| Background:Pepsinogen(PG)is a class of aspartic proteases secreted by the chief cells of the stomach.Under acidic conditions in the stomach,it is activated into active pepsin,which has the function of digesting gastric proteins.PG consists of two forms: pepsinogen A(PGA)and pepsinogen C(PGC).The PGA gene is located at 11q13 and is composed of three genes:PGA3,PGA4,and PGA5.The PGC gene is located at 6p21.1 and consists of a single gene.PGA is primarily secreted by the chief cells of the gastric fundic glands and neck mucous cells,while PGC is produced by the entire gastric gland(fundic,cardia,and antral glands)and the Brunner’s glands of the proximal duodenum.After synthesis,PGC is secreted into the gastric lumen and,under certain acidic conditions,is converted into its active proteolytic form.It is widely distributed in the gastrointestinal tract and constitutes the major proteinase in gastric fluid.Under normal circumstances,approximately 1% of PG can enter the bloodstream through the capillaries of the gastric mucosa,exhibiting good stability in the systemic circulation.PG protein appears in the late stage of embryonic development and is mainly expressed in the stomach.It is the terminal product of differentiation and maturation of gastric mucosal cells and serves as a marker for the gradual maturation of digestive function.PG is highly expressed in normal gastric mucosa,but its expression is significantly decreased or lost in gastric cancer.Currently,it is wellestablished that PG expression is strongly negatively correlated with gastric cancer,although the specific regulatory mechanisms remain unclear.Furthermore,studies have shown that although the expression of PG is mainly present in the stomach,abnormal expression of PG can also be detected to varying degrees in some extra-gastric organs that do not normally express PG.Particularly,abnormal elevation of PG and its correlation with tumor prognosis have been observed in the occurrence of tumors.For instance,PGA can exhibit abnormal expression in esophageal cancer and bile duct tumors,while PGC can show abnormal expression in prostate cancer,breast cancer,ovarian cancer,endometrial cancer,pancreatic cancer,renal cancer,bladder cancer,eyelid basal cell carcinoma,squamous cell carcinoma,and melanoma.Further research has indicated that high-level expression of PGC is associated with better prognosis and longer survival time in certain types of cancer such as prostate cancer,breast cancer,and ovarian cancer.These studies suggest that the pathogenic mechanisms underlying the abnormal expression of PG in extra-gastric tumors may differ from those in gastric tumors.In-depth investigation of the relationship between PG expression and extra-gastric tumors can help explore the functions and mechanisms of PG in these tumors and contribute to a better understanding of its role and functional mechanisms.Breast cancer is the most common malignant tumor in women worldwide,ranking first in incidence and second in mortality among female cancers.Some studies have found a correlation between PGC expression and breast cancer through immunohistochemical detection,suggesting that abnormal expression of PGC is closely related to the differentiation,biological behavior,and prognosis of breast cancer,and indicates a favorable prognosis.Currently,the mechanism of PGC expression in breast cancer tissue remains unclear,and there are no reported studies on the expression of PGA in breast cancer and its relationship with clinical pathological parameters.Regarding the physiological regulation of the stomach,studies related to PG suggest the following mechanisms: in the chief cells of the gastric mucosa,activation of adrenergic receptors promotes PG secretion through adenylate cyclase;additionally,the receptors for cholecystokinin and acetylcholine promote PGC secretion through a second messenger(inositol trisphosphate-calcium ion pathway);an increase in gastric acid or a decrease in p H can induce cholinergic reflex regulation,stimulating the chief cells to further secrete PG.Gastrin is a peptide hormone that promotes gastric mucosal growth,gastric motility,and hydrochloric acid secretion into the stomach.It also has the function of stimulating PG secretion by stimulating chief cell secretion.The main product of G cells in the gastric antrum mucosa is gastrin-17(G17),which is the major factor stimulating gastric acid secretion.The promoting factors for its secretion regulation include digestive product peptides and proteins,gastric distension,gastrin-releasing peptide(GRP)produced by the vagus nerve signal,plasma adrenaline,Ca^2+,etc.Among them,GRP produces effects by binding to its receptor(GRP-r);inhibitory factors include gastric acid and somatostatin.Currently,there are no reports on the association between gastrin,GRP,GRP-r,and PG in the breast,and the regulatory relationship between them in the breast needs further investigation.In summary,this study aims to explore the relationship and clinical significance of PG gene expression,including PGA and