الفهرس 
HEPATOCELLULAR CARCINOMAOnly 14 pages are availabe for public view
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Abstract
H
epatocellular carcinoma (HCC) is the most common type of primary liver cancers with cirrhosis being the major risk factor for its development, it occurs usually due to HBV and HCV infection. Egypt has the highest prevalence of HCV worldwide and thus has rising rates of HCC representing a major health problem in Egypt, which highlights the urgent need for novel biomarkers for early diagnosis of HCC. HCC diagnosis depends on imaging techniques, serological tumor markers analysis and histopathological confirmation. Imaging techniques include US, CT& MRI however, their sensitivities for detection of small HCC nodules might be low while the histopathological confirmation is an invasive technique and may cause tumor seeding.
As regards serological tumor markers, currently the most widely used diagnostic marker is serum AFP, which has many limitations as it is not significantly elevated in many HCC patients particularly those in early stages which is potentially curable. Moreover, AFP could be elevated in cirrhotic patients or during exacerbations of chronic viral hepatitis without HCC. Due to these limitations, AFP has been excluded from HCC surveillance or diagnosis by the American Association for the Study of Liver Diseases guidelines (AASLD). So there is urgent need for new biomarkers that would be simple, minimally-invasive and reliable for the early diagnosis of HCC.
Due to previous stated information, we used an integrative approach based on bioinformatics analysis together with clinical validation to provide great insights into the molecular mechanisms of HCC. To the best of our knowledge it is the first time to assess a competing endogenous network of Lipid Raft Linker 1 gene (RFTN1), which was selected according to public microarray databases, because this gene plays a major role in HCC and T cell signaling. To confirm the expression of RFTN1 gene (RFTN1 mRNA) in HCC cases; we searched Gene Atlas database and protein Atlas database. Next, Lnc-RNA- RP11-156p1.3, was identified, using a database of lncRNAs that act as ceRNAs (the lnCeDB database). This lncRNA acts as master regulator of the RFTN1 by competing for miR-4764-5p_1 binding RFTN1 gene.
This study was done at Medical Biochemistry Department, Faculty of Medicine, Ain Shams University during the period from July 2017 – May 2019 and included 49 HCC patients 18 CHC patients and 36 normal volunteers.
The aim of the current study was to evaluate the clinical utility of serum levels of RFTN1mRNA, miR-4764-5p_1 and Lnc-RNA- RP11-156p1.3 expression as non-invasive biomarkers in diagnosis of HCC by quantitative Real Time -PCR and to correlate the expression of these markers to the various clinico-pathological parameters of the patients. This was done in an attempt to evaluate their role in HCC assessment and their use as potential selected diagnostic biomarkers. Also to characterize the efficacy of CRISPR cas9 mediated knock out of the Lnc-RNA- RP11-156p1.3 in HepG2 cell line.
The study included 103 subjects classified into three main groups:
group 1: 49 Hepatocellular carcinoma (HCC) cases with median age 58 years.
group 2: 18 chronic hepatitis C (CHC) cases with median age 58 years.
group 3: 36 Healthy normal individuals with median age 55 years.
In this study, all participants were subjected to full clinical and radiological examination preceded by complete history taking and routine laboratory investigations including liver function tests as serum ALT,AST, serum albumin, serum total bilirubin, serum AFP and INR. Serum samples were obtained from all subjects and also liver tissue samples were taken from 20 patients with HCC along with tissue from the safety margins.
Evaluation of RFTN1-mRNA, Lnc-RNA- RP11-156p1.3 in sera and tissue samples was performed by real time PCR in relation to GAP-dh as the reference gene in all samples. The level of miR-4764-5p_1 was estimated also by real time PCR in relation to SNORD68 as an internal control in all samples. Then the results were statistically analyzed by the SPSS software.
A significant difference was observed in the positivity rates for serum Lnc-RNA RP11-156p1.3, serum miRNA-4764-5p1, and serum RFTN1 mRNA, which were 81.6%, 75.5%, and 73.5%, respectively, in the malignant group. While among the CHC patients, the positivity rates of serum Lnc-RNA RP11-156p1.3, serum miRNA-4764-5p1, and serum RFTN1 mRNA were 33.3%, 33.3% and 33.3%, respectively. However, among the normal control, the positivity rates of serum Lnc-RNA RP11-156p1.3, serum miRNA-4764-5p1, and serum RFTN1 mRNA were 8.3%, 0% and 8.3%, respectively (p<0.01).
Using ROC curve analysis, (≥1.52, ≤ 0.03727 and ≥1.31.) were set as cut off values for human serum RFTN1 mRNA, miRNA-4764-5p1 and Lnc-RNA RP11-156p1.3 respectively to discriminate malignant from non-malignant cases. They showed sensitivity of (73.3%, 75.3% and 81.6% respectively), specificity of (83.3%, 88.9% and 83.3% respectively), PPV of (80%, 86 % and 81.6% respectively), NPV of (77.6%, 80% and 83.3% respectively) and accuracy of (78.6%, 82.5% and 82.5% respectively).
There was a highly significant inverse correlation between serum miRNA-4764-5p1 and serum RFTN1 mRNA, highly significant positive correlation between serum Lnc-RNA RP11-156p1.3 and serum RFTN1 mRNA and highly significant inverse correlation between serum miRNA-4764-5p1and serum Lnc-RNA RP11-156p1.3 based on fold changes (RQs) among the three human study groups.
There was high significance difference in the expression of the 3 biomarkers between the hepatic cancer tissue and the control tissue.
There was positive correlation between the expression of each of the biomarkers in malignant tissue and its expression in the serum of patients with HCC, denoting that the source of the expressed RNAs in the serum is the liver malignant tissue.
Furthermore, the results of this study showed the effect of editing the Lnc-RNA- RP11-156p1.3 in HepG2 cell line on the cell viability, count and the expression of the studied RNAs.
There was significant decrease in cell viability among the group after CRISPR cas 9 knock out of Lnc-RNA- RP11-156p1.3 compared to the other group including cells before editing. While there was decrease in the cell count in the HepG2 cells edited with CRISPR cas9 compared with the negative control.
There was high significant difference in the expression of the 3 investigated genes between the HepG2 cells edited with CRISPR cas9 compared with the negative control. Lnc-RNA RP11-156p1.3 and RFTN1 mRNA were significantly lower (p=0.002 and p=0.004) respectively, in the HepG2 cells edited with CRISPR cas9 compared with the negative control. While miRNA -4764-5p_1 was significantly higher (p=0.001) in the HepG2 cells edited with CRISPR cas9 compared with the negative control.
On studying the protein level of TNFα and NFκβ, both proteins were significantly lower(p=0.02 and p=0.023) in the group with HepG2 cells after CRISPR cas9 knock out of Lnc-RNA RP11-156p1.3 than the group containing cells before editing.