New stem cell autophagy surrogate diagnostic biomarkers in early-stage hepatocellular carcinoma in Egypt: A pilot study

Abstract

Background: Stem cell autophagy disruption is responsible for the development of hepatocellular carcinoma (HCC). Many non-coding RNAs are linked to the activation and inhibition of certain genes. The SQSTM1 gene controls stem cell autophagy as shown in previous studies. The upregulation of SQSTM1 is associated with the inhibition of autophagy in cancerous stem cells in patients with HCC.

Aim: To determine whether serum microRNA, hsa-miR-519d, is linked to SQSTM1 gene and whether they could be used as diagnostic biomarkers for early-stage HCC.

Methods: In silico analysis was performed to determine the most correlated genes of autophagy with microRNAs. SQSTM1 and hsa-miR-519d were validated through this pilot clinical study. This study included 50 Egyptian participants, who were classified into three subgroups: Group 1 included 34 patients with early-stage HCC, Group 2 included 11 patients with chronic liver disease, and Group 3 (control) included 5 healthy subjects. All patients were subjected to full laboratory investigations, including viral markers and alpha-fetoprotein (AFP), abdominal ultrasound, and clinical assessment with the Child-Pugh score calculation. Besides, the patients with HCC underwent triphasic computed tomography with contrast to diagnose and determine the tumor site, size, and number. Quantitative real-time polymerase chain reaction was used to assess hsa-miR-519d-3p and SQSTM1 in the serum of all the study participants.

Results: Hsa-miR-519d-3p was significantly upregulated in patients with HCC compared with those with chronic liver disease and healthy subjects with an area under the curve (AUC) of 0.939, with cutoff value 8.34, sensitivity of 91.2%, and specificity of 81.8%. SQSTM1 was upregulated with an AUC of 0.995, with cutoff value 7.89, sensitivity of 97.1%, and specificity of 100%. AFP significantly increased in patients with HCC with an AUC of 0.794, with cutoff value 7.30 ng/mL, sensitivity of 76.5%, and specificity of 72.7%.

Conclusion: This study is the first to show a direct relation between SQSTM1 and hsa-miR-519d-3p; they are both upregulated in HCC. Thus, they could be used as surrogate diagnostic markers for stem cell autophagy disturbance in early-stage HCC.

Keywords: Autophagy; Hepatocellular carcinoma; SQSTM1; Stem cell; miR-519d; miRNA.

Figure 1

Bioinformatic search and validation of the newly diagnostic biomarkers. A: miR-519d-3p and SQSTM1 as a targeted mRNA according to miRDB (http://mirdb.org/cgi-bin/search.cgi?searchType=miRNA&full=mirbase&searchBox=MIMAT0002853); B: A network of 923 genes targeted by hsa-miR-519d-3p, along with focusing on SQSTM1 in the network (miRTargetLink Human) (https://ccb-web.cs.uni-saarland.de/mirtargetlink/network.php?type=miRNA&qval=hsa-miR-519d-3p); C: The expression of miR-519d in liver tissue and other tissues (https://www.genecards.org/); D: The tissue expression of SQSTM1 is low in hepatocytes of healthy liver tissue (www.proteinatlas.org); E: The expression of SQSTM1 in cancers and liver cancer specifically (www.proteinatlas.org).

Figure 2

Box-plot figures showing the mean delta–delta threshold cycle in the new diagnostic biomarkers in different groups. A: Illustration of the mean delta–delta threshold cycle (DDCT) of the quantitative real-time polymerase chain reaction (qRT-PCR) results for hsa-miR-519d in the serum of the hepatocellular carcinoma (HCC), chronic liver infection, and control groups using error bars: ± 1 [mean ± standard deviation (SD)]; B: Illustration of the mean DDCT of the qRT-PCR results for mRNA of SQSTM1 in the serum of the HCC, chronic liver infection, and control groups using error bars: ± 1 (mean ± SD). DDCT: Delta–delta threshold cycle; HCC: Hepatocellular carcinoma.

Figure 3

Receiver operating characteristic curves of the new diagnostic biomarkers studied to differentiate between hepatocellular carcinoma and chronic liver infection groups. A: Receiver operating characteristic (ROC) curve for assessing the validity of the RQ results of quantitative real-time polymerase chain reaction (qRT-PCR) for hsa-miR-519d in the serum to differentiate the hepatocellular carcinoma and chronic liver infection groups; B: ROC curve assessing the validity of the RQ results of qRT-PCR for mRNA of SQSTM1 in the serum between hepatocellular carcinoma and chronic liver infection groups. ROC: Receiver operating characteristic.

Figure 4

Receiver operating characteristic curves of the new diagnostic biomarkers studied to differentiate between the malignant and non-malignant groups. A: Receiver operating characteristic (ROC) curve assessing the validity of the RQ results of quantitative real-time polymerase chain reaction (qRT-PCR) for hsa-miR-519d in the serum among the malignant and non-malignant groups; B: ROC curve assessing the validity of the RQ results of qRT-PCR for mRNA of SQSTM1 in the serum among the malignant and non-malignant groups. ROC: Receiver operating characteristic.

Figure 5

Receiver operating characteristic curves of the alpha-fetoprotein studied to differentiate between the hepatocellular carcinoma and chronic liver disease groups/malignant and non-malignant groups. A: Receiver operating characteristic (ROC) curve to assess the validity of alpha-fetoprotein (AFP) for the differentiation between the hepatocellular carcinoma and chronic liver disease groups; B: ROC curve assessing the validity of AFP for differentiating between the malignant and non-malignant groups. ROC: Receiver operating characteristic.