PGC,with breast cancer at multiple levels such as serum and tissue,through a large sample of clinical cases combined with bioinformatics analysis.Based on this,in vitro cell experiments will be conducted to further investigate the effects of PGA and PGC expression on the biological behavior of breast cancer cells.Drawing from the physiological regulation pathways of PGA and PGC in the stomach,the study aims to explore the regulatory modes and mechanisms of abnormal expression of PGA and PGC in breast cancer tissue,in order to elucidate the functions and roles of PG beyond being a precursor of gastric protein-digesting enzymes.This will help uncover diagnostic and therapeutic methods related to the abnormal expression of PG in extragastric tissues and contribute to a comprehensive understanding of the functions and mechanisms of PG.Objectives:1.To investigate the relationship between the expression of PGA and PGC genes and the occurrence and development of breast cancer,in order to elucidate the potential roles of PGA and PGC genes in the progression of breast cancer.2.To explore the impact of PGA and PGC genes on the biological behavior of breast cancer cells and to clarify the possible regulatory mechanisms of PGA and PGC expression in breast cancer,particularly focusing on the regulatory effects based on the gastrin-releasing peptide(GRP)and its receptor(GRP-r)pathway.Materials and methods:Part Ⅰ PGA and PGC expression characteristics in different breast diseases1.Detection of serum expression levels of PGA and PGC in different breast diseases This study included a total of 1,658 participants,including 1,100 patients(516 with breast cancer and 584 with benign breast diseases)who visited and received treatment at the Department of Breast Surgery,The First Hospital of China Medical University,from December 2016 to August 2017.Additionally,558 individuals who underwent medical examinations at the hospital’s medical examination center during the same period and had normal results were included as controls.All study participants were divided into the breast cancer group,non-cancer group(comprising different benign diseases),and healthy control group.Serum samples were collected from all study participants.Basic information such as age and menstrual status of the participants was obtained through a questionnaire survey.Clinical information including tumor size,tumor grade,lymph node metastasis,TNM stage,ER,PR,HER-2,Ki-67,CK5/6,P63,E-cadherin,GATA-3,molecular subtypes,and other data were collected from the electronic medical records of the participants.This study was approved by the Ethics Committee of The First Hospital of China Medical University,and written informed consent was obtained from all patients.Fasting venous blood samples were collected from the participants after a 24-hour fasting period,and the serum was separated by centrifugation at 3,500 × g for 10 minutes.The corresponding serum concentrations of PGA and PGC were measured using an Elisa kit(Biohit,Helsinki,Finland).2.In situ expression levels of PGA and PGC in different breast diseases This study included a total of 320 patients who underwent breast surgery at the Department of Breast Surgery,The First Hospital of China Medical University,from December 2016 to August 2017.Among them,there were 130 patients with breast cancer and 190 patients with benign breast diseases.All study participants were divided into the breast cancer group and non-cancer group(comprising different benign diseases).Tissue samples were collected from all study participants.Basic information such as age and menstrual status of the participants was obtained through a questionnaire survey.Clinical information including tumor size,tumor grade,lymph node metastasis,TNM stage,and other data were collected from the electronic medical records of the participants.This study was approved by the Ethics Committee of The First Hospital of China Medical University,and written informed consent was obtained from all patients.Immunohistochemical staining was performed on the pathological tissue sections to detect the expression of PGA and PGC.The dyeing intensity was graded according to 0-3(I0-I3).Dyeing area ratio recorded as P(0 to 4):< 5%(0),6-25%(1),26-50%(2),(3),51-75%(4),76-100.HSCORE is calculated according to the following algorithm: HSCORE=Σ(I×Pi).Finally,the staining results were divided into four groups according to HSCORE:-(0);+(1--4);+ +(5--8);+ + +(9--12)3.Statistical analysisSPSS Statistics Version 24.0 was used for statistical analysis.Analysis of variance was used to compare the age of different disease groups.Kruskal Wallis Test was used to evaluate the differential expression of serum PGA and PGC in different breast disease groups.t test was used to analyze the relationship between serum expression levels of PGA and PGC,menstrual status and clinicopathological parameters.ROC curve was used to detect the diagnostic efficiency of PGA and PGC in distinguishing different breast diseases.Chi-square test was used to evaluate the relationship