRNAs although TERT RNA was partially regulated by miR-138-5p. Interestingly, overexpression of miR-432-5p diminished cell numbers induced by long-term iron exposure in FTSECs. Collectively, our global profiling approaches uncovered patterns of miRNA and proteomic alterations that may be regulated by genome-wide epigenetic alterations and contribute to functional alterations induced by chronic iron exposure in FTSECs. This study may provide a platform to identify future biomarkers for early ovarian cancer detection and new targets for therapy.

Deregulated expression of the 14q32 miRNA cluster in clear cell renal cancer cells

“…Using dimethylsulfoxide (DMSO) treated cells as controls, cells were treated with Temsirolimus (TEMS, #50-811-7, Fisher Scientific, Pittsburgh, PA) at a final concentration of 2μM, as described in our prior work ( 17 ). Likewise, as earlier described ( 24 ), 5-Azacytidine (AZA, #S1782, Selleck Chemicals, Houston, TX) was utilized at a final concentration of 1μM while Suberoylanilide hydroxamic acid (SAHA, #S1047, Selleck Chemicals, Houston, TX) was utilized at a final concentration of 50μM. Lysophosphatidic acid (LPA, as a sodium salt in chloroform, #857130C, Avanti Polar Lipids, Alabaster, AL) was utilized at a final dose of 10μM, as previously described ( 17 ).…”

“…As previously described ( 24 ), RNA was isolated from cells using the miRVana isolation kit (#AM1561, Ambion, NY) or the RNeasy mini kit (QIAGEN, Valencia, CA) following manufacturer’s instructions. RNA was quantified using NanoDrop 1000 (Fisher Scientific, Pittsburg, PA).…”

Iron Metabolism and Idiopathic Pulmonary Arterial Hypertension: New Insights from Bioinformatic Analysis

“…Iron 22 BioMed Research International metabolism has been reported to be regulated by miRNAs, which have been demonstrated to posttranscriptionally regulate the expression of genes associated with iron acquisition, iron export, iron storage, iron utilization, and coordi-nation of systemic iron homeostasis [43][44][45]. We found that some dysregulated miRNAs in IPAH patients regulate iron metabolism in other biological circumstances [44,[46][47][48]; given that both iron metabolism disorders and miRNA dysregulation are important regulators of IPAH development, we hypothesized that there is crosstalk between them in IPAH patients and then constructed a DRmiRNA-DEIMRG regulatory network and identified 12 tIMRGs. In addition, we identified 10 hub genes in DEIMRGs that are highly associated with other proteins through the construction of PPI networks.…”

PI3K/AKT/mTOR pathway mediated‐cell cycle dysregulation contribute to malignant proliferation of mouse spermatogonia induced by microcystin‐leucine arginine

“…For example, human bronchial epithelial cells exposed to tobacco particles were tested for malignant transformation, confirming the effect of tobacco particulates on lung carcinogenesis 73 . The chronic iron exposure‐induced ovarian serous carcinoma was demonstrated by continuous exposure of fallopian tube secretory epithelial cells to ferric ammonium citrate 74 . This suggests that in vitro cellular carcinogenic test is an important method to study the carcinogenic effects of toxicants.…”

“…73 The chronic iron exposure-induced ovarian serous carcinoma was demonstrated by continuous exposure of fallopian tube secretory epithelial cells to ferric ammonium citrate. 74 This suggests that in vitro cellular carcinogenic test is an important method to study the carcinogenic effects of toxicants. Therefore, this study explored the carcinogenic effect of MC-LR, a possible human carcinogen in the environment, by a malignant transformation test.…”