Exosomal circRHCG promotes breast cancer metastasis via facilitating M2 polarization through TFEB ubiquitination and degradation.

Triple-negative breast cancer (TNBC) is a highly aggressive cancer with distant metastasis. Accumulated evidence has demonstrated that exosomes are involved in TNBC metastasis. Elucidating the mechanism underlying TNBC metastasis has important clinical significance. In the present study, exosomes were isolated from clinical specimens and TNBC cell lines. Colony formation, EdU incorporation, wound healing, and transwell assays were performed to examine TNBC cell proliferation, migration, and metastasis. Macrophage polarization was evaluated by flow cytometry and RT-qPCR analysis of polarization markers. A mouse model of subcutaneous tumor was established for assessment of tumor growth and metastasis. RNA pull-down, RIP and Co-IP assays were used for analyzing molecular interactions. Here, we proved that high abundance of circRHCG was observed in exosomes derived from TNBC patients, and increased exosomal circRHCG indicated poor prognosis. Silencing of circRHCG suppressed TNBC cell proliferation, migration, and metastasis. TNBC cell-derived exosomes promoted M2 polarization via delivering circRHCG. Exosomal circRHCG stabilized BTRC mRNA via binding FUS and naturally enhanced BTRC expression, thus promoting the ubiquitination and degradation of TFEB in THP-1 cells. In addition, knockdown of BTRC or overexpression of TFEB counteracted exosomal circRHCG-mediated facilitation of M2 polarization. Furthermore, exosomal circRHCG promoted TNBC cell proliferation and metastasis by facilitating M2 polarization. Knockdown of circRHCG reduced tumor growth, metastasis, and M2 polarization through the BTRC/TFEB axis in vivo. In summary, exosomal circRHCG promotes M2 polarization by stabilizing BTRC and promoting TFEB degradation, thereby accelerating TNBC metastasis and growth. Our study provides promising therapeutic strategies against TNBC.

Integration of bioinformatics and machine learning strategies identifies APM-related gene signatures to predict clinical outcomes and therapeutic responses for breast cancer patients.

BACKGROUND: Tumor antigenicity and efficiency of antigen presentation jointly influence tumor immunogenicity, which largely determines the effectiveness of immune checkpoint blockade (ICB). However, the role of altered antigen processing and presentation machinery (APM) in breast cancer (BRCA) has not been fully elucidated. METHODS: A series of bioinformatic analyses and machine learning strategies were performed to construct APM-related gene signatures to guide personalized treatment for BRCA patients. A single-sample gene set enrichment analysis (ssGSEA) algorithm and weighted gene co-expression network analysis (WGCNA) were combined to screen for BRCA-specific APM-related genes. The non-negative matrix factorization (NMF) algorithm was used to divide the cohort into different clusters and the fgsea algorithm was applied to investigate the altered signaling pathways. Random survival forest (RSF) and the least absolute shrinkage and selection operator (Lasso) Cox regression analysis were combined to construct an APM-related risk score (APMrs) signature to predict overall survival. Furthermore, a nomogram and decision tree were generated to improve predictive accuracy and risk stratification for individual patients. Based on Tumor Immune Dysfunction and Exclusion (TIDE) method, random forest (RF) and Lasso logistic regression model were combined to establish an APM-related immunotherapeutic response score (APMis). Finally, immune infiltration, immunomodulators, mutational patterns, and potentially applicable drugs were comprehensively analyzed in different APM-related risk groups. IHC staining was used to assess the expression of APM-related genes in clinical samples. RESULTS: In this study, APMrs and APMis showed favorable performances in risk stratification and therapeutic prediction for BRCA patients. APMrs exhibited more powerful prognostic capacity and accurate survival prediction compared to conventional clinicopathological features. APMrs was closely associated with distinct mutational patterns, immune cell infiltration and immunomodulators expression. Furthermore, the two APM-related gene signatures were independently validated in external cohorts with prognosis or immunotherapeutic responses. Potential applicable drugs and targets were mined in the APMrs-high group. APM-related genes were further validated in our in-house samples. CONCLUSION: The APM-related gene signatures established in our study could improve the personalized assessment of survival risk and guide ICB decision-making for BRCA patients.

Construction of an m6A-related lncRNA model for predicting prognosis and immunotherapy in patients with lung adenocarcinoma.

