Integration of scRNA and bulk RNA-sequence to construct the 5-gene molecular prognostic model based on the heterogeneity of thyroid carcinoma endothelial cell.

Thyroid cancer (TC) is a kind of cancer with high heterogeneity, which leads to significant difference in prognosis. The prognostic molecular processes are not well understood. Cancer cells and tumor microenvironment (TME) cells jointly determine the heterogeneity. However, quite a little attention was paid to cells in the TME in the past years. In this study, we not only reveal that endothelial cells (ECs) are strongly associated with the progress of papillary thyroid cancer (PTC) using single-cell RNA-seq (scRNA-seq) data downloaded from Gene Expression Omnibus (GEO) and WGCNA, but also screen 5 crucial genes of ECs: CLDN5, ABCG2, NOTCH4, PLAT, and TMEM47. Furthermore, the 5-gene molecular prognostic model is constructed, which can predict how well a patient will do on PD-L1 blockade immunotherapy for TC and evaluate prognosis. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrates that PLAT is decreased in TC and the increase of PLAT can restrain the migratory capacity of TC cells. Meanwhile, in TC cells, PLAT suppresses VEGFa/VEGFR2-mediated human umbilical vascular endothelial cell (HUVEC) proliferation and tube formation. Totally, we construct the 5-gene molecular prognostic model from the perspective of EC and provide a new idea for immunotherapy of TC.

Proteomics Analysis of Polyphyllin D-Treated Triple-Negative Breast Cancer Cells Reveal the Anticancer Mechanisms of Polyphyllin D.

Polyphyllin D (PD), one of the important steroid saponins in traditional medicinal herb Paris polyphylla, has been demonstrated to have anticancer activity both in vitro and in vivo. However, the mechanisms through which PD exerts its anticancer effects in triple-negative breast cancer (TNBC) remain unclear. Our study was presented to evaluate the anticancer effect and the potential mechanisms of PD in two TNBC cell lines, BT-549 and MDA-MB-231. Through comprehensively comparing the liquid chromatography-tandem mass spectrometry (LC-MS/MS) data of PD-treated and untreated BT-549 and MDA-MB-231 cells, we found that PD could induce apoptosis of TNBC cells by activating oxidative phosphorylation pathway in BT-549 cells, as well as inhibiting spliceosome function alteration in MDA-MB-231 cells. These results suggested that the mechanisms underlying the pro-apoptotic effect of PD on TNBC may be cell type-specificity-dependent. Moreover, we found that nodal modulator 2/3 (NOMO2/3) were downregulated both in PD-treated BT-549 and MDA-MB-231 cells, suggesting that NOMO2/3 may be the potential target of PD. Verification experiments revealed that PD deceased NOMO2/3 expression at protein level, rather than mRNA level. Whether NOMO2/3 are the upstream modulators of oxidative phosphorylation pathway and spliceosome needs further validation. In conclusion, a comprehensive proteomics study was performed on PD-treated or untreated TNBC cells, revealing the anticancer mechanisms of PD.

Circular forms of dedicator of cytokinesis 1 promotes breast cancer progression by derepressing never in mitosis related kinase 2 via sponging miR-128-3p.

The conjecture of breast cancer is uncertain because of its explosive growth and the complicated molecular mechanisms. Circular RNAs (circRNAs) are regulatory RNA sequences present in the genome and their regulatory mechanism involves the sponging of microRNAs (miRNAs). In this study, we explored the regulation between circular forms of dedicator of cytokinesis 1 (circDOCK1) (hsa_circ_0007142) and miR-128-3p, and its implication on the pathogenesis of breast cancer modulated by never in mitosis (NIMA) related kinase 2 (NEK2). We revealed an increase in circDOCK1 and NEK2 expression, and a decrease in miR-128-3p expression in breast cancer tissues and cell lines. Bioinformatics analysis and experimental validation indicated a positive correlation between circDOCK1 and NEK2 expression but a negative correlation was recorded between miR-128-3p and circDOCK1 or NEK2, respectively. Furthermore, inhibition of circDOCK1 expression was followed by an increase in miR-128-3p and a decrease in NEK2 levels in vitro and in vivo. The luciferase assay concluded that miR-128-3p was a direct target of circDOCK1 while NEK2 was the direct target of miR-128-3p. Furthermore, circDOCK1 inhibition hindered breast cancer development by repressing NEK2 and thus promoting the increased expression of miR-128-3p both in vitro and in vivo. We therefore conclude that circDOCK1 promotes breast cancer progression by targeting miR-128-3p-mediated downregulation of NEK2 and that the circDOCK1/hsa-miR-128-3p/NEK2 axis may be a novel therapeutic target for breast cancer treatment.

