| Objective:At present,lung malignant tumors are still one of the biggest enemies threatening human health.China's cancer incidence and death statistics show that in 2015,the number of new cases of lung cancer in my country was 787,000,and the number of deaths was 631,000,ranking first among cancers.According to different clinical and pathological characteristics,lung cancer is divided into small cell lung cancer(accounting for 13%)and non-small cell lung cancer(accounting for 87%),and its 5-year overall survival rate is only 18.2%.Traditional treatment methods include surgery,chemotherapy,radiotherapy,etc.,but they are not effective for patients with advanced lung cancer.In recent years,with the rise of molecular targeted therapy,the progression-free survival and overall survival of non-small cell lung cancer have been significantly improved.For lung cancer patients with sensitive gene mutations,molecular targeted therapy is one of the most effective treatments.For patients who do not have sensitive mutations or have target gene resistance mutations during targeted therapy,discover new tumor biology Markers and new therapeutic targets are particularly important.Protamine-1(PRM1)is a kind of tumor-testis antigen(CTA).Under normal circumstances,CAT is only expressed in testicular tissues,while other tissues are not expressed or very lowly expressed,but it can be expressed when tumors appear in the body.It is highly expressed in some tumor tissues,and the tumor-testis antigen is also named.Previous research on PRM1 has mainly focused on the field of male reproduction.In recent years,with the deepening of CTA research in the field of tumors,the role of PRM1 in malignant tumors has gradually been paid attention to.Studies have found that PRM1 is a tumor-testis antigen gene related to colon cancer.RT-PCR technology is used to analyze PRM1 in colon cancer.And the expression level of adjacent tissues,found that the level of PRM1-m RNA in colon cancer tissues was significantly higher than that of adjacent tissues and normal tissues.Studies have also found that PRM1 is highly expressed in patients with chronic lymphocytic leukemia,and PRM1 is also considered to be the CTA gene of chronic lymphocytic leukemia.Other studies have shown that the abnormal expression of PRM1 in malignant tumors may be related to tumor growth and invasion.In animal experiments,subcutaneous injection of PRM1 protein into nude mice can significantly increase the incidence of intestinal tumors in mice.So far,there is no report on the expression of PRM1 in non-small cell lung cancer.The preliminary work of the research group has confirmed that the expression of PRM1 in non-small cell lung cancer tissue is significantly higher than that of normal tissues.At the same time,PRM1 can promote the proliferation of non-small cell lung cancer cells.In this study,we detected the expression of PRM1 in non-small cell lung cancer tumor tissues and peripheral blood,analyzed the correlation between PRM1 protein levels and clinical features;and evaluated the diagnostic value of PRM1 in non-small cell lung cancer;passed Overexpress and knock down the PRM1 gene to observe its effects on cell proliferation,apoptosis and cycle;establish a nude mouse metastatic model of non-small cell lung cancer,detect the expression level of PRM1 protein in the peripheral blood of the metastatic nude mouse,and observe the recombination of exogenous PRM1 The effect of protein and PRM1 antibody on the growth of metastatic tumor in nude mice.The purpose is to evaluate the diagnostic value of PRM1 as a new biological marker for non-small cell lung cancer,to study the proliferation effect of PRM1 on non-small cell lung cancer cells,and to discuss its mechanism.Methods:1.Collect 110 cases of non-small cell lung cancer tumor tissues,matched normal tissues,preoperative serum and healthy serum samples,and use RT-PCR,immunohistochemical staining and ELISA to detect PRM1 gene and protein levels,and apply Chi-square The correlation between the level of PRM1 protein in tumor tissues and peripheral blood of patients with clinical characteristics was tested and analyzed statistically.2.Assess the diagnostic value of PRM1,use SPSS software to statistically analyze the diagnostic value of PRM1 protein level in peripheral blood for non-small cell lung cancer and its subgroups lung adenocarcinoma,lung squamous cell carcinoma,and compare it with the clinically commonly used tumor markers CEA,SCC Compare with CYFRA21-1 to analyze the pros and cons of the diagnostic value of PRM1.3.Observe the effect of PRM1 expression in cells on cell proliferation.Through plasmid construction technology and si RNA transfection technology,overexpress and knock down the PRM1 gene of A549 and NCI-H460 cells,and use RT-PCR and ELISA to detect cell PRM1 gene and For protein expression level,CCK8 method and Ed U method were used to detect cell proliferation,and flow cytometry was used to detect cell cycle and apoptosis.4.Observe the effect of exogenous PRM1 recombinant protein and antibody on cell proliferation.Add human recombinant PRM1 protein and PRM1 antibody to A549 and NCI-H460 cell culture medium,and use CCK8 and Ed U methods to detect cell proliferation.5.Observe the expression of PRM1 in cells under different nutrient conditions.Culture A549 and NCI-H460 cells in a culture medium containing different concentrations of serum,and use RT-PCR and ELISA methods to detect the PRM1 gene and protein levels of the cells.6.Observe the effects of exogenous PRM1 recombinant protein and antibody on the growth of animal metastases,prepare nude mouse metastasis models,and inject non-small cell lung cancer cells A549 subcutaneously into the right side of the back of nude mice.After tumor formation:(1)apply ELISA Detect the level of PRM1 protein in peripheral blood of metastatic nude mice and healthy nude mice,and analyze their differences;(2)Inject human recombinant PRM1 protein,PRM1 antibody and the mixture of the two through the tail vein,and measure and calculate every 3 days Tumor volume,draw tumor growth curve.