Objective To analyze the expressions of thymidine kinase 1 (TK1) and nuclear-associated antigen Ki-67 in triple negative breast cancer (TNBC) and their clinical significances.Methods One hundred and twenty tumor tissue sections of patients with breast cancer who were performed breast conservation treatment or modified mastectomy in the Second Affiliated Hospital of Qingdao University Medical College from June 2009 to December 2010 were collected,and there were 60 cases with TNBC and 60 cases with non-TNBC.The expressions of TK1 and Ki-67 in different breast tissues were detected by immunohistochemistry.The relationships between the expression status and clinicopathologic features were analyzed.Results The positive expression rates of TK1 in TNBC and non-TNBC were 83.33％ and 51.67％ respectively,with a significant difference (x2 =13.713,P =0.000).The positive expression rates of Ki-67 expression in TNBC and nonTNBC were 68.33％ and 31.67％ respectively,with a significant difference (x2=16.133,P =0.000).In TNBC,the expression of TK1 was related to histological staging (x2 =6.125,P =0.013),but it was not related to onset age (x2 =0.809,P =0.369),menopausal stutas (x2 =1.615,P =0.204),tumor size (x2 =0.054,P =0.816) and lymphatic metastasis (x2 =0.672,P =0.412).In TNBC,the expression of Ki-67 was related to histological staging (x2 =13.145,P =0.000) and lymphatic metastasis (x2 =6.182,P =0.013),but it was not related to menopausal stutas (x2 =1.018,P =0.313),onset age (x2 =2.377,P =0.123) and tumor size (x2 =2.401,P =0.121).The expression of TK1 was positively correlated with that of Ki-67 (r =0.369,P =0.023).The results of survival analysis showed that the disease-free survival rates of 5-year were 28.20％ and 66.70％ in the TK1 positive group and TK1 negative group,and the disease-free survival rates of 5-year were 24.30％ and 64.30％ in the Ki-67 positive group and Ki-67 negative group,with significant differences (x2 =4.194,P=0.041;x2 =4.540,P =0.033).Conclusion TK1 and Ki-67 are highly expressed in TNBC,and their expressions are correlated with histological staging and survival,which are expected to become prognostic indicators.

Objective:To investigate the role of hepatitis B virus X protein (HBx) in glomerular podocyte immune disorder and its regulatory mechanism.Methods:Fourteen 6-week-old male hepatitis B virus (HBV) transgenic (HBV-Tg) mice were selected, and age-matched wild type (WT) mice were as controls. They were fed to different weeks, and 24 h urinary protein, blood biochemistry, renal pathology and podocyte changes under electron microscope were detected. The expression of HBx and the infiltration of immune cells in kidney tissue of HBV-Tg mice were observed by immunohistochemistry. Human podocyte cell line was transfected with pcDNA3.1/myc-HBx plasmid, and the localization of HBx and Nephrin in podocytes was detected by immunofluorescence. The expression of major histocompatibility complex Ⅱ (MHC-Ⅱ) and co- stimulatory molecule CD40 on the cell surface was detected by flow cytometry. The contents of multiple cytokines in cell culture supernatants were determined by enzyme-linked immunosorbent assay. Transcriptome sequencing (RNA-seq) was used to screen the downstream related genes regulated by HBx, and real-time quantitative PCR was used to verify their expressions. After overexpression or silencing of Notch1 gene with overexpressed plasmids or short hairpin RNA (shRNA) in podocytes, the effects on the expression of immune molecules and cytokines secretion was observed. The Notch receptor inhibitor N-[N-(3, 5-difluorophenyl-l- alanyl)]-(s)-phenylglycine tert-butyl ester (DAPT) was used to block Notch1 signaling pathway in HBV-Tg mice, and then blood biochemistry, renal pathological changes and infiltration of immune cells in kidney tissue were observed. Results:Twenty-four-hour urine protein, serum creatinine and urea nitrogen levels were markedly increased (all P<0.05) and renal pathological injury was significantly aggravated in HBV-Tg mice than those in WT mice. Also, HBx was up-regulated and immune cells infiltrated in the glomerulus of HBV-Tg mice. After transfection with HBx in podocytes, the expression of MHC-Ⅱ and CD40 on the cellular surface was up-regulated (all P<0.05), the contents of monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor -α (TNF-α) and interleukin (IL)-1β in the supernatants were increased (all P<0.05), and the secretion of IL-4 and interferon γ (IFN-γ) was unbalanced. RNA-seq screened downstream genes of HBx, such as Notch1, PLA2R, TLR4, etc; and further confirmed that HBx could promote the up-regulation of Notch1 mRNA and protein (all P<0.05). After over-expression of Notch1 gene, HBx-induced expression of MHC-Ⅱ and CD40 on the cellular surface was significantly up-regulated (all P<0.05), and the contents of MCP-1, TNF-α and IL-1β in the supernatants were obviously increased (all P<0.05), and the imbalance of IL-4/IFN-γ was further aggravated. After Notch1 gene silencing, the above results showed the opposite changes. In vivo, the results indicated that serum creatinine levels were obviously decreased (all P<0.05), renal pathological injury and immune cell infiltration were significantly alleviated in HBV-Tg+DAPT group than those in HBV-Tg+DMSO group. Conclusions:HBx protein can promote the up-regulation of Notch1 signaling pathway in podocytes. And Notch1 signaling pathway promotes the expression of immune molecules on the surface of podocytes and regulates the imbalance of cytokines, then causes glomerular injury and dysfunction of immune microenvironment.