Docetaxel-resistant triple-negative breast cancer cell-derived exosomal lncRNA LINC00667 reduces the chemosensitivity of breast cancer cells to docetaxel via targeting miR-200b-3p/Bcl-2 axis.

Development of docetaxel (TXT) resistance is a major obstacle for triple-negative breast cancer (TNBC) treatment. Additionally, chemoresistant cell-derived exosomes were able to change the chemo-response of chemosensitive recipient cells via transportation of lncRNAs. It has been shown that lncRNA LINC00667 level was significantly elevated in breast cancer tissues. Therefore, we explored whether LINC00667 level is increased in TXT-resistant TNBC cell-derived exosomes. In addition, whether exosomal LINC00667 derived from TXT-resistant TNBC cell could affect TXT sensitivity in TXT-sensitive TNBC cells was investigated as well. In the present study, exosomes were isolated from the TXT-resistant TNBC cells and from TXT-sensitive TNBC cells. Next, the level of LINC00667 in the isolated exosomes was detected with RT-qPCR. We found that LINC00667 expression was obviously elevated in TXT-resistant TNBC cell-derived exosomes compared to that in TXT-sensitive TNBC cell-derived exosomes. In addition, LINC00667 could be transferred from TXT-resistant TNBC cells to TNBC cells via exosomes. Moreover, TXT-resistant TNBC cell secreted exosomal LINC00667 markedly reduced the sensitivity of TNBC cells to TXT via upregulation of Bcl-2. Meanwhile, downregulation of LINC00667 notably enhanced the sensitivity of TXT-resistant TNBC cells to TXT through downregulation of Bcl-2. Additionally, LINC00667 was considered to be a ceRNA to sponge miR-200b-3p, thereby elevating Bcl-2 expression. Collectively, TXT-resistant TNBC cell-derived exosomal LINC00667 could decrease the chemosensitivity of TNBC cells to TXT via regulating miR-200b-3p/Bcl-2 axis. These findings suggested that LINC00667 might serve as a promising target for enhancing sensitivity of TNBC cells to TXT therapy.

Hypoxia induces docetaxel resistance in triple-negative breast cancer via the HIF-1α/miR-494/Survivin signaling pathway.

Cytotoxic chemotherapy is the major strategy to prevent and reduce triple-negative breast cancer (TNBC) progression and metastasis. Hypoxia increases chemoresistance and is associated with a poor prognosis for patients with cancer. Based on accumulating evidence, microRNAs (miRNAs) play an important role in acquired drug resistance. However, the role of miRNAs in hypoxia-induced TNBC drug resistance remains to be clarified. Here, we found that hypoxia induced TNBC docetaxel resistance by decreasing the miR-494 level. Modulating miR-494 expression altered the sensitivity of TNBC cells to DTX under hypoxic conditions. Furthermore, we identified Survivin as a direct miR-494 target. Hypoxia upregulated survivin expression. In a clinical study, the HIF-1α/miR-494/Survivin signaling pathway was also active in primary human TNBC, and miR-494 expression negatively correlated with HIF-1α and survivin expression. Finally, in a xenograft model, both miR-494 overexpression and the HIF-1α inhibitor PX-478 increased the sensitivity of TNBC to DTX by suppressing the HIF-1α/miR-494/Survivin signaling pathway in vivo. In conclusion, treatments targeting the HIF-1α/miR-494/Survivin signaling pathway potentially reverse hypoxia-induced drug resistance in TNBC.