between PGA and PGC protein expression and clinicopathological parameters of patients.4.Bioinformatics analysis of the relationship between PGA and PGC expression and breast cancer4.1 Validation of clinicopathological parameters and prognosis evaluation of PG by TCGA and GEO databaseRNA-seq data and related clinical information of PGA and PGC in breast cancer cohort were extracted from The TCGA database(The cancer Genome Atlas;(GSE41119)(https://cancergenome.nih.gov/)and GEO database Gene Expression Omnibus;https://www.ncbi.nlm.nih.gov/geo/).4.2 GO and KEGG enrichment analysis of PG function and related pathways The PG functions and related pathways were analyzed based on GO(Gene oncology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)databases.Part 2: Effects of PGA and PGC expression on the biological behavior of breast cancer cells.1.Overexpression of PG genes and preparation of control breast cancer cell lines(MDAMB-231,MCF-7) Using lentiviral transfection,cell lines with overexpression of PGA and PGC genes were constructed,and the transfection efficiency of the virus was determined using Western Blot.2.Effects of PG on the biological behavior of breast cancer cellsCCK-8 and EDU experiments were conducted to evaluate the proliferative activity of breast cancer cells.Western Blot experiments were performed to assess the expression of breast cancer molecular subtypes-related genes(Ki-67,HER-2,ER,PR).Scratch migration,Transwell chamber migration,and Transwell chamber invasion assays were conducted to evaluate the migration and invasion abilities of breast cancer cells.Flow cytometry and apoptosis detection kits were used to assess the apoptotic capability of breast cancer cells.3.Statistical analysisGraph Pad Prism 7(USA)was used for image processing.Independent sample t-tests were employed for comparing data between groups.For data that followed a normal distribution,t-tests were used,while for data that did not follow a normal distribution,the MannWhitney U test was used to assess the differences in expression levels between the two groups.A p-value of less than 0.05 was considered statistically significant.Part Ⅲ: Regulation of PGA and PGC Expression and Their Effects on Proliferation,Migration,and Invasion of Breast Cancer Cells.1.Detection of GRP and GRP-r histological expression levels in different breast diseases:Immunohistochemistry staining and scoring were performed(methods same as in Part 1).2.Correlation analysis of PGA,PGC,GRP,and GRP-r histological expression Correlation analysis was conducted on the immunohistochemical staining results of PGA,PGC,GRP,and GRP-r to explore the relationship between PGA and PGC with GRP and GRP-r.3.Effects of GRP regulation of PGA and PGC expression on breast cancer cell proliferation,migration,and invasionHuman recombinant GRP protein was used for cell stimulation at concentrations of 50 ng,50 ng and 100 ng,and a blank control.Changes in PGA and PGC expression levels and alterations in cell behavior were compared to observe the impact of GRP on the regulation of PGA and PGC expression(experimental methods same as in Part 2).4.Effects of GRP regulation of PGA and PGC expression through GRP-r receptor on breast cancer cell behaviorGRP-r antagonist was used for cell stimulation at a concentration of 4 μmol/ml.Changes in PGA and PGC expression levels and alterations in cell behavior were compared to observe the effects of the GRP and GRP-r ligand-receptor pathway on the regulation of PGA and PGC expression(experimental methods same as in Part 2).Results:Part Ⅰ PGA and PGC expression characteristics in different breast diseases1.Serum Expression Characteristics of PGA and PGC in Different Breast Diseases The serum levels of PGA and PGC were found to be higher in patients with breast cancer compared to those with non-cancerous breast diseases and healthy controls,indicating an association with the risk of breast cancer.Subgroup analysis based on age revealed that serum PGA and PGC expression levels were higher in elderly patients in all groups,showing an increasing trend with age.Furthermore,there were statistically significant differences observed after stratifying the data by age,particularly in the comparison between the 40-49 age group and the 50-59 age group.Significant differences were found in PGA levels between pre-and postmenopausal women(67.30 μg/L vs.84.45 μg/L,P<0.001),as well as in PGC levels between pre-and postmenopausal women(7.30 μg/L vs.10.00 μg/L,P=0.006).The serum levels of PGA and PGC were also correlated with menstrual status.In the disease continuum involving normal individuals,benign breast diseases,and breast cancer,the serum levels of PGA and PGC gradually increased.The median serum PGA levels were 65.80 μg/L,64.65 μg/L,and 76.20 μg/L in the normal population,benign breast disease group,and breast cancer group,respectively.The median serum PGC levels were 5.40 μg/L,6.60 μg/L,and 8.80 μg/L in the same respective groups.When comparing different pathological parameters in breast cancer,serum PGA levels were