METTL3 and STAT3 form a positive feedback loop to promote cell metastasis in hepatocellular carcinoma.

BACKGROUND: It is well-established that most Hepatocellular carcinoma (HCC) patients die of metastasis, yet the potential mechanisms orchestrating metastasis remain poorly understood. Current evidence suggests that the dysregulation of METTL3-mediated m6A methylation modification is closely associated with cancer progression. STAT3 is an oncogenic transcription factor that reportedly plays a central role in the occurrence and development of HCC. However, the relationship between METTL3 and STAT3 in HCC metastasis remains unclear. METHODS: The relationship between METTL3 expression and the survival of HCC patients was assessed by online tools GEPIA and Kaplan-Meier Plotter. Western blotting, Tissue microarray (TMA), and immunohistochemistry (IHC) staining were used to evaluate the expression levels of METTL3 and STAT3 in HCC cell lines and metastatic and non-metastatic tissues. Methylated RNA immunoprecipitation (MeRIP), MeRIP sequencing (MeRIP-seq), qRT-PCR, RNA immunoprecipitation (RIP), Western blotting and luciferase reporter gene assay were utilized to clarify the mechanism of METTL3 regulating STAT3 expression. Immunofluorescence staining, Western blotting, qRT-PCR, Co-immunoprecipitation (Co-IP), IHC staining, TMA and Chromatin immunoprecipitation (ChIP) assay were performed to explore the mechanism of STAT3 modulating METTL3 localization. Cell viability, wound healing and transwell assay, and orthotopic xenograft model were used to evaluate the role of METTL3-STAT3 feedback loop in the promotion of HCC metastasis in vitro and in vivo. RESULTS: METTL3 and STAT3 are both abundantly expressed in high-metastatic HCC cells and tissues. Moreover, a positive correlation was found between the expression of STAT3 and METTL3 in HCC tissues. Mechanistically, METTL3 could induce the m6A modification of STAT3 mRNA, and then promote the translation of m6A-contained STAT3 mRNA by interacting with the translation initiation machinery. In contrast, STAT3 promoted nuclear localization of METTL3 via transcriptionally upregulating WTAP, a vital member of the methyltransferase complex, and facilitated the methyltransferase function of METTL3. METTL3 and STAT3 form a positive feedback loop to accelerate HCC metastasis in vitro and in vivo. CONCLUSIONS: Our findings reveal a novel mechanism of HCC metastasis and uncover the METTL3-STAT3 feedback signaling as a potential target for the anti-metastatic treatment of HCC. Video Abstract.

Advances in the use of microalgal-bacterial consortia for wastewater treatment: Community structures, interactions, economic resource reclamation, and study techniques.

The rise in living standards has generated a demand for higher aquatic environmental quality. The microalgal community and the surrounding organic molecules, environmental factors, and microorganisms, such as bacteria, are together defined as the phycosphere. The bacteria in the phycosphere can form consortia with microalgae through various forms of interaction. The study of the species in these consortia and their relative proportions is of great significance in determining the species and strains of stable algae that can be used in sewage treatment. This article summarizes the following topics: the interactions between microalgae and bacteria that are required to establish consortia; how symbiosis between algae and bacteria is established; microalgal competition with bacteria through inhibition and anti-inhibition strategies; the influence of environmental factors on microalgal-bacterial aggregates, such as illumination conditions, pH, dissolved oxygen, temperature, and nutrient levels; the application of algal-bacterial aggregates to enhance biomass production and nutrient reuse; and techniques for studying the community structure and interactions of algal-bacterial consortia, such as microscopy, flow cytometry, and omics. PRACTITIONER POINTS: Community structures in microalgal-bacterial consortia in wastewater treatment. Interactions between algae and bacteria in wastewater treatment. Effects of ecological factors on the algal-bacterial community in wastewater treatment. Economically recycling resources from algal-bacterial consortia based on wastewater. Technologies for studying microalgal-bacterial consortia in wastewater treatment.

Characterization of the small RNA transcriptomes of cell protrusions and cell bodies of highly metastatic hepatocellular carcinoma cells via RNA sequencing.

Increasing evidence suggest that hepatocellular carcinoma (HCC) HCCLM3 cells initially develop pseudopodia when they metastasize, and microRNAs (miRNAs/miRs) and circular RNAs (circRNAs) have been demonstrated to serve important roles in the development, progression and metastasis of cancer. The present study aimed to isolate the cell bodies (CBs) and cell protrusions (CPs) from HCCLM3 cells, and screen the miRNAs and circRNAs associated with HCC infiltration and metastasis in CBs and CPs. The Boyden chamber assay has been confirmed to effectively isolate the CBs and CPs from HCCLM3 cells via observation of microtubule immunofluorescence, DAPI staining and nuclear protein H3 western blotting. Following high-throughput sequencing of the successfully isolated CBs and CPs, 64 pairs of miRNAs, including 23 pairs of upregulated genes and 41 pairs of downregulated genes, and 260 sets of circRNAs, including 127 upregulated genes and 133 downregulated genes, were significantly differentially expressed, using the following criteria: HP/HB ratio, fold change ≥|1.5|, P<0.05). PCR analysis verified that changes in the expression levels of hsa-let-7a-5p, hsa-let-7c-3p, hsa-miR-30c-5p, hsa_circ_0059580, hsa_circ_0067475, hsa_circ_0002100 and hsa_circ_00072309 were consistent with the sequencing results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to analyze the functions and roles of the differentially expressed miRNAs and circRNAs. The interaction maps between miRNAs and circRNAs were constructed, and signaling pathway maps were analyzed to determine the molecular mechanism and regulation of the differentially expressed miRNAs and circRNAs. Taken together, the results of the present study suggest that the Boyden chamber assay can be used to effectively isolate the somatic CBs and CPs of HCC, which can be used to screen the miRNAs and circRNAs associated with invasion and metastasis of HCC.

