Abstract
Background
Acid phosphatase type 6 (ACP6) is a mitochondrial lipid phosphate phosphatase that played a role in regulating lipid metabolism and there is still blank in the clinico-pathological significance and functional roles of ACP6 in human cancers. No investigations have been conducted on ACP6 in hepatocellular carcinoma (HCC) up to date.
Methods
Herein, we appraised the clinico-pathological significance of ACP6 in HCC via organizing expression profiles from globally multi-center microarrays and RNA-seq datasets. The molecular basis of ACP6 in HCC was explored through multidimensional analysis. We also carried out in vitro and in vivo experiment on nude mice to investigate the effect of knocking down ACP6 expression on biological functions of HCC cells, and to evaluate the expression variance of ACP6 in xenograft of HCC tissues before and after the treatment of NC.
Results
ACP6 displayed significant overexpression in HCC samples (standard mean difference (SMD) = 0.69, 95% confidence interval (CI) = 0.56–0.83) and up-regulated ACP6 performed well in screening HCC samples from non-cancer liver samples. ACP6 expression was also remarkably correlated with clinical progression and worse overall survival of HCC patients. There were close links between ACP6 expression and immune cells including B cells, CD8 + T cells and naive CD4 + T cells. Co-expressed genes of ACP6 mainly participated in pathways including cytokine-cytokine receptor interaction, glucocorticoid receptor pathway and NABA proteoglycans. The proliferation and migration rate of HCC cells transfected with ACP6 siRNA was significantly suppressed compared with those transfected with negative control siRNA. ACP6 expression was significantly inhibited by nitidine chloride (NC) in xenograft HCC tissues.
Conclusions
ACP6 expression may serve as novel clinical biomarker indicating the clinical development of HCC and ACP6 might be potential target of anti-cancer effect by NC in HCC.
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Background
According to the statistics of latest epidemic research, liver cancer poses a serous threat to human health with high incidence rate and mortality rate ranking sixth and fourth worldwide, respectively [1, 2]. An overwhelming majority of liver cancer cases were hepatocellular carcinoma (HCC), which has a high prevalence in multiple countries [3, 4]. The risk factors for HCC are complex and vary from area to area. While non-alcoholic fatty liver disease, hepatitis C virus infection and alcohol abuse were considered as the main risk elements for HCC in Japan and Western countries; the culprit behind HCC in eastern Asia and sub-Saharan Africa is hepatitis B virus (HBV) [5,6,7]. Although great achievements have been made for extending the life expectancy of HCC patients and a combination of therapies including surgical removal, radiotherapy, chemotherapy, radiofrequency ablation (RFA), and molecular targeted therapy have been applied for the treatment of HCC patients, most HCC patients were found to be in middle or later stage when diagnosed and the five-year survival rate of HCC patients remained at low level [8,9,10,11,24,25]. NC exerted significant suppressive effect on HCC xenograft tumor growth and caused notable decrease in tumor size of NC-treated group compared with control group [26,27,28,29,30,31,32,33,34]. Specifically, the contribution of angiogenesis, MAPK cascade and humoral immune response to the malignant process of HCC has been recorded in previous literature studies [35,36,37]. The serological testing and immunoblot assay by M Volkmann et al. revealed humoral immune response to p53 exclusively in HCC patients and the presence of p53 antibodies in HCC patients was not dependent on alpha-fetoprotein level [35]. The mitogen-activated protein kinase (MAPK) signaling pathways played essential roles in diverse biological events including survival, dissemination, and resistance to drug therapy of human tumor cells [38,39,40]. Wang et al. reported activation of MAPK signaling pathway during the promotion of HCC development stimulated by linc00601 upregulation [36]. HCC is rich in blood supply, and angiogenesis was indispensable for tumor growth, invasion and metastasis [41]. The work of Wang et al. disclosed that morphine could induce angiogenesis in HCC through activating PI3K/Akt/HIF-1α pathway and up-regulating VEGF expression [37]. Despite there have been no studies on the parts of ACP6 in activities of the above mentioned biological processes and pathways, the analysis results in this study provided potential presumptions of the molecular mechanism of ACP6 in HCC. Moreover, the in vitro experiments results in the present work supported the functional roles of ACP6 in proliferation and migration of HCC cells. The last high spot of this study was in vivo experiments on how NC affects the expression of ACP6 in HCC tissues. The pharmacologic mechanism of NC in tumor inhibition of HCC was far from been clarified and we conducted experiments on nude mice for further exploration. The significant suppression of NC on ACP6 in HCC tissues found in the present work might serve as a supplement to the explanations of pharmacologic actions of NC in fighting HCC.
