The diagnostic and prognostic role of RhoA in hepatocellular carcinoma

The aim of this study was to investigate the expression level of Ras homolog gene family, member A (RhoA) in patients with hepatocellular carcinoma (HCC) and to investigate the prognostic and diagnostic value of RhoA. Data mining from various data bases and wet experiments on samples from Peking Union Medical College Hospital showed that RhoA mRNA and protein expression were significantly higher in the HCC tissues than in the normal tissues. Higher expression at both the mRNA and protein levels was associated with a poorer prognosis. High sensitivity (92.5%) and specificity (90.0%) were observed in the diagnostic model based on protein level rather than mRNA level. RhoA expression was modulated by genetic amplification. The lysosome, pathogenic Escherichia coli infection, purine metabolism and pyrimidine metabolism pathways were mainly enriched in the high RhoA level group, while the hedgehog signaling, linoleic acid metabolism, olfactory transduction and taste transduction pathways were enriched in the low RhoA level group. RhoA is commonly upregulated in HCC tissues, and its expression at both the mRNA and protein levels is associated with poor prognosis. Notably, RhoA protein levels serve as a diagnostic biomarker for HCC.

AGING Ras homolog gene family, member A (RhoA) is a small GTPase protein in the Rho family containing two switch regions, Switch I and Switch II, whose conformational states are modified following the activation or deactivation of the protein.
The key amino acids of RhoA at Gly14, Thr19, Phe30, and Gln63, which are involved in the stabilization and regulation of guanosine triphosphate (GTP) hydrolysis, are highly conserved. RhoA is primarily involved in actin organization, myosin contractility, cell cycle maintenance, cellular morphological polarization, cellular development, and transcriptional control. Accumulated evidence has demonstrated that RhoA was closely associated with cancer in relation to venous invasion, microscopic satellite lesions, advanced pTNM stage, progression [5], cell differentiation [6] and disease-free survival rates [7]. However, the prognostic and diagnostic value of RhoA in HCC is still unclear, and further research is urgently needed.
Here, using bioinformatics data mining, we found that both the gene and protein levels of RhoA were differentially expressed between liver cancer tissues and adjacent normal tissues, which indicated the potential of RhoA as a diagnostic biomarker. Analysis of The Cancer Genome Atlas (TCGA) data suggested that higher RhoA gene expression was associated with poorer prognosis, and similar patterns were also observed in two additional Gene Expression Omnibus (GEO) cohorts. The protein expression, but not the mRNA level, of RhoA varied greatly between liver cancer tissues and normal tissues, making it possible to build not only a prognostic model but also a diagnostic model, which was validated in a Peking Union Medical College Hospital (PUMCH) cohort with 30 normal tissue samples and 134 HCC samples.

RESULTS mRNA and protein expression profile of RhoA in the HPA
By examining the RhoA expression profile in the HPA, we found that the RNA expression of RhoA in the normal liver tissue samples was the lowest compared with that in the other 30 human tissue samples ( Figure  1A), and the protein level of RhoA was nearly undetectable in hepatic tissues ( Figure 1B). As Figure  1C shows, the level of RhoA in the HCC tissue samples was relatively low among the 17 carcinoma types; however, the expression of RhoA was found to be significantly upregulated in HCC tissues compared with that in normal tissues ( Figure 1D). Interestingly, regarding RhoA protein expression, liver cancer exhibited a high positive rate, which ranked second among the positive expression rates of 20 common cancer types ( Figure 1E). Figure 1F displays representative IHC images of the distinct RhoA expression levels, including undetected, low, and medium. In addition, Hep G2, which is a frequently used hepatic carcinoma cell line, had a similar RhoA expression pattern as that of the liver cancer tissue shown in Figure 1C, as identified by the Cancer Cell Line Encyclopedia (CCLE) web searching tool. Hence, the RhoA protein level, rather than the gene expression level, may be a more sensitive biomarker to diagnose HCC.

