DOI: https://doi.org/10.21203/rs.3.rs-2115229/v1
Hepatocellular carcinoma (HCC) is a common malignant tumor. The application of sorafenib has brought good results to the treatment of HCC, but the drug resistance of sorafenib cannot be ignored. Celecoxib can enhance the efficacy of sorafenib, but its mechanism is still unclear. The main purpose of this study is to study the efficacy and related mechanism of celecoxib and sorafenib in the treatment of hepatocellular carcinoma.
The GSE45340 data set was retrieved from the Gene Expression Database (GEO), and the differentially expressed genes were obtained by GEO2R. Then, the differentially expressed genes were screened, analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and then analyzed by Protein-Protein Interaction (PPI) network to obtain the hub genes, which were verified in TCGA database.
Through the analysis of GEO2r, we got 2181 differentially expressed genes. We selected 50 of the most diverse genes for go and KEGG enrichment analysis, and obtained their main enrichment pathways. The protein-protein interaction network of 50 genes was further obtained. Thus, the relevant key genes were obtained, and twelve genes were screened. Twelve genes (MCM4, POLA1, MCM6, MCM3, RBBP4, DNA2, AP2B1, KIF11, KIF23, TUBA1B, KIF14, NUDT21) significantly related to the prognosis of HCC and the molecular pathways involved in these genes were screened, which explained how celecoxib enhanced the efficacy of sorafenib. Twelve genes were further enriched and analyzed, and their possible mechanism of action was obtained.
celecoxib combined with sorafenib can enhance the regulation of hepatocellular carcinoma gene and reduce the drug resistance to sorafenib, which is of great significance for the treatment of hepatocellular carcinoma.
Hepatocellular carcinoma (HCC) is a kind of primary cancer. It ranks fifth among the common cancers in the world and third among the fatal cancers1. The incidence rate of liver cancer is still rising, though the relevant treatment methods are constantly updated2. The anticancer effect of kinase inhibitors (such as sorafenib) in preclinical research shows that it has a good prospect, and has been widely used in clinical hepatocyte therapy. These inhibitors mainly work by blocking the mitogen activated protein kinase / extracellular signal regulated kinase (ERK) pathway. This pathway is up-regulated in different types of cancer during tumor progression, which is closely related to the occurrence and development of liver cancer. However, many patients who initially respond to the treatment of targeted ERK pathway will develop drug resistance later, resulting in poor treatment effect3–5.
Celecoxib has a variety of pharmacological activities and functions, among which the most surprising is that celecoxib can be involved in the treatment of cancer6. Cancer microenvironment is often accompanied by high expression of various molecules, such as inflammatory mediators such as arachidonic acid and cytokines7. In cancer cells, cyclooxygenase-2 pathway is up-regulated, resulting in an excessive increase in prostaglandin production. This leads to the proliferation of tumor cells. Many cancer treatments have been found to inhibit cyclooxygenase-2. Inhibition of cyclooxygenase-2-mediated prostaglandin excess leads to abnormal cell growth control. Cyclooxygenase-2 inhibitors act by inhibiting the overexpression of prostaglandins and act as chemopreventive agents. In animal experiments of colorectal cancer8, lung cancer9 and breast cancer10, it is found that blocking COX-2 pathway can effectively prevent tumor growth and metastasis.
Studies have shown that cyclooxygenase-2 (COX-2) plays a role in the occurrence and development of hepatocellular carcinoma, and selective COX-2 inhibitors (COX-2 inhibitors) have obvious anti proliferative and pro apoptotic effects in human HCC cell lines11–13. There are also relevant studies showing that celecoxib can enhance the efficacy of sorafenib14, but its specific mechanism is not clear. This study aims to analyze the effect of celecoxib combined with sorafenib on gene expression of hepatoma cells by bioinformatics, and then speculate the possible mechanism and pathway related to its effect.
