The Role Of Protamine 1 In The Diagnosis And Treatment Of Gastric Adenocarcinoma And The Mechanism Of Promoting Tumor Growth

Background and purpose:Gastric cancer is one of the cancers with high mortality.About 783000 patients worldwide die of gastric cancer every year.Early diagnosis and early treatment are the key to improve the survival rate of patients with gastric cancer.At present,the detection of serum tumor markers is one of the important ways for early screening of gastric cancer.Looking for tumor markers with high sensitivity and specificity is of great significance for the effective prevention and treatment of gastric cancer.Cancer / testicular antigen(CTA)is one of the tumor-associated antigens,which only exists in normal male testicular tissues under physiological conditions,but it is specifically expressed in a variety of tumor tissues and has certain immunogenicity,which brings a new opportunity for tumor diagnosis and immunotherapy.Protamine 1(PRM1)is a member of the CTA family.It is highly expressed in leukemia cells and interacts with seminal vesicle protein 1(SEMG1).It promotes the condensation of sperm chromatin together with CTA transition protein 1(TNP1)in sperm cells..In the previous work,we used transcriptomic sequencing of colorectal cancer paired tissues and found that PRM1 was positively expressed in colorectal adenocarcinoma tissues.Since both gastric mucosal epithelium and colorectal mucosal epithelium originate from the endoderm and are similar in morphology and function,serum CEA,CA724,CA50 and other tumor markers can be detected in gastrointestinal cancer tissues and serum,and they are often used in combination with gastric cancer.,Diagnosis and recurrence monitoring of patients with colorectal cancer.Based on the above research background and preliminary work foundation,X this study will conduct in-depth research on the expression,biological function and mechanism of PRM1 in gastric adenocarcinoma;combine in vivo and in vitro experiments to verify the role of PRM1 in the growth and metastasis of gastric adenocarcinoma,and Potential value in the immunotherapy of gastric adenocarcinoma.Simultaneously analyze the expression of SEMG1 and TNP1 in gastric cancer tissues,explore the diagnostic value of combined application of PRM1,SEMG1 and TNP1 in gastric adenocarcinoma,and the possibility of developing a multivalent tumor vaccine for gastric adenocarcinoma.Methods:1.Detection and analysis of PRM1 expression in gastric adenocarcinoma1.1.q PCR was used to analyze the expression of PRM1 m RNA in tumor,paracancerous and normal tissues of patients with gastric adenocarcinoma.1.2.Immunohistochemical staining was used to detect the expression and distribution of PRM1 protein in gastric adenocarcinoma,and the relationship between PRM1 expression and clinicopathological features was analyzed.2.Detection and analysis of serum PRM1 level and its correlation with Helicobacter pylori infection in patients with gastric adenocarcinoma.2.1.Enzyme-linked immunosorbent assay(ELISA)was used to detect the concentration of PRM1 protein in peripheral blood of gastric adenocarcinoma patients and healthy subjects from different centers,and the relationship between serum PRM1 level and clinicopathological features was analyzed.2.2.Western blotting was used to analyze the infection and typing of Helicobacter pylori in patients with gastric adenocarcinoma,and to explore the relationship between Helicobacter pylori infection and serum PRM1 concentration.3.In vivo and in vitro experiments to explore the biological function of PRM1 in gastric adenocarcinoma cells3.1.Cell model screening: q PCR and intracellular protein staining were used to screen the gastric adenocarcinoma cell lines with high and low expression of PRM1 in6 gastric adenocarcinoma cell lines(AGS,HGC-27,NCI-N87,KATO?,SGC-7901 and MGC-803).3.2.The proliferative activity and PRM1 secretion characteristics of gastric adenocarcinoma cell lines AGS,HGC-27,NCI-N87,KATO? and SGC-7901 were detected and analyzed at different time points(24 h,48 h and 72 h)and different serum