Isolation of CD3 T Cells
We used Hu CD3 IMag Particles-DM HIT3a to isolate CD3 T cells from the PBMCs. The results of the CD3 isolation that was performed of three times showed that the purity of the CD3+ lymphocytes was 92.95%, 92.45%, and 92.61%, respectively, as measured by flow cytometry (Fig. 1a). The activity of the isolated CD3 T cells was enough to meet the needs of the subsequent experiments using T cells.
Construction of pCDH-CISH plasmid and packaging
pCDH (up-regulated control group) and pCDH-CISH (up-regulated CISH group) plasmids were provided by Anhui Biotechnology Company following verification of the ability of the inserted CISH gene to up-regulate the expression of CISH. pCDH and pCDH-CISH were transfected into 293T cells, and the transfected cells were collected to detect the CISH mRNA by RT-PCR and the CISH protein by western blotting. The results demonstrated that the expression of CISH mRNA in the pCDH-CISH group was significantly higher when compared to the control group (Fig. 1b1). In addition, western blot analysis revealed that the expression of CISH protein in the pCDH-CISH group was significantly higher compared to the pCDH group (Fig. 1b2).
Construction of pLKO.1-shCISH plasmid and packaging
The CISH shRNA1, CISH shRNA2, CISH shRNA3 and the scrambled shRNA were provided by the Wuhan Qingke Biological company (Wuhan, China). The CISH shRNA was amplified by the PCR and cloned into the PLKO.1 vector. Escherichia coli were transformed with pLKO.1-shCISH. pLKO.1-shRNA1 and pLKO.1-shRNA3 were successfully confirmed by DNA sequencing. pLKO.1-shRNA1 and pLKO.1-shRNA3 were transfected into 293T cell lines and the cells were collected. PCR and western blotting were used to analyze the expression of CISH. The results revealed that the expression of CISH in the 293T-shRNA1 and 293T-shRNA3 group was significantly lower compared to the 293T and 293T pLKO.1-NC groups (Fig. 1c1, c2).
Transfection of the recombinant lentiviral plasmid in CD3 T cells and expression of CISH
Lentiviral supernatant was collected from 293T cells that were transfected with the recombinant plasmids for 48 and 72 h. The lentiviral supernatant was then used to infect CD3 T cells and CISH expression in the CD3 T cells was analyzed using RT-PCR and Western blotting (Fig. 1d1, d2). We found that the expression of CISH in the up-regulated group (pCDH-CISH) was significantly higher than the down-regulated group (pLKO.1-shRNA) and the control group.
Effect of CISH on T cell proliferation
The ability of the T cell to proliferate was studied using the T cell colonies (CFU-T) assays. Methylcellulose medium was prepared to cultivate the blank, pCDH, pCDH-CISH, pLKO.1-NC, and pLKO.1-shRNA. CFU-T were evaluated following 7 days of culture. The colonies were used to evaluated proliferation ability. pCDH-CISH had lower colony-forming ability (Fig 2). The pCDH-CISH group formed no colonies compared to 10 colonies in the pLKO.1-shRNA group. This indicates that CISH up-regulation inhibits T cell proliferation.
The expression of the TCRVβ subfamily members
TCR is a molecule found on the surface of T cells. It is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. The interaction of TCR with an antigen and MHC activates T cells. Gene expression and cloning of TCRVβ subfamily members is a sensitive and reliable method to detect lineage changes and antigen-specific responses of T cells. The analysis of TCRVβ gene repertoire may provide more information about the effect of CISH gene on T cell. We used RT-PCR to detect the gene expression of TRVβ3-1, TRVβ7, TRVβ9, TRVβ11, and TRVβ12. There were significant differences in the expression of TRVβ3-1 and TRVβ12 between the groups. The expression of TRVβ3-1 and TRVβ12 were up-regulated in the pCDH-CISH group (p < 0.01, p < 0.001, respectively), but down-regulated in the pLKO.1-CISH group (p < 0.01, p < 0.05, respectively). The expression of TRVβ7, TRVβ9, and TRVβ11 however, did not differ among the groups (Fig. 3).
Effect of CISH on T cell differentiation
To determine the effect of CISH on T cell differentiation, flow cytometry was used to analyze the changes of T cell subsets in each group. Seven different subgroups were detected: CD3, CD4, CD8, CD45RA, CD45RO, CD25, and CD86. CD3 is the surface marker of T cells, while CD4, CD8, CD45RA, and CD45RO are surface markers of Th cells, CTL cells, initial cells, and memory cells, respectively, and CD25 and CD86 are related to cell growth and activation. Flow cytometry results showed that the CD8 population was significantly up-regulated in the pCDH-shRNA group compared to the other groups, while CD25 and CD86 were significantly down-regulated. CD3, CD4, CD45RA, and CD45RO markers showed no significant change in the CISH overexpression group (Fig. 4).
Differentially expressed genes (DEGs) and functional enrichment analyses
A total of 33531 DEGs were identified in the CD3 T cells across the blank and up-regulated group. There were significant differences in the expression of 2124 genes between the blank and the up-regulated group (p-adjust < 0.05 and |log2 (FoldChange)| > 1). Of these, 940 genes that were up-regulated and 1184 genes that were downregulated in the up-regulated group compared to the blank group, which were described as a volcano plot and heatmaps (Fig. 5a and b)
The GO and KEGG enrichment analyses was used to understand the functions of the DEGs. A GO analysis of the DEGs in the up-regulated group and the blank group identified clusters enriched for 6648 GO terms, among them, for 154 GO terms the p-adjust values were less than 0.005. The top 10 DEGs involved in the GO terms are shown in Fig. 5c. GO terms were classified as biological process (BP), cellular component (CC), and molecular function (MF). GO analysis results showed that the DEGs were mainly involved in chromosome segregation, nuclear division, organelle fission in the BP group; chromosomal region, centrosome, and spindle in the CC group; and ATPase activity, and carbohydrate binding in the MF group.
Based on the KEGG pathway enrichment analysis, 296 KEGG pathways were enriched. A total of 14 KEGG pathways were significantly enriched (p-adjust < 0.05). The top DEGs involved in the KEGG pathways are shown in Fig. 5d, and these include the cell cycle, DNA replication, p53 signaling pathway, and PI3K-Akt signaling pathway and others.
Validation of analyses using RT-PCR
Ten significant DEGs, identified from the RNA-seq data, were randomly selected for RT-PCR validation. These included VEGFA, PDGFR-β, DUSP1, CDKN1A, CCND2, BCL2, PIM1, MDM2, BCRA1, and CKD2. RT-PCR confirmed that the DEGs had the same pattern of expression as observed in the RNA-seq (Fig 6). Therefore, the gene expression observed in the CD3 T cell was highly credible.