Signicance of HMMR Overexpression in Hepatocellular Carcinoma

Background: Hyaluronan Mediated Motility Receptor (HMMR), as one of the key surface binding proteins of HA, is up-regulated in many tumors. What’s more, the expression level of HMMR is usually correlate with tumor progression and prognosis. However, the relationship between the expression of HMMR and the prognosis and immune inltration of hepatocellular carcinoma (HCC) is still unclear. Methods: We analyzed the expression level of HMMR by TIMER database, GEO database and GEPIA database. The correlation between the HMMR expression and tumor prognosis was analyzed via the Kaplan-Meier plots. The TIMER database and GEPIA database were used to study the relationship between HMMR expression and immune inltration. GO and KEGG enrichment analysis were used to explore the potential biological functions of HMMR. Results: HMMR expression was signicantly higher in several human cancers, including HCC, than in corresponding normal tissues. High HMMR expression associated with poorer overall survival, disease-specic survival, progression-free survival and relapse-free survival in HCC patients. HMMR showed strong correlation with tumor-inltrating B cells, CD4 + and CD8 + T cells, macrophages, neutrophils, and dendritic cells. Several immune marker genes expression, including CD86, IRF5, CD11b, KIRIDL4, CD11c, IFN-γ, STAT3, STAT5B, and CTLA4, have markedly positive correlations with HMMR expression. Enrichment analysis found that HMMR is mainly involved in cell cycle, DNA replication and repair, PLK1 pathway, E2F pathway, ATR pathway and AURORA B pathway. Conclusions: HMMR is a potential prognostic biomarker that inuence tumor progression and correlated with tumor immune cells inltration in HCC.


Introduction
Hepatocellular carcinoma is one of the most malignant tumors, and its incidence rate is increasing year by year [1]. The main factors include liver cirrhosis, viral hepatitis, a atoxin and chemical carcinogens, among which chronic hepatitis virus infection is the most important factor [2][3]. Only about 20% of patients with HCC are in the early stages at the initial diagnosis and have the chance for surgery [4]. Most of the patients can't be completely cured because of complications and distant metastasis [5][6][7][8]. In addition, HCC is prone to recrudescence, which further shortens the survival time of patients. Therefore, it is particularly important to further explore the occurrence and development mechanism of liver cancer and nd a new treatment model.
Previous studies have shown that hyaluronic acid (HA) is related to the growth, metastasis and antiapoptosis of a variety of tumors [9][10]. As one of the key surface binding proteins of HA, HMMR expression level is related to the cell cycle, mainly peaking in the late G2 and early mitosis [11].
Besides, HMMR expression is usually related to tumor progression and metastasis [20][21][22]. This study aims to investigate the role of HMMR in the development of hepatocellular carcinoma Materials And Methods

HMMR expression level analysis
The expression mRNA levels of mRNA HMMR in cell lines were obtained from the HPA (https://www.proteinatlas.org/), which contains the human transcriptomic and proteomic data in cells, tissues and organs from human normal or pathological tissues via RNA sequencing (RNA-Seq) analysis [23]. The HMMR expression in several cancers including HCC were identi ed from the TIMER database (http://timer.cistrome.org/), an online analysis website for The Cancer Genome Atlas (TCGA) [24], and the GEPIA database (http://gepia.cancer-pku.cn/), an online analysis website for The Cancer Genome Atlas (TCGA) and Gene Tissue Expression (GTEX) databases [25].

Kaplan-Meier survival curve analysis
The correlation between HMMR expression and tumor prognosis in liver, gastric, breast, lung, and ovarian cancers were analyzed by the Kaplan-Meier plots (http://www.kmplot.com/analysis/), an online analysis website for survival analysis [28].

Tumor immune in ltration analysis
Using TIMER database and GEPIA database, we investigated the correlation between HMMR expression and immune in ltration levels in HCC. At the same time, we further explored the relationship between immune in ltration and prognosis of HCC patients.

Functional and pathway enrichment analysis
The top 200 differential genes in LIHC, including top 100 over-expression genes and top 100 underexpression genes, obtained from GEPIA database were analyzed by using tools in Metascape (https://metascape.org/ ) to study KEGG and GO annotation [29].

Statistical analysis
Statistical analysis was performed using SPSS Statistics 26.0. Two-sample Student's t-test was used if the values in each group were normally distributed; otherwise, the Mann-Whitney U test was used to analyze the expression of HMMR in normal and tumor samples. p <0.05 was considered as statistically difference.

HMMR overexpression in hepatoma cell line
To explore the signi cance of HMMR expression in liver cancer cells, we analyzed HMMR expression based on the RNA-Seq data obtained from cell lines by HPA databases. The results showed that HMMR was signi cantly overexpressed in hepatoma cell line compared with normal hepatocyte line (Fig. 1).

Expression of HMMR in hepatocellular carcinoma and other tumors
Using the TIMER and GEPIA databases, we subsequently tested the HMMR expression in different human tumor samples. Compared with the corresponding normal tissues, HMMR is highly expressed in a variety of tumor tissues, including bladder cancer, breast cancer, cholangiocarcinoma, colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, hepatocellular carcinoma and so on ( Fig. 2A). Similar results were obtained from GEPIA database in a variety of tumors, except in acute myeloid leukemia and testicular germ cell tumors (Fig. 2B, 2C). To verify the above results, the gene expression pro le matrix les from GSE54236 based on platform GPL6480, GSE76427 based on platform GPL10558 and GSE14520 based on platform GPL3921 were downloaded from the Gene Expression Omnibus (GEO) database. In the above three datasets, the expression of HMMR was signi cantly overexpressed compared with corresponding normal tissues in HCC (Fig. 2D, 2E, 2F). Further analysis indicated that HMMR expression level was signi cantly correlated to tumor stage and grade, as well as TP53 gene status by UALCAN database (Fig. 3A-3D).

