According to the rule of fold change, HOTAIR, HOTTIP, and H19 gene expressions were consistently upregulated, whereas miRNA-152 was downregulated in the groups investigated (Table 2) (Fig. 1).
HOTAIR, HOTTIP, and H19 expression levels were considerably higher in all studied groups compared to controls (p values < 0.01, < 0.001, and < 0.01 respectively). When cirrhotic and HCC patients were compared to non-cirrhotic patients, HOTAIR, HOTTIP, and H19 expression levels were considerably higher (p values = 0.03, 0.04, and < 0.001 respectively) for cirrhotic patients and (p values < 0.001, < 0.001, and < 0.001 respectively) for HCC patients. It also revealed a substantial increase in HOTAIR, HOTTIP, and H19 expression in HCC patients when compared to cirrhotic patients (p values < 0.001, 0.013, and 0.005 respectively) (Table 2, Fig. 1a, b, c).
On the other hand, miRNA-152 expression, decreased considerably in all studied groups as compared to the control group (p value = 0.01). When cirrhotic and HCC patients were compared to non-cirrhotic patients, miRNA-152 expression levels were significantly lower in cirrhotic patients (p values = 0.007) and HCC patients (p values 0.001). It also found a significant decrease in miRNA-152 expression in HCC patients compared to cirrhotic individuals (p values 0.001). (Table 2, Fig. 1d).
The diagnostic performances of the HOTAIR, HOTTIP, H19, and miRNA-152 genes in the examined groups were demonstrated using Receiver Operating Curves (ROC).
It was found that serum H19 and miRNA-152 at cut-off values of (> 3.758 and 0.165 respectively), had sensitivity of (48.6% and 51.4%) and specificity of (93.3% and 70.0%) with (p values 0.0001 and 0.007) for discriminating the cirrhotic group from the non-cirrhotic group. While the HOTAIR and HOTTIP genes have no role in distinguishing between cirrhotic and non-cirrhotic patients (Table 3, Fig. 2a).
For discrimination of the HCC group from the cirrhotic group, it was found that serum HOTAIR, HOTTIP, H19 and miRNA-152 at cut-off values of (> 2.000, > 14.929, > 9.318 and < 0.055), had sensitivity of (100.0%, 39.5%, 47.4% and 65.7%) and specificity of (40.0%, 97.1%, 94.3% and 68.4%) with (p values < 0.0001, 0.007, 0.003 and 0.001) respectively (Table 3, Fig. 2b).
For discrimination of the HCC group from CLD patients, it was found that serum HOTAIR, HOTTIP, H19 and miRNA-152 at cut-off values of (> 2.000, > 17.268, > 8.574, and < 0.055, had sensitivity of (100.0%, 39.5%, 57.9%, and 68.4%) and specificity of (47.7%,98.5%,89.2%, and 70.8%,) with (p values < 0.0001, < 0.0001, < 0.0001and < 0.0001) respectively (Table 3, Fig. 2c).
Cirrhosis risk based on gene expression: H19 and miRNA-152 were deemed predictive and/or prognostic characteristics for cirrhotic progression in univariate logistic regression analysis. A one-degree rise in H19 and miRNA-152 enhanced the likelihood of having cirrhosis by factors of (1.36 and 2.32) with (p values = 0.02 and 0.01) respectively. The HOTAIR and HOTTIP genes play no function in distinguishing cirrhotic from non-cirrhotic patients.
HCC risk based on gene expression: HOTAIR, HOTTIP, H19, and miRNA-152 were studied as potential predictors and/or prognostic factors for HCC advancement. A one-degree rise in HOTAIR, HOTTIP, H19, or miRNA-152 increased the probabilities of having HCC by factors of (1.12, 1.08, 1.07, and 2.54) with p values of (0.01, 0.01, 0.03, and 0.001) respectively.
Concerning CLD in general (group I: non-cirrhotic and cirrhotic), there was a 1 degree rise in HOTAIR, HOTTIP, H19, and miRNA-152, which increased the likelihood of developing HCC by factors of (1.15, 1.10, 1.08, and 3.27) with p values of (0.001, 0.001, 0.02, and 0.001) respectively (Table 4).
Overall, the correlation analysis found that miRNA152 has a significant inverse correlation with HOTAIR, HOTTIP, and H19; (r = -0.259 and p value = 0.01), (r = -0.206 and p value = 0.05), and (r = -0.218 and p value = 0.03) respectively (Fig. 3).
Simultaneously, the HOTAIR gene has a direct correlation with the HOTTIP and H19 genes (r = 0.813, p value 0.001) and (r = 0.349, p value = 0.001) respectively. Furthermore, HOTTIP and H19 have a substantial direct link (r = 0.315, p value = 0.001).
Perspectives of bioinformatics analysis:
According to results of gene networks analysis using miRNet webserver database many cascade of regulatory interactions involving the HOTAIR, HOTTIP, and H19 as lncRNA, miRNA-152, and all possible functionally target miRNAs are shown in (Fig. 4) (https://www.mirnet.ca), which reviled the target miRNAs such as miRNA-19a and miRNA-106a, some of which are intimately connected with HOTAIR, HOTTIP, and H19, whose expression variations are involved in HCC formation. Furthermore, this might reveal novel links between them as well as prospective correlations with HCC disease.