EMT-related long non-coding RNA in hepatocellular carcinoma: A study with TCGA database.

Accumulating evidence suggest that dysregulated expression of long non-coding RNA (lncRNA) plays a critical role in human tumorigenesis. However, little is known about the lncRNA implicated in the epithelial-to-mesenchymal transition (EMT) process. In this study, we performed data mining in The Cancer Genome Atlas (TCGA) hepatocellular carcinoma (HCC) data set and identified the a spectrum of differentially expressed lncRNAs implicated the EMT process of HCC, and functionally validated their roles in LM3 cells. Especially, lncRNA WDFY3-AS2-, LINC00472-, MIAT-, and MEG3-associated genes were significantly enriched in EMT-linked pathways. Loss-of-function study showed that genetic silencing of WDFY3-AS3, MIAT, and MEG3, but not LINC00472, resulted in reduced N-cadherin expression, cell migration, and cell invasion. Collectively, our results identify several lncRNAs that regulate the EMT process of HCC, which provides critical information for HCC tumorigenesis and potential therapeutic targets.

Development and validation of a quantification method for oleanolic acid and hederagenin in rat plasma: application to the pharmacokinetic study.

A rapid and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and validated for simultaneous quantification of oleanolic acid and hederagenin in rat plasma. After the two analytes were extracted with liquid-liquid extraction, chromatographic separation was performed on a C18 column with acetonitrile and water (85:15, v/v) as mobile phase at a flow rate of 0.4 mL/min. Calibration curves exhibited good linearity (r > 0.995) over the ranges of 0.41-82.0 ng/mL for oleanolic acid and 0.32-64.0 ng/mL for hederagenin, respectively. The lower limit of quantifications (LLOQs) in plasma were 0.41 ng/mL for oleanolic acid and 0.32 ng/mL for hederagenin. The established LLOQs were within the concentration needed for the assay in plasma, which met the requirements to evaluate their pharmacokinetics of oleanolic acid and hederagenin. This developed assay was successfully applied in the pharmacokinetic study of oleanolic acid and hederagenin in rats after oral administration of Rhizoma Clematidis extract.

Ratiometric co-delivery of doxorubicin and paclitaxel prodrug by remote-loading liposomes for the treatment of triple-negative breast cancer.

Liposomal delivery system that concurrent delivery of multiple drugs can improve efficacy of cancer treatments, yet remain challenging because of differential properties, fairly low loading thresholds of many drugs, and drug burst release. Here, we report a ratiometrically designed liposomal nanoplatform with synergistic efficacy, utilizing remote loading to co-encapsulate ROS-sensitive paclitaxel prodrug (PSN) and doxorubicin (DOX). This novel dual-delivery liposomes possess high two-drug encapsulation efficacy and colloidal stability, resulting in synergistic cytotoxicity, extended blood circulation, favorable biodistribution, always maintain the ratiometrically synergistic drug ratio in vivo, and potent synergistic anticancer activity. Such a combination of PSN and DOX with synergistic effects encapsulated in a safe and effective dual-delivery liposomal nanomedicine may hold promise for the further clinical translation and provides a new strategy for high-efficient combination chemotherapy.

Observation of Coexisting Dirac Bands and Moiré Flat Bands in Magic-Angle Twisted Trilayer Graphene.

Moiré superlattices that consist of two or more layers of 2D materials stacked together with a small twist angle have emerged as a tunable platform to realize various correlated and topological phases, such as Mott insulators, unconventional superconductivity, and quantum anomalous Hall effect. Recently, magic-angle twisted trilayer graphene (MATTG) has shown both robust superconductivity similar to magic-angle twisted bilayer graphene and other unique properties, including the Pauli-limit violating and re-entrant superconductivity. These rich properties are deeply rooted in its electronic structure under the influence of distinct moiré potential and mirror symmetry. Here, combining nanometer-scale spatially resolved angle-resolved photoemission spectroscopy and scanning tunneling microscopy/spectroscopy, the as-yet unexplored band structure of MATTG near charge neutrality is systematically measured. These measurements reveal the coexistence of the distinct dispersive Dirac band with the emergent moiré flat band, showing nice agreement with the theoretical calculations. These results serve as a stepstone for further understanding of the unconventional superconductivity in MATTG.

