Hypoxia-induced HMMR Promotes Cell Proliferation, Migration and Immune Inltrates in Lung Adenocarcinoma

depletion of HMMR was inhibits the cell proliferation and migration ability of NSCLC cells. These ndings suggest that HMMR could be served as a biomarker for prognosis and immune inltration in LUAD.


Introduction
Lung cancer is one of the main causes of cancer death, and It's has brought huge economic and social burden in the worldwide [1]. Lung Cancer mainly contained the small lung squamous cell carcinoma and non-small lung squamous cell carcinoma. While, the NSCLC Composed of lung adenocarcinoma and lung squamous cell carcinoma [1,2]. Due to the lack of effective diagnostic markers result in most of the patients with lung cancer have been found in advanced stage, missed the best treatment time [2,3]. Therefore, it's imperative nd speci city and sensitivity biomarkers for diagnosis and treatment of lung cancer.
HMMR, named as RHAMM, called hyaluronan mediated motility receptor, its play pivotal roles in cell growth [4]. Studies have shown that HMMR was mainly expressed in the nervous system [5]and the mouse brain [6]. The gene mutation of HMMR usually lead to the neurodevelopmental defects [7].
Research studies have shown that high HMMR expression has been associated with various cancer, such as breast cancer [8], colorectal cancer [9], stomach cancer [10], endometrial cancer [11] and prostate cancer [12]. Previous study indicated that HMMR was essential for maintains the stemness of glioblastoma stem cells [13]. However, the signi cance and potential mechanisms of HMMR in LUAD progression and tumor-in ltrating remains bewildered.
In this nding, we show that HMMR was highly expresison in lung adenocarcinoma and it's high expression was correlated with poor prognosis, tumor stages and lymph node metastasis. furthermore, up-regulated HMMR expression was markedly positive with the the in ltration levels of B cells, CD4 + T cells, CD8 + T cells, macrophages, neutrophils, and dendritic cells in lung adenocarcinoma. Importantly, the expression of HMMR was positively with the immune related gene expression, HMMR able to in uence the prognosis of lung adenocarcinoma patients partially via immune cell in ltration. We use the qRT-PCR and IHC assay show that HMMR was highly expression in NSCLC cells and lung cancer. We also found that HMMR was induced by hypoxia via HIF-1α-dependent transcriptional regulation. Finally, we show that depletion of HMMR signi cantly inhibits growth and migration of NSCLC cells. These observations indicate that HMMR may plays an crucial role in the progression and immune in ltration of lung adenocarcinoma.

Methods
The expression, prognosis, Clinical information and Immune in ltration analysis We mainly using the following databases to analysis the expression, prognosis, clinical information and Immune in ltration of HMMR in cancers. The detail information of databases used in this study are as follows: Oncomine database [14], TIMER [15],UALCAN database [16], Kaplan-Meier plotter [17] ,TISIDB The shRNA for HMMR was constructed employ pLKO.1 vector. The NSCLC related cells mainly purchased from The Kunming Institute of Zoology (KIZ) of Chinese Academy of Sciences (CAS). BEAS-2B and NSCLC related cells lines were cultured using the RPMI1640 medium, this medium contain the 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The shRNA for HMMR primer sequences are list follows: HMMR shRNA#1: AAACAGCTGGAAGATGAAGAAGGAA, HMMR shRNA#2: CAGCTGGAAGATGAAGAAGGAAGAA.

The function assays of HMMR In LUAD
The function assay of HMMR was performed as documented [25]. The function assay of HMMR mainly included the cell growth curve, colony information, transwell and wound healing experiment. The cell growth curve and colony information assay mainly employed to estimate the cell proliferation ability of NSCLC cells after the depletion of HMMR. The transwell and wound healing experiment mainly employed to estimate the cell migration ability of NSCLC cells after the depletion of HMMR.