Polyphyllin D induces apoptosis and protective autophagy in breast cancer cells through JNK1-Bcl-2 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Polyphyllin D (PD), an active component from rhizome of Paris polyphylla Sm, root and rhizome, shows a strong anti-cancer activity in several cancers. However, whether autophagy is involved in PD-induced cell death in breast cancer cells and its molecular mechanism has not yet been elucidated. AIM OF THE STUDY: To explore the anti-tumor effects of PD in breast cancer and the underlying mechanisms. MATERIALS AND METHODS: PD was isolated from P. polyphylla Sm and confirmed by HPLC and NMR. The role of PD in cell viability, apoptosis, autophagy in breast cancer cells were determined. RESULTS: PD shows significant anti-tumor activity by inhibit cell proliferation and induce caspase-dependent apoptosis in breast cancer cells. Moreover, PD treatment could induce autophagy by activation of JNK1/Bcl-2 pathway. Importantly, blocking of autophagy by using autophagy inhibitor 3-methyladenine (3-MA) dramatically increase PD-induced apoptosis as evidence by the increased percentage of apoptotic cell death. The anti-tumor effects of PD also investigated in vivo. The results showed that the combinatory treatment of PD with autophagy inhibitor significantly promote PD-induced apoptosis. CONCLUSION: PD could induce caspase-dependent apoptosis and cyto-protectvie autophagy by activation of JNK1/Bcl-2 pathway in breast cancer cells. Combination with an autophagy inhibitor significantly enhance cytotoxic effect of PD and this combination may be a promising candidate for breast cancer therapy.

DCAF13 promotes triple-negative breast cancer metastasis by mediating DTX3 mRNA degradation.

DCAF13 is firstly identified as a substrate receptor of CUL4-DDB1 E3 ligase complex. This study disclosed that DCAF13 acted as a novel RNA binding protein (RBP) that contributed to triple-negative breast cancer (TNBC) metastasis. Clinical data obtained from TCGA and our collection showed that DCAF13 was closely correlated with poor clinicopathological characteristics and overall survival, which indicated DCAF13 may serve as a diagnostic marker for TNBC metastasis. Functionally, DCAF13 overexpression or suppression was sufficient to enhance or decrease breast cancer cell migration and invasion. Mechanistically, DCAF13 functioned as an RBP by binding with the AU-rich element (ARE) of DTX3 mRNA 3'UTR to accelerate its degradation. Moreover, we identified that DTX3 promoted the ubiquitination and degradation of NOTCH4. Finally, increased DCAF13 expression led to post-transcriptional decay of DTX3 mRNA and consequently activated of NOTCH4 signaling pathway in TNBC. In conclusion, these results identified that DCAF13 as a diagnostic marker and therapeutic target for TNBC treatment. Abbreviation: DCAF13: DDB1 and CUL4-associated factor 13; DDB1: DNA-binding protein 1; CUL4: Cullin 4; CRL4, Cullin-ring finger ligase 4; RBP: RNA binding protein; TNBC: triple-negative breast cancer; ARE: AU-rich element; DTX3: Deltex E3 ubiquitin ligase 3; HER2: human epidermal growth factor receptor 2; ER: estrogen receptor; PR: progesterone receptor; PTEN: phosphatase and tensin homolog deleted on chromosome 10; EMT: epithelial-mesenchymal transition.

circFAT1(e2) Promotes Papillary Thyroid Cancer Proliferation, Migration, and Invasion via the miRNA-873/ZEB1 Axis.

Circular RNAs (circRNAs) play an extremely important regulatory role in the occurrence and development of various malignant tumors including papillary thyroid cancer (PTC). circFAT1(e2) is a new type of circRNA derived from exon 2 of the FAT1 gene, which is distributed in the cytoplasm and nucleus of PTC cells. However, so far, the role of circFAT1(e2) in PTC is still unclear. In this study, circFAT1(e2) was found to be highly expressed in PTC cell lines and tissues. circFAT1(e2) knockdown suppressed PTC cell growth, migration, and invasion. Also, circFAT1(e2) acted as a sponge for potential microRNAs (miRNAs) to modulate cancer progression. A potential miRNA target was discovered to be miR-873 which was targeted by circFAT1(e2) in PTC. The dual-luciferase assay conducted later also confirmed that there was indeed a direct interaction between circFAT1(e2) and miR-873. This study also confirmed that circFAT1(e2) inhibited the miR-873 expression and thus promoted the ZEB1 expression, thus affecting the proliferation, metastasis, and invasion of PTC cells. In conclusion, the results of this study indicated that circFAT1(e2) played a carcinogenic role by targeting the miR-873/ZEB1 axis to promote PTC invasion and metastasis, which might become a potential novel target for therapy of PTC.

Circular RNA hsa_circ_0061825 (circ-TFF1) contributes to breast cancer progression through targeting miR-326/TFF1 signalling.

