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Background: Aneuploid circulating endothelial cells (CECs) are an indicator in breast cancer (BC). Significant changes of aneuploid CECs occurred during neoadjuvant chemotherapy (NCT). The aim of this study was to explore the predictive and prognostic value of aneuploid CECs in locally advanced breast cancer (LABC) patients with different NCT responses. Methods: Breast cancer patients received an EC4-T4 NCT regimen. A novel subtraction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH) strategy was applied for detection of CECs (CD45–/CD31+/DAPI+). Receiver operating characteristic (ROC) curves were plotted to evaluate the predictive value of aneuploid CEC counts in distinguishing NCT-resistant patients from sensitive ones. All patients were observed for progression-free survival (PFS) and overall survival (OS). Results: The CEC counts at any time point did not show the ability to predict the efficacy of NCT. The difference of the CECs between post-chemotherapy level and baseline could be sufficient to distinguish chemotherapy-resistant cases from other cases in Hormone+Her-2-/+ (HR+) BC patients. Patients with reduction of CECs after all courses of NCT were associated with higher probability of PFS. Conclusion: Variation of aneuploid CECs during NCT may have some ability to predict chemotherapy response in patients with HR+ breast cancer. The decrease of aneuploid CECs number after all courses of NCT indicate better treatment outcomes in LABC patients.

Circulating tumor cells (CTCs) have been shown to be associated with the response to neoadjuvant chemotherapy (NCT) and the prognosis of locally advanced breast cancer (LABC) patients. Our study aimed to investigate whether the change of CTC status during NCT could serve as a supplement to the Response Evaluation Criteria in Solid Tumors (RECIST) in the treatment and evaluation of LABC patients.6 ml of blood samples were collected before NCT, after the first cycle of NCT and after the completion of NCT, respectively. According to the change of CTC number during NCT, the patients were divided into "CTC low-response (low-R)" group and "CTC high-response (high-R)" group. Survival data of each group of patients were obtained through long-term follow-up.A total of 35 patients diagnosed with LABC were enrolled. The median follow-up for distant metastasis was 27 months (range 7-36 months). There was no significant difference in distant metastasis-free survival (DMFS) between PR/CR group and PD/SD group (P = 0.0914), while CTC low-R group had a worse DMFS than CTC high-R group (P = 0.0199). In PR/CR subgroup, patients with CTC low-R showed a lower DMFS compared with those with CTC high-R (P = 0.0159). However, in PD/SD subgroup, there was no significant difference in DMFS between CTC low-R and CTC high-R group (P = 0.7521). In terms of assessing response to NCT, CTC change or RECIST classification alone had an AUC of 0.533 (95% CI 0.277-0.790) and 0.700 (95% CI 0.611-0.789), respectively. When combining the two, the AUC slightly increased to 0.713 (95% CI 0.532-0.895).The change of CTC number during NCT has a potential to serve as a supplement to RECIST in the assessment of NCT efficacy and the prognosis of LABC patients.

BACKGROUND: Circulating miRNAs are promising biomarkers for detection of various cancers. As a “developmental” disorder, cancer showed great similarities with embryos. OBJECTIVE: A comprehensive analysis of circulating miRNAs in umbilical cord blood (UCB) and pan-cancers was conducted to identify circulating miRNAs with potential for cancer detection. METHODS: A total of 3831 cancer samples (2050 serum samples from 15 types of cancers and 1781 plasma samples from 13 types of cancers) and 248 UCB samples (120 serum and 128 plasma samples) with corresponding NCs from Chinese populations were analyzed via consistent experiment workflow with Exiqon panel followed by multiple-stage validation with qRT-PCR. RESULTS: Thirty-four serum and 32 plasma miRNAs were dysregulated in at least one type of cancer. Eighteen serum and 16 plasma miRNAs were related with embryos. Among them, 9 serum and 8 plasma miRNAs with consistent expression patterns between pan-cancers and UCB were identified as circulating oncofetal miRNAs. Retrospective analysis confirmed the diagnostic ability of circulating oncofetal miRNAs for specific cancers. And the oncofetal miRNAs were mainly up-regulated in tissues of pan-cancers. CONCLUSIONS: Our study might serve as bases for the potential application of the non-invasive biomarkers in the future clinical.