Prognostic role of RhoA gene expression in liver cancer patients
To explore the association between RhoA mRNA expression and OS in liver cancer patients, we performed Kaplan-Meier survival analysis based on data from TCGA. The results indicated that the HCC patients with high RhoA expression had poorer prognoses ( Figure 2A). Notably, the optimal cutoff point of RhoA mRNA expression was calculated based on the X-tile method, which can produce the optimal cutoff value to predict survival time ( Figure 2B). Moreover, the tumor-promoting role of RhoA was also demonstrated by two genomic spatial event (GSE) datasets (GSE10186 and GSE 54236), in which the P values were 0.039 and 0.024, respectively ( Figure 2C and Figure 2D).
To further investigate the independent prognostic value of RhoA, univariate and multivariate Cox regression analyses were conducted. As shown in Figure 2E, vascular invasion (HR: 1.274; 95% CI: 1.092-4.243, P = 0.027; Figure 2E) and high RhoA levels (HR: 2.578; 95% CI: 1.311-5.071, P = 0.006; Figure 2E) were associated with poor OS in the univariate analysis. In addition, multivariate analysis also demonstrated that the aforementioned factors were independent risk factors of OS (P < 0.05; Figure 2E).

RhoA expression is associated with gender
Next, we divided HCC patients from TCGA into a high-risk group and a low-risk group according to the optimal cutoff value of RhoA expression mentioned before to explore the relationships of RhoA expression with different clinicopathological parameters, and we only found that male patients had significantly higher RhoA gene expression levels than female patients (Table 1, Supplementary Table 1, and Figure  3A). However, the other clinical characteristics, such as different stage levels or grade levels of HCC, seemed to have no influence on RhoA expression levels (  Figure 3B, 3C).

mRNA and protein expression validation of RhoA
We further extracted RNA from 10 frozen HCC and paired adjacent liver tissues to quantify RhoA gene expression. The HCC tissues did have a significantly higher RhoA expression, which was consistent with the pattern identified in the HPA. ( Figure 3D). Moreover, the IHC test of RhoA showed that the hepatic cells in the normal tissue were mainly stained negatively except for portions of the interstitial substance ( Figure 3E, upper parts), while tumor cells in the HCC tissue from the same section were stained positively ( Figure 3E, lower parts). In summary, although the mRNA and protein levels of RhoA were significantly higher in the HCC tissues than in the normal adjacent tissues, the RhoA protein level, rather than the mRNA level, seems to have a better distinction ability, which indicates its potential as a diagnostic biomarker.

Diagnostic model based on RhoA protein levels in liver cancer patients
To test our hypothesis, we performed additional IHC staining with 30 normal tissue samples and 134 HCC tissue samples from an HCC patient cohort from PUMCH to detect the protein expression profile of RhoA. Consistent with the previous results, the HCC samples had significantly higher RhoA protein levels ( Figure 4A). Next, we divided patients into two groups, one with a high RhoA protein level (3+) and the other with a low level (0−2+). Similar to the outcome of the analysis of the prognostic function of RhoA gene AGING expression, the HCC patients with longer OS times had lower RhoA protein levels ( Figure 4B). Considering that the protein level of RhoA has better tissue specificity than gene expression between liver tumor tissues and adjacent normal tissues, the RhoA protein expression based on the results of the IHC analysis of normal tissues was mainly undetected (0) or low (1+), while that of the cancerous tissues was usually medium (2+) or high (3+). We proposed a diagnostic model based on this phenomenon ( Figure 4C). After validation, the sensitivity and specificity of the model were 92.5% and 90.0%, respectively ( Figure 4D). The area under the curve (AUC) of the receiver operating characteristic (ROC) curve was 0.913 ( Figure 4E), which indicates the good performance of this model. To exclude false positive caused by antibody nonspecificity, we performed immunohistochemical tests on some samples with another RhoA antibody (1:50, AF6352, Affinity biosciences, USA) and the results are in line with previous one (data not shown). Overall, the diagnostic model based on the RhoA IHC results expanded our current detection approaches for HCC.

