Expression Level of Annexin A1 Contributes to the Evaluation of the Disease Progression and Treatment Effect of Lung Adenocarcinoma: A New Perspective from Retrospective Analysis

Background: Increased Annexin A1 has been showed to be related to malignant biological characteristics of tumors; the aim of this study was to evaluate the relationship between the expression level of Annexin A1 and the disease progression and treatment effect of lung adenocarcinoma (LAC). Methods: The expression level of Annexin A1 in LAC tissues and cells was detected by the methods of immunohistochemistry, Real time-PCR and western blotting. The relationship between the expression of Annexin A1 and the disease progression and treatment effect of LAC was evaluated by descriptive statistics, T test and Chi-square test. Results: The protein expression of Annexin A1 was higher in lung cancer tissues and cells than that in normal tissues and 16 human bronchial epithelial (16HBE) cells (p<0.05). The level of Annexin A1 mRNA was higher in lung cancer tissues than that in normal tissues (p<0.05). The increase of Annexin A1 protein and mRNA was associated with the lymph node metastasis, advanced clinical stage (p<0.05). However, surgical resection and chemotherapy for LAC down-regulated the serum concentration of Annexin A1 in patients (p<0.05). Conclusions: Increased Annexin A1 protein and mRNA in LAC tissues correlate with the poor differentiation, lymph node metastasis and advanced stage of LAC. Surgical resection and chemotherapy for LAC down-regulate the serum concentration of Annexin A1 in patients. The results indicate that expression level of Annexin A1 contributes to the evaluation of the disease progression and treatment effect of LAC.

expression has been up-regulated in some cancers, but down-regulated in other cancer types. These abnormal expressions all show an inherent connection with the occurrence and development of these tumors [5,7]. In breast cancer, changes in the expression level of Annexin A1 suggest a potential impact on tumorigenic and metastatic processes. However, reports of con icting results indicate that this relationship is much more complicated than initially conceptualized [5]. A more consistent understanding of Annexin A1 in cancer may lead to more biologically meaningful and clinically relevant strategies [6][7][8].
Previously, we found that Annexin A1 had a signi cant increase in LAC cells as compared with the 16 human bronchial epithelial (16HBE) cells. In this study, we investigated the relationship between the expression of Annexin A1 and the disease progression and treatment effect of LAC.

Methods
Collection of tissue specimens and production of tissue chips From January 2018 to October 2020, 102 patients from First A liated Hospital, Xi'an Medical University and Zhangye People's Hospital who had undergone lung cancer resection were included in this study ( Table 1). The study was approved by the institutional review committee (First A liated Hospital, Xi'an Medical University), and written informed consent was obtained from all subjects. The matched adjacent tissues (from the edge of tumor mass at least 3 cm away) were selected as normal control. The tissue chips was constructed using the collected tissues. The steps were as follows: (1) marking the morphologically representative tumor area; (2) using a hollow needle with a diameter of 2 mm to cut the tissue strip from the donor wax block; (3) placing the tissue strip in the recipient wax block according to the speci c allocated position; and (4) cutting 5µm thick tissue slices and drying for use.

Immunochemistry (IHC)
Immunohistochemical method was used to detect the expression level of Annexin A1 protein in LAC tissue and normal tissues adjacent to cancer. Speci c steps are as follows. The slices were dewaxed and hydrated, and soaked in 3% H 2 O 2 for 10 min. The antigen in the tissue on slices was repaired by method of microwave heating. The slices was blocked with serum, then incubated with the primary antibody (Annexin A1 rabbit anti-human polyclonal antibody 1:100; BOSTER Biological Technology co.ltd, Wuhan, China). After rinsing with PBS the next day, the slices were incubated with a secondary antibody (biotin-labeled secondary antibody 1:200). Avidin working solution was dropped on the slices to stop the reaction. The slices were dehydrated using gradient alcohol and mounted by neutral gum. The positive slices for breast cancer provided by the kit served as the positive control, PBS solution instead of primary antibody was used as a negative control. Two pathologists used a blind method to evaluate the results of IHC reference to previously published research [9]. The intensity was graded according to the following scale: 0, no staining; 1+, mild staining; 2+, moderate staining; 3+, intense staining. The extent was evaluated as follows: 0, no staining of cells in any microscopic elds; 1+, < 30% of tissue stained positive; 2+, between 30% and 60% stained positive; 3+, > 60% stained positive. A combined staining score (intensity + extension) of ≤ 2, between 3 and 4, and between 5 and 6 were considered as low, moderate, and high expression levels, respectively [10].

