Metabolic Heterogeneity and Potential Immunotherapeutic Responses Revealed by Single-Cell Transcriptomics of Breast Cancer.

BACKGROUND: Breast cancer (BC) exhibits remarkable heterogeneity. However, the transcriptomic heterogeneity of BC at the single-cell level has not been fully elucidated. METHODS: We acquired BC samples from 14 patients. Single-cell RNA sequencing (scRNA-seq), bioinformatic analyses, along with immunohistochemistry (IHC) and immunofluorescence (IF) assays were carried out. RESULTS: According to the scRNA-seq results, 10 different cell types were identified. We found that Cancer-Associated Fibroblasts (CAFs) exhibited distinct biological functions and may promote resistance to therapy. Metabolic analysis of tumor cells revealed heterogeneity in glycolysis, gluconeogenesis, and fatty acid synthetase reprogramming, which led to chemotherapy resistance. Furthermore, patients with multiple metastases and progression were predicted to benefit from immunotherapy based on a heterogeneity analysis of T cells and tumor cells. CONCLUSIONS: Our findings provide a comprehensive understanding of the heterogeneity of BC, provide comprehensive insight into the correlation between cancer metabolism and chemotherapy resistance, and enable the prediction of immunotherapy responses based on T-cell heterogeneity.

Single-cell sequencing reveals the heterogeneity of B cells and tertiary lymphoid structures in muscle-invasive bladder cancer.

BACKGROUND: Muscle-invasive bladder cancer (MIBC) is a highly aggressive disease with a poor prognosis. B cells are crucial factors in tumor suppression, and tertiary lymphoid structures (TLSs) facilitate immune cell recruitment to the tumor microenvironment (TME). However, the function and mechanisms of tumor-infiltrating B cells and TLSs in MIBC need to be explored further. METHODS: We performed single-cell RNA sequencing analysis of 11,612 B cells and 55,392 T cells from 12 bladder cancer patients and found naïve B cells, proliferating B cells, plasma cells, interferon-stimulated B cells and germinal center-associated B cells, and described the phenotype, gene enrichment, cell-cell communication, biological processes. We utilized immunohistochemistry (IHC) and immunofluorescence (IF) to describe TLSs morphology in MIBC. RESULTS: The interferon-stimulated B-cell subtype (B-ISG15) and germinal center-associated B-cell subtypes (B-LMO2, B-STMN1) were significantly enriched in MIBC. TLSs in MIBC exhibited a distinct follicular structure characterized by a central region of B cells resembling a germinal center surrounded by T cells. CellChat analysis showed that CXCL13 + T cells play a pivotal role in recruiting CXCR5 + B cells. Cell migration experiments demonstrated the chemoattraction of CXCL13 toward CXCR5 + B cells. Importantly, the infiltration of the interferon-stimulated B-cell subtype and the presence of TLSs correlated with a more favorable prognosis in MIBC. CONCLUSIONS: The study revealed the heterogeneity of B-cell subtypes in MIBC and suggests a pivotal role of TLSs in MIBC outcomes. Our study provides novel insights that contribute to the precision treatment of MIBC.

Urinary SPP1 has potential as a non-invasive diagnostic marker for focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a type of chronic glomerular nephropathy showing characteristic glomerular sclerosis, diagnosed by kidney biopsy. However, it is difficult and expensive to monitor disease progression with repeated renal biopsy in clinical practice, and thus here we explored the feasibility of urine biomarkers as non-invasive diagnostic tools. We downloaded scRNA-seq datasets of 20 urine cell samples and 3 kidney tissues and obtained two gene lists encoding extracellular proteins for bioinformatic analysis; in addition, we identified key EP-Genes by immunohistochemical staining and performed bulk RNA sequencing with 12 urine samples. We report that urine cells and kidney cells were correlated. A total of 64 EP-Genes were acquired by intersecting genes of distal tubular cluster with extracellular proteins. Function enrichment analysis showed that EP-Genes might be involved in the immune response and extracellular components. Six key EP-Genes were identified and correlated with renal function. IMC showed that key EP-Genes were located mainly in tubules. Cross verification and examination of a urine RNAseq dataset showed that SPP1 had diagnostic potential for FSGS. The presence of urine SPP1 was primarily associated with macrophage infiltration in kidney, and the pathogenesis of FSGS may be related to innate immunity. Urinary cells seemed to be strongly similar to kidney cells. In summary, SPP1 levels reflect renal function and may have potential as a biomarker for non-invasive diagnosis of FSGS.