(3)On the 33 rd day,the nude mice were sacrificed under anesthesia,the tumor tissue was taken out,the weight was measured and the tumor volume was calculated,and the influence of exogenous PRM1 recombinant protein and PRM1 antibody on the growth of metastatic tumors in nude mice was analyzed.Result:1.The m RNA and protein levels of PRM1 in non-small cell lung cancer tumor tissues are significantly higher than those in matched normal tissues.Statistical analysis shows that with the increase of PRM1 protein levels in tumor tissues,the later the tumor T stage,the higher the positive rate of lymph node metastasis,And no correlation with pathological type,age,sex,vascular and nerve infiltration;2.The level of PRM1 protein in the peripheral blood of patients with non-small cell lung cancer is significantly higher than that of healthy people.Statistical analysis shows that the level of PRM1 protein in the peripheral blood of patients with positive lymph node metastasis and a later clinical stage is higher,which is related to pathological type and age,Gender,vascular and nerve infiltration are not related.3.PPM1 has high diagnostic value for non-small cells.The statistical analysis of SPSS software showed:(1)The level of PRM1 protein in peripheral blood has high diagnostic value in non-small cell lung cancer and its subgroups of lung adenocarcinoma and lung squamous cell carcinoma.(2)In lung adenocarcinoma,the diagnostic value of PRM1 is higher than that of CEA;in lung squamous cell carcinoma,the diagnostic value of PRM1 is significantly stronger than that of SCC and comparable to CYFRA21-1;(3)The diagnosis of a combination of tumor markers containing PRM1 The value is significantly higher than the traditional combination;(4)In the early lung adenocarcinoma,the diagnostic value of PRM1 is higher than that of CEA;in the early lung squamous cell carcinoma,the diagnostic value of PRM1 is higher than that of SCC and not inferior to CYFRA21-1.It indicates that PRM1 has a higher diagnostic value for non-small cell lung cancer.4.The level of PRM1 m RNA in non-small cell lung cancer cell lines A549 and NCI-H460 is significantly higher than that of normal bronchial epithelial BEAS-2B cells;similarly,the level of PRM1 protein in A549 and NCI-H460 cells and in the culture medium is higher than that of BEAS-2B.The cells were significantly increased.5.Successfully constructed A549 and NCI-H460 cell line models with PRM1 overexpression and knockdown.Compared with the control group,overexpression of PRM1 promoted the proliferation of A549 and NCI-H460 cells,while knockdown of PRM1 inhibited cell proliferation.6.The exogenous PRM1 recombinant protein can promote the proliferation of non-small cell lung cancer A549 and NCI-H460 cells,while the PRM1 antibody inhibits their proliferation.7.There are differences in the expression of PRM1 under different nutritional conditions.The results show that with the decrease of serum concentration in the cell culture fluid,the expression level of PRM1 in A549 and NCI-H460 cells increases significantly.8.The cell cycle test results indicate that overexpression of PRM1 reduces the ratio of G0/G1 phase in the cell cycle and increases the ratio of cells in S phase;while knocking down PRM1 causes cell cycle arrest in G0-G1 phase and decreases the ratio of cells in S phase;Apoptosis results showed that knocking down PRM1 had no significant effect on the apoptosis level of A549 and NCI-H460 cells.9.Results of animal experiments:(1)Compared with the healthy control group,the level of PRM1 protein in the peripheral blood of nude mice with metastatic tumors was significantly increased,which also confirmed that the results of animal experiments and in vitro cell experiments were consistent with the results of human tissue experiments.(2)The exogenous PRM1 recombinant protein can promote the growth of metastatic tumors in nude mice,while the PRM1 antibody inhibits the growth of metastatic tumors.The experimental results suggest that compared with the control group,nude mice given exogenous PRM1 recombinant protein via the tail vein grow faster and have larger tumors,while nude mice given PRM1 antibody have slower tumor growth and smaller tumors.Conclusion:1.The specific and high expression of PRM1 in non-small cell lung cancer tumor tissue and peripheral blood,and its high diagnostic value for non-small cell lung cancer,especially early stage non-small cell lung cancer,make it a tumor of non-small cell lung cancer The potential of the marker.2.Overexpression and knockdown of the PRM1 gene have verified its proliferation-promoting effect on non-small cell lung cancer cell lines A549 and NCIH460.This effect is related to increasing the proportion of S phase cells in the cell cycle,rather than inhibiting cell apoptosis.3.The increase of PRM1 protein levels both inside and outside the cell can promote cell proliferation,suggesting that PRM1 may have multiple ways to promote proliferation,and provide clues for further research on the mechanism of PRM1.4.The expression level of PRM1 in cells increases when the nutrient supply is poor.This negative feedback regulation can explain to a certain extent the reason why non-small cell lung cancer still grows rapidly under the condition of poor blood supply.5.The effect of exogenous PRM1 recombinant protein and antibody on the growth of metastatic tumors in nude mice has further verified the role of PRM1 in promoting tumor growth in vivo.At the same time,the anti-tumor effect of PRM1 antibody also provides direct tumor treatment targets.Theoretical support. |
PDF Full Text Request