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Article Open Access

PUM2 knockdown regulates the expression and alternative splicing of genes associated with myocardial fibrosis in H9C2 cells

  • Authors:
    • Zilong Zhang
    • Hangyu Zhou
    • Diliyaer Adili
    • Wei Zhu
    • Yong Liu
    • Wenzheng Zhou
    • Zhao Wang
    • Jie Li
  • View Affiliations / Copyright

    Affiliations: Cardiac and Panvascular Medicine Diagnosis and Treatment Center, People's Hospital of Xinjiang Uygur Autonomous Region, Ürümqi, Xinjiang Uyghur Autonomous Region 830000, P.R. China, Department of Orthopaedics, People's Hospital of Xinjiang Uygur Autonomous Region, Ürümqi, Xinjiang Uyghur Autonomous Region 830000, P.R. China
    Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 94
    |
    Published online on: February 4, 2026
       https://doi.org/10.3892/etm.2026.13089
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Abstract

Myocardial fibrosis (MF) represents a major pathological alteration observed in the progression of numerous heart conditions. The presence of excessive MF is key in the progression of numerous heart diseases, which may lead to heart failure. Abnormal expression of pumilio RNA‑binding family member 2 (PUM2), an RNA‑binding protein, is a contributing factor in the development of a variety of diseases. The present study aimed to investigate how PUM2 affected the expression and alternative splicing of genes associated with MF in H9C2 cells, thus evaluating the role of PUM2 in this context. Inhibition of PUM2 expression in H9C2 cardiomyocytes was achieved through small interfering RNA transfection. Cell viability was assessed using a Cell Counting Kit‑8 assay, while apoptosis was detected through flow cytometry. RNA‑sequencing (RNA‑seq) was utilized to analyze the differential gene expression profiles and alternative splicing events (ASEs) in H9C2 cells regulated by PUM2. Sequencing results were confirmed through reverse transcription‑quantitative PCR. The results showed that a reduction in PUM2 levels inhibited cell viability without affecting apoptosis. RNA‑seq analysis revealed notable changes in the expression of various genes associated with MF, including adrenomedullin, NDRG family member 4, phospholipid phosphatase 3, integrin subunit α 8, regulator of G protein signaling 2, cadherin 11, integrin subunit α 11 and transforming growth factor β‑induced, following PUM2 knockdown. In addition, PUM2 regulated ASEs in genes associated with fibrosis progression, such as tropomyosin 1 and actinin α1. The present study revealed that PUM2 had a regulatory effect on alternative splicing and gene expression associated with MF, suggesting PUM2 as a promising molecular target for MF therapy.

View Figures

Figure 1

PUM2 knockdown inhibits viability in
H9C2 cells. (A) Reverse transcription-quantitative PCR results
after PUM2 knockdown. (B) Representative western blot bands after
PUM2 knockdown. (C) PUM2 inhibited viability at 48 and 72 h after
transfection. **P≤0.01 and ****P<0.0001.
PUM2, pumilio RNA-binding family member 2; NC, negative control;
DSI, dicer-substrate small interfering RNA.

Figure 2

PUM2 knockdown regulates gene
expression in H9C2 cells. (A) PCA based on the FPKM value of all
genes. The ellipse for each group is the confidence ellipse. (B)
Volcano plot showing all DEGs between DSI and NC samples. (C)
Hierarchical clustering heat map showing expression levels of all
DEGs. (D) Bar plot showing the enriched KEGG pathways of
upregulated DEGs. (E) Bar plot showing the enriched KEGG pathways
of downregulated DEGs. (F) Bar plots showing the expression pattern
and statistical difference of DEGs with qPCR validation (numbers
represent three biological replicate samples). Data are presented
as the mean ± standard error of the mean. ***P<0.001
and ****P<0.0001. DSI, dicer-substrate small
interfering RNA; PUM2, pumilio RNA-binding family member 2; PCA,
principal component analysis; FPKM, fragments per kilobase of
transcript per million fragments mapped; DEGs, differentially
expressed genes; NC, negative control; KEGG, Kyoto Encyclopedia of
Genes and Genomes; Adm, adrenomedullin; Itga8, integrin subunit α
8; RNA-seq, RNA-sequencing; Dim1, dimension 1; Dim2, dimension 2;
FDR, false discovery rate; ECM, extracellular matrix; Rgs2,
regulator of G protein signaling 2; Cdh11, cadherin 11; Plpp3,
phospholipid phosphatase 3; Ndrg4, NDRG family member 4; Itga11,
integrin subunit α 11; Tgfbi, transforming growth factor β-induced;
qPCR, quantitative PCR.