Single-cell transcriptome sequencing of B-cell heterogeneity and tertiary lymphoid structure predicts breast cancer prognosis and neoadjuvant therapy efficacy.

BACKGROUND: Breast cancer (BC) is a highly heterogeneous disease, and although immunotherapy has recently increased patient survival in a number of solid and hematologic malignancies, most BC subtypes respond poorly to immune checkpoint blockade therapy (ICB). B cells, particularly those that congregate in tertiary lymphoid structures (TLS), play a significant role in antitumour immunity. However, B-cell heterogeneity at single-cell resolution and its clinical significance with TLS in BC need to be explored further. METHODS: Primary tumour lesions and surrounding normal tissues were taken from 14 BC patients, totaling 124,587 cells, for single-cell transcriptome sequencing and bioinformatics analysis. RESULTS: Based on the usual markers, the single-cell transcriptome profiles were classified into various clusters. A thorough single-cell study was conducted with a focus on tumour-infiltrating B cells (TIL-B) and tumour-associated neutrophils (TAN). TIL-B was divided into five clusters, and unusual cell types, such as follicular B cells, which are strongly related to immunotherapy efficacy, were identified. In BC, TAN and TIL-B infiltration are positively correlated, and at the same time, compared with TLS-high, TAN and TIL-B in TLS-low group are significantly positively correlated. CONCLUSIONS: In conclusion, our study highlights the heterogeneity of B cells in BC, explains how B cells and TLS contribute significantly to antitumour immunity at both the single-cell and clinical level, and offers a straightforward marker for TLS called CD23. These results will offer more pertinent information on the applicability and effectiveness of tumour immunotherapy for BC.

Establishment of a risk score model for bladder urothelial carcinoma based on energy metabolism-related genes and their relationships with immune infiltration.

Bladder urothelial carcinoma (BLCA) is a common malignant tumor of the human urinary system, and a large proportion of BLCA patients have a poor prognosis. Therefore, there is an urgent need to find more efficient and sensitive biomarkers for the prognosis of BLCA patients in clinical practice. RNA sequencing (RNA-seq) data and clinical information were obtained from The Cancer Genome Atlas, and 584 energy metabolism-related genes (EMRGs) were obtained from the Reactome pathway database. Cox regression analysis and least absolute shrinkage and selection operator analysis were applied to assess prognostic genes and build a risk score model. The estimate and cibersort algorithms were used to explore the immune microenvironment, immune infiltration, and checkpoints in BLCA patients. Furthermore, we used the Human Protein Atlas database and our single-cell RNA-seq datasets of BLCA patients to verify the expression of 13 EMRGs at the protein and single-cell levels. We constructed a risk score model; the area under the curve of the model at 5 years was 0.792. The risk score was significantly correlated with the immune markers M0 macrophages, M2 macrophages, CD8 T cells, follicular helper T cells, regulatory T cells, and dendritic activating cells. Furthermore, eight immune checkpoint genes were significantly upregulated in the high-risk group. The risk score model can accurately predict the prognosis of BLCA patients and has clinical application value. In addition, according to the differences in immune infiltration and checkpoints, BLCA patients with the most significant benefit can be selected for immune checkpoint inhibitor therapy.

Urinary PART1 and PLA2R1 Could Potentially Serve as Diagnostic Markers for Diabetic Kidney Disease Patients.

Background: Diabetic kidney disease (DKD) is a chronic renal disease which could eventually develop into renal failure. Though albuminuria and estimated glomerular filtration rate (eGFR) are helpful for the diagnosis of DKD, the lack of specific biomarkers reduces the efficiency of therapeutic interventions. Methods: Based on bulk-seq of 56 urine samples collected at different time points (including 11 acquired from DKD patients and 11 from healthy controls), in corporation of scRNA-seq data of urine samples and snRNA-seq data of renal punctures from DKD patients (retrieved from NCBI GEO Omnibus), urine-kidney specific genes were identified by Multiple Biological Information methods. Results: Forty urine-kidney specific genes/differentially expressed genes (DEGs) were identified to be highly related to kidney injury and proteinuria for the DKD patients. Most of these genes participate in regulating glucagon and apoptosis, among which, urinary PART1 (mainly derived from distal tubular cells) and PLA2R1 (podocyte cell surface marker) could be used together for the early diagnosis of DKD. Moreover, urinary PART1 was significantly associated with multiple clinical indicators, and remained stable over time in urine. Conclusion: Urinary PART1 and PLA2R1 could be shed lights on the discovery and development of non-invasive diagnostic method for DKD, especially in early stages.

