MAC mediates mammary duct epithelial cell injury in plasma cell mastitis and granulomatous mastitis.

Plasma cell mastitis (PCM) and granulomatous mastitis (GM) are common inflammatory nonbacterial mastitis (NBM). However, the pathogenesis of NBM is still unclear. METHODS: In this study, we statistically analyzed the pathological features of PCM and GM using pathological HE staining and tissue transmission electron microscopy. The levels of MAC (C5b-9n), P-selectin, E-selectin, and ICAM-1 were detected through IHC, WB, ELISA, and qPCR. The expression level and location of MAC were observed by tissue immunological electron microscopy. In addition, exosomes were isolated from tissues, identified using transmission electron microscopy, and the densities were detected by Nano-FCM. Finally, the expression intensity of MAC in exosomes was detected by flow cytometry and immunoelectron microscopy. RESULTS: The damage and apoptosis of mammary duct epithelial cells are the common pathological features of PCM and GM. MAC is primarily located in the cell membrane of mammary ductal epithelial cells and is significantly expressed in PCM and GM. The density of exosomes in PCM and GM tissues was elevated, and MAC was highly expressed in exosomes. In addition, the expression of P-selectin, E-selectin, and ICAM-1 in PCM and GM was significantly higher than in the normal group. CONCLUSION: We found severe damage of the mammary duct epithelial cells in PCM and GM tissues, which was verified by relevant pathological methods. Earlier studies demonstrated that MAC is highly expressed in PCM and GM tissues and exosomes seem to play a very important role in the understanding of MAC. Furthermore, MAC is involved in inflammatory infiltration and lesion of mammary duct epithelial cells upregulated by P-selectin, E-selectin, and ICAM-1. These findings provide new insights into PCM and GM molecular mechanisms.

LncRNA AFAP1-AS1 Knockdown Represses Cell Proliferation, Migration, and Induced Apoptosis in Breast Cancer by Downregulating SEPT2 Via Sponging miR-497-5p.

Background: Long non-coding RNA actin filament-associated protein1-antisense RNA 1 (AFAP1-AS1) was confirmed to be associated with tumorigenesis. However, the role of AFAP1-AS1 in breast cancer was little known. Materials and Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the levels of AFAP1-AS1, microRNA-497-5p (miR-497-5p), and Septin 2 (SEPT2) in breast cancer tissues and cells. The cell proliferation, migration, and apoptosis were tested by Methylthiazolyldiphenyl-tetrazolium bromide (MTT), Transwell and Flow cytometry assays, respectively. The targeting relationship between genes was predicted by StarBase v.3.0 and confirmed by dual-luciferase reporter assay. Pearson's correlation coefficient was applied to examine the correlation between the two groups. SEPT2 protein expression was evaluated by Western blot. Xenograft models were established to investigate the role of AFAP1-AS1 knockdown in vivo. Results: AFAP1-AS1 was upregulated in breast cancer tissues and cells, and AFAP1-AS1 knockdown could hinder proliferation and migration of breast cancer cells, and contribute to cell apoptosis. MiR-497-5p, which was downregulated in breast cancer, was verified to be a target of AFAP1-AS1 and inversely correlated with AFAP1-AS1 expression. SEPT2, as a target gene of miR-497-5p, was negatively regulated by miR-497-5p and positively correlated with AFAP1-AS1 expression. Importantly, AFAP1-AS1 could upregulate SEPT2 expression by sponging miR-497-5p, and modulate cell progression by regulation of the miR-497-5p/SEPT2 axis in breast cancer. Conclusion: AFAP1-AS1 knockdown repressed the progression of breast cancer cells by sponging miR-497-5p and downregulating SEPT2.

Composition and plasticity of triple-negative breast carcinoma-infiltrating regulatory T cells.