Limitations of this paper were also existed. The protein expression of ACP6 in NC-treated xenograft and in Huh7 cells transfected with ACP6 siRNA should be examined by western blotting or immunohistochemistry. In vivo experiments are warranted in future work for validating the oncogenic roles of ACP6 in HCC and the molecular interactions between ACP6 and its co-expressed genes. The number of mice in the NC-treated was only two and control group was only three in the present study; both numbers of mice were too small for statistical analysis. The sufficient number of mice for subsequent statistical analysis should be eight to ten. More mice will be added in future in vivo experiments for guaranteeing the robustness of the in vivo experiment results.
Conclusions
In summary, we proved the overexpression of ACP6 in HCC and promotive effect of up-regulated ACP6 in the aggressive development of HCC. ACP6 was one of the potential targets of NC in HCC. The findings in the present study was anticipated to shed light on the molecular mechanism of HCC and lay a theoretical foundation for application prospect of ACP6 as a biomarker for HCC.
Availability of data and materials
The datasets supporting the conclusions of this article are available in TCGA (https://portal.gdc.cancer.gov/) (TCGA-LIHC), GEO (GSE31370, GSE36376, GSE36411, GSE39791, GSE57957, GSE76427, GSE114564, GSE148355, GSE63863, GSE65485, GSE73708, GSE81550, GSE87592, GSE104310, GSE63018, GSE77509, GSE94660, GSE97214, GSE140845, GSE112221, GSE101685, GSE102079, GSE107170, GSE112790, GSE121248, GSE17548, GSE19665, GSE29721, GSE33006, GSE41804, GSE45436, GSE6222, GSE62232, GSE6764, GSE99807, GSE84402, GSE14323, GSE14520, GSE17967, GSE9839, GSE57727, GSE98617, GSE113996, GSE74656, GSE101728, GSE98269, GSE12941, GSE84005, GSE45050, GSE64041, GSE117361, GSE54236, GSE87630, GSE89377, GSE25599, GSE77314, GSE67764, GSE60502, GSE124535, GSE22405, GSE25097, GSE33294, GSE46444, GSE63898, GSE50579, GSE56545, GSE57555, GSE54238, GSE76311, GSE20140, GSE115018, GSE125469, GSE166163, GSE46408, GSE22058, GSE55048, GSE59259, GSE114783) (https://www.ncbi.nlm.nih.gov/gds) and ArrayExpress (E-MTAB-8887, E-MTAB-4171) (https://www.ebi.ac.uk/arrayexpress/) databases. RNA-seq data of HCC xenograft tissues treated with or without nitidine chloride was uploaded to Mendeley data (https://data.mendeley.com/datasets/stkrjf7w7t/1). Raw data of in vitro experiments from the current study was available in FigShare (https://figshare.com/articles/dataset/Raw_data_of_in_vitro_experiments/20337021). Public access to all above databases is open.
Abbreviations
- ACP6:
-
Acid phosphatase type 6
- HCC:
-
Hepatocellular carcinoma
- SMD:
-
Standard mean difference
- AUC:
-
Area under curve
- NC:
-
Nitidine chloride
- HBV:
-
Hepatitis B virus
- FPKM:
-
Fragments per kilobase million
- TPM:
-
Transcripts per million
- TCGA:
-
The cancer genome atlas
- GTEx:
-
Genotype-tissue expression project
- GEO:
-
Gene expression omnibus
- ROC:
-
Receiver’s operating characteristics
- SROC:
-
Summarized receiver’s operating characteristics
- CI:
-
Confidence interval
- HR:
-
Hazard ratio
- tNSE:
-
T-distributed stochastic neighbor embedding
- MEGENA:
-
Multiscale embedded gene co-expression network analysis
- SD:
-
Standard deviation
- M:
-
Mean
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Acknowledgements
We sincerely thank for the technical support provided by Guangxi Key Laboratory of Medical Pathology.
