JBUON 2020; 25(6): 2570-2575 ISSN: 1107-0625, online ISSN: 2241-6293 • www.jbuon.com Email: [email protected] ORIGINAL ARTICLE Correlations of HACE1 expression with pathological stages, CT features and prognosis of hepatocellular carcinoma patients Zhiqiang Zhang1, Minmin Teng2, Zhiying Xu3, Dewei Wang4 1Radiology, Zibo Central Hospital, Zibo City, Shandong 255036, China. 2Image Center, Second People’s Hospital of Dezhou City, Dezhou City, Shandong 253000, China. 3Hematology-Oncology, Linyi County People’s Hospital, Dezhou City, Shandong 251500, China. 4Medical Imaging, People’s Hospital of Pingyi County, Linyi City, Shandong 273300, China. Summary Purpose: To explore the correlations of HECT domain and analyzing the clinical indicators that the expression level of ankyrin repeat-containing E3 ubiquitin protein ligase 1 HACE1 was considerably correlated with the tumor diam- (HACE1) expression with the pathological stages, computed eter, tumor-node-metastasis (TNM) stages and pathological tomography (CT) features and prognosis of patients with grades (p<0.05). The survival analysis revealed that the OS of hepatocellular carcinoma (HCC). patients in Low HACE1 group was shorter than that in High HACE1 group (median OS: 12.40 months vs. 15.16 months, Methods: The clinical data were randomly collected from p=0.031). Besides, as indicated by CT examination, the ex- 70 patients with primary HCC. The messenger RNA (mRNA) pression of HACE1 was not correlated with the number of and HACE1 in the cancer and paracancer tissues were deter- tumors (p>0.05), but notably associated with the size, capsule mined via real-time quantitative-polymerase chain reaction and necrosis of tumors (p<0.05). (qRT-PCR). The curve of the relationship between HACE1 expression and patients’ overall survival (OS) was plotted Conclusions: HCC tissues are significantly deficient in using the Kaplan-Meier method. Finally, the CT imaging HACE1, and the combination of HACE1 and CT images may data of patients were pooled to analyze the relationships of serve as an efficacious strategy for the clinical diagnosis and HACE1 expression with CT signs. monitoring of HCC. Results: Compared with those in the paracancer tissues, the mRNA and protein expression levels of HACE1 declined Key words: HACE1, hepatocellular carcinoma, CT, prog- significantly in HCC tissues (p<0.05). It was found through nosis Introduction Hepatocellular carcinoma (HCC) is one of the suitable treatment options for patients. However, at most common malignancies worldwide and ranks diagnosis HCC has progressed to advanced stage and second only to lung cancer among all types of tu- cannot be surgically resected in most patients [4]. mors for its morbidity rate [1,2]. There were about Considering the poor prognosis of liver cancer, novel 782,500 new cases and 745,500 deaths of liver can- treatment strategies and targets are needed, but the cer around the world in 2012 [2]. The total cases major pathogenic genes and molecular mechanism and deaths of HCC in China where liver cancer is of HCC have not yet been elucidated by studies. Ad- prevalent account for 50% of those worldwide [3]. ditionally, the incidence rate of HCC is increasing Surgery and liver transplantation remain the most with the aging of population and environmental pol- Corresponding author: Dewei Wang, BM. Medical Imaging, People’s Hospital of Pingyi County, No. 7 Jinhua Rd, Pingyi, Linyi City, Shandong 273300, China Tel: +86 15020920920, Email: [email protected] Received: 10/10/2020; Accepted: 02/11/2020 This work by JBUON is licensed under a Creative Commons Attribution 4.0 International License. HACE1 in hepatocellular carcinoma 2571 lution [5]. Therefore, searching the pathogenic genes Determination of HACE1 messenger ribonucleic acid (mRNA) and molecular mechanism of HCC is crucial for the level via qRT-PCR prevention and treatment of this disease. Following tissue homogenization, total RNAs were According to a study, HECT domain and ankyrin isolated from the tissues using TRIzol reagent (TaKaRa, repeat-containing E3 ubiquitin protein ligase 1 Tokyo, Japan). Then, first-strand complementary deoxy- (HACE1) gene, located on human chromosome 6q21, ribose nucleic acids (cDNAs) were synthesized using the is a frequently mutated locus in malignant tumors reverse transcription system kit (GeneCopoeia, Guang- [6]. HACE1 was firstly reported to be closely associ- zhou, China), and subjected to qRT-PCR using the stand- ard SYBR PCR kit in StepOnePlus system (Applied Bio- ated with the development of Wilms tumor [7]. Lat- systems, Foster City, CA, USA). HACE1 sense sequence: er, some studies have demonstrated that HACE1, an 5’-TCTTACAGTTTGTTACGGGCAGTT-3’, antisense se- important tumor suppressor gene, mediates cell au- quence: 