BackgroundBreast cancer has become the most common malignant tumor around the world. Although most breast cancer patients have a good prognosis, due to the significant heterogeneity of breast cancer and results in the difference in prognosis, it is urgent to find new prognostic biomarkers. Recently, inflammatory-related genes have been proven to play an important role in the development and progression of breast cancer, we aimed to investigate the prognostic value of inflammatory-related genes in breast cancers.MethodsWe evaluated the relationship between inflammatory-related genes (IRGs) and breast cancer by analyzing the TCGA database. Difference and univariate Cox regression analysis were performed and then screened out prognosis related differentially expressed inflammatory genes. The prognostic model was constructed through the Least Absolute Shrinkage and Selector Operation (LASSO) regression based on the IRGs. Then we used the Kaplan-Meier curves and receiver operating characteristic (ROC) curve to evaluate the accuracy of the prognostic model. And further, the nomogram model was established to clinically predict the survival rate of breast cancer patients. We also analyzed the immune cell infiltration and the activity of the immune-related pathways based on the expression of prognostic. The CellMiner database was used to study drug sensitivity. Finally, we performed functional cellular experiments to determine the biological characteristics of IRGs in breast cancer cells.ResultsIn this study, seven IRGs were selected from eleven prognosis-related differential inflammation genes through LASSO Cox regression analysis to construct a prognostic risk model. The risk score was negatively correlated with the prognosis of breast cancer patients through further analysis. ROC curve demonstrated the accuracy of the prognostic model and the nomogram could also accurately predict the survival rate. The ssGSEA analysis was used to quantify the differences between the low- and high-risk groups for the scores of tumor-infiltrating immune cells and immune-related pathways. Then we explored the relationship between the genes which were a researcher in the model and the susceptibility of drugs. Finally, we performed cellular functional experiments and validated the accuracy of the model.

Purpose： MicroRNAs (miRNAs) represent small noncoding RNAs controlling mRNA expression. MiRNA and mRNA amounts in matching specimens were utilized for identifying miRNA–mRNA interactions. Transcriptome, immunophenotype, methylation, mutation, and survival data analyses were integrated to examine the profiles of miRNAs and target mRNAs and their associations with breast cancer (BC) diagnosis.Methods: Based on the mRNA/miRNA expression microarray and RNA-Seq data sets in the GEO database and RNA-Seq data in TCGA, we analyzed differential expression profiles through GEO2R and the R language. Differentially expressed miRNAs and targeted mRNAs were screened from experimentally verified miRNA-target interactions databases by Pearson's correlation analysis. qRT-PCR was used for verification in breast cancer and benign disease samples, and logistic regression analysis was utilized to establish a diagnostic model based on miRNAs and target mRNAs. ROC curve analysis was performed to test the ability to recognize miRNA-mRNA pairs. Then, complex interactions of miRNA-mRNA regulatory pairs and phenotypic hallmarks, including tumor-infiltrating lymphocytes, tumor microenvironment, tumor mutation burden and global methylation, were described.Result: We first identified 27 dysregulated miRNAs and 135 dysregulated mRNAs based on five gene expression and 21 miRNA expression data sets from GEO and RNA-Seq data from TCGA. Utilizing Pearson's correlation analysis, ten negative miRNA-mRNA regulatory pairs were identified after screening in TarBase and miRTarBase databases, and related miRNA and target mRNA amounts were assessed in 40 breast cancer tissue and 40 breast benign disease tissue. Finally, 2 key regulatory pairs (miR-205-5p/HMGB3 and miR-96-5p/FOXO1) were selected to establish a diagnostic model. We next analyzed the functions of these four markers in breast cancer.Conclusions: A diagnostic model including two miRNAs and their respective target mRNAs was established to distinguish BCs and benign breast diseases. These markers play an essential role in the pathogenesis of BC.

BACKGROUND: Circulating microRNAs (miRNAs) prove to be potential non-invasive indicators of cancers. The purpose of this study is to profile serum miRNA expression in breast cancer (BC) patients to find potential biomarkers for BC diagnosis. METHODS: The miRNA expression patterns of serum samples from 216 BC patients and 214 normal control subjects were compared. A four-phase validation was conducted for biomarker identification. In the screening phase, the Exiqon miRNA qPCR panel was employed to select candidates, which were further analyzed by quantitative reverse transcriptase PCR in the following training, testing, and external validation phases. RESULTS: A 12-miRNA (let-7b-5p, miR-106a-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p, miR-425-5p, miR-451a, miR-92a-3p, miR-93-5p, and miR-16-5p) panel in serum was constructed. The diagnostic performance of the panel was assessed using ROC curve analyses. The area under the curves (AUCs) were 0.952, 0.956, 0.941 and 0.950 for the four separate phases, respectively. Additionally, the expression features of the 12 miRNAs were further explored in 32 pairs of BC tumor and para-tumor tissues, and 32 pairs of serum exosomes samples from patients and healthy subjects. miR-16-5p, miR-106a-5p, miR-25-3p, miR-425-5p, and miR-93-5p were highly overexpressed and let-7b-5p was conversely downregulated in tumor tissues. Excluding miR-20a-5p and miR-223-3p, the 10 other miRNAs were all significantly upregulated in BC serum-derived exosomes. CONCLUSION: A signature consisting of 12 serum miRNAs was identified and showed potential for use in non-invasive diagnosis of BC.