N6-methyladenosine (m6A)-related lncRNAs could be involved in the development of multiple tumors with an unknown role in lung adenocarcinoma (LUAD). Hence, gene expression data and clinical data of LUAD patients were acquired from The Cancer Genome Atlas Database. The prognostic m6A-related lncRNAs were identified through differential lncRNA expression analysis and Spearman's correlation analysis. The least absolute shrinkage and selection operator regression was used to establish the prognostic risk model, so as to evaluate and validate the predictive performance with survival analysis and receiver operating characteristic curve analysis. The expression of immune checkpoints, immune cell infiltration and drug sensitivity of patients in different risk groups were analyzed separately. A total of 19 prognostic m6A-related lncRNAs were identified to set up the prognostic risk model. The patients were divided into high- and low-risk groups based on the median value of the risk scores. Compared with the patients in the low-risk group, the prognosis of the patients in the high-risk group was relatively worse. The receiver operating characteristic curves indicated that this model had excellent sensitivity and specificity. Multivariate Cox regression analysis demonstrated that the risk score could be supposed as an independent prognostic risk factor. We highlighted that the risk scores were correlated with immune cell infiltration and drug sensitivity for constructing a prognostic risk model in LUAD patients based on m6A-related lncRNAs.

PUS1 is a novel biomarker for predicting poor outcomes and triple-negative status in breast cancer.

Breast cancer patients' outcomes have improved dramatically in recent years, but relapses and poor prognosis remain common due to its aggressiveness and heterogeneity. The development of reliable biomarkers is still needed for predicting prognosis and treatment effectiveness. Recently, a growing body of research suggests that pseudouridine synthases contribute to the development of many cancers, but their contribution to breast cancer remains largely unknown. Using an integrative analysis, we selected pseudouridine synthase1(PUS1) as the candidate biomarker. A tissue microarray of 131 breast cancer patients was then utilized to determine the clinical significance and prognostic value of PUS1. RNA sequencing analysis was conducted to identify downstream genes that differ between control and PUS1 knockdown cells. The effect of PUS1 on phenotypes of cells was assessed using cell proliferation, colony formation, and transwell invasion assays. We found that breast tumors overexpressed PUS1 compared with paired normal tissues. PUS1 expression was positively correlated with triple-negative breast cancer (TNBC) status (P= 0.020) and tumor grade (P <0.0001), but not with age (P= 0.736), tumor size (P= 0.608), lymph node (P= 0.742), oestrogen receptor (ER) (P= 0.162), progesterone receptor (PR) (P= 0.901), human epidermal growth factor receptor 2 (HER2) (P= 0.608) or tumor stage (P= 0.411). Comparatively, patients with high PUS1 levels had shorter overall survival time (P=0.0001) and relapse-free survival time (P = 0.0093). A univariate and multivariate survival analysis suggested that the overall survival of patients was independently influenced by the PUS1 score (Univariate Cox P <0.0001, HR=5.176, 95% CI =2.420-11.07; Multivariate Cox P = 0.001, HR = 5.291, 95% CI =1.893-14.78). RNA sequencing data revealed the PUS1 knockdown significantly affects a series of cancer related biological process such as regulation of cell proliferation and cell migration, as well as KEGG pathways including Mitophagy and PI3K-Akt signaling. In vitro, knockdown of PUS1 significantly suppressed the proliferation and colony formation abilities of MDA-MB-231 cells and BT-549 cells. Additionally, the ability of tumor cells to invade was remarkably attenuated in low PUS1 expression groups compared with the corresponding control groups. Our results suggested that PUS1 is a novel biomarker that predicts poor outcomes in patients with breast cancer and may prove to be a promising treatment target.

Hsa_circ_0001361 facilitates the progress of lung adenocarcinoma cells via targeting miR-525-5p/VMA21 axis.

