Prognostic value of members of the HSP70 family in Hepatocellular Carcinoma

BACKGROUND: Despite multiple functions in the disease, the prognosis of the heat shock protein (HSP) 70 family in Hepatocellular Carcinoma (HCC) remains unclear. METHODS: The UALCAN database has provided information about the expression level of HSP70 family members in both HCC and in normal tissues. Overall survival (OS) was conducted by Kaplan-Meier plotter (KM plotter). Gene ontology (GO) and KEGG pathway enrichment analyses were accomplished by DAVID database. GeneMANIA and STRING were applied to construct gene-gene and protein-protein interaction (PPI) networks. RESULTS: From the UALCAN database, we found that the expression levels of eight members of the HSP70 family in HCC patients were higher than in normal liver tissues. These eight members were, namely, HSPA1A, HSPA1B, HSPA1L, HSPA2, HSPA5, HSPA6, HSPA8 and HSPA9. From KM plotter database, high expression of HSPA1A, HSPA1B, HSPA6 and HSPA8 has been observed to be associated with worse OS in patients with HCC (hazard ratio [HR] =1.49, 95% condence interval [CI]: 1.03-2.15, P=0.031; HR=1.49, 95%CI: 1.05-2.12, P=0.026; HR=1.53, 95%CI: 1.06-2.2, P=0.021 and HR=1.81, 95%CI: 1.21-2.71, P=0.0036, respectively). We also found that the high expressions of HSPA1A, HSPA1B, HSPA6 and HSPA8 were related to unfavorable OS of HCC patients with other factors such as clinical stage, gender, race and hepatitis virus. From GO and KEEG analysis, we detected a close relationship between these eight genes and Biological process, Cellular component, Molecular function and cancer related signaling pathways. The PPI network and GeneMANIA results showed that there was a strong co-expression relationship between the protein homology and the genes. CONCLUSIONS: According to previous studies and analysis, when HSPA1A,


Introduction
Amongst one of the top six cancers prevalent across the world, hepatocellular carcinoma (HCC) is predicted to cause 782,000 deaths worldwide. The number of newly diagnosed HCC cases keeps growing at a rate of 841,000 as per 2018 [1].The data from Cancer Statistics (2015), have shown that HCC is ranked as the fth most aggressive and common cancer types across China, with about 466 thousand new cases and 422 thousand deaths annually. However, the prognosis of HCC patients has remained to be unsatisfactory [2]. Even though during the past few years, the cancer diagnosis and treatment techniques have advanced considerably, the ve-year OS performance of HCC patients still remains to be improved. Hence, there is an urgent need to conduct further studies on the initiation and developmental mechanisms of HCC, and to identify more effective drug targets and prognostic biomarkers to enhance the prognosis in patients using more speci c therapeutic approaches.
The HSP70 family is composed of about 70-kDa molecular chaperones and has a highly conserved domain structure: including 15-kDa substrate-binding domain, 45-kDa N-terminal ATPase domain, Cterminal domain and 10-kDa helical lid domain [3,4]. The HSP70 family is activated by stress, heat, apoptosis, and co-chaperones, which play an important role in protein folding, maintaining protein homeostasis, and increasing the survival of cells under various pressures [5,6].The main eight members of HSP70 have been studied to be closely related to tumors, including HSPA1A (also known as HSPA1), HSPA1B (also known as HSP70-2), HSPA1L, HSPA2, HSPA5, HSPA6 (also known as HSP70B), HSPA8 and HSPA9 [7]. Previous studies suggested that HSP70 was highly expressed in numerous cancer types, including liver, colorectal, breast, prostate and bladder carcinomas [8][9][10][11][12]. A prognostic effect has been detected in highly expressed HSP70 proteins [13,14]. It is evident that protein of the HSP70 family is closely associated with tumors, but whether HSP70 has a prognostic impact on HCC is still unclear.
In this study, analysis data from the KM plotter database (http://kmplot.com/analysis/index.php? p=service&cancer=liver accessed February 16, 2020) was collected to evaluate the OS performance in HCC patients in correlation with expression of HSP70. This database currently includes the clinical outcome and expression levels of several types of cancers [15][16][17][18]. The information collected from this database might as well be applied to the analysis of speci c genes which may have an in uence on the overall survival (OS) of the HCC patients. In the past, a large number of researchers have identi ed and validated numerous genes which affect the mechanism of HCC, with the usage of the KM plotter database. Therefore, the KM plotter database is applied in our study for the evaluation of HSP70's potential to be used as drug targets and prognostic biomarkers.