Corrigendum: Single-cell transcriptomics reveals the complexity of the tumor microenvironment of treatment-naive osteosarcoma.

[This corrects the article DOI: 10.3389/fonc.2021.709210.].

Identification of Immune-Related Subtypes and Construction of a Novel Prognostic Model for Bladder Urothelial Cancer.

The purpose of this study was to explore the relationship between bladder urothelial cancer (BLCA) and immunity, to screen prognosis-related immune genes (PIGs), and to construct an immune-related prognosis model (IRPM). We processed the relevant data of The Cancer Genome Atlas (TCGA-BLCA) and GSE13507 using R software and Perl. We divided BLCA into high-immunity and low-immunity subtypes. There were significant differences in the two subtypes. In addition, we identified 13 PIGs of BLCA by jointly analyzing the gene expression data and survival information of GSE13507 and TCGA-BLCA, and constructed IRPM through nine of them. The low-risk group had better survival outcome than the high-risk group. We also constructed a nomogram based on clinicopathological information and risk scores of the patients. Moreover, the prognosis of BLCA patients was significantly impacted by the expression of almost every gene used to calculate the risk score. The result of real-time fluorescence quantitative polymerase chain reaction revealed that all the genes used to calculate the risk score were differentially expressed between BLCA and adjacent normal tissues, except PDGFRA. Our research provided potential targets for the treatment of BLCA and a reference for judging the prognosis of BLCA.

Landscape of Peripheral Blood Mononuclear Cells and Soluble Factors in Severe COVID-19 Patients With Pulmonary Fibrosis Development.

Resulting from severe inflammation and cell destruction, COVID-19 patients could develop pulmonary fibrosis (PF), which remains in the convalescent stage. Nevertheless, how immune response participates in the pathogenesis of PF progression is not well defined. To investigate that question, 12 patients with severe COVID-19 were included in the study. Peripheral mononuclear cell (PBMC) samples were collected shortly after their admission and proceeded for single-cell RNA sequencing (scRNA-seq). After 14 days of discharge, the patients were revisited for chest CT scan. PF index (FI) was computed by AI-assisted CT images. Patients were categorized into FIhi and FIlo based on median of FI. By scRNA-seq analysis, our data demonstrated that frequency of CD4+ activated T cells and Treg cells were approximately 3-fold higher in FIhi patients compared with FIlo ones (p < 0.034 for all). By dissecting the differentially expressed genes, we found an overall downregulation of IFN-responsive genes (STAT1, IRF7, ISG15, ISG20, IFIs, and IFITMs) and S100s alarmins (S100A8, S100A9, S100A12, etc.) in all T-cell clusters, and cytotoxicity-related genes (GZMB, PRF1, and GNLY) in CTLs and γδ T cells in the FIhi cohort, compared with FIlo subjects. The GSEA analysis illustrated decreased expression of genes enriched in IFN signaling, innate immune response, adaptive immune response in T cells, NK cells, and monocytes in FIhi patients compared with FIlo ones. In conclusion, these data indicated that the attenuated IFN-responsive genes and their related signaling pathways could be critical for PF progression in COVID-19 patients.

Single-Cell Transcriptomics Reveals the Complexity of the Tumor Microenvironment of Treatment-Naive Osteosarcoma.