Figure 3

PUM2 regulates gene alternative
splicing in H9C2 cells. (A) Bar plot showing the number of all
notable regulated ASEs between DSI and NC samples. (B) Heat map of
splicing ratios for ASEs with hierarchical clustering. (C) Bar plot
showing the enriched GO biological process terms of RASG. (D) Reads
distribution diagram showing Actn1 (ES) and Tpm1 (MXE). Green
blocks indicate the positions where AS occurs (the connection lines
above are junction reads and splicing sites). The top shows the
absolute position of the chromosome base; the left side represents
the samples, and the vertical coordinate is the normalized reads;
the bottom provides transcript information. Bar plots showing
splicing ratio of Actn1 (ES) and Tpm1 (MXE) and quantitative PCR
validation. **P≤0.01, ***P≤0.001 and
****P<0.0001. DSI, dicer-substrate small interfering
RNA; PUM2, pumilio RNA-binding family member 2; NC, negative
control; ASE, alternative splicing event; GO, Gene Ontology, Actn1,
actinin α1; ES, exon skipping; Tpm1, tropomyosin 1; MXE, mutually
exclusive exon; intronR, intron retention; A5SS, alternative 5'
splice sites; A3SS, alternative 3' splice sites; 3pMXE, mutually
exclusive 3'UTRs; 5pMXE, mutually exclusive 5'UTRs; AS, alternative
splicing; RASG, regulated alternative splicing gene.
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Copy and paste a formatted citation
Spandidos Publications style
Zhang Z, Zhou H, Adili D, Zhu W, Liu Y, Zhou W, Wang Z and Li J: <p>PUM2 knockdown regulates the expression and alternative splicing of genes associated with myocardial fibrosis in H9C2 cells</p>. Exp Ther Med 31: 94, 2026.
APA
Zhang, Z., Zhou, H., Adili, D., Zhu, W., Liu, Y., Zhou, W. ... Li, J. (2026). <p>PUM2 knockdown regulates the expression and alternative splicing of genes associated with myocardial fibrosis in H9C2 cells</p>. Experimental and Therapeutic Medicine, 31, 94. https://doi.org/10.3892/etm.2026.13089
MLA
Zhang, Z., Zhou, H., Adili, D., Zhu, W., Liu, Y., Zhou, W., Wang, Z., Li, J."<p>PUM2 knockdown regulates the expression and alternative splicing of genes associated with myocardial fibrosis in H9C2 cells</p>". Experimental and Therapeutic Medicine 31.4 (2026): 94.
Chicago
Zhang, Z., Zhou, H., Adili, D., Zhu, W., Liu, Y., Zhou, W., Wang, Z., Li, J."<p>PUM2 knockdown regulates the expression and alternative splicing of genes associated with myocardial fibrosis in H9C2 cells</p>". Experimental and Therapeutic Medicine 31, no. 4 (2026): 94. https://doi.org/10.3892/etm.2026.13089
Copy and paste a formatted citation
x
Spandidos Publications style
Zhang Z, Zhou H, Adili D, Zhu W, Liu Y, Zhou W, Wang Z and Li J: <p>PUM2 knockdown regulates the expression and alternative splicing of genes associated with myocardial fibrosis in H9C2 cells</p>. Exp Ther Med 31: 94, 2026.
APA
Zhang, Z., Zhou, H., Adili, D., Zhu, W., Liu, Y., Zhou, W. ... Li, J. (2026). <p>PUM2 knockdown regulates the expression and alternative splicing of genes associated with myocardial fibrosis in H9C2 cells</p>. Experimental and Therapeutic Medicine, 31, 94. https://doi.org/10.3892/etm.2026.13089
MLA
Zhang, Z., Zhou, H., Adili, D., Zhu, W., Liu, Y., Zhou, W., Wang, Z., Li, J."<p>PUM2 knockdown regulates the expression and alternative splicing of genes associated with myocardial fibrosis in H9C2 cells</p>". Experimental and Therapeutic Medicine 31.4 (2026): 94.
Chicago
Zhang, Z., Zhou, H., Adili, D., Zhu, W., Liu, Y., Zhou, W., Wang, Z., Li, J."<p>PUM2 knockdown regulates the expression and alternative splicing of genes associated with myocardial fibrosis in H9C2 cells</p>". Experimental and Therapeutic Medicine 31, no. 4 (2026): 94. https://doi.org/10.3892/etm.2026.13089
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