Studies On The Expression Of HMGB1 In Breast Cancer And Its Effects On Biological Behaviors Of Breast Cancer Cell Line MDA-MB-231

Backgrounds:Breast cancer has become the most common malignant tumor of women both in developed and developing countries. Over the past 30 years, the incidence of breast cancer is significantly increased both in urban and rural areas of China. It has become a serious threat to health, work and life of women. To be the most populous country that has one fifth of the world, the number of breast cancer patients in our country also is very large. Along with the advance of medical science and technology, people for breast cancer treatment and scientific research has made great progress, the treatment of breast cancer is going into the era of "precision medicine", However, despite considerable progression in breast cancer treatment and research, the survival rate has not been improved significantly. Tumor invasion and metastasis are the key factors leading to poor prognosis and high mortality in breast cancer. Therefore, the highlight of breast cancer research is to understand the mechanisms of tumor genesis, invasion and metastasis, to investigate the target of malignant transformation and the target therapy by molecular biology.In recent years, many researchers have payed attention to the effect of high mobility group box chromosomal protein 1(HMGB) on the biological behavior of tumor.HMGB1 is a highly conservative non-histone nuclear protein which is widely expressed in the cells of leukaryotes. It has been found to be closely related to a variety of tumor biology behavior, include tumor infinite proliferation; resistance to apoptosis, angiogenesis, invasion and metastasis, and it plays an important role in tumor progression. HMGB1 has become a hotspot on cancer research, but little is known in breast cancer. It is still unclear that how it effects on the biological behavior of cancer cells and what is the role it plays in the progression of breast cancer. This study will detect HMGB1 expression in breast cancer and do research on the effects of biological behaviour human breast cancer MDA-MB-231 cell line,The aim is to investigate the potential effect of HMGB1 in the progression of breast cancer.Objectives:(1) Immunohistochemistry and ELISA were performed respectively to detect the HMGB1 expression in breast cancer tissues and serum. The relationship between HMGB1 expression and clinical pathological parameters were investigated. (2)To investigate the effects of small specific interfering RNA (siRNA) on the expression of HMGB1 and the bioactivity of MD A-MB-231 (a breast cancer cell line). (3) To investigate the effect of HMGB1 on the bioactivity of MDA-MB-231 cells in vitro and its potential regulating mechanism.Methods:(1) Immunohistochemistry (IHC) was performed to detect HMGB1 expression in breast cancer tissues and para-cancer tissues. Enzyme-linked immunosorbent assay (ELISA) was performed to detect HMGB1 expression in serum of corresponding groups and healthy donors. The relationship between HMGB1 expression and clinical pathologic parameters were investigated. (2)Three specific siRNA of HMGB1 (siRNA-Hl, siRNA-H2 and siRNA-H3) were designed and transiently transfected into MDA-MB-231 cells by use of LipofectamineTM2000. Negative control siRNA group and blank group were setted up in parallel. RT-PCR and Western blot were performed to determine the effects of HMGBl-siRNAs on HMGB1 expression. In vitro proliferation of MDA-MB-231 cells was assessed by MTT assay, apoptosis was demonstrated by flow cytometry, migration and invasive ability were determined by Transwell assay. (3)MDA-MB-231 cells were stimulated with recombinant HMGB1 (rHMGB1) or/and anti-HMGB1 for 24 hours. MTT and Transwell assay were performed to observe cell proliferation and invasive ability respectively. RT-PCR and Western blotting were performed to detect the expression of mRNA and protein of NF-icB/p65 and MMP-9 respectively.Results:(1) the level of HMGB1 expression in breast cancer tissue and normal breast tissue adjacent to carcinoma of the control group and benign breast disease control group have obvious rasied, there has statistically significant difference(P<0.05);and it was significantly associated with tumor differentiation grade, lymphatic metastasis, and different tumor-node-metastasis stage(P<0.05); Serum HMGB1 expression level in patients with breast cancer group was obviously higher than that of breast benign disease group and