Integrative analysis of microRNAome, transcriptome, and proteome

Subscriber access provided by Iowa State University | Library

Article

Integrative analysis of microRNAome, transcriptome, and proteome during the limb regeneration of Cynops orientalis Yuan Yu, Jie Tang, Jiaojiao Su, Jihong Cui, Xin Xie, and Fulin Chen J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00778 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 6, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research

Integrative analysis of microRNAome, transcriptome, and proteome during the limb regeneration of Cynops orientalis Yuan Yu1,2,3, Jie Tang1,4, Jiaojiao Su1, Jihong Cui1,2,3, Xin Xie1,2,3, Fulin Chen1,2,3*

1 Lab

of Tissue Engineering, College of Life Sciences, Northwest University, Xi’an, 710069, P.

R. China.

2

Provincial Key Laboratory of Biotechnology of Shaanxi, Xi’an, 710069, P. R. China.

3

Key Laboratory of Resource Biology and Biotechnology in Western China Ministry of

Education, Xi’an, 710069, P. R. China.

4

Shaanxi Institute of Zoology, 88 Xingqing Road, Xi’an, Shaanxi Province 710032 People’s

Republic of China

* Correspondence:

[email protected]

ACS Paragon Plus Environment

1

Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 50

ABSTRACT

Salamanders completely regenerate their limbs after amputation. Thus, these animals are unique models to investigate the mechanisms modulating the regeneration in vertebrates. To investigate the influence of microRNAs (miRNA) on the newt limb regeneration, the miRNAs and mRNAs were simultaneously profiled using Illumina HiSeq™ 2500 System during the limb regeneration of Cynops orientalis at 3, 7, 14, 30, and 42 days post amputation. A total of 203 miRNAs and 4230 mRNAs were identified to be differentially expressed. Together with the proteomic data obtained from our previous study, integrative analysis of multiple profiling datasets was performed to construct an interaction network of differentially expressed miRNAs, mRNAs, and proteins. Results of GO and KEGG analyses showed that the differentially expressed miRNA targets were mainly directed to cytoskeletal remodeling and carbohydrate metabolism. The stagespecific regulation of miRNA on their targets were analyzed by hierarchical clustering analysis and validated by qRT-PCR. The negative regulation of miR-223 and miR-133a on their targets was tested by performing dual luciferase reporter assay. The integration


2

Page 3 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


analysis will provide a powerful tool to identify the regulatory mechanisms of miRNA and their targets. The results may have implications in understanding the complex mechanisms underlying newt limb regeneration.

KEYWORDS

Cynops orientalis, microRNA, Stage-specific regulation, Limb regeneration


3


Page 4 of 50

Introduction The salamander limb, with its striking regenerative capacity, offers a unique model to investigate the regeneration in vertebrates. The characteristic steps of successful limb regeneration begin with wound healing, followed by the emergence of a proliferative zone of cells known as blastema and then a redevelopment stage, which is a recurrence of events that occur during embryonic development (1, 2). Each regenerative stage is strictly controlled and dependent on the precise intrinsic signal regulation. Transcriptome sequencing (3-5) and proteomic methods (6, 7) have identified numerous genes and proteins involved in the salamander limb regeneration. However, the upstream regulatory molecules of these genes and underlying mechanisms remain interesting and primarily ambiguous. miRNAs are important fine regulators of gene expression. These molecules are endogenous small noncoding RNAs (~22 nucleotides) that mediate the posttranscriptional regulation of protein coding genes by repressing translation, transcriptional degradation, and in some instances mRNA deadenylation (8-10). To date, miRNAs have been shown to play an important role in tissue regeneration across species. MiR-133 (11)


