A smartphone-based immunochromatographic strip platform for on-site quantitative detection of antigenic targets.

To report the testing signal of an immunochromatographic assay for on-site quantitative detection, a portable and user-friendly smartphone-based biosensing platform is developed in this study. This innovative system is composed of an ambient light sensor inherent smartphone reader and a 3D-printed handhold device, a quantitative tool capable of directly interpreting carbon nanoparticle (CNP)-conjugated immunochromatographic strips. To showcase the platform capability, the smartphone-based immunochromatography system (SPICS) reader and device were successfully used in CNP strips for rapid detection of the early pregnancy marker human chorionic gonadotropin in female urine (HCG; limit of detection [LOD]: 0.30 mIU mL-1), prostate-specific antigen in patient blood (PSA; LOD: 0.28 ng mL-1) and ampicillin residue in animal milk (AMP; LOD: 0.23 ng mL-1). The results were fully correlated with conventional commercial instruments (R2 = 0.99). The SPICS platform exhibits significant advantages, including portability, cost-effectiveness, easy operation, and rapid and quantitative detection, making it a valuable on-site diagnosis tool for use in home and community healthcare facilities.

[Characterization of sequences, expression profiling, and natural allelic variation analysis of the MYC gene family in sorghum (Sorghum bicolor)].

Sorghum aphid (Melanaphis sacchari) and head smut fungi (Sporisorium reilianum) infesting sorghum cause delayed growth and development, and reduce yield and quality. This study use bioinformatics and molecular biological approaches to profile the gene expression pattern during sorghum development and under pest infestation, and analyzed the natural allelic DNA variation of sorghum MYC gene family. The findings provide insights for potential application in breeding the stress resistant and high productivity sorghum varieties. The results indicated that there are 28 MYC genes identified in sorghum genome, distributed on 10 chromosomes. The bHLH_MYC_N and HLH domains are the conserved domains of the MYC gene in sorghum. Gene expression analysis showed that SbbHLH35.7g exhibited high expression levels in leaves, SbAbaIn showed strong expression in early grains, and SbMYC2.1g showed high expression levels in mature pollen. In anti-aphid strains at the 5-leaf stage, SbAbaIn, SbLHW.4g and SbLHW.2g were significantly induced in leaves, while SbbHLH35.7g displayed the highest expression level in panicle tissue, which was significantly induced by the infection of head smut. Promoter cis-element analysis identified methyl jasmonate (MJ), abscisic acid (ABA), salicylic acid (SA) and MYB-binding sites related to drought-stress inducibility. Furthermore, genomic resequencing data analysis revealed natural allelic DNA variations such as single nucleotide polymorphism (SNP) and insertion-deletion (INDEL) for the key SbMYCs. Protein interaction network analysis using STRING indicated that SbAbaIn interacts with TIFYdomain protein, and SbbHLH35.7g interacts with MDR and imporin. SbMYCs exhibited temporal and spatial expression patterns and played vital roles during the sorghum development. Infestation by sugarcane aphids and head smut fungi induced the expression of SbAbaIn and SbbHLH35.7g, respectively. SbAbaIn modulated the jasmonic acid (JA) pathway to regulate the expression of defensive genes, conferring resistance to insects. On the other hand, SbbHLH35.7g participated in detoxification reactions to defend against pathogens.

miR-29a-KLF4 signaling inhibits breast tumor initiation by regulating cancer stem cells.

