Transcriptome Analysis of Wild Type and FGD5-AS1 knockdown CCC-HEH-2 cells

Purpose: According to the previous analysis results of gene regulation in fetal heart tissues with tetralogy of Fallot (ToF), we constructed the ceRNA mediated network driven by lncRNAs using a causal inference framework based on the expression correlations and validated miRNA-lncRNA/mRNA evidences. Totally 4 lncRNAs were identified as hub lncRNAs in the network, and FGD5-AS1 was focused for further loss-of-function investigation. Methods: The specific shRNAs against FGD5-AS1 (sh-FGD5-AS1) as well as the corresponding negative control (sh-NC) were constructed along with lentiviral vector respectively. Then the CCC-HEH-2 human cardiac myocytes cell lines were infected with lentivirus and followed puromycin treatment for several days. The knockdown efficiencies of the FGD5-AS1 expression were validated by qPCR. Genome-wide RNA expression of control and knockdown CCC-HEH-2 cell lines were observed using RNA-Seq. Conclusions: Knockdown of this lncRNA interferes with glutamate receptor activity and metalloendopeptidase inhibitor activity. The expression of abundant CHD genes with |fold change| ≥ 2 was validated with qPCR. These results indicate that FGD5-AS1 plays an important role in CHD genes regulation networks. For CCC-HEH-2 cell line, 3 sh-NC cells and 3 sh-FGD5-AS1 cells were used and analyzed. To identify FGD5-AS1 signatures, paired t-test was performed between control shRNA and FGD5-AS1 shRNA samples.

研究目的：基于法洛四联症（tetralogy of Fallot, ToF）胎儿心脏组织的基因调控既往分析结果，本研究以长链非编码RNA（long non-coding RNAs, lncRNAs）为驱动因子，结合表达相关性与已验证的miRNA-lncRNA/mRNA关联证据，采用基于因果推断的框架构建竞争内源RNA（competing endogenous RNA, ceRNA）调控网络。最终筛选出4个核心长链非编码RNA，并选取FGD5-AS1开展后续功能丧失实验研究。
实验方法：分别构建针对FGD5-AS1的特异性短发夹RNA（short hairpin RNA, shRNA，sh-FGD5-AS1）及其阴性对照（sh-NC），并与慢病毒载体（lentiviral vector）进行重组。将慢病毒感染人心脏肌细胞系CCC-HEH-2，随后用嘌呤霉素（puromycin）处理数日。通过实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qPCR）验证FGD5-AS1的敲低效率。采用RNA测序（RNA-Sequencing, RNA-Seq）检测对照组与FGD5-AS1敲低组CCC-HEH-2细胞的全基因组RNA表达水平。
研究结论：敲低该长链非编码RNA会干扰谷氨酸受体活性与金属内肽酶抑制剂活性。通过qPCR验证了大量|折叠变化|≥2的先天性心脏病（congenital heart disease, CHD）基因的表达差异。上述结果表明，FGD5-AS1在先天性心脏病基因调控网络中发挥重要作用。本研究中共使用3份sh-NC组细胞与3份sh-FGD5-AS1组细胞进行分析。为鉴定FGD5-AS1的表达特征，对阴性对照短发夹RNA组与FGD5-AS1短发夹RNA组的样本进行配对t检验。