BACKGROUND: Lung adenocarcinoma (LUAD) is a common subtype of lung cancer with high recurrence rate and fatality. Circ_0001361 has been recognized as key regulators in various malignancies, but its roles in LUAD remain ambiguous. METHODS: Circ_0001361, miR-525-5p, and VMA21 levels were assessed by RT-qPCR. The growth and metastasis of LUAD cells were detected by MTT, colony formation, wound scratch, and transwell assays, respectively. The interaction between circ_0001361/VMA21 and miR-525-5p was detected by dual luciferase, RNA immunoprecipitation, and RNA pull-down assays. VMA21 protein level was detected by Western blotting. Nude mouse xenograft model was established to determine the role of circ_0001361 in tumor growth in vivo. RESULTS: Circ_0001361 was up-regulated, while miR-525-5p was down-regulated in LUAD tissues and cells. Functional experiments demonstrated that circ_0001361 drove LUAD cell growth and metastasis. Mechanistically, circ_0001361 functioned as a sponge of miR-525-5p to up-regulate downstream target VMA21 level. MiR-525-5p/VMA21 axis was involved in circ_0001361-mediated malignant phenotypes of LUAD cells. Finally, inhibition of circ_0001361 restrained in vivo xenograft tumor growth via regulating miR-525-5p/VMA21 axis. CONCLUSION: Our findings elucidate that circ_0001361 facilitates the tumorigenesis and development of LUAD through miR-525-5p/VMA21 axis, providing evidence for circ_0001361 as a potential prognosis biomarker and therapeutic target for clinical treatment of LUAD.

Evolution of drug-resistant mutations in HBV genomes in patients with treatment failure during the past seven years (2010-2016).

The objective of this study was to analyze the prevalence of drug-resistant HBV mutants in patients with treatment failure during the past seven years (2010-2016). 4055 HBV-infected patients who underwent HBV polymerase gene mutation test from 2010 to 2016 were enrolled. The nucleos(t)ide analogues (NAs) resistance mutation positions, including rtL180, rtA181, rtT184, rtS202, rtM204, rtI233, rtN236, rtI169, rtV173, and rtM250 were analyzed. Genotypic resistance mutations were detected in 30.8% (1248/4055) of the patients with treatment failure. Rates of drug-resistant mutations associated with LAM, ADV, ETV, and multidrug were 27.23% (1104/4055), 9.67% (392/4055), 3.69% (150/4055), and 0.79% (32/4055). Among the primary NA-resistant mutations, rtM204I (13.44%, 545/4055) occurred more frequently, followed by rtM204V, rtN236T, rtA181T, and rtA181V. For single-base mutations, rtL180M and rtA181V increased gradually during the past seven years, while rtM204I/V and rtN236T decreased after 2015. The development of drug-resistant mutations positively correlated with the consumption of ETV (r = 0.964, P = 0.002), and weakly correlated with that of LAM (r = 0.679, P = 0.109) and ADV (r = 0.429, P = 0.354). Moreover, single-base mutation rtA181V and multi-base mutations (rtL180M + M204I and rtL180M + M204V + M204I) were more common in HBV genotype C than those in genotype B (1.94% vs. 0.66%, 1.84% vs. 0.16%, 1.02% vs. 0.16%, respectively). NA-related mutations in HBV RT region increased in the past seven years, especially for LAM. Frequencies of rtL180M and rtA181T/V increased gradually in the past seven years, to which we should pay more attention.

Predictive role of GSTP1-containing exosomes in chemotherapy-resistant breast cancer.

Anthracycline/taxane-based chemotherapy regimens are usually used as neoadjuvant chemotherapies to decrease tumour size and prevent metastasis of advanced breast cancer. However, patients have a high risk of developing chemo-resistance during treatment through still unknown mechanisms. Glutathione S-transferase P1 (GSTP1), which belongs to the family of phase II metabolic enzymes, has been reported to function in detoxifying several anti-cancer drugs by conjugating them with glutathione. Previous studies have identified GSTP1 as a predictor of prognosis and chemo-resistance in breast cancer patients, but the mechanisms governing GSTP1-dependent drug resistance are still unclear. We have found that GSTP1 expression is much higher in adriamycin-resistant cells and their corresponding exosomes. The role of GSTP1-containing exosomes in conferring drug resistance was analysed through cell apoptosis and immunofluorescence staining assays. Furthermore, we analysed 42 cases of paired breast cancer tissues collected before and after anthracycline/taxane-based neoadjuvant chemotherapy by immunohistochemistry. Higher GSTP1 expression was shown in the progressive disease (PD)/stable disease (SD) group than in the partial response (PR)/complete response (CR) group both in the samples collected before and after the chemotherapy treatment. Interestingly, GSTP1 partly re-localized from the cell nucleus to the cytoplasm upon treatment, and similar results were obtained for the exosomal marker Tumour susceptibility gene 101 protein (TSG101), which also increased in the cytoplasm after chemotherapy. After analysing the serum exosomes of 30 patients treated with anthracycline/taxane-based neoadjuvant chemotherapy, we discovered that the levels of GSTP1 in exosomes from patients in the PD/SD group were significantly higher than those in the PR/CR group. Here, for the first time, we investigated a novel role for GSTP1-containing exosomes and their capability to transfer drug resistance and evaluated their clinical use in predicting chemo-resistance.