Low Foxp3+ regulatory T-cell (Treg) presence in the tumor-infiltrating lymphocytes (TILs) is considered favorable in breast cancer, and numerous CD25-targeting agents have been applied in the attempt to remove Foxp3+ Treg cells, which typically present CD4+ CD25+/hi surface phenotype. However, CD25 is not Treg-exclusive and can be upregulated by effector T cells. Hence, CD25 depletion may cause the elimination of activated T cells that are responding to tumor-specific antigens. In this study, the composition and function of CD4+ CD25+ cells inside the microenvironment of triple-negative breast carcinoma (TNBC) were investigated. Directly ex vivo, the Foxp3+ Treg cells represented a minor subset in total CD4+ CD25+ TILs. Significant differences were observed in the expression of Treg-associated molecules between CD4+ CD25+ Foxp3+ TILs and CD4+ CD25+ Foxp3- TILs. While both the CD4+ CD25+ Foxp3+ and the CD4+ CD25+ Foxp3- TILs could express CTLA-4 and LAG-3, the expression levels were significantly higher in CD4+ CD25+ Foxp3+ TILs than in CD4+ CD25+ Foxp3- TILs. Upon TCR stimulation, the expression of TGF-beta was significantly higher in CD4+ CD25+ Foxp3+ TILs, while the expression of IL-10 was significantly higher in CD4+ CD25+ Foxp3- TILs. These differences were conserved in the blood counterparts of these cells. Interestingly, the level of CD25+ Foxp3+ cells in circulating CD4+ T cells was positively correlated with the level of CD25+ Foxp3+ cells in CD4+ TILs, but the level of CD25+ Foxp3- cells in circulating CD4+ T cells was not associated with the level of CD25+ Foxp3- cells in CD4+ TILs. Th17-polarizing medium could readily remodel CD4+ CD25+ Foxp3- , but not CD4+ CD25+ Foxp3+ , T cells into RORgammat and IL-17-expressing T cells, demonstrating stronger plasticity of the former subset. Together, these data demonstrated that the CD4+ CD25+ TILs were composed of distinctive Foxp3- and Foxp3+ cells, with the former representing the major subset. The antigen specificity and effector molecule expression of the CD4+ CD25+ Foxp3- thus require further analyses.

IL-9-producing CD8+ T cells represent a distinctive subset with different transcriptional characteristics from conventional CD8+ T cells, and partially infiltrate breast tumors.

Accumulating evidence suggests that IL-9 and IL-9-producing cells exert various roles in antitumor immunity. Our study examined the IL-9 production in CD8+ T cells from breast cancer patients as compared to healthy controls. IL-9 secretion was undetectable in CD8+ T cells ex vivo, but could be readily detected following anti-TCR or PMA + ionomycin stimulation, and was higher in breast cancer patients than in healthy controls. The capacity to express IL-9 was not universal to all CD8+ T cells, but was favored in IL-9Rhigh CD8+ T cells, which were also present in breast cancer patients at significantly higher frequency than in healthy controls. Interestingly, exogenous IL-9 could significantly increase the expression of both IL-9 and IL-9R in IL-9Rhigh, but not IL-9Rlow, CD8+ T cells. IL-9Rhigh CD8+ T cells ex vivo presented lower expression of KLRG-1, PD-1, and Tim-3 than IL-9Rlow CD8+ T cells. Additionally, IL-9Rhigh CD8+ T cells following anti-TCR and PMA + ionomycin stimulation presented higher IL-2 and IL-17 expression, and lower IFN-γ expression, than IL-9Rlow CD8+ T cells. IL-9-expressing CD8+ T cells could be found in some, but not all, resected breast tumors. IL-9R expression, on the other hand, was readily present in CD8+ T cells, but with high variability from patient to patient. Patients with high intratumoral IL-9 expression also tended to present high IL-9R expression. Together, these data demonstrate that a transcriptionally distinctive IL-9-producing CD8+ T cell subset was elevated in breast cancer patients and could be found inside the tumor, with higher capacity to produce IL-2 and IL-17 and lower expression of inhibitory receptors.

miRNA-106a Promotes Breast Cancer Cell Proliferation, Clonogenicity, Migration, and Invasion Through Inhibiting Apoptosis and Chemosensitivity.

