Some stage-specific immuneCPa genes and lncRNAs were identified in colon cancer
We first identified colon cancer-specific 17 kinds of immuneCPa genes for colon cancer in all stages. More than 70% of the 15 kinds of immuneCPa genes were differentially expressed (Figure 1A). 96.97% B cells associated genes were differentially expressed in all the colon cancer patients. The result indicated that immuneCPa genes play important roles in colon cancer. Next, stage-specific immuneCPa genes were identified in diverse colon cancer stages. In each kind of immuneCPa gene, percent of differential expressed immuneCPa genes showed similar pattern (Figure 1B). Most stage-specific immuneCPa genes were presented in patients with stage II colon cancer which is a critical stage of cancer progression. The result suggested that stage II was active stage of immune system to fight cancer cells. Diverse stage also shared some common immuneCPa genes (Figure 1C). For example, four stages had 14, 9 and 10 common immuneCPa genes in B cells, Gamma delta T cell and Eosinophils. We also extracted differential expressed lncRNAs (61%) for all colon cancer patients (Figure 1D). Similar to immuneCPa genes, there were most differential expressed lncRNAs in stage II of colon cancer. Four diverse stages shared more common differential expressed lncRNAs compared with immuneCPa genes (Figure 1E). We inferred some key lncRNAs maybe functions essentially in specific colon cancer stage.
Identification of stage-specific immuneCPa gene-lncRNA regulatory pairs in colon cancer development
Two major factors including interactions and similar expression to extract stage-specific immuneCPa gene-lncRNA regulatory pairs. Experimentally verified gene-lncRNA interaction were considered as candidate regulatory paris. Then considering that interactions do not directly imply their actual regulation of immuneCPa gene-lncRNA pairs in certain conditions, exploring gene regulation of lncRNAs through co-expression analysis can offer useful information to identify active immuneCPa gene-lncRNA relationships in different colon cancer stages. PCCs were calculated for each potential immuneCPa gene-lncRNA pair based on their expression profiles at different stages (Figure 2A). Stage II and III showed most similar distribution of PCCs for gene-lncRNA pairs. Most PCCs of gene-lncRNA pairs were concentrated between 0.5 and 1 or -0.5 and -1 (Figure 2B). It indicated that most gene-lncRNA pairs showed strong co-expressed level. The numbers of co-expressed gene-lncRNA pairs were diverse in different kinds of immune cells (Figure 2C). For example, there were most co-expressed gene-lncRNA pairs in Tem and Tfh cells. In addition, most co-expressed gene-lncRNA pairs were present in stage III for almost kinds of immune cells (Figure 2D). The different sizes of these stage-specific immuneCPa gene-lncRNA pairs indicate the heterogeneity of immune associated genes and lncRNAs in the development of colon cancer.
Construction of dynamic stage-specific immuneCPa gene-lncRNA regulatory networks in colon cancer development
We used a P <0.05 as the thresholds to identify a link between immuneCPa genes and lncRNAs in the regulatory networks. There were 91,962, 106,834, 165,589 and 118,431 immuneCPa gene and lncRNA pairs in stage I, II, III, IV (Figure 3A). We extracted absolute values of PCCs > 0.9 as the thresholds to construct more tighter and closer stage-specific immuneCPa gene-lncRNA regulatory networks in colon cancer development (Figure 3B). Specifically, 19,929 edges between 408 immuneCPa genes and 3,768 lncRNAs, 75,259 edges between 408 immuneCPa genes and 4,920 lncRNAs, 52,635 edges between 409 immuneCPa genes and 3,983 lncRNAs and 118,356 edges between 407 TFs and 4,389 lncRNAs were constructed for stages I, II, III and IV colon cancer patients, respectively. In these stage-specific immuneCPa gene-lncRNA regulatory networks, there were some key hub nodes such as immuneCPa gene DCSTAMP, KIR3DL3 and SND1-IT1 could interacted with more than 400 lncRNAs. In these four stage-specific immuneCPa gene-lncRNA regulatory networks, immuneCPa gene had bigger degree than lncRNAs (Figure 3C). The degree analysis indicated that a common immuneCPa gene could be regulated by diverse and a number of lncRNAs in colon cancer development. lncRNAs maybe play specific regulatory roles for immuneCPa genes in diverse stages of colon cancer.