Purpose Chemotherapy is a comprehensive therapy for breast cancer; nevertheless, its associated adverse effects are drawing increasing attention with the continuous improvement of the efficacy. The changes in serum lipids of breast cancer patients caused by chemotherapy have been reported by previous studies, whereby the former increase the incidence rate of cardiovascular disorders. However, the variations in the changes of serum lipids with different chemotherapy regimens have seldom been reported. Methods From January 2011 to December 2017, 1740 breast cancer patients treated with chemotherapy were recruited at the First Affiliated Hospital of Nanjing Medical University. The chemotherapy regimens included anthracycline-based, taxane-based, and anthracycline-plus-taxane-based regimens, dose-dense and standard-interval regimens. Lipid profiles that contained TG (triglyceride), TC (total cholesterol), HDL-C (high-density lipoprotein cholesterol), LDL-C (low-density lipoprotein cholesterol) and Lpa (lipoprotein a) levels were collected prior to the first, second and last cycles of chemotherapy. The changes of serum lipids with the same or different chemotherapy regimens were analyzed and compared. Results It was observed that the levels of TG, TC, LDL-C and Lpa increased significantly while that of HDL-C decreased after adjuvant chemotherapy in breast cancer patients (P

Small RNAs derived from tRNAs are attracting considerable attention; however, the effects of tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs) as biomarkers have not been investigated in early-stage breast cancer (EBC). The study aimed to explore whether tRFs and tiRNAs could be detected in plasma and whether they could serve as diagnostic biomarkers. The study was conducted in four phases. Thirty tRFs and tiRNAs were selected by high-throughput sequencing in screening phase and then assessed in training, testing, and external validation phases by qRT-PCR. Six tRFs (tRF-Glu-CTC-003, tRF-Gly-CCC-007, tRF-Gly-CCC-008, tRF-Leu-CAA-003, tRF-Ser-TGA-001, and tRF-Ser-TGA-002) were found significantly downregulated in plasma samples of patients with EBC compared with normal controls, and all were derived from 5′ ends of tRNAs. Patients with HER2+ EBC with low expression levels of tRF-Glu-CTC-003 were related to worse disease-free survival and overall survival. The identified tRFs were further examined in cell supernatants, exosomes isolated from plasma, and tissues. In conclusion, our study identified six tRFs from the 5′ ends of tRNAs as novel diagnostic biomarkers for EBC, providing additional evidence for, and a better understanding of, circulating tRFs and EBC., Graphical Abstract, Wang et al. identified six tRFs from the 5′ ends of tRNAs for the diagnosis of early-stage breast cancer (EBC) through a four-phase study. They also analyzed these biomarkers in different subtypes and evaluated the expression levels in exosomes, tissues, and so on. These data provided novel strategies for the diagnosis of EBC.

The incidence of breast cancer (BC) is the highest among women. Identification of miRNAs as biomarkers may help to improve the diagnosis of BC. The purpose of this study was to assess the expression levels of miR-1976 in plasma samples and the biological functions in the progression of BC. The expression levels of miR-1976 in plasma samples and tissues were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The associations between the expression levels and clinicopathological features were studied. Cell supernatants were used to simulate circulation. The biological functions of miR-1976 were assessed in vitro and in vivo. The expression levels of miR-1976 in plasma samples were found significantly lower in patients with BC than those in healthy controls, and were associated with Ki-67. The expression levels in BC tissues were lower than those in adjacent normal tissues, and were correlated with the number of lymph nodes and Ki-67. The expression levels in BC cell supernatants and cell lines were lower than that in normal human breast epithelial cell line HBL-100. miR-1976 knockdown promoted proliferation in vitro and in vivo. miR-1976 may serve as a promising non-invasive biomarker for the diagnosis of BC in the future.