normal volunteers in the control group (P<0.05); High and low serum levels of HMGB1 expression has nothing to do with breast cancer clinical pathology index; HMGB1 high expression in tumor tissue in patients with breast cancer than lower expression, the differences between the serum levels of HMGB1 has no statistical significance (P>0.05). (2) RT-PCR and Western-blot showed that all three specific HMGB1-siRNAs significantly inhibited HMGB1 expression, with inhibition by siRNA-H1 being strongest, three specific HMGB1-siRNAs were transfected to MDA-MB-231 cells, relative expression of HMGBlmRNA were 0.448±0.045,1.148±0.078,1.118±0.038, all were significantly reduced compared with blank control group 1.393±0.070 (t value was 25.393,6.878 and 5.867 respectively, P<0.01); relative expression of HMGB1 protein were 0.143±0.040, 0.353±0.019,0.394±0.032, all were significantly reduced compared with blank control group 0.600±0.064. (t value was 13.540,7.718 and 6.900 respectively, P< 0.01), among them siRNA H1 group interference suppression effect is strongest, the inhibition rate can reach 70%. The proliferation ability of MDA-MB-231 cells after transfection siRNA H1 in 72 h,96 h,120 h was significantly inhibited compared with the blank control group,(t value was 7.130,30.972,25.679 respectively, P< 0.01), MTT results showed that cell proliferation rate of siRNA HI was significantly decreased compared with blank control group and negative group of siRNA control group (P<0.01); FCM results showed that the cell cycle distribution siRNA HI group of G0/G1 phase cell proportion increased significantly compared with blank control group and negative siRNA control group (P<0.01),while S phase and G2/M phase cells proportion is significantly lower than the blank control group and negative siRNA control group (P< 0.01); Apoptosis results showed that siRNA H1 group, early and middle-late apoptosis rate and total cell apoptosis rate were significantly higher than control group and negative siRNA blank control group (P<0.01); According to the results of transwell method,siRNA-H1 cells invasion ability were significantly lower compared with blank control group and negative siRNA control group (P<0.01). (3) MTT results showed that the absorbance of blank control group, rHMGB1 group,rHMGB1-antibody group, rHMGB1+IgG group and rHMGB1 +rHMGB1-antibody group is 0.266±0.038、0.712±0.039,0.283±0.030,0.709±0.022 and 0.201±0.027 respectively; Compared with blank control group, proliferation effect of rHMGB1 in MDA-MB-231 cells was significantly higher(P<0.05); 20 ng/mL rHMGB1 used in MDA-MB-231 cells after 24h, relative expression of NF-kappa B/p65 and MMP-9 mRNA were 1.244±0.115,1.440±0.08,while blank control group was 0.537±0.026、0.537±0.053, the difference was statistically significant (P< 0.05); rHMGB1 used in MDA-MB-231 cells after 24h, relative content of NF-kappa B/p65 and MMP-9 protein were 0.497±0.069,0.383±0.017 respectively, while blank control group were 0.402±0.050,0.050±0.014.the difference has statistically significance (P<0.05). rHMGB1 used in MDA-MB-231 cells after 24 h, Transwell experimental results showed that cell number went through matrigel membrane is 379±32 in rHMGBl group, while blank control group is 204±26, MDA-MB-231 cells stimulated by rHMGBl had a stronger invasive activity.The difference was statistically significant (P< 0.05).Conclusions:(1)HMGB1 is highly expressed in the cancer tissues and serum of breast cancer patients, and the expression level was associated with differentiation grade, lymphatic metastasis, and different tumor-node-metastasis stage, these revealed that HMGB1 could be a supportive diagnostic marker and prognostic indicator for breast cancer. Serum HMGB1 could be a useful serological biomarker of breast cancer. (2)Specific siRNA could inhibit the proliferation and invasion of MDA-MB-231 cells effectively, and induce cell apoptosis. This is related to down-regulation of HMGB 1. HMGB1 may serve as a potential target for treatment of breast cancer.(3)rHMGBl could obviously promote malignant phenotypes of MDA-MB-231 cells, and increased expression of NF-kB/p65, MMP-9mRNA may be one of the potential reasons. Anti-HMGB1 can significantly inhibit the effects of exogenous HMGB1.HMGB1 promote breast cancer MDA-MB-231 cells proliferation, invasion may be by raising NF-kappa B/p65 gene and protein expression level, and then activate MMP-9 and other related downstream signaling pathways, its specific mechanism need further research.