Identification of differentially expressed immune related lncRNAs, immune ralated genes and miRNAs
In present study, the expression profile of 331 immune related genes, 544 immune-related lncRNAs and 1881 miRNAs were extracted. We identified 273 differentially expressed lncRNAs, including 6 down-regulated and 267 up-regulated (Figure 1A and 1B), 86 differentially expressed genes, including 16 down-regulated and 70 up-regulated (Figure 1C and 1D), 251 differentially expressed miRNAs, including 22 down-regulated and 229 up-regulated (Figure 1E and 1F).
Construction of prognostic signature
According to the results of univariate cox regression analysis (P<0.05), 31 lncRNAs were identified that were related to OS (Figure 2A). LASSO analysis revealed that 11 lncRNAs were properly considered to construct a prognostic signature (Figure 3). According to the results of multivariable cox regression analysis, six OS related immune related lncRNAs were included in the prognostic signature (Figure 2B). We constructed the prognostic signature according to the expression level of these six lncRNAs and their coefficients. The formula was as follows: risk score = (0.3342 * the expression level of AC145207.5) + (0.0865 * the expression level of AL365203.2) + (0.092 * the expression level of AC009779.2)+ (0.0441 * the expression level of ZFPM2-AS1)+ (0.0801 * the expression level of PCAT6)+ (0.0266 * the expression level of LINC00942). HCC patients were categorized as high-risk group (n=185) and low-risk group(n=185) according to the median risk score.
Evaluation of prognostic signature based on OS related immune related lncRNA
Based on the results of Kaplan-Meier analysis, we found that high-risk group had a significantly poorer OS than low-risk group (P=2.094e−06, Figure 4A), the 1-, 3- and 5-year survival rates of high-risk group were 72.3%, 50.1% and 36.2%, respectively. However, in the low-risk group, the corresponding survival rates were 92.6%, 73% and 58.5%, respectively. All lncRNAs included in prognostic signature were found to be negative correlation with OS of HCC patients (Figure 5A-F).
Compared with other Clinicopathological parameters, the AUC for the prognostic signature was the highest (0.778), suggesting moderate predicting efficacy in OS monitoring (Figure 4B). A heat map about the expression profile of the six lncRNAs show that all lncRNAs were negative with OS (Figure 6A). A dot plot of survival status revealed that patients in high risk group had much higher mortality rate than those in low-risk group (Figure 6B). Figure 6C showed the rank of prognostic index and distribution of groups.
Principal component analysis (PCA) revealed that risk-score can better distinguish high-risk and low risk patients compared with all lncRNAs, immune related lncRNAs, differentially expressed immune related lncRNAs (Figure 7)
Univariate cox regression analysis revealed that pathologic stage, tumor stage, distant metastasis status and risk-score were related to OS (Figure 8A). Multivariate cox regression analysis suggested that risk-score could become an independent predictor after other parameters were adjusted, including age (≥65 years old), gender, tumor grade, pathologic stage, tumor stage, lymph node metastasis status and distant metastasis status (Figure 8B).
Analysis the clinical relevance of the prognostic signature
In order to apply the prognostic signature to the clinical, we analyzed the relevance between risk score and clinicopathologic characteristics, including age(≥65 years old), gender, tumor grade (Grade I&II VS Grade III&IV), pathologic stage (Stage I&II VS Stage III& IV), tumor stage (T1-2 VS T3-4), lymph node metastasis status (N0 VS N1) and distant metastasis status (M0 VS M1). The result showed that risk-score were relevant with distant metastasis status, pathologic stage and tumor stage (P<0.05, Figure 9). We also analyzed the relevance between these clinicopathologic characteristics and the six lncRNAs. The result showed that AC145207.5 was relevant with age, tumor grade, pathologic stage and tumor stage (Supplementary figure 1), LINC00942 was relevant with gender, distant metastasis status and lymph node metastasis status (Supplementary figure 2), AL365203.2 were relevant with age, pathologic stage and tumor stage (Supplementary figure 3A-C), ZFPM2-AS1 was relevant with age and distant metastasis status (Supplementary figure 3D).
Potential molecular mechanisms of lncRNAs included in prognostic signature
According to the results of the ceRNA network (Figure 10A), five of the lncRNAs included in prognostic signature, six differentially expressed immune related genes and 32 differentially expressed miRNAs were included in the network. Finally, 15 immune-related ceRNA aixs were constructed (Table 1).
The results of GSEA (c2.cp.kegg.v7.symbol.gmt) showed that mainly function of these six lncRNAs were related with cancer, including nine high expressed pathways (cell cycle, DNA replication, NOTCH signaling pathway, tight junction, ERBB signaling pathway, bladder cancer, pathways in cancer, rig i like receptor signaling pathway, nod like receptor signaling pathway) and three low expressed pathways (Complement and coagulation cascades, PPAR signaling pathway, Drug metabolism cytochrome P450). (Table 2 and Figure 10B). High expressed pathways were considered to promote cancer formation, invasion and metastasis, while low expressed pathways were considered to inhibit cancer formation, invasion and metastasis.