This study aimed to retrospectively evaluate the circulating free DNA (cfDNA) level in patients with locally advanced breast cancer (LABC) having different neoadjuvant chemotherapy (NCT) responses and to investigate whether dynamic changes in cfDNA level could predict the effectiveness of NCT in patients with LABC. Data on 61 patients with LABC were included. NCT responses were evaluated using the response evaluation criteria. Blood samples were collected for cfDNA detection before treatment and after the first and eighth courses of chemotherapy. The Alu 111‐bp and 260‐bp fragment levels were evaluated by polymerase chain reaction, and the predictive value of the cfDNA level in the NCT response was determined. In vitro, the MCF‐7 and MCF‐7/ADR cell lines were applied to simulate the phenomenon of drug resistance and explain the underlying mechanism. The Alu 111‐bp level increased after the first NCT course (P = .014) and then remained high after NCT in the high‐R group (P = .047), but it remained steady in the low‐R group during NCT. A similar tendency in the Alu 260‐bp level was revealed in different groups. The ∆∆Ct value of Alu 260‐bp had good diagnostic efficiency in assessing predictive ability. The area under the curve for the ∆∆Ct1 and ∆∆Ct2 of Alu 260‐bp was 0.697 and 0.647, respectively. The cfDNA level was closely related to epirubicin‐induced apoptosis and changes in the Ki‐67 index in vitro. The elevation of cfDNA after one chemotherapy cycle was mediated by the apoptosis of tumor cells and related to the improved chemotherapy response., The cfDNA level was closely related to epirubicin‐induced apoptosis and changes in the Ki‐67 index. The elevation of cfDNA after one chemotherapy cycle was mediated by the apoptosis of tumor cells and related to the improved chemotherapy response.

Background Patients with operable breast cancer have a better prognosis for recovery. However, once distant organ metastasis occurs, the chance of a long-term survival or a cure is limited. The collection and counting of circulating tumor cells (CTCs) by reliable detection techniques are of increasing importance in the diagnosis of early metastasis and prognosis of disease progression. Isolation by size of epithelial tumor cells (ISET) has the advantage of simplicity of operation and high homogeneity. It is practical for large-scale clinical detection showing cell abundance. The value of ISET in the detection of circulating breast cancers in the blood has not been determined. The purpose of this study is to explore the feasibility of applying ISET to detect CTCs by determining the detection rate of ISET in operable breast cancer and by evaluating the correlation between detection rate, cell count and clinical factors such as molecular typing and pathological staging. Methods The experiment included 193 breast cancer patients who were diagnosed by core needle biopsy before the operation. 10 mL of venous blood was collected from the patients preoperatively, and CTCs in their blood samples were counted and analyzed by ISET. Results Patients were divided into groups according to pathology and immunohistochemistry. The overall detection rate of CTCs by ISET was 41.24%. The detection rate, the number of overall CTCs and the average number of CTCs in each group were analyzed individually. No significant differences were observed between the different groups. Conclusions Although ISET has a relatively good detection rate for circulating breast cancer cells, it fails to provide more information on pathological staging, molecular classification and so forth.

Objective To determine the utility of plasma microRNAs (miRNAs) as biomarkers in cervical cancer (CC). Methods Some studies were conducted about the specific expression of plasma miRNAs in the diagnosis of CC. Plasma samples of 97 CC patients and 87 normal controls (NCs) were used to identify dysregulation of miRNAs in the training, testing, and external validation phases. Receiver operating characteristic (ROC) curves and area under the ROC curve (AUC) were used to evaluate the sensitivity and specificity of identified individual miRNAs and miRNA panels for the diagnosis of CC. Expression levels of specific miRNAs were also examined in plasma exosomes and tissue samples of CC patients. Results Four plasma miRNAs (miR-146a-5p, miR-151a-3p, miR-2110 and miR-21-5p) which showed up-regulation were identified and validated in CC patients. A panel of the four miRNAs were constructed as potential diagnostic markers for CC. The AUCs of the panel of these four-miRNAs for the training, testing, and external validation phases were 0.911, 0.774, and 0.786, respectively. miR-146a-5p and miR-21-5p levels were all up-regulated in CC tissue specimens, whereas miR-146a-5p, miR-151a-3p, and miR-2110 levels were up-regulated in plasma exosomes. Conclusion The signature of the four-miRNAs identified in peripheral plasma is a promising novel biomarker for the diagnosis of CC.

