Long non-coding RNA SAP30-2:1 is downregulated in congenital heart disease and regulates cell proliferation by targeting HAND2

doi:10.1007/s11684-020-0778-5

Front. Med.

2021, Vol. 15

Issue (1) : 91-100 https://doi.org/10.1007/s11684-020-0778-5

RESEARCH ARTICLE

Long non-coding RNA SAP30-2:1 is downregulated in congenital heart disease and regulates cell proliferation by targeting HAND2

Jing Ma^1,², Shiyu Chen², Lili Hao², Wei Sheng³, Weicheng Chen³, Xiaojing Ma³, Bowen Zhang², Duan Ma^2,³(

), Guoying Huang³(

)

¹. ENT Institute, Department of Facial Plastic and Reconstructive Surgery, Eye & ENT Hospital, Fudan University, Shanghai 200031, China
². Research Center for Birth Defects, Institutes of Biomedical Sciences, Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
³. Children’s Hospital of Fudan University, Shanghai 201102, China

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Abstract

Congenital heart disease (CHD) is the most common birth defect worldwide. Long non-coding RNAs (lncRNAs) have been implicated in many diseases. However, their involvement in CHD is not well understood. This study aimed to investigate the role of dysregulated lncRNAs in CHD. We used Gene Expression Omnibus data mining, bioinformatics analysis, and analysis of clinical tissue samples and observed that the novel lncRNA SAP30-2:1 with unknown function was significantly downregulated in damaged cardiac tissues from patients with CHD. Knockdown of lncRNA SAP30-2:1 inhibited the proliferation of human embryonic kidney and AC16 cells and decreased the expression of heart and neural crest derivatives expressed 2 (HAND2). Moreover, lncRNA SAP30-2:1 was associated with HAND2 by RNA immunoprecipitation. Overall, these results suggest that lncRNA SAP30-2:1 may be involved in heart development through affecting cell proliferation via targeting HAND2 and may thus represent a novel therapeutic target for CHD.

Keywords congenital heart disease Gene Expression Omnibus lncRNA SAP30-2:1 cell proliferation RNA immunoprecipitation HAND2

Corresponding Author(s): Duan Ma,Guoying Huang

Just Accepted Date: 16 July 2020 Online First Date: 18 August 2020 Issue Date: 11 February 2021

Cite this article:

Jing Ma,Shiyu Chen,Lili Hao, et al. Long non-coding RNA SAP30-2:1 is downregulated in congenital heart disease and regulates cell proliferation by targeting HAND2[J]. Front. Med., 2021, 15(1): 91-100.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-020-0778-5
https://academic.hep.com.cn/fmd/EN/Y2021/V15/I1/91

Fig.1 lncRNA SAP30-2:1 was screened by data mining of GEO database and bioinformatics analysis. (A) RNA-seq data for human fetal and adult hearts were extracted from the GEO database. (B) CNC network of 19 lncRNAs and 26 mRNAs with a correlation coefficient≥0.9 or≤-0.9 and P≤0.05. Blue diamonds represent lncRNAs, red circular nodes denote mRNAs, and lines indicate the gene co-expression relationship between lncRNAs and mRNAs. (C) Higher expression of lncRNA SAP30-2:1 in fetal hearts compared with those of adults. Expression values presented in red (above median) or green (below median). (D) Relative positions of lncRNA SAP30-2:1 and its nearby gene HAND2 on the chromosome. HAND2 is located at about 13 kbp downstream of lncRNA SAP30-2:1. (E) CNC network of lncRNA SAP30-2:1 and 15 mRNAs. Rose V represents lncRNA SAP30-2:1; green circular nodes denote mRNAs; node size indicates the expression level of a gene (large dot indicates high expression level). Lines represent the gene co-expression relationship between lncRNA SAP30-2:1 and mRNA (full lines represent positive correlations; dashed lines indicate negative correlations).

Fig.2 lncRNA SAP30-2:1 was significantly downregulated in damaged cardiac tissue samples from CHD patients compared with NC based on qPCR analysis. ***P<0.001.

Fig.3 Changes in lncRNA SAP30-2:1 affected the proliferation of HEK293T cells. (A,B) Efficiencies of upregulating and downregulating lncRNA SAP30-2:1 in HEK293T cells were detected by qPCR. (C, D) Positive regulation of cell proliferation by lncRNA SAP30-2:1 was revealed by CCK8 assay. (E–H) Positive regulation of cell proliferation by lncRNA SAP30-2:1 was revealed by EdU assay. (I, J) lncRNA SAP30-2:1 had no effect on cell apoptosis as shown by flow cytometry analysis. ***P<0.001.

Fig.4 Changes in lncRNA SAP30-2:1 affected proliferation of AC16 cells. (A, B) Efficiencies of upregulating and downregulating lncRNA SAP30-2:1 in AC16 cells were detected by qPCR. (C, D) Positive regulation of cell proliferation by lncRNA SAP30-2:1 was revealed by CCK8 assay. (E, F) lncRNA SAP30-2:1 had no effect on cell apoptosis as shown by flow cytometry analysis. ***P<0.001.

Fig.5 lncRNA SAP30-2:1 regulated HAND2 expression in vitro by binding to it. (A, B) lncRNA SAP30-2:1 was mainly located in the nucleus of HEK293T and AC16 cells, similar to U1. (C, D) Dysregulation of lncRNA SAP30-2:1 positively regulated HAND2 protein levels according to WB analysis. (E, F) RIP assay of lncRNA SAP30-2:1 with anti-HAND2 antibody. lncRNA SAP30-2:1 and HAND2 interaction in HAND2-RNA precipitates was revealed by qPCR analysis.

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