The dynamic activity profiles of immuneCPa gene-lncRNA regulatory pairs
Although stage-specific networks share common topological properties, the immuneCPa gene-lncRNA regulatory interactions may change in different stages of colon cancer. To evaluate the proportion of common and specific immuneCPa gene-lncRNA regulatory interactions during colon cancer progression, we explored the overlaps of immuneCPa gene-lncRNA regulatory relationships among four stage-specific networks (Figure 4A). Most immuneCPa gene-lncRNA regulatory relationships are stage specific. Stage III had maximum number of immuneCPa gene-lncRNA regulatory relationships (Figure 4B). We inferred that stage III is a key stage which lncRNA participated in immune regulation for colon cancer. For all the immuneCPa gene-lncRNA regulatory relationships, 378,658 immuneCPa gene-lncRNA regulatory relationships were only present in one stage. The four stages only shared 249 common immuneCPa gene-lncRNA regulatory relationships, indicating that relationships were temporally-specific in colon cancer (Figure 4C). The immuneCPa gene-lncRNA regulatory relationships were making dynamic changes in colon cancer development (Figure 4D). To provide an overview of all possible immuneCPa gene-lncRNA relationships and their dynamic regulatory status, we built an activity profile for immuneCPa gene-lncRNA relationships across different stages of colon cancer. Activity score is the standardized value of PPCs. Based on the activity scores, these immuneCPa gene-lncRNA relationships were grouped by the K-means clustering method (Figure 4E). Different groups of immuneCPa gene-lncRNA relationships were apparently activated at one or more stages. The patterns of immuneCPa gene-lncRNA relationships were diverse in different groups. These groups maybe could as stage-specific biomarkers for colon cancer.
Specificity score evaluates the specificity of immuneCPa gene-lncRNA relationships in colon cancer development
Specificity score was designed to evaluate the specificity of immuneCPa gene-lncRNA relationships in colon cancer development. Most immuneCPa gene-lncRNA relationships showed lower specificity scores and indicated stage specific for these relationships in colon cancer (Figure 5A). In order to extract more stage-specific immuneCPa genes, we used mean specificity scores of all immuneCPa gene-lncRNA relationships to represent the specify of each immuneCPa gene. These stage-specific immuneCPa genes could interact with diverse numbers of lncRNAs in colon cancer (Figure 5B). The mean specificity scores for each kind of immune cells were also obtained. Mast and cytotoxic cells showed the strongest stage specificity in colon cancer (Figure 5C). Specificity scores of most immuneCPa genes in these two kinds of immune cells concentrated between 0.06 and 0.08 (Figure 5D). NK CD56bright cell showed strongest stage common features in colon cancer and indicated that it maybe participate in all immune process for colon cancer development. This results also revealed that diverse kinds of immune cells were temporally-specific in colon cancer.
Some immuneCPa gene-lncRNA relationships in colon cancer development has specific prognostic potential
To evaluate the potential value of immuneCPa gene-lncRNA pairs as prognostic biomarkers in colon cancer with diverse stages, we created a risk-score formula according to the expression of each immuneCPa gene and its corresponding lncRNAs to generate OS (overall survival) prediction (see the Material and Methods section). We used median risk score as the cut-off point to test the survival of the diverse stage colon cancer patients into high-risk or low-risk groups. These three immuneCPa gene-lncRNA clusters were associated with Macrophages, iDC and Neutrophils cell populations (Figure 6A). Two stage II and one stage III specific immuneCPa gene-lncRNA clusters were significantly associated with survival, and they could serve as prognostic biomarkers (Figure 6B).