Results
The mRNA of HMMR was elevated in human cancer In order to examine the mRNA of HMMR expression pattern in multifarious cancer, we employed the TIMER tools to analysis the expression of HMMR, the nd shown that HMMR was elevated in as follow cancer than the control group, it mainly includes BLCA, BRCA, CHOL, COAD, ESCA, HNSC, KIRC, KIRP, LIHC, LUAD, LUSC, PRAD, READ, STAD, THCA and UCEC ( Figure 1A). To further verify the results, we using the combine the TCGA and GTE databases to gure out the HMMR expression. As is show in Figure 1B, the HMMR was signi cantly up-regulation in ACC, BLCA, BRCA, CESC, COAD, DLBC, ESCA, GBM, HNSC, LIHC, LUAD, LUSC, OV, PAAD, READ, SKCM, STAD, THYM, UCEC and UVM cancer than match healthy tissue, while low expression in LAML and TGCT( Figure 1B). Besides, we found that the HMMR was highly expression in NSCLC cells lines observe in CCLE network tools ( Figure 1C). Above all, our ndings indicated that the HMMR may play an crucial roles in the progression of cancers.
The prognostic value of HMMR in pan-cancers Then, through analysis the TCGA databases, we nd the expression of HMMR has the great signi cance The expression pattern of HMMR in Immune and Molecular Subtypes of cancers Previous reports shown that the cancer could accord to the molecular characteristics divided into different immune and molecular subtypes. Thus, we adopt the TISIDB tools to analysis the expression of HMMR in immune and molecular Subtypes of different human cancer. Concerning the immune subtypes, the analysis results shown that HMMR has a differential expression pattern in cancers ( Figure 4A), a quintessential example should be cited LUAD, the HMMR was elevated in C1 and C2, while, it's low expression was observe in C3. With regard to the molecular subtypes, HMMR also display a distinctive expression pattern ( Figure 4B), such as, the HMMR was highly expression in C1 of LUAD, decreased in C3. To summarize, our result indicated that the expression pattern of HMMR has the tissue dependence speci city.

HMMR was highly expression in LUAD
Considering the signi cance of HMMR in cancers, next, we want to explore the relationship between the expression of HMMR and clinical features in LUAD. First, we nd that the RNA and protein of HMMR was signi cantly elevated in LUAD by perform the UALCAN tools analysis ( Figure 5A-5B). In addition, we also nd the expression of HMMR was increased with the elevation of stage nodal metastasis ( Figure 5C). The expression of HMMR not only highly expression in Ttp53-mutant patients but also upregulated in long term smoking patients. For the gender, the expression of HMMR signi cantly elevated in male patients than female ( Figure 5D-5F). Surprisingly, we nd higher expression of HMMR has the poor overall survival, poor OS, PFS and PPS ( Figure 5G-5I). Finally, we also evaluate the connection of among the expression of HMMR and MKI67 (famous cell proliferation index), the result suggested that HMMR expression was markedly positive with the expression of mki67 ( Figure 5K). These ndings strongly imply that the HMMR may involve in the progression of LUAD.

The gene mutation of HMME analysis
For explore the gene mutation information about the HMMR, we employ the cBioportal tools preform comprehensive analysis regard to the HMMR. The result shown that the mutation rate of HMMR reached 2.9% in NSCLC ( Figure 6A), we also examine the mutation type and base mutation in NSCLC, we found that Missense substitution and base G>A reached the highest mutation rate in NSCLC ( Figure 6B-6C). The results also display the mutation of HMMR in different NSCLC molecular Subtypes ( Figure 6D), In DNA level, gain and diploid was the main reason for the HMMR high expression in NSCLC ( Figure 6E). Overall, these results emphasize the gene mutation of HMME may be contribute to the HMMR elevated in NSCLC.
Analysis DNA methylation of HMMR in LUAD Next, we further explore the connection of among the DNA methylation of and the expression of HMMR, as is shown in the ( Figure 7A), we display varies methylation sites locate in the promoter of HMMR by heat map. Through preform the comprehensive and detailed analysis about the DNA methylation state of HMMR, we nd the methylation level was signi cantly low expression in the LUAD, as well as the methylation level was gradually decreased with the elevation of pathological stage in LUAD ( Figure 7B-7C). The methylation level was gradually decreased observe in the different lymph node metastasis state, race and tp53mutation ( Figure 7D-7G). The overall survival analysis display that elevated the methylation level of HMMR has better prognosis ( Figure 7H). These nds suggested that DNA hypomethylation may be reason for the abnormal up-regulation in LUAD.