Osteosarcoma (OS), which occurs most commonly in adolescents, is associated with a high degree of malignancy and poor prognosis. In order to develop an accurate treatment for OS, a deeper understanding of its complex tumor microenvironment (TME) is required. In the present study, tissues were isolated from six patients with OS, and then subjected to single-cell RNA sequencing (scRNA-seq) using a 10× Genomics platform. Multiplex immunofluorescence staining was subsequently used to validate the subsets identified by scRNA-seq. ScRNA-seq of six patients with OS was performed prior to neoadjuvant chemotherapy, and data were obtained on 29,278 cells. A total of nine major cell types were identified, and the single-cell transcriptional map of OS was subsequently revealed. Identified osteoblastic OS cells were divided into five subsets, and the subsets of those osteoblastic OS cells with significant prognostic correlation were determined using a deconvolution algorithm. Thereby, different transcription patterns in the cellular subtypes of osteoblastic OS cells were reported, and key transcription factors associated with survival prognosis were identified. Furthermore, the regulation of osteolysis by osteoblastic OS cells via receptor activator of nuclear factor kappa-B ligand was revealed. Furthermore, the role of osteoblastic OS cells in regulating angiogenesis through vascular endothelial growth factor-A was revealed. C3_TXNIP+ macrophages and C5_IFIT1+ macrophages were found to regulate regulatory T cells and participate in CD8+ T cell exhaustion, illustrating the possibility of immunotherapy that could target CD8+ T cells and macrophages. Our findings here show that the role of C1_osteoblastic OS cells in OS is to promote osteolysis and angiogenesis, and this is associated with survival prognosis. In addition, T cell depletion is an important feature of OS. More importantly, the present study provided a valuable resource for the in-depth study of the heterogeneity of the OS TME.

Altered regulation of LncRNA analysis of human alcoholic hepatitis with Mallory-Denk Bodies (MDBs) is revealed by RNA sequencing.

Mallory-Denk Bodies (MDBs) are prevalent in a variety of liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. Long noncoding RNAs (lncRNAs) are considered as emerging new gene regulators, which participates in many functional activities through diverse mechanisms. We previously reported the mechanisms involved in the formation of liver MDBs in mouse model and in AH livers where MDBs had formed. To investigate the regulation of mRNAs expression and the probable role of lncRNAs in AH livers with MDBs, RNA-Seq analyses was further conducted to determine the mRNA and lncRNA expression profiles of the AH livers compared with the normal livers. It showed that different lncRNAs have different information contribution degrees by principal component analysis, and the integrated analysis of lncRNA-mRNA co-expression networks were linked to endocytosis, cell cycle, p53 signaling pathways in the human. Based on the co-expression networks, we identify 36 mRNAs that could be as potential biomarkers of alcoholic liver disease (ALD) and hepatocellular carcinoma (HCC). To our knowledge, this is the first report on the regulatory network of lncRNAs associated with liver MDB formation in human, and these results might offer new insights into the molecular mechanisms of liver MDB formation and the progression of AH to HCC.

Cordycepin exhibits a suppressive effect on T cells through inhibiting TCR signaling cascade in CFA-induced inflammation mice model.

Objective: Cordycepin has been shown to exhibit multiple pharmacological activities, such as antitumor, antifungi, antivirus, and immune-regulation activities, and is involved in the regulation of T cells. However, cordycepin that affects T cell activity is still not clear, and the molecular mechanism of cordycepin in regulation of TCR signaling has not yet been elucidated. In this study, the potential effect of cordycepin on T cells was observed in CFA-induced inflammation mice model, and the function of cordycepin in regulating TCR signaling cascade was investigated.Methods: A CFA-induced inflammation mice model was established for observing the effect of cordycepin on the thymus and spleen swellings, and T cell infiltration in paw tissue was detected by immunohistochemistry. The protein expression or phosphorilation was detected by western blotting, and the NFAT1 nuclear translocation was determined by fluorescence imaging. The cell proliferation, apoptosis, and IL-2 production were analyzed by CCK-8 method, flow cytometry, and ELISA.Results: In the mice model, the thymus and spleen swellings were suppressed and the T cell infiltration in paw tissue was inhibited by cordycepin at a concentration of 10 mg/kg. Although the expressions of ZAP70 and PLCγ1 were not significantly changed in the human T cell line Jurkat with cordycepin pretreatment, the CD3-antibody-induced phosphorylations of ZAP70 and PLCγ1 were markedly blocked. The protein level of p85 decreased when Jurkat cells were pretreated with cordycepin, and cordycepin blocked TCR downstream molecule Erk phosphorylation and NFAT1 nuclear translocation. Further investigation revealed that cordycepin inhibited T cell proliferation, reduced IL-2 production, and induced T cell apoptosis. Conclusions: These findings suggest that cordycepin regulates TCR signaling to inhibit excessive T cell activation in inflammation. Thus, cordycepin may be a potential therapeutic application in inflammation-associated diseases.