Real-time quantitative PCR
According to the instructions of the Total RNA extraction kit, the total RNA was extracted from lung tissues. The Gene quact colorimeter (Pharmacia Biotech, USA) was employed to determine the total RNA concentration. The agarose gel electrophoresis (1.2%) was used to detect the integrity of total RNA.
Enzyme-linked immunosorbent assay (ELISA) We collected 10 mL of cubital venous blood from patients on an empty stomach in the morning, centrifuged at 3000 rpm for 10-15 minutes, and collected the supernatant for cryopreservation at -70°C.
A rabbit anti-human Annexin A1 sandwich ELISA test kit developed by China Xitang Biotechnology Company (CK-E91728H) was used to measure the level of serum Annexin A1. The serum samples diluted in PBS (pH 7.4) containing 1% BSA were coated in 96-well plates, and then 10 ng/mL monoclonal anti-Annexin A1 antibody were dropped into the small wells and incubated at 37°C for 1 hour. The substrate reagent was reacted with avidin-coupled peroxidase at 37°C for 15 minutes. The 50µl of 2N sulfuric acid was added to each well to stop the color reaction, and then the intensity of the color was determined with an ultraviolet spectrophotometer at a wavelength of 450 nm. The measurement standard curve of the test is drawn by using the concentration of the standard sample and the corresponding OD value of each well.

Cells
The

Western blot
The total protein in the tissues and cells was extracted, and the concentration of the protein was detected by the BCA method. The quanti ed protein was loaded, electrophoresed, transferred, blocked, incubated with the primary antibody (rabbit anti-human Annexin A1 antibody, 1:500 dilution; BOSTER Biological Technology, Wuhan, China) and incubated with the second antibody (goat anti-rabbit 1:1000) step by step. After washing PBS solution, positive signal was visualized using the enhanced chemiluminescence method and exposed with X-ray lm. The exposed lm was developed with developer and xer. Finally, the Image J software used to calculate the band gray value of the lm. The test of β-actin was used as the internal control.

Statistical analysis
The data were divided into two types: counting data and measurement data. The count data were expressed using a composition ratio (%) and analyzed using chi-square test and Fisher's exact test. The measurement data was expressed by X ± S, the comparison between the two groups of means was performed by t test, and the comparison between the means of the two groups or more was determined by single factor analysis of variance. The SPSS 21.0 statistical software was used to perform the data analysis, and the p < 0.05 was con ned as meaningful statistical difference.
Expression of Annexin A1 mRNA in LAC tissues is higher than that in normal lung tissues The results from Real-time PCR showed that the expression levels of Annexin A1 mRNA in lung cancer tissues and cancer-adjacent normal tissues were 0.568 ± 1.321 2 −ΔΔCT and 0.251 ± 0.789 2 −ΔΔCT , respectively. The expression of Annexin A1 mRNA in lung cancer tissues was higher than that in canceradjacent normal tissues (t = 3.172, p = 0.039) ( Table 3; Fig. 2A).  Fig. 2D). And, the expression level of Annexin A1 mRNA was higher in LAC tissues with lymph node metastasis (0.911 ± 1.398 2 −ΔΔCT ) than that in LAC tissues with non-lymph node metastasis (0.376 ± 0.898 2 −ΔΔCT ) (t = 3.178, p = 0.028) ( Table 3; Fig. 2E).
Expression of Annexin A1 in LAC tissues and cells shows an increase compared with normal lung tissues and 16HBE cells by western blotting Compared with normal lung tissues adjacent to cancer, Annexin A1 in LAC tissues was signi cantly increased (p < 0.05) (Fig. 5A and B). Compared with 16HBE cells, the expression of Annexin A1 in LAC A549 cells (p < 0.05) (Fig. 5C and D) and PC-9 cells (p < 0.05) (Fig. 5E and F) was also signi cantly increased. In addition, the expression of Annexin A1 in lung squamous cell carcinoma H520 cells and small cell lung cancer H446 cells was also higher than that in 16HBE cells, but the increase was not as high as LAC cells.