4

Page 5 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and miR-203 (12) regulate caudal fin regeneration in zebrafish by directly targeting the FGF signaling and the Wnt signaling pathways, respectively. MiR-99/100 and Let-7a/c are key regulators of dedifferentiation and regeneration in zebrafish cardiomyocytes (13). MiR-1 and miR-206 participates to regulate skeletal muscle regeneration in mice by repressing the Pax7 gene, thereby allowing the transition from satellite cells to myogenic progenitor cells (14). After acute nerve injury in mice, miR-206 promoted the regeneration of neuromuscular synapses through histone deacetylase 4 and the FGF signaling pathways (15). In adult newts, the let-7 members contribute to lens and hair cell regeneration (16). During newt cardiac regeneration, miR-128 regulates the non-myocyte hyperplasia and deposition of extracellular matrix (ECM) and contributes to cell differentiation into cardiac cell lineages by negatively regulating Islet1 expression (17). After the spinal cord injury in axolotl, miR-125b guides the correct regeneration of axons by creating a regeneration-permissive environment at the lesion site by targeting the

Sema4D gene (18). MiR-196, identified as an essential regulator of axolotl tail regeneration, acts upstream of BMP4 and Pax7-based patterning events (19). In addition, miR-21 is detected to be overexpressed in mid-bud blastema during axolotl limb


5


Page 6 of 50

regeneration (20). Despite the important roles of miRNAs as suggested in various regeneration paradigms in salamander, only few studies have focused on the comprehensive survey of miRNAs in salamander limb regeneration (20-22). Holman et al. identified a number of miRNAs that participated in salamander limb regeneration (20). However, studies on the efficient target prediction and identification of novel miRNAs are insufficient because of the restriction of the microarray-based method. This limitation hampers

the

investigation

of

the

complex

regulatory

mechanisms

of

miRNAs. Thus, the systematic identification of miRNA-target interaction networks is essential to understand the complex mechanisms underlying the salamander limb regeneration. Integrative analyses based on miRNA-mRNA-protein expression profiles provide a rational approach to identify a set of differentially expressed (DE) miRNAs and their putative target pathways (23, 24). The present study was undertaken to use integrated time-course analyses of DE miRNAs with their target gene and proteins to comprehensively elucidate their functions during C. orientalis limb regeneration and their related regulatory pathways. Several pointers for regenerative medicine were discussed,


6

Page 7 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and a foundation was established to understand the mechanisms of regeneration and the repair of damaged tissue. Materials and methods Ethics approval and consent to participate All animal experimental protocols were approved by the Northwest university animal care and use committee. Sample preparation

C. orientalis, the Chinese fire-bellied newts, were purchased from the Xi’an aquatic market and were maintained in an individual aquarium at 22 ± 2 °C. The animals were fed with frozen Chironomidae larvae every other day. All surgical procedures were performed under general anesthesia with 0.1% MS-222 (ethyl-p-aminobenzoate; SigmaAldrich, Steinheim, Germany) in a 1-L tap water-bath. Animals about 6-8 cm in length were subjected to amputation. The regenerating limb samples were prepared as described previously (2). The regenerating limb tissues were collected at 3, 7, 14, 30 and 42 days post-amputation (dpa). These time points cover major morphological and physiological changes during limb regeneration throughout wound healing (3 dpa), limb


7


Page 8 of 50

bud formation (7 dpa), blastema cell proliferation (14 dpa), chondrogenesis (30 dpa), and up to digit formation (42 dpa). The tissue removed distal to the amputation site acted as the control. Sequencing of miRNA and mRNA Total RNA was extracted from three independent biological replicates of limb samples at each regenerating timepoint using Trizol reagent (Invitrogen, CA, USA) following the manufacturer’s procedure. The quantity and purity of total RNA were analyzed by Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0. The total mRNA and small RNA libraries were prepared using the mRNA-Seq sample preparation kit (Illumina, San Diego, USA) and Small RNA Sample Prep Kits (Illumina, San Diego, USA), respectively. Moreover, paired-end sequencing and singleend sequencing (36 bp) were performed on an Illumina Hiseq2500 at the LC-BIO (Hangzhou, China) following the vendor’s recommended protocol. Analysis of DE genes and miRNAs For the DE mRNA, the expression abundance of unigenes was calculated using the reads per kilo base per million mapped reads (RPKM) method (25). We performed


8

Page 9 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


differential expression analysis from the raw read counts using the fold change of RPKM values from the libraries of the tissue, which were calculated to identify the DE genes. The differentially expressed miRNAs and mRNAs were selected with fold change ≥2 or ≤0.5 and with statistical significance (p value

Integrative analysis of microRNAome, transcriptome, and proteome

Recommend Documents