Cordycepin Attenuates IFN-γ-Induced Macrophage IP-10 and Mig Expressions by Inhibiting STAT1 Activity in CFA-Induced Inflammation Mice Model.

Cordycepin, a natural derivative of adenosine, has been shown to exert pharmacological properties including anti-oxidation, antitumor, and immune regulation. It is reported that cordycepin is involved in the regulation of macrophage function. However, the effect of cordycepin on inflammatory cell infiltration in inflammation remains ambiguous. In this study, we investigated the potential role of cordycepin playing in macrophage function in CFA-induced inflammation mice model. In this model, we found that cordycepin prevented against macrophage infiltration in paw tissue and reduced interferon-γ (IFN-γ) production in both serum and paw tissue. Using luciferase reporter assay, we found that cordycepin suppressed IFN-γ-induced activators of transcription-1 (STAT1) transcriptional activity in a dose-dependent manner. Moreover, western blotting data demonstrated that cordycepin inhibited IFN-γ-induced STAT1 activation through attenuating STAT1 phosphorylation. Further investigations revealed that cordycepin inhibited the expressions of IFN-γ-inducible protein 10 (IP-10) and monokine induced by IFN-γ (Mig), which were the effector genes in IFN-γ-induced STAT1 signaling. Meanwhile, the excessive inflammatory cell infiltration in paw tissue was reduced by cordycepin. These findings demonstrate that cordycepin alleviates excessive inflammatory cell infiltration through down-regulation of macrophage IP-10 and Mig expressions via suppressing STAT1 phosphorylation. Thus, cordycepin may be a potential therapeutic approach to prevent and treat inflammation-associated diseases.

Single-Cell Transcriptomic Map of the Human and Mouse Bladders.

BACKGROUND: Having a comprehensive map of the cellular anatomy of the normal human bladder is vital to understanding the cellular origins of benign bladder disease and bladder cancer. METHODS: We used single-cell RNA sequencing (scRNA-seq) of 12,423 cells from healthy human bladder tissue samples taken from patients with bladder cancer and 12,884 cells from mouse bladders to classify bladder cell types and their underlying functions. RESULTS: We created a single-cell transcriptomic map of human and mouse bladders, including 16 clusters of human bladder cells and 15 clusters of mouse bladder cells. The homology and heterogeneity of human and mouse bladder cell types were compared and both conservative and heterogeneous aspects of human and mouse bladder evolution were identified. We also discovered two novel types of human bladder cells. One type is ADRA2A+ and HRH2+ interstitial cells which may be associated with nerve conduction and allergic reactions. The other type is TNNT1+ epithelial cells that may be involved with bladder emptying. We verify these TNNT1+ epithelial cells also occur in rat and mouse bladders. CONCLUSIONS: This transcriptomic map provides a resource for studying bladder cell types, specific cell markers, signaling receptors, and genes that will help us to learn more about the relationship between bladder cell types and diseases.

α-GalCer and iNKT Cell-Based Cancer Immunotherapy: Realizing the Therapeutic Potentials.

NKT cells are CD1d-restricted innate-like T cells expressing both T cell receptor and NK cell markers. The major group of NKT cells in both human and mice is the invariant NKT (iNKT) cells and the best-known function of iNKT cells is their potent anti-tumor function in mice. Since its discovery 25 years ago, the prototype ligand of iNKT cells, α-galactosylceramide (α-GalCer) has been used in over 30 anti-tumor clinical trials with mostly suboptimal outcomes. To realize its therapeutic potential, numerous preclinical models have been developed to optimize the scheme and strategies for α-GalCer-based cancer immunotherapies. Nevertheless, since there is no standard protocol for α-GalCer delivery, we reviewed the preclinical studies with a focus on B16 melanoma model in the goal of identifying the best treatment schemes for α-GalCer treatment. We then reviewed the current progress in developing more clinically relevant mouse models for these preclinical studies, most notably the generation of new mouse models with a humanized CD1d/iNKT cell system. With ever-emerging novel iNKT cell ligands, invention of novel α-GalCer delivery strategies and significantly improved preclinical models for optimizing these new strategies, one can be hopeful that the full potential of anti-tumor potential for α-GalCer will be realized in the not too distant future.