Cancer stem cells (CSCs) are known for their potent ability to drive tumor initiation and recurrence, yet the molecular mechanisms regulating CSCs are still unclear. Our study found a positive correlation between increased levels of miR-29a and better survival rates in early-stage breast cancer patients, but a negative correlation in late-stage patients, suggesting a dual function of miR-29a in regulating breast cancer. Furthermore, miR-29a showed significant downregulation in the ALDH+ breast cancer stem cell population compared to non-stem cancer cells. Overexpression of miR-29a in human breast cancer cells reduced the proportion of CSCs, suppressed their ability to form mammospheres, and inhibited the expression of stemness genes SOX2, KLF4, and hTERT in vitro. Conversely, knockdown of miR-29a in breast cancer cells showed opposite effects. Tumor xenograft experiments revealed that miR-29a overexpression significantly inhibited tumorigenesis initiated by MDA-MB-231 cell transplantation in nude mice. We further demonstrated that Krüppel-like factor 4 (KLF4), a key gene that regulates cell stemness, was a direct target of miR-29a in breast cancer cells. miR-29a suppressed the expression of KLF4 at both mRNA and protein levels. Reintroduction of KLF4 into breast cancer cells rescued the miR-29a-induced CSC suppression phenotype. In summary, our study is the first to demonstrate that miR-29a-KLF4 signaling inhibits breast tumor initiation by regulating CSCs, which provides novel therapeutic targets for preventing breast tumor initiation.

Nanoparticles carrying paclitaxel and anti-miR-221 for breast cancer therapy triggered by ultrasound.

Nanomaterials have been well demonstrated to have the potential to be used for tumor cell-targeted drug delivery. Targeted inhibition of miR-221 was proved to promote the sensitivity of triple genitive breast cancer (TNBC) cells to chemo-drugs. In order to improve the chemotherapeutic effect in TNBC, herein, we developed a novel kind of nanoparticles shelled with PLGA and loaded with perfluoropentane (PFP), paclitaxel (PTX), and anti-miR-221 inhibitor, which was named PANP. Ultrasound-triggered vaporization of PFP in PANPs was utilized for real-time imaging track of the nanoparticles in vivo. In addition, macrophages were applied for the internalization of PANPs to form RAW-PANP with strong chemotaxis to accumulate around cancer cells. Nanoparticles with different contents did not cause M2 polarization compared with the control group but caused polarization toward M1. We compared the inherent tumor-homing behavior of macrophages containing different contents with that of normal macrophages and no significant abnormalities were observed. After injection into the tumor-burden mice, RAW-PANPs showed enrichment within tumor tissues. Upon the ultrasound cavitation-triggered burst, PTX was released in the tumor. Meanwhile, the release of anti-miR-221 improved the sensitivity of tumor cells to PTX. As a result, RAW-PANPs showed high efficiency in suppressing TNBC cell proliferation in vitro and inhibiting tumor growth and progression in vivo. The treatments did not induce liver, heart, or kidney injury. In conclusion, the current study not only developed a macrophage-carried, ultrasound-triggered, cancer cell-targeted chemotherapeutic system, but also demonstrated a miRNA-based technique to promote drug sensitivity of cancer cells, which holds strong potential to treat patients with TNBC, especially for those suffering drug-resistance. The innovation of this study is to use macrophages to deliver nanoparticles to the tumors and then use ultrasound locally to burst the nanoparticles to release the miRNA and PTX.

IL11 signaling mediates piR-2158 suppression of cell stemness and angiogenesis in breast cancer.

Emerging evidence has indicated the aberrant expression of PIWI-interacting RNAs (piRNAs) in human cancer cells to regulate tumor development and progression by governing cancer cell stemness. Herein, we identified downregulation of piR-2158 in human breast cancer tumors, especially in ALDH+ breast cancer stem cells (BCSCs) from patients and cell lines, which was further validated in two types of genetically engineered mouse models of breast cancer (MMTV-Wnt and MMTV-PyMT). Enforced overexpression of piR-2158 in basal-like or luminal subtypes of breast cancer cells suppressed cell proliferation, migration, epithelial-mesenchymal transition (EMT) and stemness in vitro. Administration of a dual mammary tumor-targeting piRNA delivery system in mice reduced tumor growth in vivo. RNA-seq, ChIP-seq and luciferase reporter assays demonstrated piR-2158 as a transcriptional repressor of IL11 by competing with AP-1 transcription factor subunit FOSL1 to bind the promoter of IL11. STAT3 signaling mediated piR-2158-IL11 regulation of cancer cell stemness and tumor growth. Moreover, by co-culturing of MDA-MB-231 and HUVECs in vitro and CD31 staining of tumor endothelial cells in vivo, we demonstrated inhibition of angiogenesis by piR-2158-IL11 in breast cancer. In conclusion, the current study not only reveals a novel mechanism through which piR-2158 inhibits mammary gland tumorigenesis via regulating cancer stem cells and tumor angiogenesis, but also provides a novel therapeutic strategy in treatment of breast cancer.

