MMP1 is a Promiseful Prognostic Biomarker and Correlating with Immune Inltrates in Hepatocellular Carcinoma

Background: Recombinase-aided amplication(RAA) is a new, simple, and ultrafast isothermal molecular diagnostic technique performed within 30min at 39°C–42°C.In this study, we evaluated the clinical performance of four duplex RAA kits for hepatitis B virus(HBV), human adenovirus 3(HAdV3), human adenovirus 7(HAdV7), and Bordetella pertussis and one duplex reverse-transcription RAA (RT-RAA) kit for respiratory syncytial virus (RSV). Methods: A total of 392 sera and 374 respiratory tract samples were collected from ve institutions in four China regions. Each RAA kit’s sensitivity and specicity were compared with those of real-time quantitative polymerase chain reaction(qPCR),real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR), or sequencing. Results: Compared with qPCR or qRT-PCR, the sensitivities of HBV RAA,RSV RT-RAA, and B.pertussis RAA were 97.55%,96.67%, and 100%,respectively,and all of the specicities were 100%.The total coincidence rates were 97.78%(383/392,95%CI:95.63%–98.85%),97.70%(212/217, 95%CI:94.57%–99.16%), and 100% (60/60,95%CI:92.80%–100%),respectively. The Kappa values were 0.977,0.947, and 1,respectively(P<0.05).Regarding the sequencing, the sensitivities of HAdV3 RAA and HAdV7 RAA were 100% and 97.37%, respectively, and all specicities were 100%.The total coincidence rates were 100% (97/97,95%CI:91.58%–100%) and 98.97%(96/97,95%CI:94.39%–99.82%),and the Kappa values were 1 and 0.978 (P<0.05),respectively. Conclusions: With comparable clinical performance, these RAA kits are suitable assays for rapidly detecting pathogens in resource-limited laboratories. + T Monocytes, tumor-associated macrophages (TAMs), M1 and M2 macrophages, neutrophils, dendritic cells, Tregs and exhausted T cells. Result attained and


Introduction
Hepatocellular carcinoma (HCC), remains the third leading cause of tumor-related death worldwide [1] and a signi cant health burden across the board. Chronic viral hepatitis B induced cirrhosis leading to HCC is the most common progress pattern in liver cancer. The top three risk factors for HCC are alcohol consumption, chronic viral hepatitis C and non-alcoholic fatty liver disease [2] . Although great strides have been made in advancing early diagnosis, surgical technology [3] , targeted treatment [4,5] and immune therapy [6,7] , high rate of recurrence and mortality remain a challenge due to most patients presenting with unresectable lesions or distant metastasis at time diagnosis, leading to a poor prognosis: a 5-year overall survival rate of only 10-18% [8][9][10] .
Matrix metalloproteinases (MMPs), a family of zinc-dependent endoproteases, is signi cantly related with extracellular matrix degradation by destroying diverse structure proteins, which plays a vital role in apoptosis, angiogenesis, and immune response [11][12][13] of the tumor microenvironment. Notably, MMP-2, alongside MMP-9, are well known as the most common progression markers correlated with various tumor invasion and metastasis, especially in HCC [14,15] .
Although MMP1 has been more commonly reported to be expressed in normal liver tissues [16] , it has also demonstrated an elevated capacity of invasion and migration in HCC by extracellular matrix (ECM) degradation in the epithelial-mesenchymal transition (EMT) [17] . MMP1 can be expressed in a wide range of cells including stromal broblasts, macrophages, endothelial cells and epithelial cells, having a low positive rate under normal conditions. However, it's expression can be elevated in malignant tumors with poor prognosis in various cancers (ovarian, liver, lung, gastric, colorectal, and prostate) [18][19][20][21][22][23] . While some studies have reported the interrelation between MMP1 and HCC, its distinct role in prognosis and the associated tumor-immunity are still unclear. Additionally, tumor in ltration immune cells (TIICs) and tumor-associated immune microenvironment are key areas of interest of current research [24,25] . Immune cells may respond to the tumor progression and metastasis through a multitude of pathways and interactions in HCC. The suppression of HCC immune microenvironment facilitates immune tolerance and escape by variant mechanisms [26] . MMPs play an important role in bladder cancer metastasis promotion through the B cell induced signaling pathway [27] and their upregulation by tumor-associated macrophages contributes to tumor in ltration and metastasis in various carcinomas [28][29][30] , indicating their potential functions in tumor-immunity microenvironment. However, what and how MMP1 in uences the immune cells and tumor-associated microenvironment still needs exploration.
This research was carried out to investigate the prognostic potential of MMP1 and its relationship with TIICs' biomarkers in HCC. Several online bioinformation analysis tools of public databases were used to examine the MMP1 expression levels (Oncomine and Tumor Immune Estimation Resource (TIMER) databases) and assess its prognostic potential (Kaplan-Meier plotter and Gene Expression Pro ling Interactive Analysis (GEPIA) databases), along with the correlation between MMP1 and TIIC biomarkers (TIMER). We demonstrated that MMP1 may be a major independent predictor of prognosis of HCC and revealed the latent interrelationship and mechanism between MMP1 and TIIC biomarkers in patients with HCC.

