MBNL1 knockdown reduces the cell apoptosis.
In this article, HeLa cells were used in both cytological experiments and RNA-seq. The knocking down of MBNL1 on HeLa cells was conducted to investigate the influence on cell proliferation and apoptosis. The expression of MBNL1 was tested by RT-qPCR, both the shRNA vector and the empty vector. Results show that the expression of MBNL1 is reduced by approximately 50% (Fig. 1A). Cell proliferation was significantly increased (Fig. 1B), while cell apoptosis was significantly inhibited by the knockdown of MBNL1 (Fig. 1C,D), similar to the previous study (Wu et al., 2018). These results illustrated that the cell proliferation was increased, while apoptosis was inhibited by shMBNL1 in HeLa cells.
Gene expression profiles regulated by the shMBNL1
Two replicates for both shMBNAL1 and control groups were conducted to eliminate the experimental bias. Complementary DNA (cDNA) libraries were conducted for RNA-seq. Illumina HiSeq X Ten platform was employed for sequencing. The raw reads are 85.5±6.0 million per sample. After removing the adaptor and low-quality reads, 82.0±6.4 million reads were left. These reads were mapped to the human GRCH38 genome with TopHat2 (Kim et al., 2013). 67.2±5.0 million reads were mapped, with 64.5±4.6 uniquely mapped and 2.7±5.5 multiple mapped (Supplementary Table S1).
The uniquely mapped reads were used for gene expression analysis (Fig. 2A). FPKM (fragments per kilobase of exon model per million fragments mapped) was calculated. There were 25,677 genes detected at the level FPKM>0, and 13,121 genes at the level FPKM>1 (SupplementaryTable S2 and S3). The relative expression level by FPKM further confirmed the knockdown of MBNL1 (Fig. 2B), similar to the result by RT-qPCR (Fig. 1A). Pearson’s correlation coefficients were generated by the global gene expression levels between the knockdown and control samples. It is shown that the correlations were high between the shMBNL1 and the control, indicating the similarity between them (Fig. 2C).
EdgeR (Robinson et al., 2010) was employed to investigate the differentially expression genes (DEGs) between the shMBNL1 and control. We found 398 genes were up-regulated and 277 genes were down-regulated (fold change ≥2 or ≤0.5 and false discovery rate<0.05). The up-regulated and down-regulated genes were shown in the volcano plot (Fig. 2D). The up and down-regulated genes were 59% and 41% of the total DEGs, respectively (Fig. 2E). Detailed up and down-regulated genes can be obtained in Supplementary Table S4 and S5. The heatmap shows the significant DEGs between the shMBNL1 and control. The number of up-regulated genes is greater than that of down-regulated. The two repeated experiments showed similar results (Fig. 2F).
MBNL1 Knockdown regulating the differential expression genes enriched in cell adhesion and apoptosis
GO and KEGG analyses were employed to analyze the potential biological process of DEGs regulated by the knockdown of MBNL1. For the GO analysis, the up-regulated DEGs are enriched in 33 terms and the down-regulated DEGs in 9 terms (Supplementary Table S5). In the top 10 terms of the up-regulated terms, these DEGs are enriched in extracellular matrix organization, extracellular matrix disassembly, synaptic transmission, homophilic cell adhesion, apoptotic process, etc (Fig. 3A). The down-regulated DEGs are enriched in cell migration, nervous system development, DNA-dependent transcription etc. (Fig. 3A). For the KEGG analysis, the up-regulated DEGs are enriched in adipocytokine signaling pathway and insulin secretion (Fig. 3B), while the down-regulated in neuroactive ligand-receptor interactions and tight junctions (Fig. 3B). Some of them have been reported in previous studies, such as synaptic (Wang et al., 2012), insulin abnormality (Dansithong et al., 2005;Sen et al., 2010), but some have not.
To validate the effect of the knockdown of MBNL1, five DEGs (BMF, EDN2, IFITM1, PCDHB11, MYH13) related to cell adhesion and cell apoptotic signal pathways were randomly selected for RT-qPCR. The results showed that the first 4 genes were higher expressed, while the last one was lower expressed, consistent with the results by RNA-seq results (Fig. 3C).
MBNL1 Knockdown regulating the alternative splicing genes enriched in cell adhesion and apoptosis
As studies specified, the main function of MBNL1 is to participate in the alternative splicing of pre-mRNAs. Our results also showed that, among the uniquely mapped reads, more than one half were junction reads (Supplementary Table S1), indicating the potential effects of the alternative splicing function of MBNL1. Among the splicing events, 160,922 were known and 149,039 were novel splice junctions detected by TopHat2 (Supplementary Table S6). ABLas pipeline (Xia et al. 2017) was employed to investigate alternative splicing events (ASE). The result showed 19,727 known and 51,225 novel ASE (Supplementary Table S6).
To select significant MBNL1-regulated alternative splicing events (RASEs), two indexes were employed, including P values and changed AS ratios. The thresholds for these two indexes were ≤0.05 and ≥0.15, respectively. As a result, 504 RASEs were detected in total. The types of alternative splicing include exon skipping(ES),alternative 5’ splice site (A5SS),alternative 3’ splice site (A3SS),intron retention (IntronR), mutually exclusive 5’ UTRs (5pMXE),mutually exclusive 3' UTRs (3pMXE), Cassette Exon,A3SS&ES and A5SS&ES. Among these, the numbers of A5SS, A3SS and ES are relatively high (Fig. 4A). There were 460 MBNL1-regulated alternatively spliced genes (RASGs), only 2 of them overlapped with the DEGs (Fig. 4B). Most of the DEGs and RASGs are not regulated by MBNL1 simultaneously. Of the 504 RASEs, intron retention (IR) events accounts for 25.4%, and non-intron retention (NIR) events 74.6% (Fig. 4C and Supplementary Table S7).
To discover the function of the RASGs, GO and KEGG analysis were employed. The alternative splicing genes are enriched in the apoptotic signaling pathway, positive regulation of apoptotic process, protein ubiquitination and DNA repair by GO analysis (Fig. 4D, Supplementary Table S8). The KEGG analysis demonstrates that the RASGs are enriched in adherent junctions, fatty acid elongation, metabolic pathways (Fig. 4E, Supplementary Table S9). The results show that the knockdown of MBNL1 regulates the alternative splicing gene related to the cell apoptosis.
RT-qPCR was employed to validate the MBNL1 regulated ASEs. Five genes are randomly selected (FLNB, CASP10, PHLDB2, DIDO1, CTNND1). The validation of these genes is consistent with the RNA-seq results (Fig. 5 and Fig. 6). The ratio in the ASE changed significantly, indicating MBNL1 regulates the alternative splicing of these genes.