Droplet microfluidics for CTC-based liquid biopsy: a review.

Circulating tumor cells (CTCs) are important biomarkers of liquid biopsy. The number and heterogeneity of CTCs play an important role in cancer diagnosis and personalized medicine. However, owing to the low-abundance biomarkers of CTCs, conventional assays are only able to detect CTCs at the population level. Therefore, there is a pressing need for a highly sensitive method to analyze CTCs at the single-cell level. As an important branch of microfluidics, droplet microfluidics is a high-throughput and sensitive single-cell analysis platform for the quantitative detection and heterogeneity analysis of CTCs. In this review, we focus on the quantitative detection and heterogeneity analysis of CTCs using droplet microfluidics. Technologies that enable droplet microfluidics, particularly high-throughput droplet generation and high-efficiency droplet manipulation, are first discussed. Then, recent advances in detecting and analyzing CTCs using droplet microfluidics from the different aspects of nucleic acids, proteins, and metabolites are introduced. The purpose of this review is to provide guidance for the continued study of droplet microfluidics for CTC-based liquid biopsy.

Efficient bioparticle extraction using a miniaturized inertial microfluidic centrifuge.

Conventional bioparticle extraction requires labor-intensive operation, and expensive and bulky centrifuges. Herein, we report a miniaturized centrifuge by cascading four paralleled inertial spiral channels with a two-stage serpentine channel, allowing for the efficient washing and acquisition of concentrated bioparticles from background fluids. First, the effects of channel size and flow rate on particle focusing dynamics and solution exchange performances are explored to enable the optimization and wide application of our device. Then, the integrated device is fabricated and tested experimentally. The results indicate that 10-20 µm particles can be washed from the original samples with increased concentrations and with recovery efficiencies of >93%. Finally, to verify its versatility, we use our miniaturized centrifuge to successfully change the culture medium for cultured MCF-7 breast cancer cells, extract A549 lung cancer cells from a calcein-AM staining solution, purify white blood cells (WBCs) from lysed whole blood, and extract target cells from an unbonded magnetic microbead background. Compared with conventional centrifuges, our device has the advantages of simple fabrication, easy operation, and small footprint. More importantly, it offers outstanding capability for extracting bioparticles from various background fluids, and avoids bioparticle damage that may be caused by high-speed centrifugation. Therefore, we envision that our miniaturized centrifuge could be a promising alternative to traditional centrifuges in many applications.

A Novel Prognostic Model for Oral Squamous Cell Carcinoma: The Functions and Prognostic Values of RNA-Binding Proteins.

PURPOSE: The biological roles and clinical significance of RNA-binding proteins (RBPs) in oral squamous cell carcinoma (OSCC) are not fully understood. We investigated the prognostic value of RBPs in OSCC using several bioinformatic strategies. MATERIALS AND METHODS: OSCC data were obtained from a public online database, the Limma R package was used to identify differentially expressed RBPs, and functional enrichment analysis was performed to elucidate the biological functions of the above RBPs in OSCC. We performed protein-protein interaction (PPI) network and Cox regression analyses to extract prognosis-related hub RBPs. Next, we established and validated a prognostic model based on the hub RBPs using Cox regression and risk score analyses. RESULTS: We found that the differentially expressed RBPs were closely related to the defense response to viruses and multiple RNA processes. We identified 10 prognosis-related hub RBPs (ZC3H12D, OAS2, INTS10, ACO1, PCBP4, RNASE3, PTGES3L-AARSD1, RNASE13, DDX4, and PCF11) and effectively predicted the overall survival of OSCC patients. The area under the receiver operating characteristic (ROC) curve (AUC) of the risk score model was 0.781, suggesting that our model exhibited excellent prognostic performance. Finally, we built a nomogram integrating the 10 RBPs. The internal validation cohort results showed a reliable predictive capability of the nomogram for OSCC. CONCLUSION: We established a novel 10-RBP-based model for OSCC that could enable precise individual treatment and follow-up management strategies in the future.

LncRNA LTSCCAT promotes tongue squamous cell carcinoma metastasis via targeting the miR-103a-2-5p/SMYD3/TWIST1 axis.