Materials And Methods
Oncomine Database Based Analysis Oncomine (https://www.oncomine.org) is currently the world's largest database of oncogene chip and features an integrated data mining platform, designed to mine cancer gene information. Oncomine, which integrates RNA and DNA-seq data from GEO, TCGA and published literature sources, has the most comprehensive pro le of cancer mutations, gene expression data and related clinical information that can be used to identify new biomarkers or therapeutic targets [31] . In our study, the MMP1 gene was selected and its expression levels were analyzed in tumor and normal tissues using the Oncomine database. The difference in results was considered statistically signi cant for p-value < 0.05, with fold change > 1.5. The threshold value of gene rank was set to "top 10%" and data type to "all".

Kaplan-Meier Plotter Database Analysis
The Kaplan-Meier Plotter database (http://kmplot.com/analysis) was constructed based on gene microarray and RNA-seq data from GEO, EGA, and TCGA public databases, and assessed the effect of 54, 675 genes on survival rate in 21 cancer types [32] . In order to investigate the relationship between MMP1 expression and the prognosis of patients with liver, breast, ovarian, lung and gastric cancers, Kaplan-Meier Plotter database was utilized to do the Cox regression analysis and hazard ratios (HRs) and p-values from a log-rank test. The parameters were as follow: Affy id/Gene symbol: MMP1; patients split by auto-select best cutoff; HR: yes; 95% con dence interval (CI): yes; with the remaining settings set to their default values.

GEPIA Database Analysis
GEPIA database (http://gepia.cancer-pku.cn/index.html), a public web server for tumor and normal gene expression pro ling and interactive analysis based on the data from Genotype-Tissue Expression (GTEx) and TCGA database [33] , was utilized in this study to analyze the association between MMP1 expression and patient prognostic potential in a variety of cancers. As a complement to the analysis by the Kaplan-Meier Plotter database, we charted survival curves for overall survival (OS) and disease-free survival (DFS) via GEPIA using the log-rank and Mantel-Cox tests. The Spearman's rank correlation coe cient was calculated with default parameters.

TIMER Database Analysis
The TIMER 2.0 database (http://timer.comp-genomics.org), incorporating 10,009 samples across 23 cancer types from TCGA, is a comprehensive web resource for the systematical evaluation of the clinical relevance of tumor-immune in ltrates and differential gene correlation analysis [34] . We selected MMP1 under the gene expression module with the default parameters to obtain heatmaps visualizing the correlation of its expression with different immune in ltrate levels in different cancer types by the purity-adjusted spearman's test. Of the various TIICs, B cells, CD4 + T cells, CD8 + T cells, macrophages, neutrophils and dendritic cells [35,36] were selected for investigation in this study to assess the relationship between MMP1 and tumor-immunity in HCC based on insight from previous studies. Furthermore, correlation analysis was conducted to investigate the relationship between MMP1 expression and different biomarker genes of TIICs as reported in other papers [37,38] , including B cells, CD8 + T cells, Monocytes, tumor-associated macrophages (TAMs), M1 and M2 macrophages, neutrophils, dendritic cells, Tregs and exhausted T cells. Result delineation was attained through Spearman's rho and p-value adjusted by purity.

