Xiao-Meng Wang,Yu Chen

1Tianjin Medical University Cancer Institute and Hospital,Tianjin 300060,China.2Department of Cancer Immunotherapy,Tianjin Cancer Hospital Airport Hospital,Tianjin 300052,China.3National Clinical Research Center for Cancer,Tianjin 300060,China.4Key Laboratory of Cancer Immunology and Biotherapy,Tianjin 300060,China.5Tianjin’s Clinical Research Center for Cancer,Tianjin 300060,China.6Key Laboratory of Cancer Prevention and Therapy,Tianjin 300060,China.7Department of Traditional Chinese Medicine,Tianjin Hospital of ITCWM Nankai Hospital,Tianjin 300100,China.

Abstract Objective:To explore the expression and clinical significance of RACGAP1 gene in hepatocellular carcinoma.Methods:Data about RACGAP1 gene and clinic pathological data in liver cancer were retrieved from The Cancer Genome Atlas(TCGA).The relationship between the expression of RACGAP1 gene and clinic pathological parameters,and prognosis were analyzed by R 2.15.3 software.The association between RACGAP1 gene expression and prognosis of liver cancer patients was analyzed by Kaplan-Meier survival function analysis and Cox regression analysis.Results:TCGA database was used to collect 235 cases of liver cancer with clinical pathological parameters and their corresponding RACGAP1 expression levels.After the incomplete cases and those with no detailed pathological parameters were excluded,and it was found that RACGAP1 was highly expressed in liver cancer tissues.Meanwhile,the expression of RACGAP1 in patients with liver cancer in the TCGA tumor database was further analyzed with the matching clinical data parameters.The expression level of RACGAP1 was significantly correlated with the pathological grade and T stage of liver cancer patients(all P＜0.05),but was not significantly correlated with American Joint Committee on Cancer(AJCC)pathological stage and gender(P＞0.05).There was a significant correlation between RACGAP1 expression level and overall survival(OS)in patients with liver cancer(P＜0.05),and the overall survival time of patients with low expression was better than that of patients with high expression(P＜0.05).Cox regression was used to analyze the correlation between T stage,M stage,N stage and RACGAP1 expression in patients with hepatocellular carcinoma(HCC),and RACGAP1 became an independent prognostic factor in patients with HCC(P＜0.05).Conclusion:Based on the tumor-related gene information in the public database TCGA,RACGAP1 gene is highly expressed in liver cancer tissues and becomes an independent prognostic factor of liver cancer,which is expected to become an important therapeutic target of drug therapy for liver cancer.

Keywords:The Cancer Genome Atlas;liver cancer;RACGAP1;gene;data mining

Background

Primary liver cancer is one of the most common clinical malignant tumors,and the most common pathological type is hepatocellular carcinoma(HCC)[1].HCC has an insidious onset,difficult early diagnosis,and rapid disease progression.Most cases are difficult to be diagnosed early,and the opportunity of surgery has been lost when diagnosed,and relapse and metastasis are prone to occur,with short survival time,poor prognosis,and high mortality and recurrence rates[2].Currently,the commonly used clinical prognostic indicators of liver cancer include AFP,primary tumor size,vascular invasion,pathological grade,etc.,but there is still a lack of effective molecular markers for predicting liver cancer prognosis.Therefore,it is of great significance to actively search for key genes related to liver cancer for early diagnosis and prognosis of liver cancer.

RACGAP1(RacGTPase-activating protein 1,RACGAP1)belongs to the family of GTPase-activating proteins.RACGAP1 has been proved to be highly expressed in breast cancer,colorectal cancer,esophageal cancer,gastric cancer,lung cancer and other malignant tumor cells,and its high expression is significantly correlated with a variety of clinicopathological parameters and poor prognosis of tumors[3,4].Although there have been reports in China,Wang et al.[5]found that RACGAP1 expression was significantly increased in liver cancer tissues,and the expression level was higher in patients with high recurrence risk,suggesting that RACGAP1 may be a prognostic marker for liver cancer.However,the significance and prognosis of RACGAP1 expression in HCC are still rarely reported.The Cancer Genome Atlas(TCGA)contains comprehensive Cancer mutation spectrum,gene expression data and relevant clinical information,which is of great significance for understanding The mechanism of cancer genesis and development,and discovering new therapeutic targets and new diagnosis and treatment methods[6].

The purpose of this paper is to deeply explore the expression of RACGAP1 in HCC collected from TCGA database and analyze the expression of RACGAP1 in HCC.The expression of RACGAP1 in HCC patients and clinical characteristics of related cases were downloaded from TCGA database with R language software for analysis,to explore the expression and clinical significance of RACGAP1 in HCC,and to predict the mechanism of action of RACGAP1 in the occurrence and development of HCC tumors.

Data and methods

Data extraction from TCGA database

The mRNA Seq V2 data of HCC data set was downloaded and preprocessed from TCGA database using R 2.15.3 software Epicalc function package for relative expression analysis.Meanwhile,the relevant data of clinical data were downloaded for clinicopathological correlation analysis.We confirm that all methods were carried out in accordance with relevant guidelines and regulations.