RhoA expression is modulated by genetic amplification and related KEGG pathways
Next, we explored the mechanisms of RhoA dysregulation using deep sequencing data from the University of California, Santa Cruz. After visualizing the somatic mutation, copy number variation, DNA methylation, and expression of RhoA, we found only four HCC samples with single nucleotide polymorphisms (SNPs), which demonstrated that point mutation did not play an essential role in RhoA expression ( Figure 5A). Notably, the copy number variation and gene expression of RhoA shared a AGING coexpression pattern, and the significant association was further validated via regression analysis (r = 0.563, P < 0.05, Figure 5B). However, the DNA methylation profile of the HCC tissues showed little variation ( Figure 5A). To depict the exact relationships between DNA methylation and gene expression levels, we analyzed 405 samples with both methylation data and gene expression levels. As shown in Figure 5C, the increase in RhoA DNA methylation only slightly attenuated RhoA gene expression (r = −0.108, P < 0.05, Figure 5C). Furthermore, to detect the primary KEGG pathways in the two groups (RhoA high and low), we performed GSEA and found that samples with high levels of RhoA were enriched with genes mainly involved in the lysosome, pathogenic Escherichia coli infection, purine metabolism and pyrimidine metabolism pathways ( Figure 5D, left part), while those with low levels of RhoA had predominant genes involved in the hedgehog signaling pathway, linoleic acid metabolism, olfactory transduction and taste transduction ( Figure 5D, right part).
Therefore, it seems that the copy number amplification level of RhoA, rather than the decrease in the DNA methylation level, plays a major role in the overexpression of RhoA in HCC tissues.

DISCUSSION
As one of the leading causes of cancer deaths around the world, liver cancer continues to exhibit increasing incidence and mortality rates, especially in East Asia, Southeast Asia, Africa and southern Europe [8,9]. Radical surgery can significantly improve the prognosis of some patients with early stage HCC; however, due to a lack of effective screening approaches and noticeable symptoms, most patients are in advanced stages the time of diagnosis and miss the optimal opportunity for treatment, which leads to dismal prognosis. Consequently, considerable research efforts have been devoted to identifying diagnostic and prognostic biomarkers of HCC with high specificity and sensitivity.
RhoA is one of the prototypical members of mammalian Rho GTPases, which include 23 intracellular signaling molecules, such as Ras-related C3 botulinum toxin substrate 1 (RAC1) and cell division cycle 42 (CDC42) [10][11][12]. The change in Rho GTPase proteins between the GTP-bound active form and the GDP-bound inactive form is regulated by three sets of molecules: guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs) [10][11][12]. Activated Rho GTPases interact with downstream proteins and regulate cytoskeletal dynamics and a variety of biological processes, including cell division, survival, migration, and adhesion [10][11][12]. Considering the crucial roles of RhoA in the regulation of cell morphology, motility, and cell-cell and cell-matrix adhesion, we can easily deduce that RhoA is associated with carcinoma metastasis, which is one of the leading causes of death in patients with solid tumors [13,14]. Recent studies have reported that the invasiveness and metastasis of rat and human hepatoma cells [15,16], bladder cancer cells [17], colorectal cancer cells [18], and lung cancer cells [19] can be suppressed by inhibiting the Rho-ROCK (Rhokinase) signaling pathway. However, there is some contradictory evidence showing that RhoA inactivation promoted the migration and metastasis of triple-negative breast cancer [20] and increased tumor growth and metastasis in colorectal cancer [21], liver cancers [22], and nasopharyngeal carcinoma [13]. Further mechanistic studies are needed to better elucidate the regulation of the RhoA signaling pathway in tumor metastasis, especially in HCC.
Taking the functions of RhoA in essential signaling pathways into account, we speculate that RhoA may be a potential functional biomarker in HCC. We initially examined both the mRNA and protein expression patterns of RhoA in normal human organs, common cancer types and cell lines using an HPA web-based tool. Intriguingly, although RhoA was significantly upregulated in HCC tissues compared with its expression in normal liver tissues, the basal expression of RhoA in normal liver tissue, HCC tissue, and the HCC cell line Hep G2 was actually very low among all the specimens. Notably, the RhoA protein was not detected in normal liver tissues but was significantly enriched in most HCC samples. It is well accepted that protein is a function executor according to the central dogma. The RhoA protein expression level in HCC ranked second, next to that in glioma, in all common tumor types, which indicated that the RhoA protein, rather than the gene, may promote liver tumorigenesis as an essential functional element. Recent studies have suggested that RhoA is one of the genes that is most frequently overexpressed in various cancer cells and is involved in cell division processes, and RhoA promotes HCC growth through the RhoA/F-actin/Hippo-YAP signaling axis [23]. In addition, the coordinated expression of Rac GTPase-activating protein 1 (RACGAP1) and epithelial cell transforming sequence 2 (ECT2) upregulated RhoA activity in HCC cells [23][24][25].
To evaluate the prognostic role of RhoA, we subsequently performed Kaplan-Meier analysis in the TCGA HCC cohort and found that high RhoA expression correlated well with poor prognosis. Two additional GEO cohorts also demonstrated this phenomenon. In addition, after univariate and multivariate analyses, RhoA expression levels, together with vascular invasion, were both independent prognostic factors in HCC, which was consistent with the results of a previous study [6]. Recent studies have also proven the prognostic value of RhoA in other cancer types, such as colorectal cancer, breast cancer, and glioma. Colorectal cancer patients with positive RhoA protein expression had higher vascular invasion rates, higher clinical stages, and lower 5-year survival rates [26]. Breast cancer samples with phosphorylated RhoA (P-RhoA) were associated with poorer prognosis [27]. Lin Yu demonstrated that SND1 (Staphylococcal nuclease domain-containing protein 1) and RhoA were independent predictors of poor prognosis in glioma patients [28]. Our results suggested only a relationship between gender and RhoA gene expression.