Prognostic values of HMMR in hepatocellular carcinoma and other cancers
In addition, we explored the prognostic signi cance of HMMR expression in human cancers by using the Kaplan-Meier plotter database. The results show that higher HMMR expression was associated with  Fig. 4N-4O). However, the expression of HMMR was not related to PPS in lung cancer and ovarian cancer (Fig. 4L, 4P).

Correlation between HMMR expression and prognosis of HCC patients under different clinical characteristics
Subsequently, we analyzed the relationship between the expression of HMMR and the prognosis of Correlation between immune in ltration and expression of HMMR in HCC Tumor in ltrating lymphocyte is reported to be an independent factor for multiple tumor site healing [30].
We used the TIMER database to analyze the correlation between HMMR expression and immune in ltration levels in HCC. The results showed that several immune cell in ltration levels seemed to associate with altered HMMR gene copy numbers, including CD4+T cells, macrophages, and neutrophil in HCC (Fig. 6A). Then, we also found that HMMR expression has markedly positively correlated with in ltrating levels of B cell, CD4+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells in HCC (Fig. 6B). Finally, we further evaluated the impact of immune in ltration on the prognosis of HCC patients. The results indicated that high levels of CD4+ T cells, macrophages, and neutrophils were associated with poor prognosis of HCC patients within two years of survival (Fig. 6C). Subsequently, we further explored the relationship between HMMR expression and immune marker gene expression in HCC using the TIMER database [31], the results showed that several marker genes expression, including CD86, IRF5, CD11b, KIRIDL4, CD11c, IFN-γ, STAT3, STAT5B, CTLA4, TIM-3, have markedly positive correlations with HMMR expression (Table. 1). We also investigated the correlation between HMMR expression and the above immune marker genes in the GEPIA database in HCC, the results similar to TIMER database (Table 2). These results suggested that the level of immune in ltration was affected by the expression of HMMR.

Functional enrichment analysis
The top 200 differential genes in LIHC, including top 100 over-expression genes and top 100 underexpression genes, obtained from GEPIA database, were analyzed by using tools in Metascape to study KEGG and GO annotation (Supplement le1). The top 20 clusters of enriched sets are shown in Fig. 7. The results indicated that enrichment terms related to HMMR were mainly enriched in "cell cycle," "meiotic cell cycle," "DNA replication and repair," "PLK1 pathway,""E2F pathway,""ATR pathway," and "AURORA B pathway," in HCC. Recently, a growing number of evidence suggests that HMMR high expression of HMMR is associated with poor prognosis in a variety of tumor types, such as gastric cancer [32], head and neck squamous cell carcinoma [33], lung cancer [14], breast cancer [15]and so on. In our study, Kaplan-Meier Plotter analyses showed that high HMMR expression correlated with worse prognosis in liver, lung, breast, gastric, ovarian cancer. That is consistent with previous literature reports. Therefore, HMMR may be a potential predictor of HCC prognosis.
Additionally, we also explore the relationship between HMMR expression and in ltration status of immune cells in HCC. The result showed that several immune cell in ltration levels seemed to associate with altered HMMR gene copy numbers, including CD4+T cells, macrophages, and neutrophil, HMMR expression has markedly positively correlated with in ltrating levels of B cell, CD4+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. We also found that high in ltration levels of immune cell, especially CD4+ T cells, macrophages and neutrophils, were associated with poor prognosis within two years of survival in HCC. This suggests that HMMR plays an important role in regulating tumor immunity, and therefore in uences HCC prognosis. Finally, we found in both TIMER and GEPIA databases that several marker genes expression, including CD86, IRF5, CD11b, KIRIDL4, CD11c, IFN-γ, STAT3, STAT5B, CTLA4, have markedly positive correlations with HMMR expression.
As a HA receptor, HMMR can play a role in a variety of biological functions that lead to the development of tumors [34]. Enrichment analysis found that HMMR is mainly involved in cell cycle, DNA replication and repair, PLK1 pathway, E2F pathway, ATR pathway and AURORA B pathway, which is consistent with previous research [34][35][36][37], PLK1 pathway, E2F pathway, ATR pathway and AURORA B pathway, the role of these pathways in initiation and development of HCC has been reported in previous studies [38 -43].
In summary, our study elaborated that relationship between HMMR and HCC, and HMMR is a potential independent prognostic biomarker for HCC. In order to validate these results, we intend to establish in vivo, in vitro and animal experiments in follow-up studies. Figure 1 HMMR expression in normal and tumor cell lines. Relationship between expression of HMMR and clinicopathological characteristics in HCC (*P < 0.05, **P < 0.01, ***P < 0.001) Figure 4 Kaplan-Meier survival curve analysis of the prognostic signi cance of high and low expression of HMMR in different types of human cancers using the Kaplan-Meier plotter database (A-P)  Functional enrichment of HMMR in HCC.

Supplementary Files
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