| Breast invasive carcinoma（BRCA）is one of the most common malignant tumors threatening to female health,and its incidence is increasing year by year.According to the latest data published by the international agency for research on cancer（IARC）of the World Health Organization in 2020,there are as many as 2.26 million new cases of breast cancer,which has surpassed lung cancer to become the world’s largest cancer.In China,BRCA is also the first incidence of female malignant tumors,with an annual incidence of about304,000.Therefore,research on the prevention,diagnosis and treatment of BRCA is one of the important subjects of study in the field of medicine today.Leucine rich alpha-2-glycoprotein 1（LRG1）,one member of leucine rich repeat（LRR）family,is the first protein to be discovered that contains the LRR structure.The human LRG1 gene is localized in 19p13.3 and contains 2810base pairs,including 2 exons and 1 intron.The first exon contains 43 base pairs,and the second exon contains 1737 base pairs,encoding a total of 347amino acid residues.Because the chromosomal region of LRG1 contains the gene encoding neutrophil granzyme,which is highly expressed in the process of neutrophil differentiation,early studies suggest that LRG1 may be one marker of early neutrophil differentiation.With the continuous development of molecular biology technology,other functions of LRG1 have gradually been discovered:LRG1 not only participates in the interaction between cell proteins,but also plays an important role in cell signal transduction,cell adhesion and development.In recent years,studies have shown that LRG1 is abnormally expressed in a variety of human malignant tumor tissues,blood,cerebrospinal fluid and exosomes.LRG1 can be used as a biomarker for early diagnosis and prognosis in some tumors.Current studies have found that LRG1 plays an important role in tumor occurrence,development,invasion and metastasis,epithelial-mesenchymal transition and abnormal angiogenesis.Therefore,the role of LRG1 in the field of anti-tumor therapy is gradually being paid attention to.However,there are significant differences in the expression of LRG1 in different tumors,LRG1 may play different roles in the occurrence and development of different tumors.For example,LRG1 in exosomes is related to the occurrence and development of non-small cell lung cancer,LRG1 expression in liver cancer tissues is down-regulated compared with normal tissues.In vitro experiments show that LRG1 has no effect on the proliferation of liver cancer cells.The increase in LRG1 expression in serum and tumor tissues of patients with ovarian cancer is related to tumor stage.However,the expression,function,and mechanism of LRG1 in BRCA are still unclear.This study will be divided into three parts to study the expression and clinical significance of LRG1 in BRCA:The first part:The expression of LRG1 in different tumors and its clinical significance will be analyzed by bioinformatics methods.The second part:The expression of LRG1 protein in 330 primary breast cancer tissues will be detected by the Max Vision TM one-step method of immunohistochemistry,and the correlation between the expression of LRG1protein and the clinicopathological indicators and prognosis of breast cancer will be further analyzed.The third part:The effect of LRG1 on the biological behavior of breast cancer cells will be explored.Reverse transcription-polymerase chain reaction（RT-PCR）technology and western-blot technology will be used to detect the expression of LRG1 in different breast cancer cell lines.RNA interference technology will be used to inhibit the expression of LRG1 in breast cancer MD-MB-231 cells,and the cell proliferation ability will be detected by the CCK-8 experiment,the scratch experiment will be used to detect the cell migration ability after transfection,the transwell experiment will be used to detect the cell invasion ability.Part One Bioinformatics analysis of LRG1 expression and clinical significance in different tumorsObjective:To analyze the expression and clinical significance of LRG1in different tumors by bioinformatics.Methods:Based on the sequencing results of RNA-seq in tumor tissues and normal tissues in the cancer genome atlas（TCGA）database（http://www.tcga.org/）,we analyzed the expression of LRG1mRNA in tumor tissues and normal tissues using the TIMER2.0 online tool（https://cistrome.shinyapps.io/timer/）.The expression of LRG1 protein in different tumor tissues and normal tissues was analyzed in the human protein atlas