AGING
Furthermore, RhoA protein expression may be a diagnostic biomarker in HCC because its high liver cancer specificity was demonstrated by analyzing IHC sections from tumor resection margin areas of 9 HCC patients from PUMCH. Hence, we estimated the RhoA IHC staining intensities in a PUMCH HCC cohort of 130 liver tumor samples and 30 adjacent normal samples. The role of the RhoA protein was apparent from its expression and distribution. In addition to the prognostic ability of the gene, the RhoA protein level can clearly distinguish between normal and cancerous tissues with high sensitivity and specificity. This is the first time that the diagnostic role of RhoA was proposed and proven.
The essential position occupied by RhoA in the cancer cellular signaling pathway network has emphasized the importance of RhoA in cancer pathophysiology, and the intrinsic characteristics of RhoA make RhoA a great potential distinguished diagnostic and prognostic biomarker for liver cancer. As described before, instead of acting solely as a cancer indicator, RhoA also acts as a functional regulator. We next explored the dysregulation mechanism of gene expression via multiomics analysis. The results showed that the copy number amplification of RhoA mainly contributed to its increased gene expression, which may be a potential therapeutic target for HCC. The predominant KEGG pathways in the high RhoA level group and the low RhoA level group were further identified by using GSEA to reveal potential differential signaling pathways.
Overall, our study confirmed RhoA mRNA and protein levels as prognostic biomarkers in HCC through the analysis of multiple cohorts, and the use of RhoA as a prognostic biomarker has been reported in previous studies [5,6,23]. To the best of our knowledge, this is the first time that a diagnostic model was proposed based on RhoA IHC staining results rather than gene expression. Our estimations indicate that this model has very good performance (AUC=0.913) and high sensitivity and specificity (92.5% and 90.0%, respectively). However, due to limited HCC cohorts and RhoA IHC expression results, we could not perform external verification of this diagnostic model. In addition, future work will focus on utilizing clinical parameters along with biomarkers, which may improve the performance of the biomarkers [29]. In addition, further clarification of the exact RhoA expression regulatory mechanism is urgently needed.

Bioinformatics data mining and processing
The mRNA level and protein level of RhoA in both normal human tissues and cancerous tissues, as well as gene expression in cancer cell lines, were examined using data from the Human Protein Atlas (HPA) (http://www.proteinatlas.org/) [30,31]. Representative immunohistochemistry images of RhoA protein expression profiles in HCC samples with different staining intensities were also downloaded from the HPA. The differential mRNA expression level of RhoA between HCC tissues and normal tissues from the TCGA database and the Genotype-Tissue Expression (GTEx) project were analyzed via the Gene Expression Profiling Interactive Analysis (GEPIA) web tool (http://gepia.cancer-pku.cn/). The mRNA transcriptome data and clinical parameters of liver hepatocellular carcinoma (LIHC) patients were obtained from the TCGA data portal (https://portal.gdc.cancer.gov/). Two other HCC validation datasets (GSE10186 and GSE54236) containing mRNA expression data and corresponding survival times were retrieved and downloaded from the GEO database (https://www.ncbi. nlm.nih.gov/geo/). Histological type, somatic mutation, copy number variation, gene expression, and DNA methylation of RhoA in liver cancer were visualized via a heatmap generated from the Xena browser web tool (https://xenabrowser.net/), and data used to analyze the factors affecting RhoA gene expression were downloaded from the Xena browser web tool. Analyzing the data downloaded from the TCGA and GEO databases did not require the approval of an ethics committee. Gene Set Enrichment Analysis (GSEA) was conducted to compare the distinct Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways between the high RhoA level group and the low RhoA level group based on the TCGA HCC mRNA expression data.