Analysis The function of HMMR in LUAD
In order to exploration the potential signi cance of HMMR in LUAD progression. We rst employ the linkedomics database to examine the positive gene with HMMR. As is show in the Figures 8A-8C, we choose the most positive gene(r>0.7) display in the form of heat map. Next, we perform the GO and KEGG analysis. The biology processes mainly involve in DNA replication, chromosome segregation, cell division and protein localization (Figures 8D), for KEGG enriched results, the pathway mainly including cell cycle, P53 signaling pathway, non-small cell lung cancer and FOXO signal pathways ( Figures 8E). In addition, we analysis the most relevant gene of HMMR by employed the Genemania, the result indicated that xx gene were most relevant, these gene functions mainly involve in cell cycle ( Figure 8F). We also using STRING databases to construct the protein interaction network, the protein of interaction with HMMR mainly including PLK4, CD44, AURKA, NEK2, CDK1, FAM83D( Figure 8G). To explore the HMMR related signal pathway in progression of LUAD, we employ the GSEA software perform KEGG pathway enrich.
The analysis results show that upregulation HMMR expression mainly involve in the IL2-STAT5 signaling pathway, IL6 JAK-STAT3-signaling pathway, Interferon-γ response, TNFα signaling pathway( Figure 8H). These results suggested that HMMR plays an pivotal roles in the immune response regulation in LAUD.

Hypoxia induced the HMMR highly expression in LUAD
The GSEA enrich results show that HMMR mainly participate in hypoxia process ( Figure 9A), we guess the HMMR expression whether by hypoxia induced. To test this hypothesis, we employ the JASPAR and AnimalTFDB tools to predict the HIF1α HRE in the promoter of HMMR. Next, we analysis the relationship among HMMR expression and HIF1α expression in TCGA LUAD, the result indicated that HIF1α signi cantly positive with the expression of HMMR ( Figure 9B). In addition, we using the different condition to treat the NSCLC cells, such as hypoxia and cocl2, cocl2 as an chemical inducer of HIF1α, through these condition treat, compared with normoxia, we nd the hypoxia and cocl2 was able to increase the expression of HMMR ( Figure 9C). We further analysis the relationship among HMMR expression and HIF1α downstream targets gene expression ( eg LDHA, PGK1, SLC2A1, GBE1) in TCGA LUAD, the results suggested that HMMR expression was strongly positive with these gene (Figure 9D-9G). Beside, we nd that the RNA of HIF1α was signi cantly elevated in LUAD by perform the UALCAN tools analysis ( Figure 9H). We also nd the expression of HIF1α was increased with the elevation of pathological stage and lymph node metastasis ( Figure 9I-9J). Surprisingly, we nd higher expression of HIF1α has the poor overall survival ( Figure 9K). These ndings strongly imply that the HMMR expression may be induced by the hypoxia.

The prognostic of HMMR based on the different immune cells
To explore the prognostic of HMMR based on the different immune cells in LUAD. We using the KMplot database analysis found that elevated the HMMR expression as well as enriched the B cells, CD4+ T-cells, CD8+ T cells, macrophages, natural killer T cells, regulatory T cells cohort had a poor prognosis ( Figures   11A-L). These data indicated that the immune cell in ltration could signi cantly affect the e prognostic of HMMR in LUAD.

Depletion of HMMR inhibits the cell proliferation and migration of NSCLC cells
To further de nite the function of HMMR in NSCLC progression, we rst using the IHC and qRT-PCR assays examine the expression of HMMR in different NSCLC cells lines, the results show that HMMR signi cantly elevated in the lung cancers and NSCLC cells (Figures 12A-12B), especially in A549 and H1299 cells. Next, we construction the HMMR knockdown cell lines in A549 and H1299 cells as well as employ the qRT-PCR and Western blot examine the knock down e ciency( Figures 12C-12D), through the loss of functions shown that depletion of HMMR inhibits the cell growth and migration of NSCLC cells (Figures 12E-12H). These ndings display that HMMR boost the cell growth and migration of NSCLC cells.