Study of recombinant human interleukin-12 for treatment of complications after radiotherapy for tumor patients.

AIM: To evaluate the treatment effects of recombinant human interleukin-12 (rhIL-12) on radiotherapy complications, such as severe myelosuppression or pancytopenia, the decline or imbalance of immune function, etc. METHODS: The patients received high-dose and short-course precise radiotherapy, such as Cyber knife and image-guided radiotherapy (IGRT), which can cause myelosuppression or pancytopenia and immune function decline within a short time. One-hundred subjects were enrolled in the study, and 50 were randomized to a treatment group which used rhIL-12 and 50 were randomized to a control group which used symptomatic and supportive therapy after radiotherapy. The 50 subjects in the treatment group were further divided into five subgroups and intervened with rhIL-12 at a dose of 50, 100, 150, 200 or 250 ng/kg respectively. The dose-effect relationship was observed. RESULTS: RhIL-12 significantly attenuated the decrease of peripheral blood cells in the treatment group, and immune function was improved after treatment. Due to the different radiation doses, there was a fluctuation within 12 h after treatment but mostly showing an increasing trend. As to the clinical manifestations, 2 patients in the 250 ng/kg subgroup showed low fever after administration, 1 patient in the 200 ng/kg subgroup and 2 patients in the 250 ng/kg subgroup showed mild impairment of liver function during the observation period. CONCLUSION: RhIL-12 has effective therapeutic and protective effects on complications following radiotherapy, such as the decline of blood cells, myelosuppression and the decline or imbalance of immune function, which indicated good prospects for development and application.

The miR-30 family: Versatile players in breast cancer.

The microRNA family, miR-30, plays diverse roles in regulating key aspects of neoplastic transformation, metastasis, and clinical outcomes in different types of tumors. Accumulating evidence proves that miR-30 family is pivotal in the breast cancer development by controlling critical signaling pathways and relevant oncogenes. Here, we review the roles of miR-30 family members in the tumorigenesis, metastasis, and drug resistance of breast cancer, and their application to predict the prognosis of breast cancer patients. We think miR-30 family members would be promising biomarkers for breast cancer and may bring a novel insight in molecular targeted therapy of breast cancer.

miR-222 induces Adriamycin resistance in breast cancer through PTEN/Akt/p27kip1 pathway.

The high resistant rate of Adriamycin (Adr) is associated with a poor prognosis of breast cancer in women worldwide. Since miR-222 might contribute to chemoresistance in many cancer types, in this study, we aimed to investigate its efficacy in breast cancer through PTEN/Akt/p27 kip1 pathway. Firstly, in vivo, we verified that miR-222 was upregulated in chemoresistant tissues after surgery compared with the paired preneoadjuvant samples of 21 breast cancer patients. Then, human breast cancer Adr-resistant cell line (MCF-7/Adr) was constructed to validate the pathway from the parental sensitive cell line (MCF-7/S). MCF-7/Adr and MCF-7/S were transfected with miR-222 mimics, miR-222 inhibitors, or their negative controls, respectively. The results showed that inhibition of miR-222 in MCF-7/Adr significantly increased the expressions of PTEN and p27 kip1 and decreased phospho-Akt (p-Akt) both in mRNA and protein levels (p < 0.05) by using quantitative real-time PCR (qRT-PCR) and western blot. MTT and flow cytometry suggested that lower expressed miR-222 enhanced apoptosis and decreased the IC50 of MCF-7/Adr cells. Additionally, immunofluorescence demonstrated that the subcellular location of p27 kip1 was dislocated resulting from the alteration of miR-222. Conversely, in MCF-7/S transfected with miR-222 mimics, upregulation of miR-222 is associated with decreasing PTEN and p27 kip1 and increasing Akt accompanied by less apoptosis and higher IC50. Importantly, Adr resistance induced by miR-222 overexpression through PTEN/Akt/p27 was completely blocked by LY294002, an Akt inhibitor. Taken together, these data firstly elucidated that miR-222 could reduce the sensitivity of breast cancer cells to Adr through PTEN/Akt/p27 kip1 signaling pathway, which provided a potential target to increase the sensitivity to Adr in breast cancer treatment and further improved the prognosis of breast cancer patients.