Xinyang Wang,1,* Dandan Xue,1,* Xiaoxia Zhu,2,* Rui Geng,3 Xin Bao,2 Xiang Chen,2 Tiansong Xia1,4 1Department of Breast Surgery, The First Affiliated Hospital with Nanjing Medical University, Nanjing, People’s Republic of China; 2Department of Thyroid and Breast, Yixing People’s Hospital Affiliated to Jiangsu University, Yixing, People’s Republic of China; 3Department of Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, People’s Republic of China; 4Jiangsu Key Lab of Cancer Biomarkers, Prevention, and Treatment, Jiangsu Collaborative Innovation Center for Cancer Personalized Medicine, School of Public Health, Nanjing Medical University, Nanjing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiang ChenDepartment of Thyroid and Breast, Yixing People’s Hospital Affiliated to Jiangsu University, 75 Zhen’guan Road, Yixing, 214200, People’s Republic of ChinaEmail staff984@yxph.comTiansong XiaDepartment of Breast Surgery, The First Affiliated Hospital with Nanjing Medical University, 300 Guangzhou Road, Nanjing, 210029, People’s Republic of ChinaEmail xiatsswms@163.comPurpose: Breast cancer accounts for the highest incidence of tumors in women. Immune infiltrating of the tumor microenvironment positively correlates with the overall survival of breast cancer patients. PLAT can affect the development of many cancers, but its mechanism in breast cancer is unclear. We assessed the correlation between PLAT and immune infiltrating in breast cancer based on the TCGA database.Patients and Methods: The expression and DNA methylation of PLAT in breast cancer with different clinical characteristics was tested by Wilcoxon signed rank test and displayed by box plot. Sequentially, Kaplan–Meier plot was employed to compare the difference in overall survival rates between patients with different expressed levels. Univariate and multivariate Cox regression analyses were used to validate whether PLAT is an independent prognostic factor of breast cancer. After that, GO, KEGG, and gene-set enrichment analysis were employed to do functional enrichment analysis. Finally, TIMER, TISIDB database, and ssGSEA algorithm were used to assess the correlation between PLAT expression and various immune characteristics. The correlation between PLAT expression and DNA methylation was examined by Pearson correlation coefficient.Results: PLAT displays differential expression levels in breast cancer patients with various clinical characteristics. As an independent protective factor for breast cancer, PLAT may significantly correlate with the immune status of breast cancer by adjusting many immune molecules and affecting the immune infiltration in the tumor microenvironment. DNA methylation of PLAT downregulates the gene expression and affects the prognosis of breast cancer.Conclusion: PLAT can be considered a potential biomarker to predict breast cancer prognosis and might contribute to the development of immunological treatment strategies.Keywords: tPA, prognosis, immune infiltration, bioinformatic

Background: Chemoresistance is a major obstacle in the neoadjuvant chemotherapy (NCT) of locally advanced breast cancer (LABC). Identification of miRNAs as prognostic biomarkers may help overcome chemoresistance of breast cancer (BC). The aim of this study was to evaluate the expression level of miR-1275 in plasma samples and the biological functions in the chemoresistance of BC.Methods: The expression levels of miR-1275 in plasma samples and cells were measured by RT-qPCR. The associations between the expression levels of miR-1275 and clinicopathological features were studied. CRISPR/Cas9-mediated gene editing was used to construct miR-1275 knock-out cells. CCK-8, colony formation assays, epirubicin accumulation assay and xenograft tumor models were used to detect the sensitivity of breast cancer cells to epirubicin in vitro and in vivo. Mammosphere formation assay, flow cytometry analysis and western blot analyses were used to evaluate the effects of miR-1275 on cancer stem cells (CSCs) characteristics in BC cells. Dual-luciferase reporter, RNA pulldown, ELISA and IHC were used to verify the relationship between the expression of miR-1275 and Midkine (MDK). Results: We found that miR-1275 was significantly downregulated in plasma from patients resistant to chemotherapy with LABC and in chemoresistant breast cancer cell lines, while patients with low levels of miR-1275 show poor overall survival. Mir-1275 knock-out promoted chemoresistance in breast cancer cells by increasing the properties of cancer stem cells in vitro and in vivo. Mechanistically, we identified that Midkine was determined to be direct downstream protein of miR-1275 which initiated PI3K/Akt signaling in breast cancer cells.Conclusions: We demonstrated that the high expression level of miR-1275 in plasma predicted better response to NCT. The reduction of miR-1275 promoted BC cells chemoresistance by increasing CSC properties via targeting MDK/AKT axis. The potential of miR-1275 as a new prognostic biomarker and therapeutic target of breast cancer patients was identified.