Funding
This work was supported by Fund of National Natural Science Foundation of China (NSFC82160762), Innovation Project of Guangxi Graduate Education (YCSW2021121), Guangxi Medical High-level Key Talents Training "139" Program (2020), Guangxi Higher Education Undergraduate Teaching Reform Project (2020JGA146, 2021JGA142), Guangxi Educational Science Planning Key Project (2021B167), Guangxi Medical University Training Program for Distinguished Young Scholars (2017) and Medical Excellence Award Funded by the Creative Research Development Grant from the First Affiliated Hospital of Guangxi Medical University (2016). All funding sources had no involvement.
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Authors and Affiliations
Contributions
LG: data analysis and writing of original manuscript. DDX: conduction of in vivo experiments. XY: literature searches and providing predicted targets of nitidine chloride. JDL: collecting microarrays and RNA-seq datasets of HCC. RQH and ZGH: process of microarrays and RNA-seq datasets. ZFL: providing nitidine chloride and editing of manuscript. LML: improving our understanding of the anti-cancer effect of nitidine chloride on cancers. JYL: plotting figures. XFD: advice on method design for the manuscript. JHZ and MFL: fund raising for the manuscript. SHL: explaining the use of software. YWD: data check for the manuscript. Professor GC: design and revision of the manuscript. All authors have read and approved the manuscript.
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The in vivo experiments in the current study adhered to the ethical standards proposed by Guide for the Care and Use of Laboratory Animals (the Shanghai SLAC Laboratory Animal of China, 2015) and were approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University. Animal experiments were carried out under the guidelines of Ethics Committee of the First Affiliated Hospital of Guangxi Medical University. The study was carried out in compliance with the ARRIVE guidelines.
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Supplementary Information
Additional file 1:
Figure 1. Flowchart of the selection process of eligible RNA-seq datasets or microarrays for expression analysis.
Additional file 2:
Figure 2. The expression pattern of ACP6 in HCC and non-cancer liver samples. Differential expression of ACP6 between HCC (marked in red) and non-cancer liver samples (marked in blue) was displayed in a panel of violin plot. N: non-cancer liver samples. T: HCC samples.
Additional file 3:
Figure 1. Flowchart of the selection process of eligible RNA-seq datasets or microarrays for expression analysis.
Additional file 4:
Figure 4. The overall expression trend of ACP6 in HCC and its discriminatory capacity. A. Forest plot of SMD. SMD: standard mean difference; SD: standard deviation. B. SROC curves. AUC: area under curve. SENS: sensitivity; SPEC: specificity.
Additional file 5:
Figure 5. The associations between ACP6 expression and clinico-pathological variables of HCC patients. The violin plots showed ACP6 expression in HCC patients with different groups of adjacent hepatic tissue inflammation (A), history of hepatistis B (B), Ishak fibrosis scores (C) and histologic grades (D).
Additional file 6:
Figure 6. Prognostic value of ACP6 expression for HCC patients. Kaplan-Meier survival curves were created based on prognostic data of HCC patients in E-TABM-36 (A), GSE76427 (B) and TCGA database (C). The forest plot of HR value summarized the overall effect of ACP6 expression on overall survival of HCC patients (D). HR: hazard ratio.
Additional file 7:
Figure 7. Genetic alteration profile of ACP6 in HCC samples. HCC cases with genetic alterations of ACP6 were marked in different colors. GISTIC: genomic identification of significant targets in cancer.
Additional file 8:
Figure 8. Functional annotations for genes co-expressed with ACP6. A. Network of enriched biological process or pathway terms colored by ID. B. Network of enriched biological process or pathway terms colored by p value.
Additional file 10:
Supplementary Table 1. Detailed information of all included RNA-seq dataset and microarrays for the current work.
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Gao, L., **ong, DD., Yang, X. et al. The expression characteristics and clinical significance of ACP6, a potential target of nitidine chloride, in hepatocellular carcinoma. BMC Cancer 22, 1244 (2022). https://doi.org/10.1186/s12885-022-10292-1
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DOI: https://doi.org/10.1186/s12885-022-10292-1