Obtaining data from GEO database and screening differential genes
The data set of related studies was retrieved from geo database (https://www.ncbi.nlm.nih.gov/), and GSE45340 data set was downloaded. The platform used was Agilent-014850 whole human genome microarray 4x44k g4112f (feature number version). The data set contained four samples (GSM1102674: HepG2 cells_ SOR + CELE treated_ 48h vs untreated HepG2_ rep1, GSM1102675: HepG2 cells_ SOR + CELE treated_ 48h vs untreated HepG2_ rep2 (dye swap), GSM1102676: Huh7 cells_ SOR + CELE treated_ 48h vs untreated HepG2 cells_ rep1, GSM1102677: Huh7 cells_ SOR + CELE treated_ 48h vs untreated HepG2 cells_ Rep2 (dye swap)) and then use GEO2R to screen the differentially expressed genes(DEGs), and then further visualize and statistically analyze the DEGs.
Functional Annotation and Protein–Protein Interaction Network of the DEGs
Metascape15 (http://metascape.org/gp/index.html#/main/step1) is a simple and powerful tool for gene function annotation analysis, which can help users apply the current popular bioinformatics analysis methods to batch gene and protein analysis, so as to realize the cognition of gene or protein function. Metascape not only includes the enrichment analysis of biological pathways, the analysis of protein-protein interaction network structure and abundant gene annotation functions, but also presents the results in a high-quality graphic language that biologists can easily understand. So as to screen out the hub genes and related enrichment pathways.
Analysis and screening of key genes with UALCAN
Uarcan16 is a comprehensive, user-friendly, interactive web resource for analyzing cancer data. It is built on Perl CGI, with high-quality graphics using JavaScript and CSS. Uarcan design,) provides convenient access to open cancer histochemical data (TCGA, met500 and cptac), b) allows users to identify biomarkers or perform potentially interested genes verified in silicon, c) provide graphic and plot descriptions as expression profiles of encoded proteins and patient survival information, miRNA coding and lincrna coding genes, d) epigenetic regulation of gene expression evaluation by promoter methylation, e) For the analysis of Pan oncogene expression, f) provide more information about the selected gene / target by connecting HPRD, genecards, PubMed, targetscan, the human protein atlas, drugbank, open targets and GTEX. These resources enable researchers to collect valuable information and data on genes / objectives of interest.
Acquisition of DEGs
Through the analysis of GEO2r, we got the differentially expressed genes, and drew the volcano map (Fig. 1A). Further screening in the analysis results of GEO2R (logFc > 2 or logFc <-2), we got 2181 differentially expressed genes. The 50 genes with the highest expression multiple were selected and the expression heat map (Fig. 1B) was drawn.
Analysis of DEGs in metascape
Metascape is a software for enrichment analysis. We uploaded the differential genes obtained in the above steps to this software for analysis, and obtained the enrichment analysis results of KEGG and GO (Fig. 2A). Network is a very important means of expression. The results of enrichment analysis can be further analyzed to obtain the correlation between them (Fig. 2B).
Metascape is a powerful software that can visually analyze various data. One of the important functions is PPI analysis. After analysis, we get relevant results (Fig. 3A). The MCODE networks identified for individual gene lists have been gathered and are shown in Fig. 3B. 44 hub genes were screened out.
Analysis of hub gene in UALCAN
The 44 genes were analyzed in UALCAN one by one, and the analysis showed that 12 genes were differentially expressed in HCC (MCM4(Fig. 4A), POLA1(Fig. 4B), MCM6(Fig. 4C), MCM3(Fig. 4D), RBBP4(Fig. 4E), DNA2(Fig. 4F), AP2B1(Fig. 4G), KIF11(Fig. 4H), KIF23(Fig. 4I), TUBA1B(Fig. 4J), KIF14(Fig. 4K), NUDT21(Fig. 4L)), and their expression was closely related to the overall survival rate of HCC patients. The 12 genes were further enriched (Fig. 5B) and the expression heat map (Fig. 5A) was drawn. It is mainly enriched in E2F pathway and DNA replication pathway.