database（https://www.proteinatlas.org）.Based on the clinical characteristics and survival data of patients in the Kaplan-Meier plotter database（http://kmplot.com/analysis）,the expression of LRG1mRNA in different tumors and overall survival（OS）and recurrence-free survival（RFS）were analyzed and the Kaplan-Meier curve was drawn.Results:1.The expression of LRG1mRNA in breast invasive carcinoma（BRCA,1097 cases）,renal clear cell carcinoma（KIRC,533 cases）,lung adenocarcinoma（LUAD,515 cases）,uterine corpus endometrial carcinoma（UCEC,545 cases）and thyroid carcinoma（THCA,501 cases）were significantly higher than adjacent normal tissues（P<0.001）.2.The expression of LRG1 protein in breast invasive carcinoma,renal clear cell carcinoma,lung adenocarcinoma,uterine corpus endometrial carcinoma and thyroid carcinoma was similar to LRG1mRNA,which was significantly higher than adjacent tissues.3.The expression of LRG1mRNA in 566 cases of Luminal breast invasive carcinoma was 37.462（9.930-74.197）,higher than HER-2 and triple negative breast invasive carcinoma（P<0.01）.According to the further analysis of lymph node metastasis,the expression level of 1646 patients with positive lymph nodes was higher than 2415 patients with negative lymph nodes,and the difference was statistically significant（P<0.0001）.The expression of LRG1mRNA in 17 cases of mucinous adenocarcinoma was significantly lower than other invasive carcinomas（invasive ductal carcinoma,invasive lobular carcinoma,and mixed carcinoma）,the difference was statistically significant（P<0.0001）.Using the Kaplan-Meier plotter online tool to analyze the survival data in the TCGA database,the expression of LRG1mRNA has no significant relationship with the OS of Luminal A,Luminal B,and HER-2 positive breast cancers（all P>0.05）,but in Basal-like breast invasive carcinoma,LRG1 was significantly correlated with OS,and the patients with low expression of LRG1 had higher OS（HR=3.12,95%CI=1.54～6.29,P<0.001）.4.In KIRC,with the increase of clinical stage,the expression of LRG1mRNA showed an upward trend.The expression of LRG1 in stage IV patients was significantly higher than that in stage I and II patients,and the difference was significant（P<0.01）.In different histological grades,the same trend was shown.（P<0.01）.Kaplan Meier plotter was used to analyze OS and RFS of 530 KIRC patients in TCGA database and draw survival curve.It was found that OS of 367 patients with high LRG1 expression was lower than 163patients with low LRG1 expression,and LRG1 expression was significantly correlated with OS（HR=1.71,95%CI=1.18～2.47,P=0.0042）.The relationship between LRG1 expression and RFS was consistent with OS.The RFS of patients with high LRG1 expression was lower than patients with low LRG1 expression（HR=3.57,95%CI=1.26-9.87,P=0.0089）.In the data of kidney renal papillary cell carcinoma（KIRP）patients,it was also found that the expression of LRG1 could predict OS and RFS.The OS and RFS in the high expression group were significantly lower than the low expression group（OS:HR=2.19,95%CI=1.2～4.01,P=0.0091.RFS:HR=5.32,95%CI=1.2～22.54,P=0.011）.5.The expression of LRG1mRNA in LUAD tissues was significantly higher than that in normal tissues.In normal lung tissue,the median expression of LRG1mRNA was 25.277（20.394-30.683）,but in LUAD tissue was 35.431（20.538-53.567）,the difference was statistically significant（P<0.001）.By analyzing the data of 503 cases of lung squamous cell carcinoma（LUSC）and 52 cases of normal tissues,the expression level of LRG1mRNA in normal tissues was significantly higher than that of LUSC（26.701vs11.612,P<0.001）.There is a significant difference in the expression of LRG1mRNA in LUAD and LUSC.In the LUAD group,the OS of 222patients in the LRG1 high expression group was lower than that in the 282 low expression group,and the expression of LRG1mRNA was significantly correlated with OS（HR=1.34,95%CI=1-1.8,P=0.045）.6.The expression level of LRG1mRNA in uterine corpus endometrial carcinoma was 24.37（10.368-48.392）,which was significantly higher than normal endometrial tissue 10.072（2.357-14.296）（P<0.001）.There was no significant correlation between the expression level of LRG1 and age,clinical stage,menstrual status and prognosis.7.The expression of LRG1mRNA in thyroid cancer increased with the number of lymph node metastasis,which was statistically significant（P=0.027）.In terms of prognosis,OS of 261 patients with LRG1 high expression group was better than 241 patients with low expression group,and there is a significant difference（HR=0.11,95%CI=0.02-0.48,P=0.00035）.Conclusions:1.The