Identification of autophagy-related lncRNAs in HCC patient tissue samples
TCGA HCC transcriptome profiling contained 14142 lncRNAs and 19658 mRNAs transcripts. A total of 226 lncRNAs and 22688 mRNAs were identified by analyzing RNA-seq data of HCC patient tissue samples from the ICGC database.
The expression level of 232 autophagy genes were also extracted from the TCGA and ICGC databases, respectively. Pearson correlation analysis was performed to identify autophagy-related lncRNAs using |R| > 0.5 and P < 0.050 as the selection criteria. Finally, 349 and 67 autophagy-related lncRNAs were selected from the TCGA and ICGC databases, respectively. After autophagy-related lncRNAs were intersected from the two cohorts, 19 autophagy-related lncRNAs were obtained (Fig. 1A).
Identification of prognostic risk expressed autophagy-related lncRNAs in HCC patients from TCGA database
According to KM method and univariate Cox proportional hazards model, survival analysis of each autophagy-related lncRNA was done to screen prognostic biomarker with both significant P values less than 0.050. Figure 1B showed that a total of 9 autophagy-related lncRNAs might have prognostic value for patients with HCC. Subsequently, Multivariate Cox regression analysis showed that 4 of 9 autophagy-related lncRNAs were well candidates to construct a prognostic risk signature on the basis of the lowest Akaike information criterion (AIC). The four autophagy-related lncRNAs (BACE1-AS, SNHG3, MIR210HG, and ZEB1-AS1) were confirmed to be unfavorable factors for HCC (Fig. 2).
Construction and evaluation of the autophagy-related lncRNAs prognostic signature
The above lncRNAs were performed to construct a prognostic lncRNAs signature as follows: Risk Score = (0.142×expression level of BACE1-AS) + (0.032×expression level of SNHG3) + (0.067×expression level of MIR210HG) + (0.112×expression level of ZEB1-AS1).
According to the prognostic signature, each patient obtained a risk score in connection with the patient’s prognosis. All patients were divided into high- (n = 171) or low-risk groups (n = 172) by the median risk score, respectively. KM survival analysis demonstrated that the OS of HCC patients with low-risk score had significantly longer than those with high-risk score (shown in Fig. 1C). The 1-, 3-, and 5-year survival rates for the high-risk group patients were 75.60%, 49.90%, and 41.50%, respectively, whereas 1-, 3-, and 5-year survival rates for the low-risk group patients were 93.40%, 76.30% and 57.00%, respectively. Distribution of patients’ risk scores in different groups was ranked in Fig. 1D. The scatter dots plot showed that the correlations of dead status with the risk score (Fig. 1E). Those results demonstrated that patients with a higher risk score suffered from a shorter survival time and lower survival rates. Moreover, heat map showed different expression levels of the autophagy-related lncRNAs in the high- and low-risk groups. As shown, patients with high-risk score also expressed higher levels (Fig. 1F). Time-dependent ROC curve analysis further showed that the AUC value for the prognostic signature was 0.728 (Fig. 1G).
The correlation of the autophagy-related lncRNA prognostic signature with clinicopathological features
Subsequently, analysis was done to investigate the clinical value of the prognostic signature in different subgroups stratified by patients’ clinicopathological characteristics. As shown in Table 1, patients with high riskscore were prone to find in those with higher level of creatinine or AFP. Interesting, riskscore tends to increase in the advanced T stage and TNM staging, suggesting that the autophagy-related lncRNA prognostic signature might be significantly associated with HCC progression. No significant difference of the other clinicopathological variables with the prognostic signature was found.