Sorafenib, lenvatinib and atezolizumab bevacizumab are FDA approved first-line drugs for the treatment of advanced liver cancer. At present, atezolizumab bevacizumab is the first line, sorafenib and lenvatinib are the second line. It is a multi-target kinase inhibitor of Raf kinase, vascular endothelial growth factor and platelet-derived growth factor receptor. Compared with the placebo group, sorafenib improves the survival time, and the median OS rate is 6.5 months17. However, patients with advanced HCC are mainly resistant to sorafenib, and their survival benefits are limited to 3–5 months, accompanied by serious side effects, which limit the clinical efficacy of sorafenib and greatly reduce the therapeutic effect of patients with advanced liver cancer18.Therefore, we need to find more effective methods. Celecoxib is a surprise, but its specific role in tumor needs further study, especially in hepatocellular carcinoma. Our results of Shengxin analysis show that celecoxib combined with sorafenib significantly inhibits the expression of liver cancer related genes, which provides help for the prediction of new targets for liver cancer treatment.
Another comparative study showed that celecoxib had the most significant carcinogenic effect compared with traditional NSAIDs. Harri's model describes chemoprevention of celecoxib and Bloven in a rat model of breast cancer. The data showed that the incidence of tumor was reduced by 68% with celecoxib, 40% with ibuprofen, 86% with celecoxib, 52% with ibuprofen, followed by tumor volume, 81% with celecoxib and 57% with buprofen19. The antitumor effect of cyclooxygenase-2-derived prostaglandin E2 (PGE2) is based on a variety of mechanisms, including direct stimulation of cancer cell growth and neovascularization (neovascularization)20,21. The best goal of chemotherapy is to prevent the growth of abnormal cells without any effect on the function of normal cells. Therefore, as a COX-2 inhibitor (celecoxib), it plays an important role in tumorigenesis. Its toxicity is lower than that of conventional non steroidal anti-inflammatory drugs20, and its safety and efficacy parameters have been strictly determined for long-term treatment.
Our study further confirmed that celecoxib can significantly improve the drug resistance of sorafenib, improve the efficacy of sorafenib, and identify the possible genes and mechanisms of their combination. From our analysis results, the 12 genes (MCM4, POLA1, MCM6, MCM3, RBBP4, DNA2, AP2B1, KIF11, KIF23, TUBA1B, KIF14, NUDT21) in HCC tissues have significant expression differences, and also affect the overall survival rate of patients. These genes are mainly concentrated in DNA replication pathway, DNA replication stress should be regarded as a marker of cancer, because it may promote the development of cancer, and it is very common22. The results show that these genes are also enriched in E2F pathway, which is a pathway of many kinds of cancer, and is very important for the treatment of cancer. Cdk-rb-e2f pathway is very important for the regulation of cell proliferation. This suggests that celecoxib enhances the efficacy of tumor cells by regulating related genes to influence cell cycle pathways. Recently, studies have highlighted the additional role of this pathway, especially E2F transcription factor itself, in tumor progression, angiogenesis and metastasis. Specific E2Fs has prognostic value in breast cancer and has nothing to do with clinical parameters23.
Celecoxib has been explored over many centuries and has potential dual functional roles in inflammation and cancer dynamics. Celecoxib blocking COX-2 is an effective treatment for hepatocellular carcinoma. It is hoped that the progress of celecoxib's journey will bring admirable constructive and fruitful results in all areas of treatment, including cancer-related pain signals, pathophysiological diseases.
Hepatocellular carcinoma
Gene Expression Integrated Database
The Cancer Genome Atlas
Kyoto Encyclopedia of Genes and Genomic Pathways
Gene Ontology
Differentially expressed genes
Biological Processes
Molecular Functions
Cellular Components.
Ethics approval and consent to participate
All methods were carried out in accordance with relevant guidelines and regulations.
Human and Animal Ethics
Not applicable.
Consent for publication
Not applicable.
Availability of data and materials
The data is available from public databases, and the dataset can be retrieved from the GEO database (GSE45340). You can also contact the author via the author email for data.
Competing interests
The authors declare that they have no competing interests.
Funding
The authors declare that they have no funding source.
Authors’ contributions
WG wrote the manuscript. CZ provided expert advice. All authors read and approved the final version.
Acknowledgements
Gu Wang was very grateful for Han Yu's help.
Authors' information
Wang Gu1；Chao Zhang2，1Hepatological surgery department , The First Affiliated Hospital of Anhui Medical University；218 Jixi Road, Shushan District, Hefei City, Anhui Province, China. 2 Hepatological surgery department ,The First Affiliated Hospital of Anhui Medical University；218 Jixi Road, Shushan District, Hefei City, Anhui Province, China.