Results
High expression of eight members of the HSP70 family in HCC patient.
The UALCAN web portal was applied to detect the expression levels of the eight members of the HSP70 family in normal and in HCC tissues, respectively. Through the UALCAN, we observed the high expression of HSPA1A, HSPA1B, HSPA1L, HSPA2, HSPA5, HSPA6, HSPA8 and HSPA9 mRNA in 371 patients with liver cancer, as compared to their lower expression in 50 normal liver tissues, which was statistically signi cant (P<0.05, Fig. 1A-H).
Prognostic value of members of HSP70 in HCC from KM plotter database.
Firstly, the prognostic effect of HSPA1A was assessed using the information collected from the dataset, with the valid gene ID 3303(HSPA1A). Curves that described the survival performance were drawn as demonstrated in Fig. 2A (n=364). It turned out that highly expressed HSPA1A would signi cantly lead to a worse OS performance in patients with HCC ([hazard ratio (HR) =1.49; 95% con dence interval (CI): 1.03-2.15; P=0.031]).
Then, the prognostic effect of HSPA1B was examined using the information collected in this database, with the valid gene ID 3304(HSPA1B). It turned out that highly expressed HSPA1B would signi cantly lead to a worse OS performance in patients with HCC (HR=1.49; 95%CI: 1.05-2.12; P=0.026; Fig. 2B).
Subsequently, the prognostic effect of HSPA6 was also examined using the information collected from the database, with the valid gene ID 3310 (HSPA6). It turned out that highly expressed HSPA6 would signi cantly lead to a worse OS performance in patients with HCC (HR=1.53; 95%CI: 1.06-2.2; P=0.021; Fig. 2F).
Furthermore, the prognostic effect of HSPA8's was examined using the information collected from the database, with the valid gene ID 3312(HSPA8). It turned out that highly expressed HSPA8 would signi cantly lead to a worse OS performance in patients with HCC (HR=1.81; 95%CI: 1.21-2.71; P=0.0036; Fig. 2G).
In contrast, no signi cant correlation has been detected between the high expression of HSPA1L, HSPA2, HSPA5 and HSPA9 and OS performance in patients with HCC. Their ID, HR, CI and Pvalues are as follows: The HSPA1L gene ID was 3305, HR =1.2 (95% CI: 0.84-1.72), P=0.32 ( Figure.  In summary, a signi cant correlation was detected between the members of the HSP70 family, showing high expressions of HSPA1A, HSPA1B, HSPA6 and HSPA8 were associated with OS of HCC patients, while this correlation was not signi cant between the OS performance of HCC patients and the high expression of HSPA1L, HSPA2, HSPA5 and HSPA9.
Prognostic value of members of HSP70 in HCC from clinicopathological characteristics .
Besides, we further analyzed the related factors in uencing the prognosis of HCC from clinicopathological characteristics. Including clinical stage (  table 3 illuminated that high expression of HSPA6 and HSPA8 was related to unfavorable OS of HCC patients with AJCC-T 1; High expression of HSPA1A with AJCC-T 1 showed a similar outcome. As shown in Table  4, High expression of HSPA8 was relevant to poor OS of HCC patients with no vascular invasion; On the contrary, High expression of HSPA8 showed a good OS from HCC patients with micro vascular invasion.
As presented in Table 5, High expression of HSPA1A, HSPA1B, HSPA1L and HSPA6 had poor prognosis in female HCC patients; High expression of HSPA8 was associated with a poor prognosis for HCC male patients; In contrast, high expression of HSPA8 was associated with a better prognosis for HCC female patients. In GO functional annotation analysis and KEGG pathway analysis for members of HSP70.
Functional enrichment analysis together with the corresponding genes was investigated for the members of HSP70, with the usage of KEGG and GO analyses, conducted with DAVID, in which the biological processes (BPs), cellular components (CCs) and molecular functions (MFs) were predicted via GO analysis. According to the analysis, BPs included such as GO:1903265  with each other in a complex manner. Besides, the co-expression of the HSP70 sub-members was also implied.