Discussion
The development and progression of LAC have been con rmed to be a multi-gene, multi-step, multi-stage process. It has been gradually realized that lung cancer may be a disease that develops from external causes through internal causes. The external factors can induce malignant transformation of cells and irreversible genetic changes, including the activation of proto-oncogenes, the inactivation of tumor suppressor genes, the activation of self-feedback secretion loops and the inhibition of cell apoptosis, leading to uncontrolled cell growth [2][3][4]. The proliferation activity of tumor cells is closely related to the biological behavior of tumors. Uncontrolled cell proliferation is the most basic feature of the occurrence, invasion and metastasis of malignant tumors. This process is related to the abnormal expression of proteins translated by many oncogenes [2,11]. In this study, we found that Annexin A1 was highly expressed in LAC tissues. The results indicated that Annexin A1 may have played a role in the progression of LAC, thus correlating with the occurrence and development of LAC. Some previous studies have suggested that the increase in Annexin A1 is related to the tumorigenesis of lung cancer, including lung squamous cell carcinoma and small cell lung cancer [11][12][13][14]. Our study showed that the expression of Annexin A1 was related to the malignant biological behavior of LAC, including poor differentiation, lymph node metastasis and advanced clinical stage of LAC, suggesting that the expression of Annexin A1 can be considered as a tumor marker to predict the disease progression and prognosis of LAC. Poor differentiation, lymph node metastasis, and advanced disease all indicate the malignant characteristics of tumors and have a worse prognosis. Our research suggests that Annexin A1 can be used to predict the degree of malignancy of LAC, and has certain prognostic evaluation value. Studies have shown that Annexin A1 is closely related to a variety of malignant tumors. It may be involved in the occurrence and development of tumors, and is closely related to tumor differentiation, proliferation, invasion, metastasis and tumor prognosis [15].
We especially observed that the expression of Annexin A1 mRNA in LAC tissue is also higher than that in normal lung tissue. And the high level of Annexin A1 mRNA was related to the poor differentiation of LAC, lymph node metastasis and advanced clinical stage. The mRNA is a general term for a large class of RNA molecules, which are responsible for transmitting genetic information from DNA to ribosomes, and then producing proteins encoded by genes on the ribosomes. Then, RNA polymerase transcribes genes into mRNA precursors. The mRNA precursors are processed into mature RNAs and translated into speci c proteins [16]. Although mRNA and protein levels are a coupled process, there is no necessarily consistent trend between the two. The increase in mRNA level represents the activation of promoters or enhancers, or the activation of upstream transcription factors, while the increase in protein levels is mostly related to functional enhancement. The transcription of mRNA and the process of protein translation are coupled with each other, so verifying the relationship between the two is quite powerful for con rming the activation of a certain gene [16,17]. Our research showed that the expressions of Annexin A1 mRNA and protein were consistent, which illustrates that this gene plays a certain role in the occurrence and development of LAC.
We found that the concentration of Annexin A1 in the patient's serum was related to the poor differentiation of LAC, lymph node metastasis and later clinical stage. More importantly, the serum Annexin A1 concentration in LAC patients one month after surgery was signi cantly lower than that before surgery. Surgical removal of tumors plays an important role in reducing tumor burden, restoring the body's immune function, and improving the effect of immunotherapy. The expressions of various costimulatory molecules were signi cantly reduced. Surgical removal of tumors can reduce the adverse effects of many tumor-related substances on the human body, and the long-term effect is to improve the body's immune function [18][19][20]. Our ndings indicate that Annexin A1 is closely related to the malignant biological behavior of LAC. The serum concentration drops after tumor resection, which indirectly indicates the bene t of the rapid reduction in the body's tumor load after surgery. We also found that pemetrexed combined with cisplatin chemotherapy further suppressed the concentration of serum Annexin A1. Chemotherapy plays an important role in killing the remaining tumor cells after surgery. Our nding further indicates that Annexin A1 is related to the progression of LAC. After chemotherapy, the serum concentration of Annexin A1 further reduced, which can also re ect the therapeutic effect of chemotherapy on tumors from the side. Studies show that Annexin A1 is a multifunctional protein characterized by its role in regulating innate and adaptive immune responses. Evidence of increased expression of Annexin A1 in many human tumors has aroused interest in its functional role in cancer biology [5,7]. Annexin A1 can regulate the polarization and activation of macrophages, and acts through its receptor FPR2 to promote the polarization of macrophages. Annexin A1-de cient mice showed a decrease in tumor growth rate, which may be due to the increase of M1 macrophages in the tumor microenvironment [21]. Pemetrexed is a multi-target folate antagonist that inhibits cell replication by disrupting the key folate-dependent metabolic process necessary for cell replication. The chemotherapy regimen of pemetrexed plus cisplatin is the rst-line treatment regimen for advanced LAC, which is better than the chemotherapy regimen of gemcitabine plus cisplatin, paclitaxel plus cisplatin, and docetaxel plus cisplatin [22,23]. The nding that serum concentration of Annexin A1 further decreases after chemotherapy can re ect that Annexin A1 is related to the progress of LAC, and its down-regulation after chemotherapy implies that the further killing degree of LAC cells can to be correlated by the serum level of Annexin A1. Patients with metastatic renal cell carcinoma are usually treated with the tyrosine kinase inhibitor sunitinib. A study suggests that Annexin A1-negative tumors can get longer treatment bene ts, and the overall survival of patients with Annexin A1-negative tumors is also signi cantly better. This study shows that the cytoplasmic expression of Annexin A1 is a negative predictor of sunitinib treatment in patients with metastatic renal cell carcinoma [24]. A study suggests that the overexpression of Annexin A1 is signi cantly related to the advanced clinical stages of LAC and lymph node metastasis, an increase in recurrence rate and overall survival. In addition, siRNA interference to Annexin A1 signi cantly inhibites the invasion ability of LAC cells A549 in vitro [25].
We further veri ed the expression of Annexin A1 in tissues and cells by Western blotting. Compared with normal lung tissue adjacent to cancer and 16HBE cells, Annexin A1 expression in LAC tissues and cells was signi cantly increased. These results further reinforces this evidence that Annexin A1 expression is increased in LAC, and it also further strengthens the idea that it may be used as a molecular marker of LAC. Some basic research at the cellular level has gradually consolidated the chain of evidence linking Annexin A1 to the proliferation and invasion of malignant tumors. Studies used RNA interference technology to down-regulate the expression of Annexin A1 in A549 and H1299 cells of NSCLC showed that the knockdown of Annexin A1 gene inhibited the proliferation, migration and invasion of NSCLC cells [5,25]. The Annexin A1 overexpression plasmid was used to transfect esophageal squamous cell carcinoma (ESCC) Eca109 cells, which increased the expression of Annexin A1 in Eca109 cells. Subsequent observations showed that the proliferation, migration and invasion of Eca109 cells increased signi cantly and proved that Annexin A1 promoted the expression of Snail but inhibited the expression of E-cadherin, thereby enhancing the migration and invasion of ESCC cells [26]. In the process of tumorigenesis, how to control the rate of tumor cell migration, invasion and apoptosis is a focus of current research on tumor molecular biology. Annexin A1 has a certain effect on the migration, invasion and apoptosis of LAC cells, which may provide research ideas for the development of molecular targeted therapy based on it. Some of the shortcomings of this study are as follows. This study did not conduct studies on Annexin A1 functions at the level of LAC cells and model animals. We plan to carry out research in this area in the future. The initial design of this study was limited to the study of LAC, and no study on Annexin A1 in squamous cell carcinoma and small cell lung cancer was carried out. This also suggests that we can expand the scope of research in the next step.