Regulation of ERα-dependent breast cancer metastasis by a miR-29a signaling.

Malignant breast cancer (BC) remains incurable mainly due to the cancer cell metastasis, which is mostly related to the status of Estrogen receptor alpha (ERα). However, our understanding of the mechanisms through which ERα regulates cancer cell metastasis remains limited. Here we identified a miR-29a-PTEN-AKT axis as a downstream signaling pathway of ERα governing breast cancer progression and metastasis. Two estrogen response element (ERE) half sites were identified in the promoter and enhancer regions of miR-29a, which mediated transcriptional regulation of miR-29a by ERα. Low level of miR-29a showed association with reduced metastasis and better survival in ERα+ luminal subtype of BC. In contrast, high level of miR-29a was detected in ERα- triple negative breast cancer (TNBC) in association with distant metastasis and poor survival. miR-29a overexpression in BC tumors increased the number of circulating tumor cells and promoted lung metastasis in mice. Targeted knockdown of miR-29a in TNBC cells in vitro or administration of a nanotechnology-based anti-miR-29a delivery in TNBC tumor-bearing mice in vivo suppressed cellular invasion, EMT and lung metastasis. PTEN was identified as a direct target of miR-29a, inducing EMT and metastasis via AKT signaling. A small molecular inhibitor of AKT attenuated miR-29a-induced EMT. These findings demonstrate a novel mechanism responsible for ERα-regulated breast cancer metastasis, and reveal the combination of ERα status and miR-29a levels as a new risk indicator in BC.

Dual Function of CCAT2 in Regulating Luminal Subtype of Breast Cancer Depending on the Subcellular Distribution.

Breast cancer is the most common cancer in women around the world. Emerging evidence has indicated the important roles that non-coding RNAs play in regulating tumor development and progression in breast cancer. Herein, we found a dual function of long non-coding RNA (LncRNA) CCAT2 in the luminal subtype of breast cancer, depending on its subcellular distribution. CCAT2 showed an overall downregulation in the tumor tissues from luminal breast cancer patients. Transient overexpression of CCAT2 in the luminal subtype of breast cancer cell MCF-7 or T47D significantly suppressed cell proliferation in vitro and inhibited tumor growth in vivo. Gene expression analysis of cancer stem cell markers including OCT4, NANOG, h-TERT, SOX2 and KLF4; flow cytometry analysis of breast cancer stem cell population, and mammosphere formation assay demonstrated inhibition of cancer cell stemness with transient transfection of CCAT2 in which exogenous CCAT2 mainly distributed in the cytoplasm and regulated miR-221-p27 signaling via RNA sequence interaction. However, overexpression of CCAT2 in MCF-7 cells through pMX retroviral nuclear expression vector accumulated CCAT2 in the nucleus, leading to upregulation of OCT4-PG1, a pseudogene of stem gene OCT4, thereby promoting the cancer cell stemness. In conclusion, the current study, for the first time, revealed a dual function of lncRNA CCAT2 as a tumor suppressor or oncogene depending upon its subcellular distribution. It also demonstrated the regulatory mechanism of cytoplasmic CCAT2 in suppressing tumorigenesis in the luminal subtype of breast cancer.

A circulating miR-19b-based model in diagnosis of human breast cancer.