Table 1
Clinical value of the autophagy-related lncRNA prognostic signature for HCC
Characteristics
|
Group
|
Risk score
|
n
|
mean
|
SD
|
t
|
P value
|
Age (years)
|
< 60
|
157
|
1.154
|
0.891
|
0.271
|
0.787
|
|
≥ 60
|
186
|
1.183
|
1.038
|
|
|
Gender
|
Male
|
233
|
1.114
|
0.921
|
1.560
|
0.120
|
|
Female
|
110
|
1.289
|
1.066
|
|
|
Alcohol consumption
|
Present
|
109
|
1.106
|
0.749
|
0.704
|
0.482
|
|
Absent
|
218
|
1.185
|
1.045
|
|
|
Liver cirrhosis
|
Present
|
131
|
1.254
|
1.032
|
0.931
|
0.353
|
|
Absent
|
65
|
1.112
|
0.984
|
|
|
HBV
|
Present
|
98
|
1.223
|
1.166
|
0.800
|
0.424
|
|
Absent
|
229
|
1.131
|
0.853
|
|
|
HCV
|
Present
|
51
|
1.079
|
0.678
|
0.642
|
0.521
|
|
Absent
|
276
|
1.173
|
0.999
|
|
|
Family history
|
Present
|
104
|
1.063
|
0.789
|
1.394
|
0.165
|
|
Absent
|
194
|
1.216
|
0.958
|
|
|
Histological grade
|
G1 + G2
|
211
|
1.159
|
0.892
|
0.387
|
0.699
|
|
G3 + G4
|
138
|
1.202
|
1.109
|
|
|
Albumin (g/dl)
|
< 4.0
|
128
|
1.055
|
0.958
|
0.478
|
0.633
|
|
≥ 4.0
|
153
|
1.105
|
0.799
|
|
|
Creatinine (mg/dl)
|
< 1.1
|
189
|
0.926
|
0.375
|
2.842
|
0.005
|
|
≥ 1.1
|
92
|
1.169
|
1.044
|
|
|
BMI (kg/cm2)
|
< 25
|
143
|
1.163
|
1.035
|
0.285
|
0.776
|
|
≥ 25
|
152
|
1.196
|
0.987
|
|
|
AFP (ng/ml)
|
≤ 200
|
187
|
0.977
|
0.635
|
2.806
|
0.006
|
|
> 200
|
73
|
1.408
|
1.251
|
|
|
ECOG
|
= 0
|
156
|
1.094
|
0.954
|
1.297
|
0.196
|
|
> 0
|
117
|
1.253
|
1.059
|
|
|
T stage
|
I + II
|
252
|
1.042
|
0.675
|
3.111
|
0.002
|
|
III + IV
|
88
|
1.551
|
1.483
|
|
|
TNM stage
|
I + II
|
238
|
1.025
|
0.659
|
3.329
|
0.001
|
|
III + IV
|
83
|
1.576
|
1.457
|
|
|
Note: Bold font represents P < 0.05 and the relevant variables are statistically significant
Abbreviations: HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; AFP, alpha-fetoprotein; BMI, body mass index; ECOG: Eastern Cooperative Oncology Group; TNM, tumor node metastasis; SD, standard deviation.
Prognostic value of the autophagy-related lncRNA prognostic signature in different subgroups
Subgroup analysis was done to investigate the prognostic value of the autophagy-related lncRNA prognosis signature among different subgroups stratified by clinicopathological variables. As shown in Table 2, the risk scores prognostic signature showed better performance in male patients without liver cirrhosis, the history of HBV or HCV infection as well as family history, whereas obese patients with poor physical condition could benefit more in prognosis based on the autophagy-related lncRNA prognostic signature. Considering laboratory index, the prognostic signature seemed more applicable to patients with relatively lower expression levels of serum AFP, albumin, and creatinine. Besides, the prognostic signature showed strong predictive power independent of clinicopathological features including gender, age, the history of alcohol consumption, tumor stage and histological grade.
