Functional And Mechanistic Studies Of HMGA2, A Prognostic Biomarker In Breast Cancer

Breast cancer is currently the most common malignancy that endangers women's health.With the advance of diagnosis and treatment,the morbidity and mortality of breast cancer are gradually reduced.Chemotherapy is still the best choice for treatment in patients with advanced or metastatic breast cancer.However,the chemotherapy resistance is the leading cause of treatment failure,with more than 90%of deaths due to chemotherapy resistance leading to tumor recurrence or metastasis.Breast cancer can be divided into four major molecular subtypes:Luminal A,Luminal B,HER2 and triple negative breast cancer(TNBC).Endocrine therapy is the main treatment for Luminal type breast cancer,HER2 targeted therapy is the favorable treatment for those with HER2-positive breast cancer,however,TNBC tends to have poor prognosis because of lacking of conventional chemotherapy for sustained response.At present,scholars at home and abroad still continue to strive for new and effective biomarkers that can be used to accurately predict drug resistance.The high mobility group protein HMGA2 belongs to the non-histone chromosome protein,which binds to the AT-enriched region of DNA to promote or inhibit the expression of the relevant gene.HMGA2 can act as a transcription factor to regulate the expression of downstream genes and maintain the undifferentiated state of cells during embryogenesis or tumorigenesis.HMGA2 is considered a kind of carcinoembryos which is highly expressed in the embryonic period,but not expressed in normal mature cells.However,HMGA2 is highly expressed in malignant tumors such as pancreatic cancer,liposarcoma,bladder cancer and so on.HMGA2 expression is associated with the degree of tumor differentiation and prognosis.Our previous studies have shown that increased expression of HMGA2 is associated with distant metastasis and poor prognosis of colorectal cancer,and HMGA2 expression is associated with radiotherapy sensitivity.It has been reported that HMGA2 mRNA levels are elevated in breast cancer tissues,which is associated with poor pathological types.In the peripheral blood of patients with breast cancer can also be detected the increased HMGA2 mRNA levels.However,there is no systematic report on the correlation between HMGA2 protein expression and prognosis in breast cancer.Therefore,in order to further understand the relationship between the expression of HMGA2 and breast cancer prognosis,in the first part of this paper,we collected 273 breast cancer cases from the Second Affiliated Hospital of Zhejiang University and 310 breast cancer specimens in the Shanghai Biochip National Engineering Center,and stained HMGA2 expressionby immunohistochemical.Kaplan-Meier analysis and Cox proportional hazards model were performed for prognostic analysis.In the second part,we carried out MCF-7 and SKBR-3 breast cancer cells transfected with HMGA2 for study.CCK8 assay,cell scratches,TRANSWELL and other experimental studies were conducted to study the carcinogenic effect of HMGA2in breast cancer cells.Part 1:The relationship between the expression of HMGA2 and the prognosis of breast cancer patientsPurpose:The expression of HMGA2 was detected in clinical tissue samples,and the relationship between HMGA2 expression and the prognosis of breast cancer patients was analyzed.Method:We collected 583 breast cancer samples from the Second Affiliated Hospital of Zhejiang University and Shanghai Bio-chip Super National Engineering Center.All samples were followed up to obtain the follow-up data.The expression of HMGA2 in breast cancer samples was obtained by immunohistochemical staining.Result:1.The expression rate of HMGA2 in breast cancer tissues was about 50%-65%,and the highest expression rate of HMGA2 was in G3 breast cancer tissues.2.The prognosis of breast cancer patients with HMGA2 expression was significantly worse than that of non-HMGA2-expressing breast cancer.COX regression analysis showed that the HR of HMGA2 expression was 4.24(1.17,15.37)and 2.06(1.21,3.49).Conclusion:1.The expression of HMGA2 in breast cancer is related to the degree of tumor differentiation.2.HMGA2 expression is associated with poor prognosis of breast cancer,HMGA2 is an independent risk factor for poor prognosis of breast cancer patients.Part 2:HMGA2 promotes the migration and invasion of breast cancer cells,HMGA2 enhancesdoxorubicin resistance by promoting phosphorylation of p53(ser15)Purpose:To study the carcinogenicity of HMGA2 in breast cancer at the cellular level,and to explore the mechanism of HMGA2 on the effect of doxorubicin.Method:Breast cancer cells were transfected by lentivirus to obtain the breast cancer cell lines with high expression of HMGA2.CCK-8 test was used to detect the effect of HMGA2 on the growth of breast cancer cells.Transwell assay was used to detect the effect of HMGA2 on the migration and invasion of breast cancer cells.Result:1.There was no significant difference in the proliferation of breast cancer cells with HMGA2 expression or not,but the results of Transwell assay showed that HMGA2 overexpressing breast cancer cells were more likely to pass through Matrigel cell and E-cadherin was significantly downregulated in HMGA2 overexpressing cells.2.The phosphorylation of p53(ser15)in HMGA2 overexpressing breast cancer cells were significantly higher than that in non-HMGA2 overexpressing cells after 24 hours of doxorubicin treatment.Conclusion:1.HMGA2 promotes the migration and invasion of breast cancer cells by inducing epithelial-mesenchymal transition(EMT).2.HMGA2 promotes the phosphorylation of p53(ser-15)to increase the resistance of doxorubicin to breast cancer cells.