In the original publication of this article [1], the molecular weight of RBMS3 was incorrectly noted as 38 KDa within Fig 1A, Fig 2A and Fig 2B. The figures have been updated to list the correct molecular weight of RBMS3 as 41 KDa.

Breast cancer (BC) is one of the most common cancers in females. Since early detection can bring prognosis benefit, discovery of novel noninvasive biomarkers for BC diagnosis is in urgent need. In this four‐phase study, we profiled miRNA expression in plasma samples from a total of 257 BC patients and 257 normal controls (NCs). Exiqon miRNA qPCR panel was used to select candidate miRNAs in the screening phase which were further analyzed using qRT‐PCR in the following training, testing and external validation phases. Finally, we identified five plasma miRNAs (let‐7b‐5p, miR‐122‐5p, miR‐146b‐5p, miR‐210‐3p and miR‐215‐5p) whose expression levels were significantly different between BC patients and NCs. A 5‐miRNA panel in plasma with high sensitivity and specificity was then constructed to detect BC. The areas under the receiver‐operating characteristic curves (AUCs) of the panel were 0.683, 0.966, 0.978 for the training, testing and external validation sets, respectively. Expression of the identified miRNAs was further analyzed among 32 pairs of BC tissue and the adjacent normal tissue samples as well as plasma‐derived exosome samples from 32 BC patients vs 32 NCs. Let‐7b‐5p was contrarily down‐regulated in BC tissue. In exosomes samples, only miR‐122‐5p was significantly up‐regulated as in plasma for BC patients. In conclusion, we identified a 5‐miRNA plasma panel (let‐7b‐5p, miR‐122‐5p, miR‐146b‐5p, miR‐210‐3p and miR‐215‐5p) that could serve as a promising biomarker for BC detection., We identified five plasma miRNAs (let‐7b‐5p, miR‐122‐5p, miR‐146b‐5p, miR‐210‐3p and miR‐215‐5p) by multiple‐phase validation which could serve as novel noninvasive biomarkers for BC diagnosis. Our findings may contribute to the early detection of BC and be beneficial to BC patients.

Drug resistance, including epirubicin-based therapeutic resistance, is one of the major reasons responsible for the unfavorable prognosis of patients diagnosed with breast cancer (BC). Acquired chemoresistance and metastatic properties have been identified to be closely associated with the process of epithelial-mesenchymal transition (EMT). Recently, dysregulation of long non-coding RNAs (lncRNAs) have been increasingly reported to perform promotive or suppressive functions in chemoresistance and EMT process in multiple cancers. However, relevant novel lncRNA participating in epirubicin resistance and EMT and its underlying molecular mechanisms remain unknown in BC. Herein, we established the epirubicin-resistant breast cancer cell subline (MCF-7/ADR), which presented mesenchymal phenotype and increased metastatic potential. A panel of differentially expressed lncRNAs, including 268 upregulated and 49 downregulated lncRNAs, were identified by high-flux microarray investigation in MCF-7 and MCF-7/ADR cells. The novel lncRNA NONHSAT101069 was significantly overexpressed in BC specimens, BC cell lines, and epirubicin-resistant cell sublines. The knockdown of NONHSAT101069 significantly repressed, whereas overexpression of NONHSAT101069 promoted the epirubicin resistance, migration, invasion and EMT process of BC cells both in vitro and in vivo. Further mechanism-related researches uncovered that NONHSAT101069 functioned as a ceRNA (competing endogenous RNA) via sponging miR-129-5p. Twist1 was a direct downstream protein of NONHSAT101069/miR-129-5p axis in BC cells. To conclude, NONHSAT101069 was upregulated in BC tissues and promoted epirubicin resistance, migration and invasion of BC cells via regulation of NONHSAT101069/miR-129-5p/Twist1 axis, highlighting its potential as an oncogene and a therapeutic biomarker for BC.