Objective: Breast cancer (BC) is becoming the leading cause of cancer-related death in women all over the word. Identification of diagnostic biomarkers for early detection of BC is one of the most effective ways to reduce the mortality. Methods: Plasma samples from BC patients (n = 120) and normal controls (n = 50) were collected to determine the differentially expressed circulating miRNAs in BC patients. Binary logistic regression was applied to develop miRNA diagnostic models. Receiver operating characteristic (ROC) curves were applied to calculate the area under the curve (AUC). MMTV-PYMT mammary tumor mice were used to validate the expression change of those circulating miRNAs. Plasma samples from patients with other tumor types were collected to determine the specificity of the model in diagnosis of BC. Results: In the screening phase, 5 circulating miRNAs (miR-16, miR-17, miR-19b, miR-27a, and miR-106a) were identified as the most significantly upregulated miRNAs in plasma of BC patients. In consistence, the 5 miRNAs showed upregulation in the circulation of additional 80 BC patients in a tumor stage-dependent manner. Application of a tumor-burden mice model further confirmed upregulation of the 5 miRNAs in circulation. Based on these data, five models with diagnostic potential of BC were developed. Among the 5 miRNAs, miR-19b ranked at the top position with the highest specificity and the biggest contribution. In combination with miR-16 and miR-106a, a miR-19b-based 3-circulating miRNA model was selected as the best for further validation. Taken the samples together, the model showed 92% of sensitivity and 90% of specificity in diagnosis of BC. In addition, three other tumor types including prostate cancer, thyroid cancer and colorectal cancer further verified the specificity of the BC diagnostic model. Conclusion: The current study developed a miR-19b-based 3-miRNA model holding potential for diagnosis of BC using blood samples.

Diagnostic potential of a circulating miRNA model associated with therapeutic effect in heart failure.

Heart failure (HF), as the leading cause of death, is continuing to increase along with the aging of the general population all over the world. Identification of diagnostic biomarkers for early detection of HF is considered as the most effective way to reduce the risk and mortality. Herein, we collected plasma samples from HF patients (n = 40) before and after medical therapy to determine the change of circulating miRNAs through a quantitative real-time PCR (QRT-PCR)-based miRNA screening analysis. miR-30a-5p and miR-654-5p were identified as the most significantly changed miRNAs in the plasma of patients upon treatment. In consistence, miR-30a-5p showed upregulation and miR-654-5p showed downregulation in the circulation of 30 HF patients, compared to 15 normal controls in the training phase, from which a two-circulating miRNA model was developed for HF diagnosis. Next, we performed the model validation using an independent cohort including 50 HF patients and 30 controls. As high as 98.75% of sensitivity and 95.00% of specificity were achieved. A comparison between the miRNA model and NT-pro BNP in diagnostic accuracy of HF indicated an upward trend of the miRNA model. Moreover, change of the two miRNAs was further verified in association with the therapeutic effect of HF patients, in which miR-30a-5p showed decrease while miR-654-5p showed increase in the plasma of patients after LVAD implantation. In conclusion, the current study not only identified circulating miR-654-5p for the first time as a novel biomarker of HF, but also developed a novel 2-circulating miRNA model with promising potentials for diagnosis and prognosis of HF patients, and in association with therapeutic effects as well.

Evolution of gene expression signature in mammary gland stem cells from neonatal to old mice.

During the lifetime of females, mammary epithelial cells undergo cyclical expansion and proliferation depending on the cyclical activation of mammary gland stem/progenitor cells (MaSCs) in response to the change of hormone level. The structural shrink of mammary duct tree and the functional loss of mammary gland occur along with inactivation of MaSCs in old females, even leading to breast cancer occasionally. However, the gene expression signature in MaSCs across the lifespan remains unclear. Herein, we tested the tissue regeneration ability of CD24+CD49fhigh MaSCs over six time points from neonatal (4-day-old) to aged mice (360-day-old). Further RNA-seq analyses identified four clusters of gene signatures based on the gene expression patterns. A subset of stemness-related genes was identified, showing the highest level at day 4 of the neonatal age, and the lowest level at the old age. We also identified an aging-related gene signature showing significant change in the old mice, in which an association between aging process and stemness loss was indicated. The aging-related gene signature showed regulation of cancer signaling pathways, as well as aging-related diseases including Huntington disease, Parkinson disease, and Alzheimer disease. Moreover, 425, 1056, 418, and 1107 gene variants were identified at D20, D40, D90, and D180, respectively, which were mostly reported to associated with tumorigenesis and metastasis in cancer. In summary, the current study is the first to demonstrate the gene expression shift in MaSCs from neonatal to aging, which leads to stemness loss, aging, aging-related diseases, and even breast cancer in old mice.