Expression of GOLPH3 in patients with non-small cell lung cancer and xenografts models.

Increased expression of Golgi phosphoprotein 3 (GOLPH3) has been reported to be associated with several types of human cancer. Patient-derived cancer xenograft models have demonstrated great potential in preclinical studies. In the present study, the link between GOLPH3 expression and survival was examined in patients with non-small cell lung cancer (NSCLC). Patient-derived lung cancer xenograft models were established with two different methods. Lastly, the association between GOLPH3 expression and establishment of the xenograft models was explored. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry analysis were used to examine GOLPH3 expression in 60 NSCLC tissues and matched adjacent non-cancerous tissues (ANT). In addition, tumor pieces from the 60 NSCLC tissues were implanted in the subcutaneous layer and in the subrenal kidney capsule of nude mice. RT-qPCR, histopathology and immunohistochemistry were used to confirm the human origin of the xenograft tumors. RT-qPCR was also used to research the mutation status of GOLPH3 in the xenograft tumors. The results demonstrated that NSCLC tissues had higher expression of GOLPH3, at the mRNA and protein level, compared with ANT. High expression of GOLPH3 correlated with poor survival in patients with NSCLC. Successful engraftment was established for 27 tissues in the subrenal kidney capsule and for 16 in the subcutaneous layer of nude mice. The subrenal kidney capsule group demonstrated significantly higher engraftment rates than the subcutaneous layer group. In addition, higher GOLPH3 expression in the tumor tissues was significantly correlated with higher engraftment rates in mice. In both groups, few xenografts lost the GOLPH3 mutation. In summary, GOLPH3 may be an important diagnosis and prognosis indicator in patients with NSCLC. The genotype and phenotype of the xenograft tumors derived from patient lung cancer tissues exhibited significant similarities to the originating primary tumors. High GOLPH3 expression may promote the successful establishment of xenograft models for NSCLC.

GOLPH3: a novel biomarker that correlates with poor survival and resistance to chemotherapy in breast cancer.

The association between Golgi phosphoprotein 3 (GOLPH3) and clinical pathological characteristics, as well as the clinical outcomes of both neoadjuvant and adjuvant chemotherapies in breast cancer, remain largely unknown. In this study, we investigated the biological role and clinical significance of GOLPH3 in breast cancer. We found that GOLPH3 expression in tumor tissue was higher than that in adjacent noncancerous tissue (ANT) and fibroadenoma. GOLPH3 silencing reduced the migration, invasion, and proliferation of breast cancer cells and promoted apoptosis of the cells. Importantly, patients with high GOLPH3 expression had worse disease-free survival (DFS) and overall survival (OS), and GOLPH3 expression was correlated with clinical pathological characteristics such as molecular subtype, tumor-node-metastasis classification, and age but was not associated with surgery type. Patients with high GOLPH3 expression had poor DFS and OS in every molecular subtype, and an increase in tumor invasion and lymph node metastasis. The risk of recurrence increased with age in patients with high GOLPH3 expression, and surgery type had no influence on patient survival. This is the first study to investigate the correlation between GOLPH3 and response to chemotherapy in breast cancer. Patients with high GOLPH3 expression showed resistance to neoadjuvant and adjuvant chemotherapies, and GOLPH3 overexpression indicated a high risk of recurrence in patients who received adjuvant chemotherapy. These data suggest that GOLPH3 may be a novel biomarker that correlates with poor survival and resistance to chemotherapy in breast cancer.

Overexpression of microRNA-21 strengthens stem cell-like characteristics in a hepatocellular carcinoma cell line.