concentrations(0%,2.5%,5%,10%,15% and 20%).3.3.GSEA gene enrichment analysis predicted the biological function of PRM1 in gastric cancer.3.4.AGS,HGC-27,NCI-N87 and KATO? cells were cultured with human PRM1 recombinant protein and anti-PRM1 antibody,and their effects on cell proliferation were detected by CCK-8 and Ed U.3.5.The PRM1 overexpression plasmid was transfected into NCI-N87 and Kato? cells by Lipofectamine transfection reagent,and si RNA-PRM1 was transfected into AGS and HGC-27 cells.The cell proliferation was detected by CCK-8 and Ed U.3.6.Cell cycle was detected by 7-AAD single staining and apoptosis was detected by Annexin-V/7-AAD double staining.3.7.Cell scratch test was used to detect the migration ability of gastric cancer cells,and Transwell test was used to detect the invasive ability of gastric cancer cells.3.8.The effect of PRM1 on the phosphorylation of key factors of PI3K/Akt/m TOR/4E-BP1 pathway was evaluated by Western blot.3.9.The subcutaneous transplantation tumor model of NCI-N87 gastric adenocarcinoma in nude mice was established.The tumor-bearing nude mice were randomly divided into control group and anti-PRM1 antibody treatment group.The control group was injected with nano-gel mixed with the same amount of PBS,and the treatment group was injected with nano-gel mixed with anti-PRM1 antibody330?g/kg/5 days for 4 times.The tumor volume was measured every other day to draw the tumor growth curve.4.To explore the relationship between the expression of PRM1,SEMG1 and TNP1 proteins in gastric adenocarcinoma cells.Immunohistochemical staining was used to detect the expression of SEMG1 and TNP1 protein in the same gastric adenocarcinoma tissue,and to explore the XII correlation of PRM1,SEMG1 and TNP1 protein expression in gastric adenocarcinoma tissue,so as to provide data basis for the development of multivalent tumor vaccine.Results:1.High expression of PRM1 in gastric adenocarcinoma.1.1.q PCR results showed that PRM1 m RNA was positive in gastric adenocarcinoma,with a positive rate of 93.8%.1.2.PRM1 protein was positive in gastric cancer and atypical hyperplasia(precancerous lesions),and the positive rates were 81.7% and 61.6%,respectively.Positive staining could be seen in the glandular lumen of gastric cancer,but not in normal tissues,suggesting that tumor cells may express and secrete PRM1.The positive rate of PRM1 protein expression was positively correlated with malignant degree,clinical stage,vascular invasion and nerve invasion of gastric cancer,while the expression level of PRM1 protein was positively correlated with nerve invasion(0.039).2.Serum PRM1 plays an important role in the diagnosis of gastric adenocarcinoma.2.1.The results of ELISA detection showed that the protein concentration of serum PRM1 in patients(1029.31 ± 824.47pg/m L)was significantly higher than that in healthy controls(94.30 ± 88.70pg/ m L)(P < 0.001).ROC curve showed that serum PRM1 had high sensitivity and specificity(AUC=0.991)in the diagnosis of gastric adenocarcinoma,and the positive rate was 95.95%.It also has excellent diagnostic ability(AUC=0.985)for early patients,and the positive rate of diagnosis is 94.19%(81 / 86).The verification group also confirmed that the patient's serum PRM1 protein is an effective and reliable diagnostic marker of gastric cancer with repeatability.The content of serum PRM1 protein in patients after radical resection of gastric cancer was significantly lower than that before operation(P < 0.001),suggesting that serum PRM1 protein was directly secreted by tumor.2.2.The level of serum PRM1 in patients with gastric adenocarcinoma was positively correlated with Helicobacter pylori infection,tumor markers CA724 and AFP,and negatively correlated with tumor volume.3.Biological role of PRM1 in gastric adenocarcinoma cells3.1.The results of q PCR and intracellular protein staining showed that the expression level of PRM1 was the highest in AGS and HGC-27 cells,and the lowest in NCI-N87 and KATO? cells.Gastric adenocarcinoma cells could secrete PRM1 protein into the supernatant of cell culture,and the ability to secrete