Sangerbox database analysis
Sangerbox database (http://www.sangerbox.com) is a tool to facilitate a variety of biological information analysis, visualization mapping and convenient data download functions, based on TCGA and GEO databases [39] . We investigated the correlation between MMP1 expression levels and the tumor mutational burden (TMB) and microsatellite instability (MSI) in pan-carcinomas using pearson's method.

Statistical analysis
All statistical analysis and graphing was performed with R (version 4.0.3) and STATA software (College Station, TX: Stata Corp LP, USA). Survival curves were created by the GEPIA and Kaplan-Meier plots database based on R. Forest plots were generated by meta-analysis in metan module of STATA. The relationship between MMP1 expression and TMB and MSI in pan-cancers was evaluated by Pearson's correlation, while the sperman's correlation was used to assess the relationship between MMP1 expression and TIICs, as well as biomarkers. A p value < 0.05 was considered statistically signi cant. The correlations were de ned as follows: 0.00-0.19 (none), 0.20-0.39 (low), 0.40-0.59 (moderate), 0.60-.079 (high), ≥ 0.80 (very high) [40] .

MMP1 expression in HCC and other carcinomas
MMP1 expression inhibition would ultimately contribute to prevention of HCC progression, according to a previous study [41] . In order to con rm the carcinogenesis of MMP1 in HCC, we utilized the Oncomine database to evaluate the levels of MMP1 expression in both tumor and normal tissues of various cancers. Except kidney cancer as well as brain and central nervous system cancer which have a reduced expression of MMP1, together with leukemia, liver cancer and myeloma with censored data, the expression levels of MMP1 gene were upregulated signi cantly in other types of tumor tissues compared to their respective adjacent normal tissues; including bladder, breast, cervical, colorectal, esophageal, gastric, head and neck, lung, lymphoma, melanoma, ovarian, pancreatic, prostate, and sarcoma cancer tissues (Fig. 1A). Although no discernible relationship between MMP1 expression and HCC was observed using Oncomine, further assessment of MMP1 RNA-seq expression in HCC using TIMER 2.0 based on the TCGA database, indicated that a high expression of MMP1 in 22 types of cancers was detected, including HCC, p-value < 0.001 (Fig. 1B). These results strongly suggested that the overexpression of MMP1 might play a positive role in the progression of different neoplasms, especially in HCC.

Evaluation of MMP1 expression and HCC prognosis
The Kaplan-Meier plotter and GEPIA database were used to evaluate the prognostic potential of MMP1 transcriptional levels in HCC tissues. Interestingly, we found that higher expression of MMP1 was associated with poorer prognosis in patients with HCC ( Fig As a supplement, we assessed the relationship between MMP1 transcriptional level and the other tumor tissues via GEPIA database. The high expression of MMP1 was associated with poor OS in head and neck squamous cell carcinoma (HNSC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), pancreatic adenocarcinoma (PAAD) (Fig. 4B, E, F), poor DFS in glioblastoma multiforme (GBM) (Fig. 4A), and both in kidney chromophobe (KICH) and kidney renal papillary cell carcinoma (KIRP) (Fig. 4C, D).

Correlation analysis between MMP1 expression and clinical characteristics of patients with HCC
Furthermore, we evaluated the correlation between MMP1 expression levels and clinicopathological factors in HCC using STATA based on the data of Kaplan-Meier plotter database (Table.1

