3.1 GNL3 promotes cell proliferation in HeLa cells
In order to verify the effect of knockdown GNL3 on cell reproduction and
apoptosis, cytological experiments were carried out. After 48 hours of
transfection, the green fluorescent protein (GFP) fluorescence
expression after shRNA transfection revealed that the cell transfection
was successful (Figure 1A). The reproduction and apoptosis of cells in
the experimental and the control groups were detected by MTT assay and
apoptosis kit, respectively. The results showed that cell reproduction
was inhibited while cell apoptosis was enhanced in the GNL3-knockdown
group when compared with the control group (Figure 1B and C).
RNA-seq analysis of GNL3-regulated genes in HeLa cell
In this experiment, cDNA libraries and high-throughput sequencing of
samples have been successfully constructed. The library was constructed
using Illunima HiSeq X Ten sequencing platform and paired with end
sequencing, and high quality transcriptome data were obtained. In this
study, after knocking down GNL3, the GNL3 expression in the experimental
group decreased significantly (Figure 2A and B), which indicated that
the modeling was successful.
To ensure the reliability of the experimental data, quality control on
the sequencing results were conducted (Table 2). As shown in table 2,
raw reads per sample were generated and clean reads per sample were
retained by the removal of adaptors and contaminating sequences. Among
these, the data of Clean Per in these four groups were all more than
95%, which means that the polluted possibility of this sequencing was
little.
The samples in this experiment were from HeLa cell line, Homo sapiens,
and the reference genome version was GRCh38. We selected software TopHat
2 [28]. After a certain tolerance rate was set, clean reads was
matched to the reference genome (Table 3). Total mapped indicates the
number and proportion of reads that can be located in the genome. In
transcriptome sequencing, if there is no contamination and the reference
genome is selected appropriately, the proportion of this part of data is
usually higher than 70%. The data in the four groups of this experiment
were all above 90% which indicated that the data in this experiment was
reliable. As for the data analysis on the downstream, only this batch of
reads was used to ensure the reliability of the experimental results.
In order to compare the differences in gene expression among different
samples, FPKM value was used to represent gene expression. FPKM
(expected fragments per kilobase of transcript per million fragments
mapped) represents the number of reads per million base lengths from a
gene. In this research, most genes were at an expression level of FPKM ≤
20 (Figure 2D). Then the differential expression of transcriptome
between samples were analyzed (DEG). First, inter-sample clustering
analysis on the correlation between samples was conducted (Figure 2C) to
determine the correctness of sampling.
Based on the detections above, samples were compared and differentially
expressed genes (DEG) were obtained. Software edgeR was selected to
analyze the differential expression between two or more samples. The
fold change (FC) and false discovery rate (FDR) were used to determine
whether a gene was differentially expressed. The evaluation criteria for
significant difference expression were FC ≥ 2 or ≤ 0.5,FDR <
0.05. Through the comparison between the experimental group and the
control group, it is indicated that GNL3 extensively regulated gene
transcription. Heatmap analysis of the expression patterns of partial
DEGs in RNA-seq samples showed a high consistency of the GNL3-mediated
transcription in both data sets (Figure
2E).
Functional clustering of GNL3-regulated genes
In order to reveal the biological role of these DEGs, a taxonomic
enrichment analysis of the differentially expressed genes on GO and KEGG
was performed (Figure 3A and B). In the Figure 3, the top ten
representative events enriched in the biological processes subset of GO
and pathways of KEGG were selected. As shown in Figure 3 after knockdown
of GNL3, the down-regulated gene functions are concentrated in the
aspects of signal transduction, apoptotic, transcription, cell
proliferation and primary immunodeficiency. Through enrichment analysis
on GO and KEGG, it was speculated that GNL3 plays an important role in a
series of biological activities such as inflammation and angiogenesis.
Interestingly, it was found that GNL3 knockdown significantly affected
the expression of many genes including IL24 gene and PTN gene (Figure 3C
and D). Q-RT-PCR was performed to verify the expression of IL24 and PTN
(Figure 3E and F). The results showed that the expression of IL24 and
PTN was down-regulated when GNL3 was knocked down.
Expression level of GNL3, IL24 and PTN gene in the OA
patients
To further verify the results, femoral head specimens from 20 patients
with osteoarthritis and 20 control samples were collected (Figure 4A and
B). The results showed that the expression level of GNL3, IL24 and PTN
gene in specimens were significantly up-regulated compared with the
control group (Figure 4C and D). GNL3 is abundantly expressed in bone
marrow mesenchymal stem cells and correlates with chondrocytes
differentiation. IL24 induces apoptosis and PTN promotes angiogenesis
respectively in the pathogenesis of osteoarthritis. So, the expression
of IL24 and PTN genes were up-regulated with OA.