Immune and nonimmune mechanisms mediate the mental stress-induced tumor growth in a xenograft model of breast cancer.

Excess mental stress may harm health, and even accelerate cancer initiation and progression. One fourth of breast cancer patients suffer mental stress including anxiety, sadness, or depression, which negatively affect prognosis and survival. However, the regulatory mechanism is yet to be determined. Herein, we applied unpredictable stress stimuli to the breast tumor-bearing mice to establish a xenograft model of breast cancer suffering mental stress, followed by behavioral tests, tumor growth tracking, immune analysis, miRNA screening, and tumor cell proliferation analysis as well. As a result, increased stress hormone levels in serum, decreased percentage of T and NK cells in both blood and tumor samples and accelerated tumor growth in vivo were observed in the mice exposed to mental stress. Promoted cell proliferation was observed in both primary tumor cells derived from the stressed mice and 4T1 breast cancer cells treated with stress hormone corticosterone. In addition, a subset of miRNAs including miR-326, 346, 493, 595, 615, and 665 were identified through a miRNA screening with downregulation in tumors of the stressed mice. CCND1 was identified as a common target gene of miR-346 and miR-493, the top two most significantly downregulated miRNAs by stress exposure. The stress-miRNA-CCND1 signaling regulation of the tumor cell proliferation was further validated in 4T1 cells treated with corticosterone in vitro. GO terms and KEGG pathways analyses on the target genes of miR-346 and miR-493 revealed their involvement in the regulation of human cancer and neuron system, indicating the importance of non-coding genome in mediating the mental stress-induced cancer regulation. In conclusion, this study not only explored immune and nonimmune mechanisms through which mental stress exposure contributes to tumor growth in breast cancer, but also suggested a new therapeutic strategy for cancer patients suffering mental stress.

Piwi-Interacting RNAs: A New Class of Regulator in Human Breast Cancer.

P-element-induced wimpy testis (Piwi)-interacting RNAs (piRNAs) are a class of germline-enriched small non-coding RNA that associate with Piwi family proteins and mostly induce transposon silencing and epigenetic regulation. Emerging evidence indicated the aberrant expression of Piwil proteins and associated piRNAs in multiple types of human cancer including breast cancer. Although the majority of piRNAs in breast cancer remains unclear of the function mainly due to the variety of regulatory mechanisms, the potential of piRNAs serving as biomarkers for cancer diagnosis and prognosis or therapeutic targets for cancer treatment has been demonstrated by in vitro and in vivo studies. Herein we summarized the research progress of oncogenic or tumor suppressing piRNAs and their regulatory mechanisms in regulating human breast cancer, including piR-021285, piR-823, piR-932, piR-36712, piR-016658, piR-016975 and piR-4987. The challenges and perspectives of piRNAs in the field of human cancer were discussed.

Reck-Notch1 Signaling Mediates miR-221/222 Regulation of Lung Cancer Stem Cells in NSCLC.

Cancer stem cells (CSCs) contribute to the cancer initiation, metastasis and drug resistance in non-small cell lung cancer (NSCLC). Herein, we identified a miR-221/222 cluster as a novel regulator of CSCs in NSCLC. Targeted overexpression or knockdown of miR-221/222 in NSCLC cells revealed the essential roles of miR-221/222 in regulation of lung cancer cell proliferation, mammosphere formation, subpopulation of CD133+ CSCs and the expression of stemness genes including OCT4, NANOG and h-TERT. The in vivo animal study showed that overexpression of miR-221/222 significantly enhanced the capacity of lung cancer cells to develop tumor and grow faster, indicating the importance of miR-221/222 in tumorigenesis and tumor growth. Mechanistically, Reck was found to be a key direct target gene of miR-221/222 in NSCLC. Overexpression of miR-221/222 significantly suppressed Reck expression, activated Notch1 signaling and increased the level of NICD. As an activated form of Notch1, NICD leads to enhanced stemness in NSCLC cells. In addition, knockdown of Reck by siRNA not only mimicked miR-221/222 effects, but also demonstrated involvement of Reck in the miR-221/222-induced activation of Notch1 signaling, verifying the essential roles of the miR-221/222-Reck-Notch1 axis in regulating stemness of NSCLC cells. These findings uncover a novel mechanism by which lung CSCs are significantly manipulated by miR-221/222, and provide a potential therapeutic target for the treatment of NSCLC.