Gene analysis for MMP1 and Immune cell subset biomarkers
Besides verifying the positive correlation between MMP1 expression and several TIICs, we proceeded to test the correlation of MMP1 expression with levels of speci c biomarker genes in the select TIICs or their subsets in HCC tissues vias TIMER database and explore MMP1-involved carcinogenic mechanisms and ltrate promising therapeutic targets in the future. In detail, the correlation analysis was conducted with with/without adjustment by purity, indicating signi cantly positive correlation of co-expression of MMP1 and biomarker genes of TIICs or subsets of them (Fig. 7). What we found was that almost all the biomarker genes we investigated were positively related to MMP1 expression, including CD19 (B cell biomarker, R = 0.147, p = 6.35e-03), CD79A (B cell biomarker, R = 0.155, p = 3.8e-03) (Fig. 7A), CD8A (CD8 + T cell biomarker, R = 0.209, p = 9.55e-05), CD8B (CD8 + T cell biomarker, R = 0.239, p = 7.09e-06) (Fig. 7B)

Expression levels of MMP1 related to TMB and MSI in HCC
Studies have increasingly reported that TMB [42] and MSI [43] could be used as predictive biomarkers for cancer immunotherapy, which might be the one of most popular methods to predict the therapeutic e ciency of immunotherapy on carcinomas. Therefore, we investigated the correlation between MMP1 expression and TMB/MSI in 32 types cancers via SangerBox. Contrary to what would be expected, there was no signi cant correlation between MMP1 expression and TMB/MSI in HCC patients. It was only positively related to TMB in lung adenocarcinoma (LUAD) (p = 0.0024), prostate adenocarcinoma (PRAD) (p = 0.046), sarcoma (SARC) (p = 0.013), breast invasive carcinoma (BRCA) (p = 7.3e-05), colon adenocarcinoma (COAD) (p = 0.0017) (Fig. 8A) and MSI in testicular germ cell tumors (TGCT) (p = 0.00095), kidney renal clear cell carcinoma (KIRC) (p = 6e-04), and COAD (p = 3.2d-07) (Fig. 8B). In addition, negative correlation was only observed between MMP1 expression and TMB in head and neck squamous cell carcinoma (HNSC) (p=-0.0079) (Fig. 8A) and MSI in pancreatic adenocarcinoma (PAAD) (p=-0.023). This might indicate that HCC patients with neither high nor low expression of MMP1 could equally bene t from TMB/MSI targeted immunotherapy.