Table 2
Prognostic value of the autophagy-related lncRNA prognostic signature in different subgroups stratified by clinicopathological variables
Characteristics
|
Group
|
Low /High
|
HR (95% CI)
|
P value
|
Overall
|
|
172/171
|
2.249 (1.559–3.256)
|
< 0.001
|
Age
|
< 60
|
76/81
|
2.832 (1.586–5.056)
|
< 0.001
|
|
≥ 60
|
95/91
|
1.754 (1.079–2.853)
|
0.023
|
Gender
|
Male
|
124/109
|
3.125 (1.925–5.072)
|
< 0.001
|
|
Female
|
47/63
|
1.290(0.7285–2.285)
|
0.383
|
Alcohol consumption
|
Present
|
60/49
|
3.718 (1.921–7.194)
|
< 0.001
|
|
Absent
|
106/112
|
1.794 (1.136–2.833)
|
0.012
|
Liver cirrhosis
|
Present
|
26/39
|
2.108 (1.010–4.396)
|
0.047
|
|
Absent
|
70/67
|
1.922 (0.962–3.836)
|
0.064
|
HBV
|
Present
|
48/20
|
1.650 (0.720–3.780)
|
0.237
|
|
Absent
|
118/111
|
2.513 (1.653–3.820)
|
< 0.001
|
HCV
|
Present
|
28/23
|
3.242 (0.805–13.057)
|
0.098
|
|
Absent
|
138/138
|
2.206 (1.496–3.251)
|
< 0.001
|
Family history
|
Present
|
63/41
|
1.828 (1.019–3.277)
|
0.043
|
|
Absent
|
83/111
|
2.944 (1.688–5.133)
|
< 0.001
|
Histological grade
|
G1 + G2
|
102/109
|
2.154 (1.338–3.466)
|
0.002
|
|
G3 + G4
|
66/61
|
2.549 (1.411–4.607)
|
0.002
|
Albumin(g/dl)
|
< 4.0
|
76/52
|
1.908 (1.040–3.501)
|
0.037
|
|
≥ 4.0
|
75/78
|
1.644 (1.893–3.025)
|
0.110
|
Creatinine(mg/dl)
|
< 1.1
|
98/91
|
1.721 (1.034–2.862)
|
0.037
|
|
≥ 1.1
|
53/39
|
1.736 (0.825–3.656)
|
0.147
|
BMI (kg/cm2)
|
< 25
|
70/73
|
1.670 (0.917–3.040)
|
0.093
|
|
≥ 25
|
77/75
|
3.805 (2.105–6.787)
|
< 0.001
|
AFP (ng/ml)
|
≤ 200
|
116/171
|
1.985 (1.156–3.408)
|
0.013
|
|
> 200
|
21/52
|
2.041 (0.757–5.506)
|
0.159
|
ECOG
|
= 0
|
91/65
|
1.878 (0.924–3.817)
|
0.081
|
|
> 0
|
53/64
|
3.016 (1.679–5.416)
|
< 0.001
|
T stage
|
I + II
|
140/112
|
2.064 (1.280–3.329)
|
0.003
|
|
III + IV
|
29/59
|
1.926 (1.054–3.520)
|
0.033
|
TNM stage
|
I + II
|
135/103
|
1.915 (1.155–3.174)
|
0.012
|
|
III + IV
|
26/57
|
2.025 (1.046–3.921)
|
0.036
|
Note: Bold font represents P < 0.050 and the relevant variables are statistically significant
Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; AFP, alpha-fetoprotein; BMI, body mass index; ECOG: Eastern Cooperative Oncology Group; TNM, tumor node metastasis; HR, hazard ratio.
Validation of the autophagy-related lncRNA prognostic signature in the ICGC database
Next, we further assessed the predictive power of the autophagy-related lncRNA prognosis signature in the ICGA database. A total of 230 HCC patients were enrolled. All the patients were separated into low and high-risk groups based on the median value of the risk score. Consistent with the results derived from the TCGA database, KM survival curve analysis demonstrated that the OS of HCC patients in the low-risk group was significantly higher than that of the patients in the high-risk group (Fig. 3A). The expression of each autophagy-related lncRNA showed that those lncRNAs were down-regulated in the low-risk group and up-regulated in the high-risk group (Fig. 3B). Time-dependent ROC curve analysis further demonstrated that the AUC value for the autophagy-related lncRNA prognostic signature was 0.685 in ICGC database, which further confirmed the robust prediction of the signature among HCC patients (Fig. 3C). The distribution of risk score and survival status was shown in Fig. 3D-E. Those results further confirmed that the signature could accurately predicted the prognosis of HCC patients.