OBJECTIVES: Circular RNAs (circRNAs) are RNA transcripts that belong to non-coding RNAs (ncRNAs), whose implication in human cancers has been recently demonstrated. However, the specific role of multiple circRNAs in breast cancer remains unidentified. MATERIALS AND METHODS: Microarray analysis and bioinformatics analysis were applied to select circRNA and miRNA, respectively. The loop structure of circ-TFF1 was confirmed using RNase R treatment, divergent primer PCR and Sanger sequencing. qRT-PCR and Western blot were employed for gene expressions. In vitro and in vivo experiments were conducted to assess the function of circ-TFF1 in biological processes in breast cancer cells. FISH and subcellular separation indicated circ-TFF1 cellular distribution. Luciferase reporter and RIP assays and Pearson's correlation analysis were performed to evaluate relationships between genes. RESULTS: Circ-TFF1 and TFF1 were both upregulated and positively associated with each other in breast cancer. Knockdown of circ-TFF1 hindered breast cancer cell proliferation, migration, invasion and EMT in vitro and controlled tumour growth in vivo. Circ-TFF1 acted as a ceRNA of TFF1 by sponging miR-326, and its contribution to breast cancer progression was mediated by miR-326/TFF1 axis. CONCLUSIONS: Circ-TFF1 is a facilitator in breast cancer relying on TFF1 by absorbing miR-326, providing a novel promising target for BC treatment.

KLF8 is associated with poor prognosis and regulates glycolysis by targeting GLUT4 in gastric cancer.

Krüppel-like transcription factor (KLF) family is involved in tumorigenesis in different types of cancer. However, the importance of KLF family in gastric cancer is unclear. Here, we examined KLF gene expression in five paired liver metastases and primary gastric cancer tissues by RT-PCR, and immunohistochemistry was used to study KLF8 expression in 206 gastric cancer samples. The impact of KLF8 expression on glycolysis, an altered energy metabolism that characterizes cancer cells, was evaluated. KLF8 showed the highest up-regulation in liver metastases compared with primary tumours among all KLF members. Higher KLF8 expression associated with larger tumour size (P < 0.001), advanced T stage (P = 0.003) and N stage (P < 0.001). High KLF8 expression implied shorter survival outcome in both TCGA and validation cohort (P < 0.05). Silencing KLF8 expression impaired the glycolysis rate of gastric cancer cells in vitro. Moreover, high KLF8 expression positively associated with SUVmax in patient samples. KLF8 activated the GLUT4 promoter activity in a dose-dependent manner (P < 0.05). Importantly, KLF8 and GLUT4 showed consistent expression patterns in gastric cancer tissues. These findings suggest that KLF8 modulates glycolysis by targeting GLUT4 and could serve as a novel biomarker for survival and potential therapeutic target in gastric cancer.

Catalpol ameliorates diabetic atherosclerosis in diabetic rabbits.

Catalpol, isolated from the roots of Rehmanniaglutinosa, Chinese foxglove, is an iridoid glycoside with antioxidant, anti-inflammatory and anti-hyperglycemic agent. The present study was to investigate the effects of catalpol on diabetic atherosclerosis in alloxan-induced diabetic rabbits. Diabetes was induced in rabbits by a hyperlipidemic diet and intravenous injection of alloxan (100 mg/kg). Rabbits were treated for 12 weeks. The fasting blood glucose, insulin, homeostasis model of insulin resistance, total cholesterol and triglyceride were measured. The thoracic aorta was excised for histology. The plasma and vascular changes including some markers of oxidative stress, inflammatory cytokines and fibrosis factors were examined. Plasma levels of fasting blood glucose, insulin and homeostasis model of insulin resistance were significantly decreased in catalpol group. Catalpol treatment ameliorated diabetic atherosclerosis in diabetic rabbits as demonstrated by significantly inhibited neointimal hyperplasia and macrophages recruitment. Catalpol treatment also enhanced the activities of superoxide dismutase, glutathione peroxidase, and increased the plasma levels of total antioxidant status, meanwhile reduced the levels of malondialdehyde, protein carbonyl groups and advanced glycation end product. Furthermore, catalpol also reduced circulating levels of tumor necrosis factor-α, monocyte chemotactic protein-1 and vascular cell adhesion molecule-1. Catalpol also decreased transforming growth factor-ß1 and collagen IV mRNA and protein expressions in the vessels. Catalpol exerts an ameliorative effect on atherosclerotic lesion in alloxan-induced diabetic rabbits. The possible mechanisms may be related to inhibition of oxidative stress inflammatory response and anti-fibrosis and reduced aggregation of extracellular matrix.