Correlation  between  expression  levels  of  protein-­‐coding  and  non-­‐protein  coding

As described in chapter 3.5 the expression of both protein-coding and non-protein coding genes changes during NK cell development from stage 1 to stage 5. To investigate further relations between these two gene groups, a correlation analysis of their expression levels was performed. The sample set was the same as in chapter 3.5 and included 56 ex vivo NK samples from stages 1 to 5; the gene set included the top 100 differentially expressed protein-coding genes and the top 100 non-protein coding genes, described for this sample set above. Only correlations between protein-coding and non-protein coding genes were taken into account. Correlations with the value of coefficient >0.8 or <-0.8 and p<0.05 were considered to be significantly strong and are summarized in the Appendix 14.

Out of the top 100 non-protein coding genes, 26 showed a significant strong positive (Figure 27) or negative (Figure 28) correlation to at least one of the top 100 protein-coding genes; 42 out of the top 100 protein-coding genes were involved in these correlations (Appendix 14).

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Figure 26 Correlation of gene expression levels between consequent developmental stages based on expression of protein-coding and non-protein coding genes

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Two genes among these 26 genes, RP11-973H7.1 and RP11-20I20.4, are expressed specifically in stages 4 and 5 and had the highest number of correlations: 17 and 20 respectively. Both of them correlated to following genes: S1PR5 (correlation coefficients 0.85 and 0.91 respectively), TGFBR3 (0.85 for both), GZMA (0.84 and 0.8), GZMB (0.92 for both), GZMM (0.82 for both), FGFBP2 (0.86 and 0.82 respectively), KLRD1 (0.82 and 0.81), TBX21 (0.82 and 0.83), PYHIN (0.87 and 0.86), PDZD4 (0.86 and 0.84), PRSS23

Figure 27 Correlation network of gene expression between protein-coding and non-protein coding genes within ex vivo NK samples

Only correlations with Spearman’s coefficient>0.8, p<0.05 are shown.

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coefficients 0.88 and 0.87 respectively). Consistently, all of these protein-coding genes were also expressed by samples from stage 4 and/or 5.

The expression of 3 more genes typical for mature NK cells, IFNG, GZMH and B3GAT1, correlated with the expression of RP11-973H7.1 (with coefficients 0.84, 0.8 and 0.81 respectively), but not with RP11-20I20.4. At the same time, expression of RP11-20I20.4, but not of RP11-973H7.1, strongly correlated with the expression of DTHD1 and BNC2 (coefficients 0.8 and 0.82 respectively). In addition, expression of RP11-20I20.4 was negatively correlated with the expression of SPINK2 (coefficient -0.82), MAP7 (-0.83), MYB (0.82) and VWDE (-0.8). Notably, while MAP7, MYB and VWDE were expressed by stages 1 to 3, SPINK2 gene was expressed mainly by stage 4 NKs.

RP11-1094M14.5, the gene with the third highest number of correlations, was also expressed within stage 4 and 5 samples, but also in samples from stage 3. Strong positive correlations were observed for genes TGFBR3 (correlation coefficient 0.8), DTHD1 (0.84),

correlation network

Figure 28 Correlation network of gene expression between protein-coding and non-protein coding genes within ex vivo NK samples

Only correlations with Spearman’s coefficient<-0.8, p<0.05 are shown.

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PYHIN1 (0.9), FCGR3A (0.82), KLRF1 (0.83), GZMA (0.85), BNC2 (0.8), and FEZ1 (0.8);

all these genes were also highly expressed by mature NK cells of stages 4 and 5. The same was the case for another non-protein coding gene expressed within stages 4 and 5, RP3-467K16.4. Its correlation also correlated to those of mature NK cell markers: GZMB (coefficient 0.83), GZMM (0.82), IFNG (0.82), PDZD4 (0.81), PRF1 (0.8).

Three more lncRNA genes, RP11-330A16.1 (expressed mainly in stage 4 samples), RP11-563D10.1 (expressed in stage 3 samples from tonsils and cord blood) and RP1-206D15.6 (expressed in stage 3 samples, but also among some samples from stage 4) possessed four positive correlations each. Expression levels of RP11-330A16.1 correlated with expression of ENPP1 (correlation coefficient 0.82), CSPG4 (0.83), IL7R (0.82), and LTB (0.82). RP11-563D10.1 expression correlated expression levels of ENPP1 (correlation coefficient 0.82), LIF (0.84), RORC (0.82), and KLRF2 (0.8). Expression of RP1-206D15.6 correlated with expression levels of LIF (correlation coefficient 0.83), IL1R1 (0.8), LPAR1 (0.83), and MYO7A (0.83). Expression of AC005083.1, a gene encoding processed transcript and expressed also mainly in stage 3 samples, correlated positively to expression of IL1R1 (correlation coefficient 0.82) and MYO7A (0.81) and negatively to S1PR5 (-0.81).

KIAA0125 (expressed in stages 1 to 4, but not in any stage 5 sample) and RP11-686D22.10 (expressed mainly in stages 4 and 5, but also stage 3 samples from tonsils) had four correlation connections each as well. The expression of KIAA0125 positively correlated with that of SPINK2 (correlation coefficient 0.84) and MAP7 (0,81), and correlated negatively with two genes expressed within stages 4 and 5, S1PR5 and GZMB (-0.8 for both). The expression of RP11-686D22.10 positively correlated to that of TGFBR3, PYHIN1 and FEZ1 (correlation coefficients 0.81, 0.86 and 0.82 respectively) and negatively with that of FLT3 (-0.82).

Genes RP6-91H8.3, RP11-796E2.4 and RP11-326C3.2 were expressed in stages 3 and 4, while the latter two of them were also expressed in stage 5, but at lower levels. The expression of all three genes correlated significantly with that of LTB (correlation coefficient 0.8, 0.81 and 0.82 respectively), while RP6-91H8.3 expression also correlated with that of MYO7A (0.83), the expression of RP11-796E2.4 with that of XCL1 (0.85), and expression of 326C3.2 correlated with that of DLL1 (0.84). The expression of RP11-222K16.2, a gene expressed strictly in stage 4 and 5, correlated with that of typical mature NK marker genes, KLRF1 and GZMA (correlation coefficient 0.84 and 0.86 respectively).

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Finally, the other eight non-protein coding genes correlated to only one gene each.

Two genes, LINC00892 and RP11-520A21.1, were expressed by samples from stages 3 and 4. While LINC00892 expression correlated with that of ENPP1 (correlation coefficient 0.84), RP11-520A21.1 expression correlated with IL4I1 (0.86). One more gene, LINC00309, which was also expressed at stage 3 and at lower level in stages 4 and 5, correlated as well with IL4I1 expression (0.84).

From another three genes expressed mainly at stages 4 and 5, but also partly at stage 3, the expression of FTH1P22 correlated significantly with that of KLRF1 (correlation coefficient 0.82), RP11.121A8.1 correlated with PRF1 (0.83), and RP11.463J10.2 correlated with BNC2 (0.84). In addition, RP11-693N9.2 was expressed in stages 3 to 5 and correlated with PRF1 (correlation coefficient 0.83).

The last identified non-protein coding gene, SCART1, was expressed ubiquitously within all analyzed samples and its expression correlated with that of ENPP1 (correlation coefficient 0.82).