BACKGROUND: Liver cancer stem cells (LCSCs) have been shown to express higher levels of microRNA-21 (miR-21). Here, we examine the possible contributions of miR-21 to the phenotype of LCSCs in culture and in xenograft tumors in nude mice. METHODS: The hepatocellular carcinoma cell line MHCC-97H was stably transformed with a retroviral vector to establish cells overexpressing miR-21, while a cell line transformed with empty vector served as a negative control. RT-PCR and Western blotting were used to evaluate the effects of miR-21 overexpression on the expression of various LCSC markers, a Transwell assay was used to assess the effects on cell migration and invasion, and a spheroid formation assay was used to examine the effects on clonogenesis. The effects of miR-21 overexpression were also examined in tumors in nude mice. RESULTS: An MHCC-97H cell line was constructed that stably overexpresses miR-21 at 7.78 ± 1.51-fold higher levels than the negative control cell line. Expression of the LCSC markers CD13, Ep-CAM, CD90, and OCT4 was significantly higher in the miR-21-overexpressing cell line than in the negative control at both mRNA and protein levels. The overexpressing cell line formed larger, tighter, and more numerous spheroids. Overexpression of miR-21 was associated with greater cell migration and invasion. Tumors of overexpressing cells in nude mice had a significantly larger mean volume after 34 days of growth (773.62 ± 163.46 mm3) than tumors of negative control cells (502.79 ± 33.94 mm3, p = 0.048), as well as greater mean weight (0.422 ± 0.019 vs. 0.346 ± 0.006 g, p = 0.003). CONCLUSIONS: Overexpression of miR-21 strengthens the phenotype of LCSCs, facilitating invasion, migration, and tumorigenesis in hepatocellular carcinoma.

Side population in hepatocellular carcinoma HCCLM3 cells is enriched with stem-like cancer cells.

Substantial evidence implicates that low-abundance cancer stem cells (CSCs) are responsible for tumor metastasis and recurrence in hepatocellular carcinoma (HCC). Side population (SP) cells possess typical CSCs-like features, and are frequently considered as a special subpopulation in which CSCs are enriched and in studies may be considered as a substitute for CSCs. The aim of the present study was to examine the abundance of SP cells in human HCC cell lines with different metastatic potentials and compare their CSC-like, tumorigenic and invasive properties with those of the main population (MP) cells. An experimental system is described for identifying SP cells and analyzing their CSC-like properties. The relative abundance of SP cells correlated directly with the metastatic potential of the HCC cell line: HCCLM3, 16.3±2.2%; MHCC97-H, 8.4±0.7%; MHCC97-L, 4.7±0.5%; and Huh7, 1.0±0.3% (P<0.05). SP cells isolated from HCCLM3 cultures showed significantly higher proliferation rates and clonogenicity than the corresponding MP cells, in addition to higher migration and invasive abilities in vitro and greater tumorigenicity in mice. Expression levels of all CSC-associated genes tested, except EpCAM and Oct4, were significantly higher in SP cells. The findings revealed that the proportion of SP cells correlates with metastatic potential, and SP cells demonstrated the characteristics expected of CSCs, implicating them in HCC metastasis. Further studies on the identification and characterization of SP cells using clinical HCC specimens will contribute to the understanding of how SP cells are involved in these disease processes.

Characterization of Amphioxus IFN Regulatory Factor Family Reveals an Archaic Signaling Framework for Innate Immune Response.

The IFN regulatory factor (IRF) family encodes transcription factors that play important roles in immune defense, stress response, reproduction, development, and carcinogenesis. Although the origin of the IRF family has been dated back to multicellular organisms, invertebrate IRFs differ from vertebrate IRFs in genomic structure and gene synteny, and little is known about their functions. Through comparison of multiple amphioxus genomes, in this study we suggested that amphioxus contains nine IRF members, whose orthologs are supposed to be shared among three amphioxus species. As the orthologs to the vertebrate IRF1 and IRF4 subgroups, Branchiostoma belcheri tsingtauense (bbt)IRF1 and bbtIRF8 bind the IFN-stimulated response element (ISRE) and were upregulated when amphioxus intestinal cells were stimulated with poly(I:C). As amphioxus-specific IRFs, both bbtIRF3 and bbtIRF7 bind ISRE. When activated, they can be phosphorylated by bbtTBK1 and then translocate into nucleus for target gene transcription. As transcriptional repressors, bbtIRF2 and bbtIRF4 can inhibit the transcriptional activities of bbtIRF1, 3, 7, and 8 by competing for the binding of ISRE. Interestingly, amphioxus IRF2, IRF8, and Rel were identified as target genes of bbtIRF1, bbtIRF7, and bbtIRF3, respectively, suggesting a dynamic feedback regulation among amphioxus IRF and NF-κB. Collectively, to our knowledge we present for the first time an archaic IRF signaling framework in a basal chordate, shedding new insights into the origin and evolution of vertebrate IFN-based antiviral networks.