Discussion
Normal hypoxic environments play a key role in pathologic and physiologic conditions [26]. Previous study have indicated that hypoxia can induce the expression of varies oncogene and promote the cancer development [26]. Hypoxia was necessary for the self-renewal of cancer stem cells [27]. In addition, numerous results have shown that HIF-1α can also directly upregualtion the expression of VEGFR1 and activation cancer-related signaling pathway, result in the progression of cancers. While, the expression of HMMR whether induced by hypoxia need to be further explored.
HMMR was report plays crucial roles in the progression of human cancer [13] . For example, studies have shown that HMMR was elevated in gbm, high expression of HMMR could boost the self-renewal of GSC [13]. In breast cancer, elevated the levels of HMMR are related to poor outcome [28]. At the present time, important evidence is still lacking about the signi cance of HMMR in the progression of LUAD. We also adopt STRING databases to construct the protein interaction network, the protein of interaction with HMMR mainly including PLK4, CD44, AURKA, NEK2, CDK1 and FAM83D. It has been show that PLK4 was elevated in NSCLC and high expression was related to the tumor size and lymph node metastasis [29]. Recent one study found that CD44 able to elevated the expression of PDL1 by regulation the CD274 transcription, result in restrain the tumor-intrinsic function of PD-L1 [30]. It has been report that the alisertib, as an inhibitor of AURKA, combined with PD-L1 blockade able to employ treat the mammary tumors [31]. NEK2 was report that elevated in lung cancer, regulated by EGFR mutation, over-expression of NEK2signi cantly promotes the cell growth and induce the cell cycle progression in NSCLC cells [32]. The above results indicate that HMMR may plays a central regulate role in the cancer progression.
In this study, we employ varies bioinformatics database conduct a comprehensive and detailed analysis about the expression, prognosis, clinical signi cance, Immune in ltration and biological function in LUAD. We nd the HMMR was highly expression in various cancer tissues, such as ACC, BRCA, KIRC, KIRP, LIHC, LUAD, LUSC, OV, SKCM, TGCT, THCA and THYM high expression of HMMR was signi cantly correlated with the poor prognosis, tumor stages and lymph node metastasis of LUAD.
Hypoxia microenvironment and epigenetic modication play an crucial role in control gene expression [33]. Therefore, Hypoxia microenvironment and DNA hypomethylation may be a cause for HMMR elevated in LUAD. Here, we rst revealed that elevated the HMMR expression was relation to the immune in ltration of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells. Moreover, HMMR expression was also positively relation to various immune check points related gene was observed in lung cancer. Interestingly, HMMR affect the existence state of LUAD patients partially via immune cell in ltration. Our GSEA enrichment analysis results showed that HMMR is closely associated with cell immune-related pathways such as interferon γ response, TNF α signaling pathway, IL6-JAK-STAT3 signaling pathway and IL2-STAT5 signaling pathway. These ndings suggested that HMMR could be as a immune-related biomarker in LUAD. Finally, we nd that knockdown of HMMR signi cantly reduced the ability of proliferation and migration of NSCLC cells. These results demonstrate that HMMR promote the cell growth and migration of NSCLC cells.
In our study, we demonstrated HMMR was elevated in LUAD and positively relation to poor prognosis. We nd the hypoxia microenvironment and DNA hypomethylation able to up-regulation of the HMMR expression. Moreover, our nding also suggested that HMMR promote the LUAD progression by boosts the ability of cell proliferation and migration in LUAD.

Data Availability
The original data was display in this study are included in the article.       KEGG Pathways Networks of HMMR in LUAD (a)The differentially expressed gene was related to display by Volcano plot (b-c) The positively and negatively related to h HMMR display by Heat maps (d) Analysis the Biology process of HMMR (e) Analysis the KEGG pathway of HMMR (f) Employed the GeneMania to construction the gene interaction network of HMMR (g) Employed the STRING to construction the protein interaction network of HMMR (h) The signaling pathway enriched employed the GSEA software.  Prognostic Potential of HMMR Expressions in Different Tumors Based on Immune Cells (a-l) The relationship between the HMMR expression and OS based on immune cell subgroups emploted the KMplot *p < 0.05, **p < 0.01, ***p < 0.001.