Experimental validation with samples from Peking Union Medical College Hospital
From January 2004 to August 2016，thirty paired HCC and para-cancerous normal tissues and 104 HCC tissues from patients who underwent surgery and pathologically diagnosed as HCC at PUMCH were collected and made into four formalin-fixed paraffin-embedded (FFPE) tissue chips, the remaining tissues were frozen in liquid nitrogen and then stored at −80°C. Among these patient samples, the differential RhoA expression of ten frozen HCC tissues and paired adjacent normal tissues were validated by quantitative polymerase chain reaction (qPCR) as described previously [32]. The primer sequences were as follows: RhoA forward primer: 5′-AGCCTGTGGAAAGACATGCTT-3′; RhoA reverse primer: 5′-TCAAACACTGTGGGCACATAC-3′; β-actin forward primer: 5′-GCCGGGACCTGACTGACTAC-3′; β-actin reverse primer: 5′-CGGATGTCCACGTCACA CTT-3′. Immunohistochemistry (IHC) sections of four FFPE tissue chips were stained with the RhoA antibody (EPR18134, RabMAb, UK) at a dilution of 1:100 to AGING detect their protein profiles. In addition, nine IHC sections of the cancer margin with both normal and tumor components were used to elucidate the differential protein expression level of RhoA. Interpretation of immunohistochemical results was performed by two independent experienced pathologists based on the proportion of positive cells without considering dyeing intensity. Histochemical score from 0 to 3 as follows: 0, 1-5% positive cells; 1, 6-25% positive cells; 2, 26-75% positive cells; and 3, >76% positive cells. Samples with discordant scores from the two pathologists were discussed and re-scored.

Statistical analysis
Statistical analysis was performed using R software v.3.5.2. Continuous variables are reported as the means ± standard deviations (SDs). Differences between groups were compared by unpaired Student's t-test and visualized by GraphPad Prism v.8.0 (GraphPad Inc.). The associations between RhoA expression and clinicopathological parameters were evaluated using the χ 2 test. The OS curves of the above three HCC patient cohorts were generated based on the best cutoff value (Xtile algorithm) with the survival package in R. Univariate and multivariate Cox regression analyses were performed to identify independent prognostic variables based on RhoA level and other clinical characteristics, including age, gender, body mass index (BMI), stage, grade, fibrosis, AFP, extent of inflammation, vascular invasion, and family history. Unless noted otherwise, P < 0.05 was considered statistically significant. Linear regression analyses were conducted to assess the correlations between RhoA gene expression and copy number variation and between RhoA gene expression and DNA methylation. |R|>3 and P < 0.05 were considered relevant and statistically significant.

CONCLUSIONS
RhoA is commonly upregulated in HCC tissues, and both high mRNA expression and high protein expression levels are associated with poor prognosis. Notably, RhoA protein levels serve as a diagnostic biomarker for HCC.

AUTHOR CONTRIBUTIONS
YB and FCX conceived the study and performed bioinformatics analyses. JZL, DXW, XY, JB, and FM downloaded and organized the clinical and gene AGING expression data. YB and SH designed and executed experiments. FCX, FM, JYL, JZM and HCH performed the statistical analyses. YB and FCX wrote the manuscript. XW, YLM, XTS, and HTZ critically revised the article for essential intellectual content and provided administrative support. All authors read and approved the final version of the manuscript. All authors also reviewed and revised the manuscript. HTZ was the guarantor of this study.