expression of LRG1mRNA and protein is significantly up-regulated in BRCA,KIRC,LUAD,UCEC and THCA.LRG1 may play an important role in the development of tumors.2.The expression level of LRG1mRNA in Luminal breast invasive carcinoma is higher than that of HER-2 and triple negative.LRG1 is negatively correlated with OS in Basal-like breast invasive carcinoma.3.The expression of LRG1mRNA in KIRC can predict OS and RFS,and the expression of LRG1 is negatively correlated with OS and RFS.4.LRG1mRNA expression is up regulated in LUAD and negatively correlated with OS.5.The expression of LRG1mRNA is up-regulated in UCEC,but it has no significant correlation with clinical features and prognosis.6.The expression of LRG1mRNA in THCA is positively correlated with the number of lymph node metastasis and OS.Part Two The relationship between the expression of LRG1 protein and clinical features,survival and prognosis in breast invasive carcinomaObjective:To investigate the relationship between the expression of LRG1 protein and the clinical features,survival and prognosis of breast invasive carcinoma.Methods:The expression of LRG1 protein in 330 cases of primary breast invasive carcinoma was detected by immunohistochemical maxvisionTMone-step method,and the correlation between LRG1 and age,menstruation,tumor size,clinical stage,lymph node metastasis,histopathological grade,molecular type and prognosis were discussed.Results:1.The expression of LRG1 protein in primary breast invasive carcinoma.Among 330 patients with primary breast invasive carcinoma,58.48%（193cases）showed positive expression of LRG1.The staining part of LRG1 is mainly located in the cytoplasm,and the positive signal appears as a brown granular appearance.2.The relationship between LRG1 and clinicopathological parameters.In the 0-3 lymph node metastasis group,128 cases had positive expression of LRG1,121 cases had negative expression,and the positive expression rate was 51.41%,in the group with more than 3 lymph node metastasis,65 cases had positive expression of LRG1,16 cases had negative expression,and the positive expression rate was 80.25%.There was a significant difference in the positive expression rate of LRG1（χ2=20.939,P<0.001）.The expression of LRG1 was positively correlated with the load of lymph node metastasis.As the number of lymph node metastases increased,the positive expression rate of LRG1 increased.There were 124 cases with LRG1 positive expression and125 cases with negative expression in stageⅠ-Ⅱpatients,with a positive expression rate of 62.96%,in stageⅢgroup,69 cases with positive expression of LRG1 and 12 cases with negative expression,with a positive expression rate of 85.19%.The difference in the positive expression rate of LRG1 between the two groups was statistically significant（χ2=31.520,P<0.001）.The expression of LRG1 was positively correlated with tumor burden.As the disease progresses,the positive expression rate of LRG1increased.3.The relationship between the expression of LRG1 protein and DFS,OS in breast invasive carcinoma.After a median follow-up of 72 months,92patients had local recurrence or distant metastasis,and 80 patients died.The72-month DFS of the LRG1 positive expression group was 61.14%（118/193）,and the negative group was 87.59%（120/137）.There was a significant difference in DFS between the two groups（χ2=27.123,P<0.001）.The72-month OS was similar to the DFS results.There were 80 deaths,and the72-month OS of the overall population was 75.76%.The 72-month OS of the LRG1 positive expression group was 66.32%（128/193）,the LRG1 negative group was 89.05%（122/137）,and the difference between the two groups was significant（χ2=22.864,P<0.001）.4.Multi-factor analysis of COX regression model affecting DFS and OS.COX multivariate analysis showed that LRG1 expression,TNM pathological staging and molecular subtypes were independent risk factors affecting DFS and OS in breast invasive carcinoma patients（P<0.05）.There was no significant correlation between tumor size,histopathological grade,age,menstrual status and DFS,OS（P>0.05）.The hazard ratio of LRG1 expression,TNM pathological stage and molecular subtype to DFS were 2.090,3.214 and1.796,respectively,and to OS were 2.112,2.628 and 1.755,respectively.Conclusions:1.The expression of LRG1 protein in breast invasive carcinoma is not related to age,menstrual status,tumor size,histological grade and molecular type.2.The expression of LRG1 in breast invasive carcinoma is positively correlated with the number of lymph node metastasis.As the number