piRNA-823 Is Involved in Cancer Stem Cell Regulation Through Altering DNA Methylation in Association With Luminal Breast Cancer.

Cancer stem cells (CSCs) are believed to be the main source of cancer relapse and metastasis. PIWI-interacting small non-coding RNAs (piRNAs) have been recently recognized to be relevant to cancer biology. Whether and how piRNAs regulate human CSCs remain unknown. Herein, upregulation of piR-823 was identified in tested luminal breast cancer cells, especially in the luminal subtype of breast CSCs. Enforced expression or targeted knockdown of piR-823 demonstrated its oncogenic function in regulating cell proliferation and colony formation in MCF-7 and T-47D breast cancer cells. In addition, piR-823 induced ALDH (+) breast CSC subpopulation promoted the expression of stem cell markers including OCT4, SOX2, KLF4, NANOG, and hTERT, and increased mammosphere formation. Tail vein injection of magnetic nanoparticles carrying anti-piR-823 into the mammary gland of tumor-burdened mice significantly inhibited tumor growth in vivo. DNA methyltransferases (DNMTs) including DNMT1, DNMT3A, and DNMT3B were demonstrated to be the downstream genes of piR-823, which regulate gene expression by maintaining DNA methylation. piR-823 increased the expression of DNMTs, promoted DNA methylation of gene adenomatous polyposis coli (APC), thereby activating Wnt signaling and inducing cancer cell stemness in the luminal subtype of breast cancer cells. The current study not only revealed a novel mechanism through which piRNAs contribute to tumorigenesis in breast cancer by regulating CSCs, but also provided a therapeutic strategy using non-coding genomes in the suppression of human breast cancer.

Role of core protein mutations in the development of occult HBV infection.

BACKGROUND & AIMS: Occult HBV infection (OBI) is associated with transfusion-transmitted HBV infection and hepatocellular carcinoma. Studies on OBI genesis have concentrated on mutations in the S region and the regulatory elements. Herein, we aimed to determine the role of mutations in the core region on OBIs. METHODS: An OBI strain (SZA) carrying 9 amino acid (aa) substitutions in the core protein/capsid (Cp) was selected by sequence alignment and Western blot analysis from 26 genotype B OBI samples to extensively explore the impact of Cp mutations on viral antigen production in vitro and in vivo. RESULTS: A large panel of 30 Cp replicons were generated by a replication-competent pHBV1.3 carrying SZA or wild-type (WT) Cp in a 1.3-fold over-length of HBV genome, in which the various Cp mutants were individually introduced by repairing site mutations of SZA-Cp or creating site mutations of WT-Cp by site-directed mutagenesis. The expression of HBcAg, HBeAg, and HBsAg and viral RNA was quantified from individual SZA and WT Cp mutant replicons in transfected Huh7 cells or infected mice, respectively. An analysis of the effect of Cp mutants on intracellular or extracellular viral protein production indicated that the W62R mutation in Cp had a critical impact on the reduction of HBcAg and HBeAg production during HBV replication, whereas P50H and/or S74G mutations played a limited role in influencing viral protein production invivo. CONCLUSIONS: W62R and its combination mutations in HBV Cp might massively affect HBcAg and HBeAg production during viral replication, which, in turn, might contribute to the occurrence of OBI. LAY SUMMARY: Occult hepatitis B virus infections (OBIs) have been found to be associated with amino acid mutations in the S region of the HBV, but the role of mutations in the core protein (Cp) remains unclear. In this study, an OBI strain (SZA) carrying 9 amino acid substitutions in Cp has been examined comprehensively in vitro and in vivo. The W62R mutation in Cp majorly reduces HBcAg and HBeAg production during HBV replication, potentially contributing to the occurrence of OBI.

miR-301a-PTEN-AKT Signaling Induces Cardiomyocyte Proliferation and Promotes Cardiac Repair Post-MI.