significantly lower in high-grade(grade III)breast cancer compared to the combined group of intermediate and low-grade(grades II + I)tumors(P=0.040).Similarly,serum PGC levels were significantly lower in high-grade(grade III)breast cancer compared to the combined group of intermediate and low-grade(grades II + I)tumors(P=0.044).ROC curve analysis was performed to evaluate the diagnostic performance of serum PGA and PGC.The cut-off values for PGA and PGC were determined as 90.45 μg/L and 4.85 μg/L,respectively.The sensitivity and specificity were 37%,78.5% for PGA,and 87.4%,44.6%for PGC,respectively.The corresponding AUC values were 0.582 for PGA and 0.689 for PGC.Subgroup analysis based on age further revealed that PGA had better diagnostic performance in the subgroup aged ≥60 years compared to the overall population(sensitivity 0.701 and specificity 0.521),while PGC had better diagnostic performance in the 40-49 age subgroup compared to the overall population(sensitivity 0.783 and specificity 0.593).2.In situ expression characteristics of PGA and PGC in different breast diseases The high expression of PGA and PGC in breast cancer tissues is higher than that in noncancerous breast disease tissues and normal control tissues,which is associated with the risk of breast cancer.Compared with the control group,the expression intensity of PGA and PGC in breast cancer group was significantly increased(P<0.001).Clinicopathological analysis showed that the expression intensity of PGA was significantly increased in the patients with larger tumors(P=0.023)and E-cadherin positive expression group(P<0.001),while the expression intensity of PGC was only significantly increased in the E-cadherin positive expression group(P<0.001).The overall positive rate of PGA was 51.25%,35.26%in benign breast diseases and 74.62% in breast cancer after the cut-off value of ROC curve was used as the negative positive boundary.The overall positive rate of PGC was 42.19%,28.42% in benign breast diseases and 62.31% in breast cancer.3.Expression characteristics of PG transcription level based on TCGA and its relationship with prognosis of breast cancerThe breast cancer m RNA expression data in the TCGA database showed that the expression level of PGA in breast cancer was lower than that in the normal control group,while the expression level of PGC in breast cancer was higher than that in the normal control group.Survival analysis results showed that PGA and PGC did not achieve statistical difference.GO analysis showed that PGA was involved in the regulation of nuclear division,lipid metabolism and organelle fission,and PGC was involved in the formation of extracellular matrix and extracellular skeleton.In KEGG enrichment analysis,PGA was closely related to PPAR signaling pathway and AMPK signaling pathway,and PGC was closely related to the interaction of cytokine-cytokine receptor,viral protein-cytokine and cytokine receptor pathway.Part Ⅱ The effect of PGA and PGC expression on the biological behavior of breast cancer cells1.PGA and PGC appear to have a potential promotional effect on the proliferation of breast cancer cells,although statistical significance was not reached.The results of the CCK-8 experiment showed that in MDA-MB-231 cells,the overexpression of PGA and PGC exhibited an enhancing trend in cell viability compared to the control group,but without statistical significance.The Ed U assay confirmed that in MDA-MB-231 cells,both the overexpression of PGA and PGC led to an enhanced cell proliferation trend compared to the control group.In MCF-7 cells,the overexpression of PGA demonstrated an inhibitory trend in cell proliferation compared to the control group,while PGC showed a tendency to promote cell proliferation.2.PGA and PGC had no significant effect on apoptosis of breast cancer cells In MDA-MB-231 cells,compared with the control group(21.59±0.69 Vs.21.28±1.46,P=0.860),compared with the control group(20.81±0.13 Vs.21.28±1.46,P=0.762),There was no significant difference in cell apoptosis.Similarly,there was no significant difference in apoptosis in MCF-7 cells between the PGA exposed group and the control group(5.79±0.13 VS 6.10±0.14,P=0.170)and the PGC exposed group and the control group(5.80 ±0.14 VS 6.10±0.14,P=0.197).3.PGA and PGC can inhibit the migration and invasion ability of breast cancer cells Scratch test showed that in MDA-MB-231 cells and MCF-7 cells,the difference between0 h cell spacing and 24 h cell spacing in the PGA and PGC groups was significantly smaller than that in the control group,and there was a significant difference in cell mobility between the two groups(P<0.05),suggesting that PGA and PGC had an inhibitory effect on the migration ability of breast cancer cells.In Transwell experiment,the number of breast cancer cells passing through the chamber in PGA and PGC passing group was significantly less than that in control group,suggesting that PGA and PGC inhibited the migration and invasion ability of breast cancer cells(P<0.05).4.PGA and PGC can affect the expression of molecular