Discussion
As discussed previously, hepatocellular carcinoma (HCC) is a common cancer type, characterized with high incidence rates and severe aggressiveness. HCC patients generally have poor performance in veyear OS [1]. HSP70 was reported to be highly expressed in a number of cancer types, like esophagus, bladder, prostate, lung and colorectal cancers [9,12,[31][32][33]. Chuma et al have found that HSP70 had the potential to be applied in the development of a sensitive marker to diagnose HCC in an early stage, from non-cancerous liver tissues to pre-cancerous lesion [34]. Gehrmann et al found that amongst 143 patients with serum HSP70, including 47 liver cancer patients, 46 cirrhosis patients and 50 chronic hepatitis patients, a signi cantly higher expression level of HSP70 was detected in the tissues of patients with liver diseases as compared to healthy individuals [35]. Our study also found the eight major sub-members of the HSP70 family (HSPA1A, HSPA1B, HSPA1L, HSPA2, HSPA5, HSPA6, HSPA8 and HSPA9) were highly expressed in 371 patients with HCC, which were consistent with the above reports. It is therefore suggested that members of HSP70 family may be used as a diagnostic indicator for treating HCC in the future.
We previously found that eight members of the HSP70 family were highly expressed in HCC. Next, we will discuss the prognostic value of the high expression of HSP70 family members in HCC. First, HSPA1A has been shown to be a biomarker for tumor prediction. Jakobsson et al analyzed 53 patients with high-grade serous ovarian cancer using a protein mass spectrometer and found that unmethylated HSPA1 may be a prognostic marker for high-grade serous ovarian cancer patients[36]. Chen et al analyzed the expression of LLIM and SH3 protein 1 (LASP1) and HSPA1A in head and neck squamous cell carcinoma (HNSCC) by quantitative PCR and Western blot, and found that LASP1 and HSPA1A were highly expressed in HNSCC and were associated with poor OS of HNSCC patients[37]. HSPA1A has not been reported in the prognosis of HCC. In our present study, the high expression of HSPA1A in HCC patients tend to present a worse OS performance, and especially for HCC patients with Stage I+II, Grade 1, AJCC-T 2, white and female. Previous researches have reported the relationship between HSPA1B expression and prognosis in malignant tumor patients. Song's study indicated that Hepatitis B virus regulated HCC cell growth through microrna-340-5p activation of transcription factor 7 (ATF7)/HSPA1B axis, leading to increased cell proliferation and decreased apoptosis in HCC [38]. Another study also showed the relationship between lung cancer patients labeled with single nucleotide polymorphisms and the OS performance of 330 nonsmall cell lung cancer patients was evaluated, and the ndings indicated the association between the variation of functional HSPA1B and the incidence risk and prognosis of lung cancer [39]. In our study, a high expression of HSPA1B was markedly related to an unfavorable OS in HCC patients and in particular for female and white patients, but was not associated with Stage I-IV, Grade 1-4 , AJCC-T 1-4 and hepatitis virus HCC patients. Some studies had found that HSPA6 mRNA expression was signi cantly related to the prognosis of malignant tumors. Yang et al showed the high expression of HSPA6 with earlier recurrence of HBV-related HCC [40]. In our present study, high expression of HSPA6 mRNA was associated with worse OS for HCC patients, especially for HCC patients with Stage I, Grade 2, AJCC-T 1, female and hepatitis virus. So far, there have been few reports on the prognosis of HSPA8.In our study, a high expression of HSPA1B was connected with a poor OS in patients with HCC in particular for male, white, hepatitis virus, Stage I, Grade 3 and AJCC-T 1. Overall, it is fair to conclude that highly expressed HSPA1A, HSPA1B, HSPA6 and HSPA8 mRNA were signi cantly likely to lead to poor OS performance in patients with HCC.
After discussing the prognostic value of HSP70 family members, we next study the functional enrichment analysis and biological behavior of HSP70 family members. HSP70 is secreted from tumor cells and is of great importance in signaling, especially when it comes to immune and in ammatory responses [41]. HSP70 proteins have been shown to have regulating effects on apoptosis and proliferation of cancer cells, which means they can be applied to the progression and prognosis of HCC as well as other types of malignant tumors [34,42]. According to the studies of Udono et al, tumor-speci c antigenic polypeptides in the cells of malignant tumors are bound via HSP70, so that the immune system can recognize them more easily [43]. From GO enrichment analysis, we found that HSP70 was involved in cell composition, biological functions, molecular function and signaling pathway by regulating how cells respond to heat and oxidative stress, the apoptosis of cells, the ATP metabolic process, antigen processing and presentation and so on. Our report is also consistent with the above research.