To explore the effect of miR-106a in breast cancer cell behavior and sensitivity to chemotherapeutic agents. Tumor tissue and adjacent normal tissue were derived from 40 breast cancer patients, and miR-106a expression was measured by reverse transcription-qPCR. Breast cancer cells, MDA-MB-231 and MCF-7, were treated with miRNA-106a mimic (MM) or miRNA-106a inhibitor (MI) and negative controls. Cell proliferation was measured by MTT assay. Clonogenicity was measured by colony-forming assay. Cell migration and invasion ability were measured by scratch test and transwell assay, respectively. Apoptosis was determined by flow cytometry, and chemosensitivity to cisplatin was measured by MTT assay. Finally, protein expression of p53, Bax, Bcl-2, RUNX3, and ABCG2 was quantified by western blot. miR-106a expression was significantly upregulated in human breast cancer tissue relative to adjacent normal tissue. Upregulation of miR-106a enhanced breast cancer cell proliferation, colony-forming capacity, migration, and invasion of cultured breast cancer cells. Additionally, miR-106a overexpression significantly decreased breast cancer cell apoptosis and sensitivity to cisplatin. Finally, we showed miR-106a overexpression upregulated the levels of Bcl-2 and ABCG2, and downregulated the expression of P53, Bax, and RUNX3. miR-106a promotes breast cancer cell proliferation and invasion through upregulation of Bcl-2, ABCG2, and P53, and downregulation of Bax and RUNX3.

Osteopontin versus alpha-fetoprotein as a diagnostic marker for hepatocellular carcinoma: a meta-analysis.

BACKGROUND: Several studies have reported that osteopontin (OPN) is a promising marker for the diagnosis of hepatocellular carcinoma (HCC); however, some studies emerged with conflicting results. Therefore, we provide a systematic review to evaluate the diagnostic performance of OPN for HCC. METHODS: Studies that investigated the diagnostic value of OPN and alpha-fetoprotein (AFP) in HCC were collected from PubMed and Embase. Sensitivity, specificity, and other parameters about the diagnostic accuracy of serum OPN and AFP in HCC were pooled using STATA 12.0 software. The summary receiver operating characteristic curve (sROC) and other parameters were used to summarize the overall test performance. RESULTS: Twelve studies were included in our meta-analysis. Pooled sensitivity, specificity, and diagnostic odds ratio were 0.813 (95% CI: 0.671-0.902), 0.874 (95% CI: 0.778-0.932), and 30.047 (95% CI: 8.845-102.067) for OPN; 0.639 (95% CI: 0.538-0.729), 0.959 (95% CI: 0.909-0.982), and 41.518 (95% CI: 13.688-125.929) for AFP; and 0.856 (95% CI: 0.760-0.918), 0.738 (95% CI: 0.630-0.823), and 16.718 (95% CI: 7.950-35.156) for OPN+AFP, respectively. The area under the sROC for OPN, AFP, and OPN+AFP was 0.91, 0.88, and 0.85, respectively. For diagnosis of early HCC, pooled sensitivity of serum OPN, AFP, and OPN+AFP was 0.493 (95% CI: 0.422-0.563), 0.517 (95% CI: 0.446-0.587), and 0.732 (95% CI: 0.666-0.791), respectively. CONCLUSIONS: OPN is a comparable marker to AFP for the diagnosis of HCC, and the sensitivity of OPN was higher than that of AFP. A combination of AFP and OPN can elevate the sensitivity of diagnosis for early HCC.

Prognostic value of osteopontin in patients with hepatocellular carcinoma: A systematic review and meta-analysis.