The differential expression of RACGAP1 in HCC and normal tissues was further analyzed in TCGA database,and the relationship between RACGAP1 expression and clinicopathological characteristics(tumor stage,gender,etc.),as well as the relationship between RACGAP1 expression and HCC survival and prognosis were analyzed.

Statistical analysis

T test was used to compare the difference in RACGAP1 expression between normal tissues and HCC tissues.Kaplan-Meier method and Cox regression were used to calculate the relationship between RACGAP1 expression and HCC prognosis.SPSS 18.0 software was used for all statistical analysis and Graphpad Prism 5.0 software was used for plotting.P＜0.05 was considered to be statistically significant.

Results

RACGAP1 expression difference between HCC and normal tissues in different HCC study chips

The expression of RACGAP1 in different liver cancer tissues was analyzed in TCGA database,and it was found that RACGAP1 was highly expressed in liver cancer tissues compared with normal tissues(P＜0.05),and the difference was statistically significant.The increased expression of RACGAP1 contributes to the development of liver cancer,and it acts as an oncogenic gene in the development of liver cancer(Figure 1).

Figure 1 Expression of RACGAP1 in normal liver and liver cancer tissue

Correlation between RACGAP1 expression and clinicopathological parameters

A total of 235 HCC cases with complete clinicopathological parameters were included in the TCGA data set,including 111 males and 124 females,aged 35-88 years,with a median age of 65 years.The prognostic factors of HCC patients were assigned in this data set.Statistical analysis showed that RACGAP1 expression level was significantly correlated with the pathological grade(Figure 2A)and T stage(Figure 2B)of HCC patients(P＜0.05),but not with AJCC pathological stage(Figure 2C)and gender(Figure 2D)(P＞0.05).

Relationship between RACGAP1 expression and survival prognosis of HCC patients

The expression level of RACGAP1 is elevated in liver cancer.To further clarify the relationship between RACGAP1 expression and prognosis of liver cancer,in this study,clinicopathological parameters of RACGAP1 were extracted from TCGA liver cancer data set and analyzed by Kaplan-Meier model.As shown in Figure 3,the expression level of RACGAP1 was correlated with the prognosis of HCC patients in overall survival(OS)(P＜0.05),and the overall survival time of patients with low expression was better than that of patients with high expression(P＜0.05).Cox regression analysis showed that T stage,M stage,N stage and RACGAP1 expression were all correlated with the prognosis of HCC patients Figure 4,and RACGAP1 was an independent prognostic factor of HCC patients(P＜0.05)(Table 1 and 2).

Table 1 The relationship between RACGAP1 expression and survival prognosis of patients with liver cancer

Table 2 The influence of different pathological features on survival and prognosis of patients with liver cancer by univariate and multivariate Cox analysis

Figure 2 Expression of RACGAP1 in patients with liver cancer.(A)with different pathological grade;(B)with different T stages;(C)different pathological stages;(D)with different genders.

Figure 3 The relationship between RACGAP1 expression and survival prognosis of patients with liver cancer

Figure 4 The relationship between RACGAP1 expression and survival prognosis of patients with liver cancer

Discussion

At present,there are about 782,000 new cases of liver cancer every year worldwide,ranking sixth in the number of new cases of all malignant tumors,and the number of deaths related to liver cancer is up to 745,000 every year,ranking third in the number of malignant tumor-related deaths[1-3].The number of liver cancer-related deaths in China is about 383,000 annually,accounting for 51% of the total number of liver cancer-related deaths worldwide[7].There are generally no obvious symptoms in the early stage of liver cancer,and the 5-year overall survival rate is about 7%.The natural survival period of patients in advanced stage without any treatment is only 3-6 months[8].Most patients with liver cancer have lost the opportunity of surgery when they were diagnosed.In addition to high morbidity and mortality,liver cancer is also characterized by strong invasiveness,easy recurrence and easy spread.Therefore,in clinical practice,how to make a more effective and accurate early diagnosis of liver cancer and prognosis judgment has become the key to the treatment of liver cancer.

