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Identification of miR-16/29 targets in adipocytes by LC-MSMS

ABSTRACT: Cancer cachexia is a multifactorial metabolic syndrome defined by the rapid loss of skeletal muscle mass and the loss of fat mass. Up 80% of cancer patients at the late stage with cachexia suffer from progressive atrophy of adipose tissue. Unlike studies on skeletal muscle wasting, there is limited research on fat loss in cachexia. It was noted that most patients suffer from fat loss as cancer progress. Fat loss precedes muscle loss, is associated with shorter survival, and is variable to timing and intensity in various cancer populations. Increased lipolysis may be the leading cause of fat loss in cancer cachexia. miRNAs are a class of non-coding RNAs of 19~25 nucleotides that regulate gene silencing by interacting with the 3’ untranslated region (UTR) of target mRNA to cause mRNA degradation and translational repression. miRNAs play multifaceted roles in pancreatic cancer proliferation, survival, metastasis, and chemoresistance. Aberrant expression of miRNA in circulating exosomes may play potential roles in modulating fat loss in cancer cachexia. We identified 2 miRNAs, miR-16 and miR-29, which have 2-fold higher expression existed in at PDAC cells. To explore which genes in adipogenesis and lipolysis were directly affected by miR-16-5p or/and miR-29a-3p, we analyzed the targets which were down-regulated in both miR-16-5p and miR-29a-3p-transfected 3T3-L1 cells by mass analysis.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Sui-Chih Tien

LAB HEAD: Chun-Mei Hu

PROVIDER: PXD037998 | Pride | 2024-06-13

REPOSITORIES: Pride

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Project description:We tested the hypothesis that a panel of placental mammal-specific miRNAs and their targets play important to establish receptivity to implantation and their dysregulated expression may be a feature in women with early pregnancy loss. Relative expression levels of miR-340-5p, −542-3p, and −671-5p all increased following treatment of Ishikawa cells with progesterone (10 μg/ml) for 24 hrs (p < 0.05). RNA sequencing of these P4-treated cells identified co-ordinate changes to 6,367 transcripts of which 1713 were predicted targets of miR-340-5p, 670 of miR-542-3p, and 618 of miR-671-5p. Quantitative proteomic analysis of Ishikawa cells transfected with mimic or inhibitor (48 hrs: n=3 biological replicates) for each of the P4-regulated miRNAs was carried out to identify targets of these miRNAs. Excluding off target effects, mir-340-5p mimic altered 1,369 proteins while inhibition changed expression of 376 proteins (p < 0.05) of which, 72 were common to both treatments. A total of 280 proteins were identified between predicted (mirDB) and confirmed (in vitro) targets. In total, 171 proteins predicted to be targets by mirDB were altered in vitro by treatment with miR-340-5p mimic or inhibitor and were also altered by treatment of endometrial epithelial cells with P4. In vitro targets of miR-542-3p identified 1,378 proteins altered by mimic while inhibition altered 975 a core of 200 proteins were changed by both. 100 protein targets were predicted and only 46 proteins were P4 regulated. miR-671-mimic altered 1,252 proteins with inhibition changing 492 proteins of which 97 were common to both, 95 were miDB predicted targets and 46 were also P4-regulated. All miRNAs were detected in endometrial biopsies taken from patients during the luteal phase of their cycle, irrespective of prior or future pregnancy outcomes Expression of mir-340-5p showed an overall increase in patients who had previously suffered a miscarriage and had a subsequent miscarriage, as compared to those who had infertility or previous miscarriage and subsequently went on to have a life birth outcome. The regulation of these miRNAs and their protein targets regulate the function of transport and secretion, and adhesion of the endometrial epithelia required for successful implantation in humans. Dysfunction of these miRNAs (and therefore the targets they regulate) may contribute to endometrial-derived recurrent pregnancy loss in women.

Project description:To investigate the potential role of miRNAs in disease mechanisms, we conducted a thorough analysis by comparing the entire miRNome of 18 vEDS dermal fibroblasts to those of 18 control cells. To improve statistical reliability, we sequenced each sample from both groups as biological replicates. By applying a log2 fold change threshold of greater than 1 or less than -1, and an adjusted p-value of less than 0.05, we identified 137 DE-miRNAs in patient vs. control fibroblasts. Among these, 71 miRNAs exhibited decreased expression, while 66 miRNAs were upregulated. To explore the functional relationship between DE-miRNAs and the altered gene expression profile of vEDS cells, we conducted an extensive review of the literature and consulted several bioinformatics databases. Our search involved utilizing data from four miRNA-target prediction databases (microT-CDS, Targetscan, miRTarBase, and miRwalk) in addition to relevant literature sources providing information on the functional roles of these miRNAs. We thus focused our research on a specific subset of DE-miRNAs, known as miR-15b-5p, miR-16-5p, miR-21-3p, miR-24-3p, miR-29a-3p, miR-29b-3p, miR-138-5p, miR-145-5p, and miR-195-5p, which may play a biological role in dysregulated molecular mechanisms underlying disease pathogenesis. Our initial emphasis was on miR-29a-3p and miR-29b-3p due to their involvement in regulating critical cellular processes such as ECM remodeling into the vascular network, autophagy, cell proliferation, and apoptosis. To study how miR-29a-3p and miR-29b-3p contribute to the dysregulated cellular processes observed in patient cells, we performed GO and pathway enrichment analyses on their predicted target DEGs. GO analysis revealed that both these miRNAs may disturb fundamental biological functions, such as regulation of transcription by RNA polymerase II, negative gene expression regulation, miRNA-mediated gene silencing, translation regulation, heterochromatin organization, and ECM organization. This comphrensive transcriptome analysis of a large cohort of dermal fibroblasts from patients with vEDS reveals deregulated biological functions potentially involved in the disease pathogenesis. Taken together, the abnormal expression of several miRNAs suggests the presence of epigenetic control in vEDS cells, which could influence the transcriptional regulation of numerous target DEGs and related biological processes, potentially influencing disease mechanisms. This work was supported by Fondazione Telethon and the “Associazione con Giacomo contro vEDS”, (Grant number: GSA21F001, Seed Grant Fall 2021 vEDS) to Nicola Chiarelli.

			Action	DRS
	Lipo_29.raw	Raw
	Lipo_NC.raw	Raw
	allSpectra.CID.ITMS.iso_0.apl	Other
	allSpectra.CID.ITMS.iso_1.apl	Other
	allSpectra.CID.ITMS.iso_2.apl	Other

Dataset Information

Identification of miR-16/29 targets in adipocytes by LC-MSMS

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