Adult hearts are hard to recover after cardiac injury due to the limited proliferative ability of cardiomyocytes. Emerging evidence indicates the induction of cell cycle reentry of cardiomyocytes by special treatment or stimulation, which offers adult heart regenerative potential. Herein, a microRNA (miRNA) screening in cardiomyocytes identified miR-301a enriched specially in the neonatal cardiomyocytes from rats and mice. Overexpression of miR-301a in primary neonatal cardiomyocytes and H9C2 cells induced G1/S transition of the cell cycle, promoted cellular proliferation, and protected cardiomyocytes against hypoxia-induced apoptosis. Adeno-associated virus (AAV)9-mediated cardiac delivery of miR-301a to the mice model with myocardial infarction (MI) dramatically promoted cardiac repair post-MI in vivo. Phosphatase and tensin homolog (PTEN)/phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway was confirmed to mediate miR-301a-induced cell proliferation in cardiomyocytes. Loss of function of PTEN mimicked the miR-301a-induced phenotype, while gain of function of PTEN attenuated the miR-301a-induced cell proliferation in cardiomyocytes. Application of RG7440, a small molecule inhibitor of AKT, blocked the function of miR-301a in cardiomyocytes. The current study revealed a miRNA signaling in inducing the cell cycle reentry of cardiomyocytes in the injured heart, and it demonstrated the miR-301a/PTEN/AKT signaling as a potential therapeutic target to reconstitute lost cardiomyocytes in mammals.

Cancer stem cell property and gene signature in bone-metastatic Breast Cancer cells.

The majority of the deaths from breast cancer is due to metastasis. Bone is the most common organ to which breast cancer cells metastasize. The mechanism regulating the bone-metastatic preference remains unclear; there is a lack of a gene signature to distinguish bone-metastatic breast cancer cells. Herein, florescence-labeled MDA-MB-231 cells were transplanted into the fat pads of of the mammary gland in nude mice to generate breast tumors. Tumor cells invaded into the circulation were tracked by in vivo flow cytometry system. Metastatic tumor cells in the bone were isolated using fluorescent-activated cell sorting technique, followed by assays of cell colony formation, migration and invasion, mammosphere formation in vitro, mammary gland tumorigenesis in vivo, and Next-Generation Sequencing analysis as well. Through tumor regeneration and cell sorting, two bone-metastatic cell sublines were derived from MDA-MB-231 cells; which showed higher abilities to proliferate, migrate, invade and epithelial-to-mesenchymal transit in vitro, and stronger ability to regenerate tumors and metastasize to the bone in vivo. Both cell sublines exhibited cancer stem cell-like characteristics including higher expression levels of stem cell markers and stronger ability for mommaspheres formation. Furthermore, a Normal Distribution-like pattern of the bone-metastatic cells invading into circulation was firstly identified. Deep-sequencing analysis indicated upregulation of multiple signaling pathways in regulating EMT, cell membrane budding and morphologic change, lipid metabolism, and protein translation, which are required to provide adequate metabolic enzymes, structural proteins, and energy for the cells undergoing metastasis. In conclusion, we established two bone-metastatic breast cancer cell sublines, carrying higher degree of stemness and malignancy. The gene signature distinguishing the bone-metastatic breast cancer cells holds therapeutic potentials in prevention of breast cancer metastasis to the bone.

Starvation stress attenuates the miRNA-target interaction in suppressing breast cancer cell proliferation.