Discussion
Although the incidence and mortality of HCC have decreased in South-Eastern Asia due to hepatitis vaccination progress, it is still one of the leading causes of cancer related deaths world wide [1] . Lack of e cient prognostic factors leads to delayed diagnosis and intervention, which in turn considerably contribute to poor HCC patient survival. In this study, we demonstrated that MMP1 expression was elevated in HCC and was signi cantly correlated with worse prognosis utilizing a bioinformatic analysis method based on public databases resources. The MMP1 expression had a positive relationship with differential TIICs and various immune-related gene biomarkers in HCC. All the ndings suggested an underlying mechanism of MMP1 expression in remodulating the tumorimmunity microenvironment and immune escape. To our knowledge, our study is the rst one to comprehensively reveal the prognostic value of MMP1 and its relationship with immune in ltration in HCC. MMP1, as a member of MMPs, participated in the EMT which was identi ed as a strict programmed shift playing a crucial role in tumor invasion and metastasis [44] . MMPs could auto-activate and lead to a cascade of interactional activation between each other to enhance their in uence in the EMT [19] . For invasive HCC, overexpression of MMP1 has been con rmed to correlate with an elevated capacity of invasion and migratory in HCC cells, by the most likely mechanism of ECM degradation promoting the transmembrane migration of tumor cells [17] . This speculation explained the outcomes of high expression of MMP1 in tumor tissues and poor prognosis in HCC patients to a certain extent. On the other hand, MMP1-mediated HCC progression could be suppressed by circularDLC1 [41] , miR-526b [45] , ETV4-MMP1 axis [46] and ERK/MMP1 signaling pathway [47] , indicating multiple pathological pathways of MMP1 carcinogenesis on HCC.
Two databases were utilized for mutual con rmation of the positive relationship between HCC prognosis and MMP1 expression, including OS, PFS, DSS, RFS, and DFS. High expression of MMP1 is closely related to poor survival in various of tumors, which is consistent with our nding thus highlighting the signi cance of monitoring MMP1 expression level to in attempts to detect and prevent early recurrence. However, MMP1 expression was not highly correlated with all clinicopathological characteristics, subgroups indicating some unknown factors might in uence the prognosis in conjunction with these subgroups.
According to resent studies, the integration of clinicopathologic characteristics and TIICs can be a clinical predictive model for the e ciency of immunotherapy [48,49] . The genesis and development of tumors can involve large numbers of immune in ltrating cells and in ammatory mediators. Although MMP1 is involved in the tumor-immune-related progression of some carcinomas, there is barely any studies regarding the interaction between MMP1 expression and TIICs in HCC proliferation and migration. Our study, presents a positive interaction between MMP1 expression and TIICs, signifying the probable utility of MMP1-based prediction in HCC patients associated with immune in ltration. However, the absence of an apparent correlation between MMP1 and tumor purity indicated that MMP1 was expressed from cells in the tumor microenvironment, most likely from TIICs. Although the function of TIICs in carcinogenesis is still controversial, a cluster of studies have reported that MMP1 alongside TIICs plays a vital role in tumor progression [50][51][52] . For further investigation of the interrelationship between MMP1 expression and immune cells in ltration in HCC, we analyzed the data via cox regression. B cells, CD8+/CD4 + T cells and dendritic cells are speci c immune cells playing major roles in activating the body's immune response with antitumor effects [53,54] . On the contrary, reports on TAMs have been focused on their function of modulating the tumor microenvironment and promoting tumor proliferation and angiogenesis [55] . In our study, all of the immune cells showed a high degree of in ltration in HCC, with almost opposite pro-and anti-tumor activity. The results suggest that high expression of MMP1 may overall promote HCC carcinogenesis via increasing in ltration of TAMs, partially counteracting the antitumor activity of other immune cells. This discovery might contribute to the development of innovative immunotherapeutic agents for HCC patients who do not respond to the current immunosuppressive checkpoint inhibitors. Moreover, the results of TMB/MSI evaluation suggested the patients with HCC might not readily bene t from the treatment of PD-1, necessitating the exploration of inhibitors targeted to new immunosuppressive site.
Coupled with the rapid development of immunotherapy in the origination and progression of cancer, the function and mechanism of tumor-immunity microenvironment have been the frontier area for research to screen out related genes that can serve as innovative biomarkers for diagnosis and prognosis or therapeutic targets [56] . We interestingly found that almost all the expression levels of related gene biomarkers in TIICs were positively correlated with MMP1 expression, as well positive correlation between TIICs and MMP1 expression. Furthermore, in spite of the anti-tumor activity of gene biomarkers in B cells, CD8 + T cells, dendritic cells and M1 Macrophages, large numbers of biomarkers in Tregs and exhausted T cells which exerted pro-tumor activity of immuneescaping and angiogenesis were observed. This consequence can explain how the MMP1 expression in uence the TIICs and related gene biomarkers to promote the progression of HCC to a certain extent, indicating the complex mechanisms of interaction and tumorigenesis in tumor microenvironment.
There were several notable limitations to our study. First, the data in our study being collected and analyzed based on the big data from authoritative databases as comprehensively as possible, it can only provide a primary theoretical basis and needs further veri cation by animal experiments and clinical trials. Second, different statistics methods used in data analysis might lead to differentials in results. Furthermore, the correlation coe cients between MMP1 and TIICs or their biomarker genes were not very strong, suggesting the important direction and terra incognita for further research.
In conclusion, our study revealed that the high expression of MMP1 was closely related with poor prognosis in HCC patients, so is the positive correlation with tumor-immune cell in ltration. Although some mechanisms of interactive network are unknown, we still have reason to believe that MMP1 is a prospective prognostic biomarker in HCC.

Declarations Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the research ethics committee of Lihuili hospital a liated to Ningbo University at which the studies were conducted (Approval no. KY2021PJ045) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Since this was an observational but not prospective intervention study, the Ethics Committee provided a waiver of informed consent.

Con ict of interest
The authors have declared that no con ict of interest exists.