The role of miRNAs in drug resistance and prognosis of breast cancer formalin-fixed paraffin-embedded tissues.

PURPOSE: Chemoresistance mediated by miRNAs in breast cancer have been already validated by previous studies in vitro, while little is known concerning the expression of them in vivo. The aim of this study was to investigate the role of miR-222, miR-29a, miR-34a, miR-130a, miR-90b, miR-200b, miR-452, miR-197, miR-138, miR-210, miR-423, miR-4298, miR-4644, miR-139, miR-1246, miR-1268a, miR-140, miR-149, miR-3178, miR-3613, miR-4258, miR-574, miR-671, miR-6780b, miR-7107, miR-744 and miR-7847 linked to drug resistance in breast cancer formalin-fixed paraffin-embedded tissues and the association of prognosis with miRNAs, thus providing effective targets in chemotherapy, as well as potential biomarkers for guiding effective treatments of breast cancer. METHODS: The relationship between the expression of diverse miRNAs and drug resistance was detected by RT-qPCR using 55 breast cancer FFPE tissues containing 26 paired FFPE specimens. RESULTS: MiR-222, miR-29a, miR-34a, miR-423, miR-140, miR-3178, miR-574, miR-6780b and miR-744 exhibited significantly higher expression levels in surgically-resected specimens compared with pre-neoadjuvant chemotherapy biopsies. Evidently high expression of miR-222, miR-29a, miR-140, miR-574, miR-6780b, miR-7107 and miR-744 were found in ineffective group comparing with effective group. Further investigations revealed the significant association between several miRNAs in breast cancer patients. CONCLUSIONS: This study highlights the role of numerous miRNAs in prediction of therapeutic responses and suggests that specific miRNAs could serve as valuable sources for biomarker detections and optimal chemotherapeutic choices for breast cancer patients.

miR-4443 Participates in the Malignancy of Breast Cancer.

PURPOSE: Chemo-resistance is the leading cause of failure in cancer therapy, however, much remains to be understood about the intrinsic mechanisms. In the present study, we discovered the novel miR-4443 that regulated malignancy of breast cancer both in vitro and in vivo. METHODS: We examined the expression of miR-4443 in MDA-MB-231/S and MDA-MB-231 Epirubicin-resistant cell lines with 76 breast cancer formalin-fixed paraffin-embedded tissues by real-time PCR. Also, we investigated the loss- and gain-functions of miR-4443 by MTT assay and flow cytometry. Furthermore, we detected miR-4443 mediated tissue inhibitor of metalloproteinase 2 expression in cells by TargetScan, RT-qPCR and western blot. RESULTS: We identified the up-regulated expression of miR-4443 in Epi-resistant cell lines versus MDA-MB-231/S cell(Epi versus S) and in post-chemotherapy FFPE tissues, along with statistically differential expressions in PR(partial response) versus SD(stable disease)/PD(progressive disease) patients. Overexpression of miR-4443 increased the IC50 value of Epi for the target cells transfected, while inhibition of miR-4443 could restored sensitivity of the target cells to Epi. Besides, down-regulation of endogenous miR-4443 by miRNA-inhibitors significantly enhanced Epi-induced apoptosis while up-regulation of miR-4443 by miRNA-mimics lead to less Epi-induced apoptotic cells. Consequently, changes in TIMP2 mRNA and protein expression revealed that miR-4443 mimics suppressed expression of TIMP2 and induced migration in breast cancer cells. Furthermore, TIMP2 expression associated with better prognosis(HR = 0.721, 95%CI: 0.529-0.983). CONCLUSIONS: We revealed that miR-4443 induced malignancy of breast cancer mainly in chemo-resistance aspect for the very first time, providing a novel biomarker in breast cancer diagnosis and therapy.