Calcium-sensing receptor antagonist NPS-2143 suppresses proliferation and invasion of gastric cancer cells.

NPS-2143 is a calcium-sensing receptor (CaSR) antagonist that has been demonstrated to possess anticancer activity. To date, the effects of NPS-2143 on gastric cancer (GC) cell growth, motility, and apoptosis have not been investigated. In the present study, we firstly investigated the expression of CaSR in GC tissues using immunohistochemistry and western blotting. Then Cell Counting Kit-8 and colony formation assays were conducted to explore the effect of the NPS-2143 on the proliferation of GC cell line AGS. Transwell invasion and migration assays were performed to test the effect of NPS-2143 on AGS cell motility. We determined the percentage of apoptotic cells by flow cytometry and explored the changes of apoptosis-related protein by western blotting. Furthermore, we constructed a CaSR knockdown AGS cell line to determine whether NPS-2143 acted via inhibition of CaSR. We found that the protein expression level of CaSR was higher in GC tissues compared with the paired adjacent normal tissues. In addition, NPS-2143 treatment caused an inhibitory effect on the proliferation, invasion, and migration of AGS cells and a promoting effect on AGS apoptosis. The expression of Bcl-2 was decreased while the levels of Bax and active caspase 3 were enhanced in AGS cells after NPS-2143 treatment. Mechanistically, NPS-2143 lead to a significant decrease in the expression of phosphorylated (p)-AKT, phosphorylated mechanistic target of rapamycin (p-mTOR), p70, and cyclin D1. Knockdown of CaSR also suppressed cell proliferation, invasion, and migration and promoted cell apoptosis. No significant difference was observed between CaSR-silenced AGS cells with and without NPS-2143 treatment. These results confirmed that NPS-2143 has an inhibitory influence on AGS cell growth via inhibiting CaSR, which then suppresses the PI3K/Akt signaling pathway.

Schisandrin A reverses doxorubicin-resistant human breast cancer cell line by the inhibition of P65 and Stat3 phosphorylation.

BACKGROUND: Multidrug resistance (MDR) in breast cancer therapy occurs frequently. Thus, anti-MDR agents from natural products or synthetic compounds were tested extensively. We have also explored the reverse effect and mechanism of Schisandrin A (Sch A), a natural product, on MCF-7 breast cancer doxorubicin (DOX)-resistant subline MCF-7/DOX. METHODS: MTT assay was performed to measure the viability of MCF-7 cells to assess the reverse effect of Sch A. Western blot analysis was used to study the protein levels. Laser scanning confocal microscopy was performed to detect the intercellular DOX and Rhodamine 123 accumulation. The qRT-PCR was used to analysis the target gene expression. Dual-luciferase reporter assay was performed to test the transcriptional activity of P-glycoprotein (P-gp). RESULTS: Sch A, at the concentration of 20 µM, showed selective reverse effect (better than the positive control, verapamil at 5 µM) on MCF-7/DOX cell line but not on BEL-7402/DOX, Hep G2/DOX, and K-562/DOX cells. In addition, Sch A enhanced DOX-induced cleavage of Caspase-9 and PARP levels by increasing intracellular DOX accumulation and inhibiting P-gp function. Furthermore, Sch A selectively suppressed P-gp at gene and protein levels in MCF-7/DOX cells which express high level of MDR1 but not MRP1, MRP3, or BCRP. Besides, Sch A showed inhibitory effect on P-gp transcriptional activity. Sch A significantly reduced p-IκB-α (Ser32) and p-Stat3 (Tyr705) levels which mediate P-gp expression. In addition, Stat3 knockdown enhanced the reverse effect of siP65. The combined effect of siStat3 and siP65 was better than Sch A single treatment in MCF-7/DOX cells. CONCLUSION: Sch A specifically reverses P-gp-mediated DOX resistance in MCF-7/DOX cells by blocking P-gp, NF-κB, and Stat3 signaling. Inhibition of P65 and Stat3 shows potent anti-MDR effect on MCF-7/DOX cells.