of lymph node metastases increases,the positive expression of LRG1 increases significantly.3.The expression of LRG1 protein in breast invasive carcinoma is related to the pathological TNM staging.The later the staging,the higher the positive expression of LRG1.4.The expression of LRG1 protein,TNM pathological staging and molecular subtypes are independent risk factors affecting DFS and OS of breast invasive carcinoma.The expression level of LRG1 is negatively correlated with DFS and OS.Part Three The expression of LRG1 in breast cancer cells and its effect on biological behavior of breast carcinoma cellsObjective:To verify whether the expression of LRG1 in breast cancer cell lines is consistent with that in breast carcinoma tissues,and to provide theoretical support for the role of LRG1 in the occurrence,development,invasion and metastasis of breast carcinoma.Methods:RNA interference technology,RT-PCR,Western-blot technol-ogy were mainly used to observe the expression of LRG1 in different breast cancer cell lines.CCK-8 assay,cell scratch experiment and transwell assay were mainly used to observe the changes of proliferation,migration and invasion after inhibiting the expression of LRG1.Results:1.The expression of LRG1mRNA in MDA-MB-231,MCF-7 and SK-BR-3 cells was detected by reverse transcription polymerase chain reaction（RT-PCR）.In three different breast carcinoma cells（MDA-MB-231,MCF-7 and SK-BR-3）,the transcription level of LRG1mRNA was significantly different（P<0.05）.The relative expression of LRG1mRNA in MCF-7 cells（0.1260±0.0070）was significantly higher than MDA-MB-231cells（0.1103±0.0081）and SK-BR-3 cells（0.0703±0.0027）.2.The differential expression of LRG1 protein in MDA-MB-231,MCF-7and SK-BR-3 cells.The expression of LRG1 protein in three different cell lines was significantly different（P<0.05）,the relative expression level of LRG1 protein in MCF-7 cells was the highest（10.9510±0.6721）,which was significantly different from MDA-MB-231 cells（7.8492±0.1327）and SK-BR-3 cells（4.3906±0.1914）.3.RNA interference technology was used to inhibit the expression of LRG1 in MD-MB-231 cells.The expression of LRG1 protein in MDA-MB-231 cells after successful transfection was detected using western-blot technology.The results showed that the expression of LRG1protein in the transfection group（MDA-MB-231-Si）（0.4105±0.1906）was significantly lower than that in the control group（MDA-MB-231）（0.8295±0.1295）,and the difference was significant（P<0.05）.4.Cell proliferation was detected by CCK-8 assay.After successful transfection,the OD value of each group of cells at 450 nm after 24h,48h,72h and 96h was detected by CCK-8 assay.The results showed that as time went by,the proliferation rate of the transfection group（MDA-MB-231-Si）was significantly lower than that of the transfection negative control group（MDA-MB-231-NC）and the control group（MDA-MB-231）,this showed that the proliferation ability of MDA-MB-231 cells was inhibited after the inhibition of LRG1mRNA expression.5.Cell migration was detected by cell scratch test.The migration of the cultured cells in control group,negative control group and transfection group（MDA-MB-231,MDA-MB-231-NC and MDA-MB-231-Si）were detected by scratch test.The scratch area measured at 0 hours of scratching were:control group cells（MDA-MB-231）,transfection group cells（MDA-MB-231-Si）,transfection negative control cells（MDA-MB-231-NC）respectively:15.054±0.122,14.679±0.142,13.407±0.112.The area of the scratched area measured 24 hours after the scratch were:6.714±0.131,9.559±0.122,5.217±0.132.The ratios of area reduction were:55.40%,34.88%,61.08%.The migration rate of MDA-MB-231-Si cells at 24 hours was significantly lower than that of the control group（MDA-MB-231）and the transfection negative control group（MDA-MB-231-NC）.The differences were significant（P<0.05）.6.Transwell assay was used to detect cell invasion ability.Transwell invasion assay showed that the number of transmembrane cells in MDA-MB-231-Si group was less than that in MDA-MB-231-NC group and control group（MDA-MB-231）（P<0.05）.This showed that cell invasion ability decreased when the expression of LRG1 was inhibited.Conclusions:1.The relative expression of LRG1mRNA in MCF-7 cells is significantly higher than that of MDA-MB-231 cells and SK-BR-3 cells.2.The relative expression of LRG1 protein in MCF-7 cells is significantly higher than that in MDA-MB-231 cells and SK-BR-3 cells.3.The proliferation,migration and invasion of MDA-MB-231 cells in vitro are attenuated by interference of LRG1 with si-RNA. |