Declarations
Ethics approval and consent to participate The study was approved by the Ethics Committee, the First Hospital, Xi'an Medical University. All research specimens from patients were authorized by the patients and signed informed consents. This study was performed strictly following the Statement of Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). We declare that all research strategies and methods used in this study were performed in accordance with the relevant guidelines and regulations.

Consent to publish
Not applicable.

Availability of data and materials
The datasets supporting the conclusions of this article are included within the article. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
None.

Funding
This study was supported by grants from the Medical Project of the Xi'an Science and Technology Bureau (XA2020-YXYJ-0163). The fund provider did not play any role in this study, nor did it participate in any matters in this study.
Authors' contributions BX R, HL Y, G W, K L, M L, CX R, and W L participated in the design and coordination of the study, carried out the critical appraisal of studies, statistical analysis of studies and wrote the manuscript. All authors have read and approved the manuscript, and ensure that this is the case. E. The expression of Annexin A1 in LAC tissues was higher than that in normal lung tissues (p<0.01). F. The expression of Annexin A1 was higher in the poor differentiated LAC tissues than in moderately and well differentiated LAC (p<0.01). G. The expression of Annexin A1 was higher in the non-lymph node metastasis LAC tissues than in lymph node metastasis LAC tissues (p<0.01); H. the expression of Annexin A1 was higher in IIB-IIIA stage LAC tissues than in IIA stage tissues (p<0.01). LAC, lung adenocarcinoma.