5’-CAATCCACTTCCACCCATGAT-3’. GAPDH tophagy, and ubiquitination of RAC1 to play a vital sense sequence: 5’-GGGAGCCAAAAGGGTCAT-3’, anti- role in suppressing tumors [8,9]. Moreover, HACE1 sense sequence: 5’-GAGTCCTTCCACGATACCAA-3’. This is implicated in multiple biological processes, such experiment was performed in triplicate and the fold as cardioprotection, anti-oxidative stress and cell change of gene expression was calculated using 2−ΔΔCt. dynamics [10]. A recent study uncovered that low Measurement of HACE1 protein level via IHC expression or mutation of HACE1 is associated with various human malignancies, including breast can- The paraffin-embedded tissues were sliced into 4 cer, colorectal cancer and lymphoma [11]. However, µm-thick sections, baked in an oven at 60°C for 30 min, de-paraffinized in xylene and hydrated. Then, the sections there have been few data on the biological role and were permeabilized by 5% Triton for 30 min and added clinical significance of HACE1 in HCC. with citric acid buffer (pH=6.0), followed by 15 min of Therefore, in the present study 70 HCC patients microwave antigen retrieval. Subsequently, endogenous were randomly recruited and the differences in peroxidases were blocked using 3% H2O2-containing the transcriptome and protein expression levels of deionized water. The resulting sections were sealed by HACE1 between the cancer and paracancer tissues 5-10% normal goat serum diluted by phosphate buffered were detected using real-time quantitative poly- saline (PBS) and incubated at room temperature for 10 merase chain reaction (qRT-PCR) and immunohis- min, and then with 100 µL of HACE1 primary antibody tochemistry (IHC), respectively. Besides, the corre- (1:200, product No.: ab133637, Abcam, Cambridge, MA, USA) at 4°C overnight, with the PBS as the negative con- lations of HACE1 with the clinical indicators of HCC trol of the primary antibody. On the next day, the slides and its role in the prognosis of HCC patients were were rinsed using PBS, added dropwise with horserad- evaluated. Importantly, this study firstly combined ish peroxidase-labeled secondary antibody, incubated at HACE1 expression with the computed tomography room temperature for 30 min and stained by the GTVi- (CT) signs of patients to comprehensively explore sion anti-mouse/rabbit antibody complex method and GT- the role of HACE1 in HCC. Vison detection system kit (Gene Tech Co., Ltd., Shanghai, China). The presence of yellow particles in cells indicated positive expression. Finally, images of each section were Methods randomly captured in 10 randomly selected different fields of view under a microscope, and the positive cells Clinical data of samples were counted, followed by calculation of the positive rate. The clinical data and the paired specimens of tumor Semi-quantitative scoring was performed for posi- tissues and adjacent non-tumor tissues (5 cm away from tive cells whose cytoplasm was stained brown or tan from the tumors) were randomly collected from 70 patients the following two aspects: (1) Degree of staining: 0 points with primary HCC, who had undergone conventional he- for no stain, 1 point for slightly staining, 2 points for patectomy in our hospital from May 2011 to July 2019. moderately staining and 3 points for deeply staining. (2) Fresh tissues were divided into 2 portions: one was Percentage of stained cells: 0 points for no stained cells, 1 cryopreserved in liquid nitrogen and the other was im- point for <25% stained cells, 2 points for 25-50% stained mediately fixed in 4% paraformaldehyde overnight and cells and 3 points for >50% stained cells. The total score embedded in paraffin. Among these patients, there were was obtained by adding the points from these two aspects 46 males and 24 females, aged 30-78 years old, with me- (0-6 points). 0-2 points represented negative expression, dian age of 61 years old. The histology of tumor tissues while over 2 points indicated positive expression. was evaluated independently by two pathologists. The CT examination clinicopathological data, including age, gender, levels of serum AFP and ALT, tumor size and number, vascular Double spiral CT scanning was performed using the invasion, HBsAg, tumor differentiation and cirrhosis and CT-Twin scanner (Elscint, Israel) at the reconstruction CT imaging data were collected from all the patients. matrix of 512×512, the conventional slice thickness of 10 This study was approved by the Ethics Committee of mm, and an increase of 5 mm for thin slice scanning in Zibo Central Hospital. Signed informed consents were affected zones, and contrast-enhanced scans were adopt- obtained from all participants before the study entry. ed for the affected zones in some patients. In the present JBUON 2020; 25(6): 2571 2572 HACE1