Next, we study the signaling pathways that are mainly involved in HSP70 family members. It has been reported previously that HSP70 is involved in several pathways, including MAPK signaling pathway, necrosis tumor factor-mediated signaling pathway, the apoptotic signal pathway, etc. Stankiewicz et al found that by primarily oppressing the activation of Bax, HSP70 inhibited cell apoptosis induced by extracellular heat, thus preventing mitochondrias from releasing pro-apoptotic factors [44]. Other study showed two major mechanisms through which the cell apoptosis in colon and pancreatic cancers was oppressed by HSP70: lysosomal stabilization and cytosolic calcium attenuation [45]. Another report showed that in the TNF-induced cell apoptosis of primary culture of human broblasts, IMR90, there was speci c interference of HSP72 with the Bid-dependent apoptotic pathway by preliminarily inhibiting the stress kinase c-jun N-terminal kinase (JNK)[46]. Zhe et al had showed that by activating TLR4 and TLR2, and subsequently activating the intracellular JNK1/2/MAPK signaling pathway, the cell proliferation of HCC tumors can be promoted by extracellular HSP70/HSP70-peptide complexes (HSP70-PCs) [47]. Our study found that HSP70 was involved in the negative regulation of mitochondrial outer membrane permeabilization, involved in apoptotic signaling pathway, negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway, negative regulation of extrinsic apoptotic signaling pathway in absence of ligand, positive regulation of tumor necrosis factor-mediated signaling pathway and MAPK signaling pathway. It is basically consistent with the signaling pathways involved by HSP70 as reported above.
Finally, we further discuss the potential therapeutic value of the HSP70 family. It has been also reported that the vaccine constructed by HSP70 is used to treat liver cancer in mice model expressing alphafetoprotein(AFP). Such as, constructing a therapeutic peptide vaccine through connecting AFP epitope with HSP70 function peptide to get HSP70-P/AFP-P. Then, the HSP70-P/AFP-P was administered into BALB/c mice. The natural killer cells and CD8 + T cells in BALB/c mice reacted strongly and had a protective effect on the AFP-producing tumors. Therefore, it is safe to assume that HSP70/AFP-P has a huge potential in the application of the treatment of patients diagnosed with AFP-expressing malignant tumors [48].
Certainly, there are a number of limitations in the present study. Firstly, the number of patients included in liver cancer research is just over 300, which is a relatively small sample size. Secondly, the research only focused on the differences between tissues of HCC patients and normal tissues, and did not investigate further based on gender, age, etiology and clinical stage of patients with HCC. Therefore, a strati ed survival analysis for HCC should be conducted with larger and separate samples in the future studies. Our result for this study is solely obtained by using bioinformatics tools, without any experimental veri cation, so it needs to be veri ed by large random multi-center sample studies.
Taken together, it can be concluded that HSP70 members, HSPA1A, HSPA1B, HSPA6 and HSPA8, have the potential to be used as effective biomarkers to enhance the OS in patients with HCC. Nevertheless, this result will also require further con rmation and validation, as it has been obtained solely with the usage of bioinformatics approaches.

Authors' contributions
Haixing Jiang designed the study and performed bioinformatics analysis. Ziyu Liang analyzed the data and wrote the manuscript. Shanyu Qin and Kang Liu analyzed the data. All authors read and approved the nal manuscript.

Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Ethical approval and consent to participate Not applicable.

Patient consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests      PPI and gene-gene interaction networks were constructed by STRING and GeneMANIA (A-B). It showed a strong co-expression relationship between the protein homology and the genes.