PURPOSE: The prognostic value of tissue and serum osteopontin (OPN) in hepatocellular carcinoma (HCC) remain controversial. The aim of present meta-analysis was to evaluate the prognostic value of OPN in patients with HCC. METHODS: Eligible studies were systematically searched by PubMed, EMBASE, and Google scholar. A meta-analysis of 12 studies included 2117 cases was performed to estimate the association between OPN level and overall survival (OS), disease-free survival (DFS) in HCC patients. Subgroup analyses were also performed in the meta-analysis. RESULTS: The pooled data of studies showed that high OPN level was significantly associated with poor OS (hazard ratios [HR] 1.84; 95% confidence intervals [CI] 1.54-2.20; P = .000) and DFS (HR 1.67; 95% CI 1.40-1.98; P = .000) in HCC. Furthermore, in subgroup analysis, high tissue based OPN by immunohistochemistry detection and serum-based OPN by enzyme-linked immunosorbent assay (ELISA) detection were both significantly associated with OS (tissue: HR 1.88; 95% CI 1.53-2.31; P < .0001; serum: HR 2.38; 95% CI 1.58-3.59; P < .0001). Simultaneously, we also found that OPN expression was positively associated with stage (odds ratios [OR] 5.68; 95% CI 3.443-7.758), tumor size (Size≤5 cm vs >5 cm; OR 2.001; 95% CI1.036-3.867). CONCLUSION: The current evidence indicates that OPN could serve as a prognostic biomarker and a potential therapeutic target for HCC.

Th9 cells promote antitumor immunity via IL-9 and IL-21 and demonstrate atypical cytokine expression in breast cancer.

Breast cancer is a major cause of cancer-related death in women. Antitumor T cell responses play critical therapeutic roles, including direct cytotoxicity mediated by CD8+ T cells and immunomodulatory roles mediated by CD4+ T cells. The IL-9-expressing Th9 cells are recently found to present antitumor immunity in melanoma and lung adenocarcinoma. In this study, we found that IL-9 expression in the serum and in circulating CD4+ T cells were significantly upregulated in breast cancer patients compared to healthy controls. The IL-9-expressing Th9 cells were enriched in the CCR4-CCR6-CXCR3- subset. Upon TCR stimulation, this subset also presented potent IL-10 and IL-21 expression in addition to IL-9 expression. CCR4-CCR6-CXCR3- CD4+ T cells also assisted in the killing of autologous tumor cells by CD8+ T cells, but did not initiate cytotoxicity by themselves. This enhancement in CD8+ T cell-mediated cytotoxicity was dependent on IL-9 as well as on IL-21. Interestingly, the tumor-infiltrating Th9 cells presented comparable IL-9, reduced IL-10, and elevated IL-21 expression compared with their counterparts in the peripheral blood. Together, these results demonstrated that IL-9-expressing Th9 cells were upregulated in breast cancer patients and potentially possessed antitumor roles by enhancing CD8+ T cell-mediated cytotoxicity.

Long Noncoding RNA H19 Inhibits Cell Viability, Migration, and Invasion Via Downregulation of IRS-1 in Thyroid Cancer Cells.

Thyroid cancer is a common endocrine gland malignancy which exhibited rapid increased incidence worldwide in recent decades. This study was aimed to investigate the role of long noncoding RNA H19 in thyroid cancer. Long noncoding RNA H19 was overexpressed or knockdown in thyroid cancer cells SW579 and TPC-1, and the expression of long noncoding RNA H19 was detected by real-time polymerase chain reaction. The cell viability, migration, and invasion were determined by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide assay, Transwell assay, and wound healing assay, respectively. Furthermore, cell apoptosis was analyzed by flow cytometry, and expressions of some factors that were related to phosphatidyl inositide 3-kinases/protein kinase B and nuclear factor κB signal pathway were measured by Western blotting. This study revealed that cell viability and migration/invasion of SW579 and TPC-1 were significantly decreased by long noncoding RNA H19 overexpression compared with the control group ( P < .05), whereas cell apoptosis was statistically increased ( P < .001). Meanwhile, cell viability and migration/invasion were significantly increased after long noncoding RNA H19 knockdown ( P < .05). Furthermore, long noncoding RNA H19 negatively regulated the expression of insulin receptor substrate 1 and thus effect on cell proliferation and apoptosis. Insulin receptor substrate 1 regulated the activation of phosphatidyl inositide 3-kinases/AKT and nuclear factor κB signal pathways. In conclusion, long noncoding RNA H19 could suppress cell viability, migration, and invasion via downregulation of insulin receptor substrate 1 in SW579 and TPC-1 cells. These results suggested the important role of long noncoding RNA H19 in thyroid cancer, and long noncoding RNA H19 might be a potential target of thyroid cancer treatment.

