Liver-Specific microRNA Identification in Farmed Carp, Labeo bata (Hamilton, 1822), Fed with Starch Diet Using High-Throughput Sequencing

Abstract

The liver is an important central organ, which controls carbohydrate metabolism through maintaining glucose homeostasis by a tightly regulated system of genes or enzymes. The microRNAs are small non-coding RNAs playing an important role in the regulation of genes associated with developmental biology, physiology, metabolism, etc. Thus, in this study, we have intended to detect liver-specific microRNAs in farmed carp, Labeo bata, upon being fed a diet with different levels of carbohydrates. Here, we have conducted the experiment for 45 days using fingerlings of farmed carp fed with 20% (control), 40%, and 60% gelatinized starch levels. The liver tissues were collected from each treatment and processed for RNA isolation, small RNA library preparation, and high-throughput sequencing using Illumina NexSeq500. Through sequencing, 15,779,417 reads in 20% CHO, 13,959,039 in 40% CHO, and 13,661,950 in 60% CHO reads were generated for control and treated fishes using three small RNA libraries. We have investigated 445 novel and 231 conserved microRNAs in 20%, 40%, and 60% carbohydrate (CHO), respectively, through computational analysis. The differential expression analysis of miRNAs was carried out between different treatments compared with control and this study depicted 117 known and 114 novel miRNA genes involved in carbohydrate metabolic pathways. Further, target prediction and gene ontology analysis revealed that miRNAs were involved in several pathways such as signaling pathway, G protein pathway, complement receptor–mediated pathway, dopamine receptor signaling pathway, epidermal growth factor pathway, and notch signaling pathway. The predicted miRNA sites in targeted genes were associated with cellular activities, developmental biology, DNA binding, Golgi apparatus, extracellular region, catalytic activity, MAPK cascade, etc. Overall, we have generated a vital resource of liver-specific miRNAs involved in metabolic gene regulation. These studies further will help develop miRNA inhibitors to study their role during carbohydrate metabolism in farmed carp.

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Fig. 1

Abbreviations

NGS:

Next-generation sequencing

qRT PCR:

Real-time quantitative reverse transcription PCR

nt:

Nucleotides

LN2 :

Liquid nitrogen

TPM:

Transcripts per million

CHO:

Carbohydrate

DEMs:

Differentially expressed miRNAs

UTR:

Untranslated region

GO:

Gene ontology

AMPK:

AMP-activated protein kinase

G6PC:

Glucose-6-phosphatase

PK:

Pyruvate kinase

PCK1:

Phosphoenolpyruvatecarboxykinase

JAK/STAT:

Janus kinase/signal transducers and activators of transcription

MAPK:

Mitogen-activated protein kinases

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Acknowledgments

We are thankful to Director, ICAR-Central Institute of Freshwater Aquaculture, Bhubaneswar and Director, ICAR-IASRI, New Delhi, for providing the facility to undertake this work.

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Correspondence to Jitendra Kumar Sundaray.

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Electronic Supplementary Material

Supplementary Fig. S1
figure2

Abundance of reads mapped to Rfam (Family Distribution) (PNG 31730 kb)

Supplementary Fig. S2
figure3

Real-time PCR studies for selected miRNAs. (PNG 133 kb)

Supplementary Fig. S3
figure4

Venn diagram illustrations common and unique miRNA present in 20 (control), 40 and 60% of high CHO from the liver tissue of L. bata (PNG 6793 kb)

Supplementary Fig. S4
figure5

GO enrichment analysis of target genes by miRNAs depicted in the bar chart, which indicates enriched GO terms with y-axis represent the total number of differentially expressed miRNAs and x-axis represents different GO terms such as biological process, cellular component and molecular function. (PNG 659 kb)

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Rasal, K.D., Iquebal, M.A., Jaiswal, S. et al. Liver-Specific microRNA Identification in Farmed Carp, Labeo bata (Hamilton, 1822), Fed with Starch Diet Using High-Throughput Sequencing. Mar Biotechnol 21, 589–595 (2019). https://doi.org/10.1007/s10126-019-09912-y

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Keywords

  • MicroRNA
  • Liver
  • Labeo bata
  • Next-generation sequencing