Determination of the autophagy-related lncRNA prognostic signature as an independent prognostic factor
Subsequently, univariate, and multivariate Cox regression analyses were performed to assess whether the prognostic significance was a prognostic factor independent of clinicopathological features in the TCGA and ICGC databases. As shown in Fig. 4A, Univariate analysis indicated that ECOG [HR: 2.390; 95% CI: 1.894–3.016; P < 0.001], TNM stage [HR: 1.784; 95% CI: 1.446–2.202; P < 0.001], HBV [HR: 1.587; 95% CI: 1.006–2.504; P = 0.047], HCV [HR:2.446; 95% CI: 1.239–4.828; P = 0.010], liver cirrhosis [HR: 2.426; 95% CI: 1.516–3881.; P < 0.001], and autophagy-related lncRNAs prognostic riskscore [HR: 1.539; 95% CI: 1.339–1.769; P < 0.001]were significantly prognostic factors in HCC patients cohort from the TCGA database. To avoid the occurrence of collinearity of TNM stage with T stage, T stage was not enrolled into multivariate Cox regression modeling because TNM stage was calculated based on T stage, N stage, and M stage. Multivariate analysis further screened out ECOG [HR: 1.589; 95% CI: 1.101–2.294; P = 0.013] and autophagy-related lncRNAs prognostic riskscore [HR: 1.376; 95% CI: 1.114–1.699; P = 0.003] as independent prognostic parameters (Fig. 4B). Besides, based on univariate and multivariate analyses, autophagy-related lncRNAs prognostic riskscore was also proven to be an independent prognostic factor in the ICGC validation cohorts (Fig. 4C-D).
Constructing and Evaluating a nomogram to predict OS in patients with HCC
We constructed a predictive nomogram to accurately predict the 1-, 3-, and 5-year OS rate by using riskscore calculated form the autophagy-related lncRNA prognostic signature and clinicopathological parameters including age, gender and AJCC stage in the TCGA cohort (Fig. 5A). Calibration plots further identified that the nomogram performed well in predicted 1-, 3-, and 5-year survival probabilities with an ideal model, which indicated that the nomogram was perfectly calibrated to predict OS at assessing the performance characteristics (Fig. 5B-D). The C-index value for the nomogram was 0.734. Therefore, internal validation demonstrated that the nomogram was accurate and reliable.
Subsequently, external validation was investigated in the ICGC external validation cohort. The C-index of the nomogram was 0.701 in validation cohort. The calibration plots showed excellent agreement between the actual observation and the nomogram prediction in terms of the 1- ,3-, and 5-year survival probabilities (Fig. 5E–6G). Hence, external validation from ICGC testing cohort further confirmed the robustly predictive power of the nomogram.
Construction of the prognostic autophagy-associated lncRNA–mRNA co-expression network and functional enrichment analysis
To figure out the potential functions of the four-prognostic autophagy-associated-lncRNAs in HCC, we analyzed the lncRNAs mediated mRNA network by using cytoscape 3.6.1. As showed in Fig. 6A, 4 lncRNAs, 91 mRNAs and 141 lncRNA-mRNA pairs were shown in lncRNA-mediated genes network. Sankey plot also showed the detail relationships of prognostic autophagy-associated lncRNAs with autophagy-related-genes and risk types (Fig. 6B). Then, GO and KEGG pathway analyses further demonstrated that these genes were main correlated with autophagy and enriched in pathways in cancer (Fig. 6C-D).
Gene set enrichment analysis
Further functional annotation was performed by GSEA. GSEA results showed the altered gene sets in the high-risk group were mainly found to be directly involved in the tumorigenesis and progression of malignant tumor (Fig. 7A). Besides, the differentially expressed genes between the two groups were main enriched in the autophagy-associated and tumor-related pathways including ERBB signaling pathway, MAPK signaling pathway, mTOR signaling pathway, VEGF signaling pathway, WNT signaling pathway, and P53 signaling pathway (Fig. 7B). In addition, the altered gene sets in the high-risk group were also found to be involved in the physical actions of autophagy (Fig. 7C). Whereas, the protective pathways involved in metabolism were significantly enriched in the low-risk group (Fig. 7D).