Study on the value of C1q in the diagnosis of Esophageal cancer

Background: Esophageal cancer was hardly diagnosed in early stage, and more potential biomarkers should be found. Methods: 252 patients and normal controls which recruited in Renmin Hospital of Wuhan University, were divided into esophageal carcinoma group (105 cases), disease control group (75 cases) and the control group of healthy people (72 cases). Moreover, TISIDB and GEPIA databases were used to investigate the different expression of EC and normal tissues, and explore the roles of C1q in tumor-immune system interactions in EC. Results: The concentration of serum C1q in EC group is 196.8(180~219.4) mg/L, which is higher than the level of DC [178.10(153.70~200.85) mg/L]and HC [183.00(167.75~201.00) mg/L] (P<0.05). A higher expression level of C1q was observed in III and IV grades [214(192~237.3) mg/ml] than grades I and II [180.95(172.03~193.85) mg/L] (P<0.05). C1q was positively correlated with eosinophils, active CD8 T cells, myeloid derived suppressor cells, natural killer cells, monocytes and macrophages (r = 0.373; r = 0.659; r = 0.846; r = 0.760; r = 0.499; r = 0.757; P<0.05). Conclusion: The concentrations of C1q increased in EC and related to the severity of EC, which had potential value of diagnosis of EC. There were correlations in C1q and tumor-infiltrating lymphocytes.

difference(P>0.05). The gender and ages of each group also have no difference in statistics(P>0.05), while PLT, NE, LY, MO are higher than DC and HC(P<0.05).
Table1. Clinical characteristics of the participants.

Elevated Expression of C1q mRNA in EC
We investigated the differential expression of C1q using the GEPIA databases. In GEPIA, C1q mRNA expression between tumors and their paired normal tissues across 32 TCGA cancer types were compared. C1q mRNA showed differences in all 17 types cancers. (Figure 1

Elevated Expression of Serum C1q in ESCA Group
To further validate these results, we test the concentrations of C1q in serum of all patients. As shown in Table  1

The Regulation of Immune Molecules by C1q
C1q mediates a series of immunoregulatory functions which are regarded as key points in autoimmunity. Consequently, we explored whether C1q related to immune infiltration using TISIDB databases. We found that C1q expression was correlated with several kinds of tumor-infiltrating lymphocytes (TILs) in ESCA patients in TISIDB databases. TILs were shown great correlation with C1q, that included eosinophils (Spearman: r = 0.373, P =2.06e-07), active CD8 T cells (Spearman: r

Correlation Analysis of PLT, NE, LY , MO and C1q in EC
As shown in Table 1, the levels of PLT and LY were lowest in EC group than DC group and HC group respectively. The level of NE was highest in EC groups (Figure 4). There was no correlation between C1q and anyone of those.

Discussion
EC is the sixth most common cause of cancer death worldwide, which cause a huge global health challenge. Most patients with EC were discovered in advanced stages, and had adverse therapeutic effect. The common treatments were chemotherapy, chemoradiotherapy and surgical resection [15,16] . Reflux disease is associated with an increased risk of esophagitis, esophageal strictures, Barrett esophagus, and esophageal adenocarcinoma, and it has the similar symptoms to EC [17,18] . Using endoscopy can distinguish EC from reflux disease, though some patients refuse this way to check their esophagus [19] .As a result, a blood biomarker which can help to diagnose EC from reflux disease come to be important.
Our study showed that C1q expressed higher in serum of EC patients which indicate that C1q may have potential diagnosis of EC. Further, along with the growth of the grades, the concentrations of C1q were much higher which indicated that C1q may become a warning signal of progression of EC. Besides, PLT and lymphocytes expressed differently in EC from HC which indicated that inflammatory cells may play key role in EC. Although C1q has no correlation with inflammatory cells in serum, tumor-infiltrating lymphocytes were correlated with C1q. The level of C1q in serum is elevated in patients with atherosclerosis and acute ischemic stroke, and a similar observation has been documented for glimoas and sepsis [20,21,22,23] . Although it was observed reduced in patients with multiple myeloma [24] . In this study, we showed that for the first time that C1q elevated in EC, and rose with evaluation of grades. It should be noted that different cancer cells respond to C1q in different ways, and it is a proor anti-tumorigenic factor have not been confirmed yet. Hong et al. reported that C1q active the tumor suppressor WW-domain containing oxidoreductase (WOX1) to affect growth procession and apoptosis of human prostate cells [25] . C1q can promote adhesion, proliferation and migration of mice melanoma cells which indicated that C1q may play a role as tumor-promoting factor in cancers, which by complement dependent way or not [26] . Yuri et al. reported that C1q express higher in normal tissue than in Barrett's esophagus specimens and specimens with adenocarcinoma [27] . We speculate that blood circulating C1q may be different from C1q expressed in tissues.
A great blood biomarker of cancer should possess several features, such as high sensitivity and can be tested in early stage [28,29] . However, such a perfect biomarker has not been found. As a result, the combination of different blood biomarkers come to be important. Nowadays, CEA is widely used in lab to diagnose EC, and its sensitivity ranged from 8% to 70%, specificity ranged from 57% to 100% [30] . Meanwhile, SCC and Cyfra21-1 also commonly used for diagnosis. Our findings may be useful in adding a new biomarker to potential circulating blood molecules for detecting EC. Because we examined only 252 patients, our results may have due to chance. The results must be confirmed in a larger study. Besides, the molecular mechanism of C1q in EC should be research in further study.
In summary, our study found that the concentrations of serum C1q in patients with EC is higher than patients with reflux esophagitis and healthy people, and it elevated with growth of grades, which may have potential to diagnose EC in early stage. Besides, C1q may play a role in inflammatory effects, which should be explored further. We suggest that scholars can focus on molecular mechanism of C1q in EC.

Author contributions statement:
Conceptualization, C, X. and L, H.; methodology, C, X.; software, L, H.; validation, C, J.J. and T, D.L.; formal analysis, C, X.; investigation, Z, P.A.; resources, Z, P.A.; writing-original draft preparation, C, X.; writing-review and editing, Z, P.A. All authors have read and agreed to the published version of the manuscript.

Funding:
This research was funded by the National Natural Science Foundation of China, grant number 81773444.