Abnormal expression of long-noncoding RNA is involved in the tumorigenesis and progression of various cancers, but the potential molecular regulatory mechanisms are unclear. Microbial flora and chronic inflammation, such as periodontitis, which is associated with oral cancer, affect the occurrence and progression of tumors. Accordingly, we stimulated the tongue squamous cell carcinoma (TSCC) cell lines CAL27 and SCC15 with a low concentration of lipopolysaccharide (LPS) from Porphyromonas gingivalis (P.g) for 6 days and then performed LncRNA sequencing on P.g-LPS-treated CAL27 cells and untreated CAL27 cells. LTSCCAT was upregulated in P.g-LPS-treated CAL27 cells compared with untreated CAL27 cells. LTSCCAT induced epithelial-mesenchymal transition and promoted the invasion and metastasis of TSCC in vitro and in vivo. LncRNA LTSCCAT was upregulated in TSCC patients with periodontitis and was correlated with metastasis and poor prognosis. We predicted through an online database and confirmed by dual-luciferase reporter assays that LTSCCAT is a competitive endogenous RNA for the regulation of miR-103a-2-5p. Another dual-luciferase reporter assay confirmed that miR-103a-2-5p has a binding site at the 3'-UTR of the histone methylation transferase SMYD3 and inhibits its translation. Chromatin immunoprecipitation experiments demonstrated that SMYD3 binds directly to the promoter region of TWIST1 and promotes its transcription, which is related to H3K4 trimethylation. The effect of pcDNA/LTSCCAT on expression was attenuated by miR-103a-2-5p mimics. The RF and SVM classifier predicts that LTSCCAT can bind to SMYD3, whereas the RNA immunoprecipitation (RIP) assay confirms that it cannot. In addition, we predicted the combination of LTSCCAT and SMYD3 through software, but the RIP assay confirmed that LTSCCAT could not be combined with SMYD3. For the first time, we showed that periodontitis promotes the invasion and metastasis of TSCC and clarified the molecular mechanism of LTSCCAT to promote invasion and metastasis of TSCC, providing a potential therapeutic target for clinical treatment of TSCC.

Long non-coding RNA maternally expressed gene 3 inhibits osteogenic differentiation of human dental pulp stem cells via microRNA-543/smad ubiquitin regulatory factor 1/runt-related transcription factor 2 axis.

OBJECTIVE: The aim of the present study was to investigate the biological roles and underlying mechanism of the long non-coding RNA maternally expressed gene 3 (MEG3) on osteogenic differentiation of human dental pulp stem cells (hDPSCs). METHODS: The expression levels of MEG3, microRNA-543 (miR-543), osterix, osteopontin, osteocalcin and runt-related transcription factor 2 (RUNX2) were measured by quantitative real-time PCR (qRT-PCR). Alkaline phosphatase (ALP) activity assay and alizarin red S staining (ARS) were used to measure the impacts exerted by MEG3, miR-543 on osteogenic differentiation. Cell proliferation was measured by MTT assay. In addition, the targeted relationships between miR-543, MEG3, and Smad ubiquitin regulatory factor 1 (SMURF1) were assessed through dual luciferase reporter assay. RESULTS: During osteogenic induction, the expression of MEG3 was gradually reduced, whereas the expression of miR-543, osterix, osteopontin, osteocalcin and RUNX2 were gradually increased. Functional analysis implied that MEG3 overexpression or miR-543 inhibition reduced the cell proliferation, ALP activity, ARS levels, and decreased the expression of osteoblast-related proteins. Moreover, MEG3 promoted SMURF1 expression by directly targeting miR-543 as a competing endogenous RNA. Furthermore, overexpression of miR-543 or silencing SMURF1 could reverse the inhibitory effects of MEG3 on the osteogenic differentiation of hDPSCs. CONCLUSIONS: In conclusion, our study revealed that overexpression of MEG3 inhibited hDPSCs osteogenic differentiation via miR-543/SMURF1/RUNX2 regulatory network, which may contribute to the functional regulation and clinical applications of hDPSCs.

[Contents of organochlorine pesticides in water, pore water and sediment in Minjiang River Estuary of China].

The concentrations of 18 organochlorine pesticides in water, pore water and surface sediment from Minjiang River Estuary were analyzed by GC-ECD, and confirmed by GC-MSD. The range of organochlorine pesticides was 0.532-1.82 micrograms/L at water, 4.54-13.7 micrograms/L at pore water, and 28.79-52.07 micrograms/kg at sediment. Compared with the results of other estuaries and gulfs (Pearl River Estuary, Jiulong River Estuary and so on), it showed that the pollution of organochlorines in Minjiang was some moderate. The concentration difference in surface water, middle water, bottom water, pore water and sediment, due to the higher affinity of these hydrophobic compound for sedimentary phase than to water and a potential flux of pollutants from sediment to overlying water. The predominance of beta-HCH, DDE, Heptachlor, Endosulfan II and Methoxychlor in all water, pore water and sediment were clearly observed. On average, DDE, beta-HCH, Endosulfan II occupied the most part of total DDTs, HCHs and Endosulfans respectively, indicated the fate of these parent compounds or congeners. At the same time, it was found that the positive relationship among the individual organochlorine components (HCHs, DDTs and Endosulfans), which indicated that the similar of the source and behavior for organochlorine pesticides.