Most N6-methyladenosine (m6A) associated regulatory proteins (i.e., m6A writer, eraser, and reader proteins) are involved in the pathogenesis of various cancers, mostly in m6A-dependent manners. As a component in the m6A ‘writers’, KIAA1429 is reported to be an RNA-binding protein and involved in the m6A modification, mRNA splicing and processing. Till now, the functions of KIAA1429 in tumorigenesis and related mechanism have not been reported. In the present study, we found KIAA1429 was highly expressed in breast cancer tissues, but frequently down-regulated in non-cancerous breast tissues. The overall survival of breast cancer patients with high-expression KIAA1429 was significantly shorter than those with low-expression KIAA1429. Then, we demonstrated that KIAA1429 was associated with breast cancer proliferation and metastasis in vivo and in vitro. The potential targeting genes of KIAA1429 in breast cancer were identified by RNA immunoprecipitation sequencing. One of these genes is cyclin-dependent kinase 1 (CDK1), which plays an oncogenic role in cancers. Furthermore, we confirmed that KIAA1429 played its oncogenic role in breast cancer by regulating CDK1 by an m6A-independent manner. 5′-fluorouracil was found to be very effective in reducing the expression of KIAA1429 and CDK1 in breast cancer. These findings indicated that KIAA1429 could promote breast cancer progression and was correlated with pathogenesis. It may represent a promising therapeutic strategy on breast cancer, especially in combination with CDK1 treatment.

Background Metastasis remains the biggest obstacle for breast cancer treatment. Therefore, identification of specific biomarker of metastasis is very necessary. The RNA binding protein 3 (RBMS3) acts as a tumor suppressor in various cancers. Whereas, its role and underlying molecular mechanism in breast cancer is far from elucidated. Methods Quantitative real-time PCR and western blots were carried out to determine the expression of RBMS3 in breast cancer cells and tissues. Transwell and in vivo metastasis assay were conducted to investigate the effects of RBMS3 on migration, invasion and metastasis of breast cancer cells. Transcriptome sequencing was applied to screen out the differential gene expression affected by RBMS3. RNA immunoprecipitation assay combined with luciferase reporter assay were performed to explore the direct correlation between RBMS3 and Twist1 mRNA. Results RBMS3 was downregulated in breast cancer and ectopic expression of RBMS3 contributed to inhibition of cell migration, invasion in vitro and lung metastasis in vivo. Furthermore, RBMS3 negatively regulated Twsit1 expression via directly binding to 3′-UTR of Twist1 mRNA, and thereby decreased Twist1-induced expression of matrix metalloproteinase 2 (MMP2). Additionally, Twist1-induced cell migration, invasion and lung metastasis could be reversed by the upregulation of RBMS3. Conclusions In summary, our study revealed a novel mechanism of the RBMS3/Twsit1/MMP2 axis in the regulation of invasion and metastasis of breast cancer, which may become a potential molecular marker for breast cancer treatment.

Background: Breast cancer stem cells (BCSCs) play an essential role in facilitating breast cancer relapse and metastasis. The underlying mechanism, however, remains incompletely understood. In the current study, we investigated the clinical significance, biological function and mechanism of a long noncoding RNA CCAT1 (LncCCAT1) in BCSCs. Methods: Firstly, lncRNAs expression in poorly differentiated breast cancer tissues and BCSCs were measured by lncRNA microarray and confirmed in breast cancer tissues and cell lines. The functional roles and mechanisms of LncCCAT1 were further investigated by gain and loss of function assays in vitro and in vivo. Results: LncCCAT1 is markedly upregulated in breast cancer tissues BCSCs and is correlated with poor outcomes in breast cancer patients. Overexpression of LncCCAT1 contributes to the proliferation, stemness, migration and invasion capacities of BCSCs. Mechanistic investigation suggests that LncCCAT1 can interact with miR-204/211, miR-148a/152 and Annexin A2(ANXA2), then upregulate T-cell factor 4 (TCF4) or promote translocation of β-catenin to the nucleus where it activates TCF4, leading to the activation of wingless/integrated (Wnt) signaling. Furthermore, TCF4 can also bind to the promoter of LncCCAT1 to promote LncCCAT1 transcription, thus forming a positive feedback regulatory circuit of LncCCAT1-TCF4-LncCCAT1 in BCSCs. Conclusions: LncCCAT1 plays an important role in breast cancer progression and may serve as a novel target for breast cancer diagnosis and therapy.

Liquid biopsy is noninvasive and convenient to detect cancer-derived materials in blood or other body fluids. The aim of this study was to identify tRNA-derived small RNAs (tsRNAs) in plasma that could distinguish patients with breast cancer (BC) from healthy controls. Basing on high-throughput sequencing, 15 significantly upregulated tsRNAs were selected and assessed in cell supernatants and cell lines. 6 tsRNAs were identified and verified in a large cohort of 120 patients with BC and 112 healthy controls. tRF-Arg-CCT-017, tRF-Gly-CCC-001, and tiRNA-Phe-GAA-003 could serve as novel diagnostic biomarkers. Meanwhile, tRF-Arg-CCT-017 and tiRNA-Phe-GAA-003 could also act as prognostic biomarkers. Target genes of these tsRNAs were related to the development of cancers. These results suggested that specific tsRNAs in plasma might serve as diagnostic and prognostic biomarkers of BC.