Through the exploration of molecular biological mechanisms related to liver cancer,it is found that the molecular pathogenesis of liver cancer is very complex.The most common examples are:abnormal growth factor activation,abnormal neovascularization and abnormal cell signaling pathways,cell signaling pathway activation,anti-apoptotic signaling pathway disorder,etc.Hepatitis virus infection and environmental toxins can induce gene mutations in hepatocytes,and many gene mutations promote the occurrence,development and metastasis of hepatocellular carcinoma.RACGAP1 gene exists in chromosome 12q13 and plays different roles in intermitosis and mitosis[09,10].Currently,studies have shown that RACGAP1 gene is overexpressed in chromosome 12q13 of non-small cell lung cancer(NSCLC)cells,which is related to cell cycle regulation,cytokinesis and signal transduction.Recent studies have also proved that some gene loci on chromosome 12q13 are associated with colorectal cancer,polycystic ovary syndrome,sarcoidosis and other diseases,indicating that there may be genes with extensive biological significance on chromosome 12q13,and indirectly indicating that RACGAP1 may have extensive biological effects[11].Shi-Jun Mi et al.[12]showed that RACGAP1 expression was higher in patients with advanced uterine carcinosarcoma,and its high expression was significantly correlated with extrauterine metastasis of uterine carcinosarcoma.These results further demonstrated that RACGAP1 regulates the migration and invasion of cancer cells through the STAT3-survival signaling pathway,thus promoting the formation of aggressive phenotypes in uterine carcinosarcoma.In addition,the specifically increased expression of RACGAP1 in cancer tissues has also been found in a variety of malignant tumors,such as breast cancer,gastric cancer and bladder cancer[13,14].Its high expression was also found to be significantly correlated with clinicopathological parameters such as primary tumor size,degree of necrosis,vascular invasion,clinical stage,degree of lymphatic invasion,and distant spread,which could indirectly reflect tumor proliferation and increased aggressiveness.All of these indicated that RACGAP1 expression may be correlated with the occurrence and progression of HCC,and also suggested that RACGAP1 may be involved in the functional regulation of tumor cells.

HCC-related in vitro experiments showed that cells with high RACGAP1 expression(MHCC97-H and Hep3B)had higher mobility compared with cells with low RACGAP1 expression(HepG2 and PLC/PRF5).SiRNA blocking RACGAP1 expression in HCC cells with high RACGAP1 expression can reduce cell migration and invasion activities and increase DNA fragmentation leading to cell death[6].Studies have shown that RACGAP1 may promote hepatitis C virus(HCV)activation replication by enhancing the activity of viral protein NS5B polymerase,thus mediating the silencing of RACGAP1 expression,and thus inhibiting the antiviral effect of HCV replication[15].The potential anti-HCV effect of RACGAP1 expression silencing,combined with the significant correlation between RACGAP1 high expression and hepatocellular carcinoma invasiveness and postoperative recurrence,suggests that RACGAP1 is expected to be an effective molecular target for the prevention of postoperative recurrence of HCV-associated HCC.High RACGAP1 expression,along with vascular invasion and cirrhosis,is an independent prognostic factor associated with HCC recurrence.HCC patients with high RACGAP1 expression are 2.7 times more likely to have early tumor recurrence.This suggests that the high expression of RACGAP1 may be an independent prognostic factor for predicting early tumor recurrence after radical HCC resection[6].

In this study,the differential expression of RACGAP1 in liver cancer tissues and normal liver tissues was analyzed based on TCGA liver cancer data,suggesting that RACGAP1 was highly expressed in liver cancer tissues.Meanwhile,further analysis of RACGAP1 expression level in HCC patients in TCGA tumor database and matched clinical data parameters showed that RACGAP1 expression level was significantly correlated with pathological grade and T stage of HCC patients(P＜0.05).There was no significant correlation with AJCC pathological stage,gender(P＞0.05).Kaplan-Meier model was used for analysis,the expression level of RACGAP1 was correlated with the prognosis of HCC patients in overall survival(OS)(P＜0.05),and the overall survival time of patients with low expression was better than that of patients with high expression(P＜0.05).Cox regression analysis showed that T stage,M stage,N stage and RACGAP1 expression were correlated with the prognosis of HCC patients,and RACGAP1 was an independent prognostic factor of HCC patients(P＜0.05).Based on TCGA cancer database,this study explored the expression of RACGAP1 in liver cancer and its prognostic significance,laying a theoretical foundation for further elucidating the role and mechanism of RACGAP1 in the development and development of liver cancer,and also providing potential targets for early diagnosis and treatment of clinical liver cancer.

In our study,there is a lack of data sample collection to further authenticate the views in the paper.In our next study,it is worthwhile to further explore and study the expression of RACGAP1 in liver cancer and its prognostic significance.Our subsequent study will collect the sequencing data of the samples to further authenticate the views in the paper.

In summary,RACGAP1 gene is involved in a variety of cellular functions, such as regulating cytokinesis, proliferation,transformation,migration,invasion and metastasis,and it is highly expressed in a variety of malignant solid tumor cells,such as breast cancer,gastric cancer,colorectal cancer,HCC and lung cancer[3,4,13,14].In addition,the high expression of RACGAP1 in a variety of malignant tumors has been proved to be significantly correlated with the clinicopathological parameters such as tumor size,clinical stage,vascular invasion,lymph node metastasis,distant metastasis,and poor prognosis of patients.However,the specific molecular biological mechanism of RACGAP1 regulation and overexpression has not been clarified,and its expression and clinical significance in different tumor tissues remain to be further studied.With the progress of the expression,mechanism and clinical significance of RACGAP1 in malignant tumors,RACGAP1 is expected to become a new prognostic factor and a potential molecular target for the design of therapeutic strategies in the future.