The expressions of NEDD9 and E-cadherin correlate with metastasis and poor prognosis in triple-negative breast cancer patients.

BACKGROUND: Neural precursor cell expressed, developmentally downregulated 9 (NEDD9), a member of Crk-associated substrate family, is involved in cancer cell adhesion, migration, invasion, and epithelial-mesenchymal transition. E-cadherin is a key event in the cellular invasion during the epithelial-mesenchymal transition mechanism. The aim of this study was to evaluate the association among NEDD9 expression, E-cadherin expression, and survival in triple-negative breast cancer (TNBC) patients. METHODS: NEDD9 and E-cadherin expressions were analyzed by immunohistochemistry in 106 TNBC patients and 120 non-TNBC patients. And the association of clinicopathological factors with survival was analyzed using Kaplan-Meier analysis and Cox regression in TNBC patients. RESULTS: The results revealed that the rate of increased expression of NEDD9 and reduced expression of E-cadherin was significantly higher in TNBC group than that in non-TNBC group (P<0.001, both). Comparison of features between TNBC and non-TNBC groups showed that histological type (P=0.026) and lymph node metastasis (P=0.001) were significantly different. Correlation analysis showed that positive NEDD9 expression and negative E-cadherin expression were significantly correlated with lymph node metastasis and tumor-node-metastasis stage (P<0.05). In addition, the enhanced NEDD9 expression was significantly associated with a reduced 5-year survival for TNBC patients (overall survival [OS]: P=0.013; disease-free survival [DFS]: P=0.021). Negative E-cadherin expression showed a significantly worse 5-year OS and DFS (OS: P=0.011; DFS: P=0.012). Multivariate analysis showed that lymph node metastasis (OS: P=0.006; DFS: P=0.004), tumor-node-metastasis stage (OS: P=0.012; DFS: P=0.001), NEDD9 (OS: P=0.046; DFS: P=0.022), and E-cadherin (OS: P=0.022; DFS: P=0.025) independently predicted a poor prognosis of OS and DFS. Moreover, patients with NEDD9-positive/E-cadherin-negative expression had a significantly worse outcome than other groups (OS: P=0.004; DFS: P=0.001). CONCLUSION: Our finding demonstrated the potential value of NEDD9 and E-cadherin expression levels as prognostic molecular markers and a target for new therapies for TNBC patients.

MicroRNA-26a inhibits proliferation by targeting high mobility group AT-hook 1 in breast cancer.

OBJECTIVES: To investigate the crucial role of miR-26a in breast cancer and to validate whether miR-26a could regulate proliferation of breast cancer cells by targeting high mobility group AT-hook 1 (HMGA1). METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the expression levels of miR-26a in breast cancer and adjacent non-cancerous breast tissues. MTT, cell migration and invasion assay were carried out to characterize the miR-26a function. Finally, to validate the target gene of miR-26a, luciferase reporter assay was employed, followed by RT-PCR and Western blot confirmation. RESULTS: Compared with normal tissues, a significant down-regulation of miR-26a expression was observed in breast cancer tissues (P=0.002). miR-26a suppresses MDA-MB-231 and Mcf-7 breast cancer cell lines proliferation and motility. The luciferase activity was significantly decreased after co-transfection with psiCHECK-2/HMGA1 3'-UTR and miR-26a mimics in comparison with control cells, and qRT-PCR and Western blotting analysis found that HMGA1 expression at the mRNA and protein levels decreased in the miR-26a mimic-treatment group relative to NC. MTT assay showed that down regulation of HMGA1 by siRNA could significantly enhance the tumor-suppressive effect of miR-26a (P < 0.05). CONCLUSIONS: The results of the present study indicate that miR-26a may be associated with human breast carcinogenesis, which inhibits tumor cell proliferation by targeting HMGA1.