BACKGROUND: Emerging evidence has demonstrated the limited access to metabolic substrates as an effective approach to block cancer cell growth. The mechanisms remain unclear. Our previous work has revealed that miR-221/222 plays important role in regulating breast cancer development and progression through interaction with target gene p27. RESULTS: Herein, we determined the miRNA-mRNA interaction in breast cancer cells under induced stress status of starvation. Starvation stimulation attenuated the miR-221/222-p27 interaction in MDA-MB-231 cells, thereby increased p27 expression and suppressed cell proliferation. Through overexpression or knockdown of miR-221/222, we found that starvation-induced stress attenuated the negative regulation of p27 expression by miR-221/222. Similar patterns for miRNA-target mRNA interaction were observed between miR-17-5p and CyclinD1, and between mR-155 and Socs1. Expression of Ago2, one of the key components of RNA-induced silencing complex (RISC), was decreased under starvation-induced stress status, which took responsibility for the impaired miRNA-target interaction since addition of exogenous Ago2 into MDA-MB-231 cells restored the miR-221/222-p27 interaction in starvation condition. CONCLUSIONS: We demonstrated the attenuated interaction between miR-221/222 and p27 by starvation-induced stress in MDA-MB-231 breast cancer cells. The findings add a new page to the general knowledge of negative regulation of gene expression by miRNAs, also demonstrate a novel mechanism through which limited access to nutrients suppresses cancer cell proliferation. These insights provide a basis for development of novel therapeutic options for breast cancer.

Cyclin D1 promotes secretion of pro-oncogenic immuno-miRNAs and piRNAs.

The molecular mechanisms governing the secretion of the non-coding genome are poorly understood. We show herein that cyclin D1, the regulatory subunit of the cyclin-dependent kinase that drives cell-cycle progression, governs the secretion and relative proportion of secreted non-coding RNA subtypes (miRNA, rRNA, tRNA, CDBox, scRNA, HAcaBox. scaRNA, piRNA) in human breast cancer. Cyclin D1 induced the secretion of miRNA governing the tumor immune response and oncogenic miRNAs. miR-21 and miR-93, which bind Toll-Like Receptor 8 to trigger a pro-metastatic inflammatory response, represented >85% of the cyclin D1-induced secreted miRNA transcripts. Furthermore, cyclin D1 regulated secretion of the P-element Induced WImpy testis (PIWI)-interacting RNAs (piRNAs) including piR-016658 and piR-016975 that governed stem cell expansion, and increased the abundance of the PIWI member of the Argonaute family, piwil2 in ERα positive breast cancer. The cyclin D1-mediated secretion of pro-tumorigenic immuno-miRs and piRNAs may contribute to tumor initiation and progression.

Long non-coding RNA CCAT2 promotes oncogenesis in triple-negative breast cancer by regulating stemness of cancer cells.

Triple-negative breast cancers (TNBC) are more aggressive due to lacking receptors for hormone therapy and maintaining stemness features in cancer cells. Herein we found long non-coding RNA CCAT2 overexpressed specially in TNBC, and in breast cancer stem cells (BCSC) as well. Enforced overexpression and targeted knockdown demonstrated the oncogenic function of CCAT2 both in vitro and in vivo. CCAT2 promoted the expression of stemness markers including OCT4, Nanog and KLF4, increased mammosphere formation and induced ALDH+ cancer stem cell population in TNBC. A chromosomally adjacent gene OCT4-PG1, as a pseudogene of OCT4, was upregulated by CCAT2, and positively regulated the stemness features of TNBC cells. miR-205 was identified as a target gene of CCAT2 in TNBC. Point-mutation in CCAT2 impaired the sponge inhibition of miR-205. Overexpression of miR-205 rescued the oncogenic phenotypes induced by CCAT2. In addition, Notch2, as a target gene of miR-205, was downregulated by miR-205 and upregulated by CCAT2 in TNBC. Collectively, the current study revealed a novel function of CCAT2 in promoting tumor initiation and progression in TNBC through upregulating OCT4-PG1 expression and activating Notch signaling. These findings not only demonstrated a lncRNA-based therapeutic strategy in treatment of TNBC, but also added a node to the regulatory network of CCAT2 that controls aggressiveness of breast cancer stem cells.