HuR promotes breast cancer cell proliferation and survival via binding to CDK3 mRNA.

HuR, a ubiquitously expressed RNA-binding protein, stabilizes mRNA and regulates its translation. HuR expression was increased at all stages of breast cancer and correlated with poor clinical outcome. However, the detailed mechanisms remain unclear. Here we reported that overexpression of HuR increased CDK3 mRNA stability and thus its protein expression in MDA-MB-231 and MCF-7 cells. Mechanistically, CDK3 mRNA was identified as a target of HuR via bioinformatics and RNA binding protein immunoprecipitation (RIP) assays. Furthermore, treatment with HuR shRNA decreased CDK3 expression, inhibited cell proliferation and promoted cell apoptosis in breast cancer. More importantly, overexpression of CDK3 reversed the suppressive effects of HuR knockdown on cell growth in both MDA-MB-231 and MCF-7 cells. Finally, HuR and CDK3 expression levels were positively correlated and significantly up-regulated in breast cancer samples. And overexpression of HuR attenuated the chemotherapeutical efficiency of breast cancer. Therefore, our results indicate that ectopic expression of HuR promotes breast cancer cell proliferation and survival by directly binding to and stabilizing CDK3 mRNA.

The Efficacy of Sodium Aescinate on Cutaneous Wound Healing in Diabetic Rats.

This study is aimed to evaluate the potential effects of sodium aescinate (SA, the sodium salt of aescin) on wound healing in streptozotocin-induced diabetic rats. An excision skin wound was created in diabetic rats, and the wounded rats were divided into three groups: I) control group, II) gel-treated group, and III) SA-treated group. The control group wounds received topically normal saline once daily for 19 days. The gel-treated and SA-treated wounds received topically 400 µl of pluronic F-127 gel (25%) and 400 µl of SA (0.3%) in pluronic gel, respectively, once daily for 19 days. SA application in diabetic rats increased the wound contraction and significantly decreased the level of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α) in comparison to the gel-treated group and control group. SA application in diabetic rats also resulted in a marked increase in the level of anti-inflammatory cytokine interleukin-10 (IL-10) and activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) compared to the other groups. Histopathologically, SA-treated wounds showed better granulation tissue dominated by marked fibroblast proliferation, and wounds were covered by thick regenerated epithelial layer. Additionally, the application of only pluronic gel produced some beneficial effects in some parameters in comparison to control group, but most of them were not significantly different. These findings demonstrated that SA may effectively control and improve wound healing in diabetic rats via its anti-inflammatory and antioxidant activities.

[Construction of GJB2 mutations common in Chinese EGFP fusion protein vectors].

OBJECTIVE: To construct GJB2 gene mutations common in Chinese EGFP fusion protein vectors, and to search for better way to study the mechanism of deletion mutations in GJB2 gene. METHOD: Non-fusion protein vectors of 235delC, 299-300 del AT and 176 del 16 bp were first made by point mutation methods in vitro. Then expression part of the upper 3 mutations were amplified by PCR and the PCR products were cloned into TA cloning vector. After cutting by restriction enzymes EcoRI/BamHI, three deletion mutations were inserted into pEGFP-N1 vector. Sequencing was used to verify the validity of the fusion protein vectors. HEK293 cells were transfected with the recombinant DNA samples by the liposome complex method. RESULT: The recombined plasmids were highly expressed in HEK293 cells. Green fluorescence signals were distributed uniformly in cytoplasm. CONCLUSION: GJB2 mutations common in Chinese EGFP fusion protein vectors were constructed successfully. It may provide a better way to explore the reasons of nonsyndromic hearing loss common in Chinese.