Background The activation of the NF-κB pathway plays a crucial role in the progression of breast cancer (BCa) and also involved in endocrine therapy resistance. On the contrary to the canonical NF-κB pathway, the effect of the noncanonical NF-κB pathway in BCa progression remains elusive. Methods BCa tumor tissues and the corresponding cell lines were examined to determine the correlation between RelB and the aggressiveness of BCa. RelB was manipulated in BCa cells to examine whether RelB promotes cell proliferation and motility by quantitation of apoptosis, cell cycle, migration, and invasion. RNA-Seq was performed to identify the critical RelB-regulated genes involved in BCa metastasis. Particularly, RelB-regulated MMP1 transcription was verified using luciferase reporter and ChIP assay. Subsequently, the effect of RelB on BCa progression was further validated using BCa mice xenograft models. Results RelB uniquely expresses at a high level in aggressive BCa tissues, particularly in triple-negative breast cancer (TNBC). RelB promotes BCa cell proliferation through increasing G1/S transition and/or decreasing apoptosis by upregulation of Cyclin D1 and Bcl-2. Additionally, RelB enhances cell mobility by activating EMT. Importantly, RelB upregulates bone metastatic protein MMP1 expression through binding to an NF-κB enhancer element located at the 5′-flanking region. Accordingly, in vivo functional validation confirmed that RelB deficiency impairs tumor growth in nude mice and inhibits lung metastasis in SCID mice.

Triple-negative breast cancer (TNBC), characterized by high aggression and invasiveness, has a worse prognosis than other subtypes of breast cancer. Establishing a novel animal model is helpful to understand the mechanisms involved in the progress of TNBC metastasis. In a self-established mouse model consisting normal human breast tissues and normal human bone tissues, TNBC cell line SUM-1315 could spontaneously form species-specific bone metastasis. The expression level of miR-1976 in SUM-1315-bo (derived from metastatic bone tumor) was found lower than that in SUM-1315-br (derived from orthotopic breast tumor). MiR-1976 was found to be downregulated in TNBC tissues, and lower expression of miR-1976 was correlated with worse overall survival in a patient cohort obtained from TCGA database. MiR-1976 knockdown promoted epithelial–mesenchymal transition (EMT) and cancer stem cell (CSC) properties in vitro and in vivo. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PIK3CG) was verified as a target gene by sequencing, biotinylated miRNA pull-down, and luciferase reporter assay. Moreover, overexpression and suppression analysis implicated PIK3CG as a mediator of the biological effects of miR-1976. Our study demonstrated that miR-1976 knockdown could promote EMT and CSCs by PIK3CG. These findings may reveal mechanisms of TNBC metastasis, and represent a potential treatment target for patients with TNBC.

Background: Neoadjuvant chemotherapy (NCT) is the standard treatment for patients with locally advanced breast cancer (LABC). However, the predictive value of circulating tumor cells (CTCs) with different molecular subtypes in NCT response has not yet been determined, which was, therefore, the aim of this study. Methods: All patients were staged as LABC and received an EC×4 –T×4 NCT regimen. Blood samples were collected from patients at the time of biopsy, and after the first and eighth NCT courses. Patients were divided into High responders (High-R) and Low responders (Low-R) according to Miller-Payne system and changes in Ki-67 levels after NCT treatment. A novel SE-i•FISH strategy was applied to detect CTCs. Subtypes were successfully analyzed in LABC patients undergoing NCT, for the first time. Results: Total CTCs increased continuously and were higher for Low-R patients; while in the High-R group, total CTCs increased slightly during NCT before returning to baseline levels. Triploid and tetraploid chromosome 8 as well as the proportion of each, increased for Low-R but not High-R patients. The number of small CTCs in the Low-R group increased significantly until the last sample, however, remained constant in the High-R group. The patients with more CTCs had shorter PFS and OS than those with less CTCs after the 8th course of NCT. Conclusions: Total CTCs as well as individual subtypes within peripheral blood following NCT were predictive of patient responses to NCT. More detailed characterization of CTC blood profiles may improve predictive capacity and lead to improved LABC treatments.