MiRNAs-mediated cisplatin resistance in breast cancer.

Cisplatin is a widely used chemotherapeutic agent in breast cancer treatments with inevitable rapidly acquired resistance or intrinsically resistance. Enormous evidence points to the bioprocesses of resistant formation consisting of diverse miRNAs direct and indirect actions on relevant encoding genes. In this report, we overview detailed information on the miRNAs effect on cisplatin-induced resistance, including alterations in cell survival, modification of DNA damage response, changes in cellular uptake or efflux of the drug, altered DNA methylation, and perturbations in the miRNA biogenesis pathway. This will provide potential miRNA-targeted strategies for the treatment of breast cancer therapy and requires further clinical application.

miR-197: A novel biomarker for cancers.

microRNAs (miRNAs) are small noncoding RNAs that could regulate post-transcription level through binding to 3' untranslated region (3'UTR) of target messenger RNAs (mRNAs), which were reported to be related with the incidence and development of diverse neoplasms. Among them, miR-197 was confirmed to play a vital role of oncogene or anti-oncogene in different cancers via targeting key tumorigenic or tumor-suppressive genes. Additionally, miR-197 had extensively been studied in carcinogenesis progression of cancers through various mechanisms, including apoptosis, proliferation, angiogenesis, metastasis, drug resistance and tumor suppressor, and also played a role in prognosis of cancers. In this review, we summarized the roles of miR-197 in cancers and considered it as a potentially novel biomarker for different cancers, offering an alternatively secure and effective tool in molecular targeting cancer treatment.

miR-222 confers the resistance of breast cancer cells to Adriamycin through suppression of p27(kip1) expression.

Adriamycin (Adr) is a potent chemotherapeutic agent for chemotherapy of breast cancer patients. Despite impressive initial clinical responses, some developed drug resistance to Adr-based therapy and the mechanisms underlying breast cancer cells resistance to Adr are not well known. In our previous study, in vitro, we verified that miR-222 was upregulated in Adr-resistant breast cancer cells (MCF-7/Adr) compared with the sensitive parental cells (MCF-7/S). Here, miR-222 inhibitors or mimics were transfected into MCF-7 cell lines. RT-qPCR and western blot were used to detect the expression of p27(kip1). Immunofluorescence showed that miR-222 altered the subcellular location of p27(kip1) in nucleus. MTT was employed to verify the sensitivity of breast cancer cell lines to Adr. Flow cytometry showed the apoptosis and cell cycles of the cells after adding Adr. The results showed that downregulation of miR-222 in MCF-7/Adr increased sensitivity to Adr and Adr-induced apoptosis, and arrested the cells in G1 phase, accompanied by more expressions of p27(kip1), especially in nucleus. Furthermore, overexpressed miR-222 in MCF-7/S had the inverse results. Taken together, the results found that miR-222 induced Adr-resistance at least in part via suppressing p27(kip1) expression and altering its subcellular localization, and miR-222 inhibitors could reverse Adr-resistance of breast cancer cells. These results disclosed that the future holds much promise for the targeted therapeutic in the treatment of Adr-resistant breast cancer.

Perioperative propofol-paravertebral anesthesia decreases the metastasis and progression of breast cancer.

Propofol-paravertebral anesthesia (PPA) is a unique combination of paravertebral nerve blocks (PVBs) and propofol that regulates the cellular microenvironment during surgical period. Growing evidence points to its ability to attenuate perioperative immunosuppression of cancers. Abundant studies show that cancer patients who undergo perioperative PPA exhibit less recurrence as well as metastasis. Breast cancer remains a leading cause of cancer-induced death in women. Over the last decades, increasing concerns have been put on the promotional role of PPA in the prognosis of breast cancer patients. Among them, PPA participates in several bioprocesses in the development of breast cancer, including inhibiting hypoxia-inducible factor (HIF) activity, elevating serum concentration of nitric oxide index (NOx), depression of the neuroepithelial cell transforming gene 1 (NET1) signal pathway, blocking the nuclear factor kappa B (NF-κB) pathway following an decreased expression of matrix metalloproteinase (MMP), increasing NK cytotoxicity, and affecting transforming growth factor (TGF)-ß-targeted ras and HER2/neu gene pathways. In this review, we discuss the effect of PPA on breast cancer metastasis and progression. This will provide an alteration pattern of surgical anesthesia technique in breast cancer patients with poor prognosis.