typing related indicators of breast cancerOverexpression of PGA and PGC inhibited the expression of PR and ER in MDA-MB-231 and MCF-7 cells(both P<0.05).Part Ⅲ Regulation of PG gene expression and its effect on proliferation,migration,and invasion of breast cancer cells1.Histological expression levels of GRP and GRP-R in different breast diseases and their relationship with clinicopathologic parameters of breast cancerThe immunohistochemical detection results of GRP and GRP-R in different breast diseases indicated that GRP and GRP-R were highly expressed in breast cancer tissues.GRP expression intensity was correlated with breast cancer grade(P=0.022),P63 expression(P=0.032)and E-cadherin expression(P <0.001).GRP-r expression was correlated with Her2 expression(P =0.023),E-cadherin expression(P <0.001)and GATA-3 expression(P=0.015).2.Correlation between the expression of PGA,PGC and GRP and GRP-R in breast cancer tissuesIn breast cancer tissues,the co-expression relationship between PGA,PGC,GRP and GRPR exists.The expressions of PGA and PGC were positively correlated with GRP and GRPR.The correlation coefficient between PGA and GRP was 0.442(P<0.001),and that between PGA and GRP-R was 0.237(P<0.001).The correlation coefficient between PGC and GRP was 0.302(P<0.001),and 0.174(P=0.002)with GRP-R.3.The effect of GRP regulation on the expression of PGA and PGC on the proliferation,migration and invasion of breast cancer cellsAfter the stimulation of MDA-MB-231 and MCF-7 breast cancer cells by GRP protein,the expression levels of PGA and PGC were detected in the supernatant.The results showed that the expressions of PGA and PGC were up-regulated(P<0.001 for both),suggesting that GRP significantly promoted the expression levels of PGA and PGC.The results of CCK-8 experiment showed that GRP protein could promote the proliferation of breast cancer cells after stimulation(P=0.009 in MDA-MB-231,P=0.021 in MCF-7).The results of Edu experiment also showed a trend of promoting proliferation(P=0.006 in MDA-MB-231,P=0.090 in MCF-7).Scratch assay results showed that GRP inhibited migration of breast cancer cells after stimulation(P<0.001 in MDA-MB-231 and P<0.001 in MCF-7).These results suggest that GRP protein stimulation can up-regulate the expression of PGA and PGC proteins in breast cancer cells,and inhibit the migration of breast cancer cells.4.GRP regulates the expression of PGA and PGC through GRP-R receptor,thus affecting the biological behavior of breast cancer cellsAfter the stimulation of MD-MB-231 and MCF-7 breast cancer cells by GRP-r inhibitor,the expression levels of PGA and PGC were detected in the cell supernatant.The results showed that the expressions of PGA and PGC were significantly down-regulated,suggesting that the expression of PGA and PGC was inhibited by GRP-r inhibitor after stimulation.That is,GRP-r may reverse activate the expression of PGA and PGC(PGA:P<0.001 in MDA-MB-231,PGC: P<0.001;PGA: P=0.025,PGC: P=0.006)in MCF-7.The proliferative activity of breast cancer cells was detected by CCK-8 after the addition of GRP-r inhibitor,and the results showed that the proliferative activity of MDA-MB-231 cells was significantly inhibited(P<0.05).The results of Edu experiment showed that the proliferation ability of MDA-MB-231 and MCF-7 cells could be significantly inhibited after the addition of GRP-r inhibitor,and the proliferation rate of the inhibitor group was lower than that of no-load control group(P<0.01).Scratch test results showed that the addition of GRP-r inhibitor could significantly inhibit the migration ability of MDA-MB-231 and MCF-7 cells,and the mobility of the inhibitor group was lower than that of the no-load control group(both P<0.05).The addition of inhibitor and PGA or PGC group at the same time produced a recovery effect compared with only the addition of inhibitor group,suggesting that GRP-r inhibitor may inhibit the proliferation and migration ability of the two types of breast cancer cells by down-regulating the expression of PGA and PGC proteins.Conclusions:1.PGA and PGC are highly expressed in serum and tissues of breast cancer patients.2.Low serum expression of PGA and PGC is associated with high grade(Ⅲ)breast cancer.The high histological expression of PGA and PGC is related to the positive expression of E-cadherin,and the positive expression of PGA is related to the larger tumor.3.Serum and tissue expression of PGA and PGC can differentiate between benign and malignant breast diseases,with a significantly higher positivity rate in breast cancer compared to benign diseases,suggesting their potential as auxiliary diagnostic biomarkers for breast cancer.4.Overexpression of PGA and PGC has a tendency to promote proliferation of breast cancer cells;It can inhibit the migration and invasion of breast cancer cells,and inhibit the expression of ER and PR.5.GRP regulates the expression of PGA and PGC through GRP-R ligand receptor pathway,and then changes the biological behavior of breast cancer cells. |
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