PRM1 was enhanced under the condition of low serum.3.2.Bioinformatics analysis predicted that PRM1 was related to the proliferation,metastasis and cycle of gastric adenocarcinoma cells.The results of CCK-8 and Ed U experiments showed that human PRM1 recombinant protein incubation and PRM1 gene overexpression could significantly promote the proliferation of AGS,HGC-27,NCI-N87 and KATO? cells,while anti-PRM1 antibody incubation and PRM1 gene knockout could significantly inhibit the proliferation of AGS,HGC-27,NCI-N87 and KATO? cells.The results of flow cytometry showed that PRM1 could significantly increase the proportion of S phase and promote the progression of G1 / S phase of gastric adenocarcinoma cells,but did not affect the apoptosis of gastric adenocarcinoma cells.Western blot experiments showed that PRM1 could significantly increase the level of phosphorylation of PI3K/Akt/m TOR/4E-BP1 pathway,thus promoting the proliferation of gastric adenocarcinoma cells.3.3.Scratch assay showed that human PRM1 recombinant protein could significantly enhance the migration ability of gastric adenocarcinoma cell line AGS,HGC-27,anti-PRM1 antibody could also inhibit the migration ability of gastric adenocarcinoma cell line AGS,HGC-27,and Transwell assay showed that PRM1 protein could significantly promote the invasive ability of gastric adenocarcinoma cell line.3.4.The results of animal experiments showed that the tumor growth rate of the anti-PRM1 antibody group was significantly lower than that of the control group,and the tumor growth was inhibited.4.Analysis of the correlation between the expression of PRM1 protein and XIV SEMG1,TNP1 protein in gastric adenocarcinoma.4.1.SEMG1 protein was widely expressed in gastric adenocarcinoma,and the positive rate was 73.33%(88 / 120).The positive rate was positively correlated with clinical stage(Pause 0.021),lymph node metastasis(P < 0.001),vascular invasion(P< 0.007)and nerve invasion(P < 0.05).4.2.TNP1 protein is widely expressed in gastric adenocarcinoma,and the positive rate is 48.33%(58 /120).The positive rate is positively correlated with clinical stage(P= 0.022),tumor size(P=0.046)and EGFR expression(P=0.027).4.3.The expression of PRM1 protein was positively correlated with the expression of SEMG1 and TNP1 protein in gastric adenocarcinoma(P < 0.001).The expression correlation of the three proteins had the characteristics of CTA co-expression pattern.The positive expression of TNP1 protein can predict the expression of PRM1 protein.Conclusions:1.PRM1 was highly expressed in gastric adenocarcinoma,and the positive rate was 81.7%(98 / 120).It was positively correlated with the prognosis of gastric cancer,clinical stage,vascular invasion and nerve invasion,and negatively correlated with the differentiation of gastric adenocarcinoma cells.2.The level of serum PRM1 protein in patients with gastric adenocarcinoma is significantly higher than that in healthy controls,which shows high sensitivity and specificity in the early and late diagnosis of gastric cancer,and is expected to become a potential marker for blood diagnosis of gastric adenocarcinoma.3.PRM1 promotes the migration and invasion of gastric adenocarcinoma cells,and promotes the progression of G1 / S phase by activating PI3K/Akt/m TOR/4E-BP1 signaling pathway,thus promoting the proliferation of gastric adenocarcinoma cells.In vivo and in vitro,anti-PRM1 antibodies have been proved to significantly inhibit tumor growth,and PRM1 can be used as a potential target for immunotherapy of gastric adenocarcinoma.4.The expression of PRM1 protein in gastric adenocarcinoma was positively correlated with SEMG1 and TNP1 protein,and had the common characteristics of CTA co-expression pattern.PRM1,SEMG1 and TNP1 are expected to be potential targets for multivalent tumor vaccines.In summary,this study provides theoretical basis and data support for PRM1 as a new gastric cancer marker,provides new methods and ideas for early diagnosis,immunotherapy and tumor vaccine target selection of gastric cancer in the future,and has important clinical application value.