Exosomes in development, metastasis and drug resistance of breast cancer.

Transport through the cell membrane can be divided into active, passive and vesicular types (exosomes). Exosomes are nano-sized vesicles released by a variety of cells. Emerging evidence shows that exosomes play a critical role in cancers. Exosomes mediate communication between stroma and cancer cells through the transfer of nucleic acid and proteins. It is demonstrated that the contents and the quantity of exosomes will change after occurrence of cancers. Over the last decade, growing attention has been paid to the role of exosomes in the development of breast cancer, the most life-threatening cancer in women. Breast cancer could induce salivary glands to secret specific exosomes, which could be used as biomarkers in the diagnosis of early breast cancer. Exosome-delivered nucleic acid and proteins partly facilitate the tumorigenesis, metastasis and resistance of breast cancer. Exosomes could also transmit anti-cancer drugs outside breast cancer cells, therefore leading to drug resistance. However, exosomes are effective tools for transportation of anti-cancer drugs with lower immunogenicity and toxicity. This is a promising way to establish a drug delivery system.

Simultaneous detection of hepatitis B virus genotypes and mutations associated with resistance to lamivudine, adefovir, and telbivudine by the polymerase chain reaction-ligase detection reaction

OBJECTIVES: Detection of mutations associated to nucleos(t)ide analogs and hepatitis B virus (HBV) genotyping are essential for monitoring treatment of HBV infection. We developed a multiplex polymerase chain reaction-ligase detection reaction (PCR-LDR) assay for the rapid detection of HBV genotypes and mutations associated with lamivudine, adefovir, and telbivudine resistance in HBV-infected patients. METHODS: HBV templates were amplified by PCR, followed by LDR and electrophoresis on a sequencer. The assay was evaluated using plasmids that contained wild-type or mutant HBV sequences and 216 clinical samples. RESULTS: The PCR-LDR assay and sequencing gave comparable results for 158 of the 216 samples (73.1 percent) with respect to mutation detection and genotyping. Complete agreement between the two methods was observed for all the samples (100 percent) at codon 180 and codon 204. Concordant results were observed for 99.4 percent of the 158 samples at codon 181 and 98.7 percent at codon 236. The genotyping results were completely concordant between the PCR-LDR assay and sequencing. The PCR-LDR assay could detect a proportion of 1 percent mutant plasmid in a background of wild-type plasmid. CONCLUSION: The PCR-LDR assay is sensitive and specific for detection of HBV genotypes and drug resistance mutations, and could be helpful for decision making in the treatment of HBV infection.

Simultaneous detection of hepatitis B virus genotypes and mutations associated with resistance to lamivudine, adefovir, and telbivudine by the polymerase chain reaction-ligase detection reaction.

OBJECTIVES: Detection of mutations associated to nucleos(t)ide analogs and hepatitis B virus (HBV) genotyping are essential for monitoring treatment of HBV infection. We developed a multiplex polymerase chain reaction-ligase detection reaction (PCR-LDR) assay for the rapid detection of HBV genotypes and mutations associated with lamivudine, adefovir, and telbivudine resistance in HBV-infected patients. METHODS: HBV templates were amplified by PCR, followed by LDR and electrophoresis on a sequencer. The assay was evaluated using plasmids that contained wild-type or mutant HBV sequences and 216 clinical samples. RESULTS: The PCR-LDR assay and sequencing gave comparable results for 158 of the 216 samples (73.1%) with respect to mutation detection and genotyping. Complete agreement between the two methods was observed for all the samples (100%) at codon 180 and codon 204. Concordant results were observed for 99.4% of the 158 samples at codon 181 and 98.7% at codon 236. The genotyping results were completely concordant between the PCR-LDR assay and sequencing. The PCR-LDR assay could detect a proportion of 1% mutant plasmid in a background of wild-type plasmid. CONCLUSION: The PCR-LDR assay is sensitive and specific for detection of HBV genotypes and drug resistance mutations, and could be helpful for decision making in the treatment of HBV infection.