Figure 2
Expression of Annexin A1 mRNA in LAC tissues correlates with malignant biological characteristics of LAC A. the expression of Annexin A1 mRNA in LAC tissues was higher than that in normal lung tissues (p<0.05); B. the expression of Annexin A1 mRNA in lung cancer tissues has nothing to do with the gender of the patient (p>0.05); C. the expression of Annexin A1 mRNA in lung cancer tissues has nothing to do with the smoking status of the patients (p>0.05); D. the expression of Annexin A1 mRNA was higher in the poor differentiated LAC tissues than in moderately and well differentiated LAC (p<0.05); E. the expression of Annexin A1 mRNA was higher in LAC tissues with lymph node metastasis than in those without lymph node metastasis LAC tissues (p<0.05); F. the expression of Annexin A1 mRNA was higher in IIB-IIIA stage LAC tissues than in IIA stage tissues (p<0.05). LAC, lung adenocarcinoma.

Figure 3
Relationship between the serum concentration of Annexin A1 and the resection of LAC A. the serum concentration of Annexin A1 was higher in the poor differentiated LAC patients than in moderately and well differentiated patients (p<0.05); B. the serum concentration of Annexin A1 was higher in LAC patients with lymph node metastasis than in those without lymph node metastasis (p<0.05); C. the serum concentration of Annexin A1 was higher in IIB-IIIA stage LAC tissues than in IIA stage tissues (p<0.05); D.
Regardless of the degree of differentiation of LAC, the serum concentration of Annexin A1 in patients 1 month after LAC surgery was lower than before surgery (p<0.05); E. Regardless of the status of lymph node metastasis, the serum concentration of Annexin A1 in patients 1 month after LAC surgery was lower than before surgery (p<0.05); F. Regardless of the clinical stage of LAC, the serum concentration of Annexin A1 in patients 1 month after LAC surgery was lower than before surgery (p<0.05). LAC, lung adenocarcinoma.

Figure 4
Relationship between the serum concentration of Annexin A1 and chemotherapy of LAC A. before chemotherapy, the serum concentration of Annexin A1 was higher in the poor differentiated LAC patients than in moderately and well differentiated patients (p<0.05); B. before chemotherapy, the serum concentration of Annexin A1 was higher in LAC patients with lymph node metastasis than in those without lymph node metastasis (p<0.05); C. before chemotherapy, the serum concentration of Annexin A1 was higher in IIB-IIIA stage LAC tissues than in IIA stage tissues (p<0.05); D. Regardless of the degree of differentiation of LAC, the serum concentration of Annexin A1 in patients after chemotherapy was lower than before chemotherapy (p<0.05); E. Regardless of the status of lymph node metastasis, the serum concentration of Annexin A1 in patients after chemotherapy was lower than before chemotherapy (p<0.05); F. Regardless of the clinical stage of LAC, the serum concentration of Annexin A1 in patients after chemotherapy was lower than before chemotherapy (p<0.05). LAC, lung adenocarcinoma.

Figure 5
Expression of Annexin A1 in lung adenocarcinoma tissues and cells by Western Blot A and B. Compared with normal lung tissues adjacent to cancer, Annexin A1 expression in LAC tissues was signi cantly increased (p<0.05). C and D. Compared with 16HBE cells, the expression of Annexin A1 in LAC A549 cells was signi cantly increased (p<0.05); the expression of Annexin A1 in lung squamous cell carcinoma H520 cells was also higher than that in 16HBE cells, but the increase was not as high as LAC cells. E and F. Compared with 16HBE cells, the expression of Annexin A1 in LAC PC-9 cells was signi cantly increased (p<0.05); the expression of Annexin A1 in small cell lung cancer H446 cells was also higher than that in 16HBE cells, but the increase was not as high as LAC cells. LAC, lung adenocarcinoma; 16HBE, 16 human bronchial epithelial.