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 find 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 figure out the HMMR expression. As is show in Figure 1B, the HMMR was significantly 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 findings 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 find the expression of HMMR has the great significance for assessment the prognostic value of different cancers. High expression of HMMR was associated with poor OS in ACC, COAD, KICH, KIRC, KIRP, LGG, LIHC), LUAD, MESO, PAAD, PCPG, PRAD, THYM and UVM (Figure 2A), and related to poor DSS in KIRP, LIHC, MESO, SARC and THCA (Figure 2B), and related to poor DFS in ACC, KICH, KIRC, KIRP, LGG, LIHC, LUAD, MESO, PRAD, THCA and UVM (Figure 2C), and linkage to poor PFS in ACC, KICH, KIRC, KIRP), LGG, LIHC, LUAD, MESO, PCPG and UVM (Figure 2D).
Analysis the pathological stage in different cancer
We employ the GEPIA tools to examine the relationship between the expression of HMMR and the human cancers pathological stage. Interestingly, we find that the expression of HMMR was markedly positive with the pathological stage of ACC, BRCA, ESCA, NHCS, KICH, KIRC, KIRP, LAML, LAML, LIHC, LUAD and LUSC (Figures 3). These results suggested that HMMR may as an oncogene in human cancers.
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 specificity.
HMMR was highly expression in LUAD
Considering the significance of HMMR in cancers, next, we want to explore the relationship between the expression of HMMR and clinical features in LUAD. First, we find that the RNA and protein of HMMR was significantly elevated in LUAD by perform the UALCAN tools analysis (Figure 5A-5B). In addition, we also find 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 significantly elevated in male patients than female (Figure 5D-5F). Surprisingly, we find 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 findings 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 find the methylation level was significantly 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 finds 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 significance of HMMR in LUAD progression. We first 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 significantly 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 find 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 find that the RNA of HIF1α was significantly elevated in LUAD by perform the UALCAN tools analysis (Figure 9H). We also find the expression of HIF1α was increased with the elevation of pathological stage and lymph node metastasis (Figure 9I-9J). Surprisingly, we find higher expression of HIF1α has the poor overall survival (Figure 9K). These findings strongly imply that the HMMR expression may be induced by the hypoxia.
Analysis the correlation between the expression of HMMR and Immune infiltration in LUAD
We employ the TIMER databases analysis examine the relationship between the expression of HMMR and Immune infiltration in LUAD, the results show that gene copy number change of HMMR was significantly affect the immune infiltration level of B cells, CD4+ T cells, CD8+ T cells, Macrophage cells, dendritic cells and neutrophils in LUAD (Figure 10A). Next, we used TIMER to analyze the relationship between the HMMR level and immune infiltration levels in LUAD. The results showed that HMMR expression is markedly positively associated with B cells (r=0.47, p=7.69e-30), CD4+ T cells (r=0.71, p=1.71e-78), CD8+ T cells (r=0.51, p=1.22e-35), Macrophage cells(r=0.44, p=8.88e-26), dendritic cells (r=0.84, p=5.94e-139) and neutrophils (r=0.77, p=3.54e-103) in LUAD (Figure 10B). To evaluate the relationship among HMMR expression and immune checkpoints related gene, for instance: CD274, CD279, CTLA4, LAG3, PDCD1LG2, TIGIT and HAVCR2. The results show that HMMR expression was memorably positively relation to CD274(r=0.34, P=9.3e-15), CTLA4(r=0.556, P=5), PDCD1(r=0.56, P=0), TIGIT(r=0.71, P=0), PDCD1LG2 (r=0.67, P=0), HAVCR2 (r=0.71, P=0) and LAG3 (r=0.39, P=0) in LUAD (Figure 10C). These results memorably indicated that HMMR play an crucial roles in tumor immune infiltration regulator in LUAD.
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 infiltration could significantly affect the e prognostic of HMMR in LUAD.
Depletion of HMMR inhibits the cell proliferation and migration of NSCLC cells
To further definite the function of HMMR in NSCLC progression, we first using the IHC and qRT-PCR assays examine the expression of HMMR in different NSCLC cells lines, the results show that HMMR significantly 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 efficiency(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 findings display that HMMR boost the cell growth and migration of NSCLC cells.