Structural requirement of G-quadruplex/aptamer-combined DNA macromolecule serving as efficient drug carrier for cancer-targeted drug delivery.

Photodynamic therapy (PDT) as a clinical cancer treatment method has been used to treat carcinomas in different organs, and G-quadruplex-based DNA nanocompartments serving as the carriers of cationic porphyrin photosensitizers, especially combined with cell-targeting aptamers, is considered to offer new opportunities for future cancer treatment. However, the structural features of G-quadruplex/aptamer complexes suitable for the capsulation of photosensitizers and target cell recognition is unexplored so far. In this study, unimolecular (UM), bimolecular (BM) and tetramolecular (TM) G-quadruplex structures were used as the drug loading compartments and grafted onto tumor cell-targeting aptamer Sgc8, constructing several targeting drug delivery vehicles (T-GMVs). Besides the binding affinity of resulting DNA architectures for target cells and cell recognition specificity were explored in a comparative fashion, the drug loading capability and cancer therapy efficacy were evaluated using TMPyP4 as the model porphyrin-based drug. The experimental results show that only TM G-quadruplex structure is suitable to combine with Sgc8 for the development of drug delivery vehicle and the as-prepared T-GMV- TMPyP4 complexes display the desirable cancer therapy efficacy, holding the potential application in the future cancer therapy. More importantly, T-GMV- TMPyP4 is expected to lay the scientific groundwork for the successful development of G-quadruplex-based photosensitizer drug delivery carriers for the targeted cancer therapy.

Investigation of Alpha-Fetoprotein Antibody Modified Fluorescent Magnetic Probe on HepG2 Cell and Cancer Model Mouse.

To construct a dual-targeting (magnetic targeting and antibody targeting) multi-functional magnetic fluorescent liposome probe with a good biocompatibility and high specificity for early liver cancerdiagnosis. 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG2000-NH2) and Cyanine7 N-hydroxysuccinimide ester (Cy7-NHS) were measured and reacted for forming DSPE-PEG2000-Cy7. Egg yolk lecithin, cholesterol, DSPE-PEG2000-Cy7 and Fe3O4 were added in orderly into the reaction solution for synthesis of fluorescent ferroferric oxide (Fe3O4) liposome. The Magnetic liposome (MLP) was coupled with Alpha-Fetoprotein (AFP) polyclonal antibody and the ending AFP antibody modified magnetic liposome was analyzed and studied on human hepatoma cell HepG2 and cancer model mouse. The fluorescent ferroferric oxide liposome appeared as a sphericalmorphology and they were about 150 nm in mean hydrodynamic diameter with negative charge. The fluorescent magnetic probe had a little toxic effect on hepG2 cells and increased the specificity liver cancer diagnosis by AFP antibody target imaging in model mouse of liver cancer. In brief, the AFP antibody conjugated fluorescent magnetic probe is a promising multi-functional tool with good biocompatibility and high sensitivity and specificity for clinical cancer diagnosis and therapy.

Label-free detection of cancer related gene based on target recycling and palindrome-mediated strand displacement amplification.

STAT3 plays an important role in regulating gene expression and is closely related with cancer. Thus, the sensitive and specific detection of the STAT3 biomarker is of great importance for disease diagnosis and therapeutics. In this study, by combining the target recycling amplification (TRA) with strand displacement amplification (SDA), we have developed a label-free and highly sensitive method for the dual-amplified detection of STAT3. The assay system consists of polymerization primer and label-free hairpin probe (HP) containing palindromic fragment and nicking site. In the presence of STAT3, the stem of the HP is opened, followed by the primer binding to initiate TRA and SDA with the help of Klenow Fragment (KF) and nickase. After multiple replication, nicking, and strand displacement, STAT3 was released and initiated the next round of reactions, generating a large number of terminal palindrome-contained fragments. Subsequently, the intermolecular hybridization between palindromic fragments occurred and the bidirectional extension by polymerase takes place, forming the dsDNAs. The double-stranded DNA products can be quantified by measuring the fluorescence intensity of SYBR Green I. The proposed strategy shows the excellent specificity and high sensitivity with a detection limit as low as 50 pM. In addition, this designed protocol can be successfully applied to detect the STAT3 in human serum, indicating great potential for the practical application in early diagnosis and prognosis.

TDP-43 induces EMT and promotes hepatocellular carcinoma metastasis via activating Wnt/ß-catenin signaling pathway.

The trans-activation response DNA-binding protein of 43 kDa (TDP-43) is a nuclear protein that has been shown to be involved in the growth and metastasis of breast cancer, neuroblastoma, and melanoma. However, the effect of TDP-43 on hepatocellular carcinoma (HCC) metastasis remains unclear. Here, we demonstrated that TDP-43 was highly upregulated in both clinical samples and cell lines of HCC. Moreover, knockdown and overexpression of TDP-43 efficiently affected the proliferation and metastasis of HCC cells as well as the expression of some proteins associated with epithelial-mesenchymal transition (EMT) and Wnt/ß-catenin signaling pathway. Furthermore, activation of the Wnt/ß-catenin pathway by LiCl restored the effect of TDP-43 knockdown on EMT and HCC cells, whereas inhibition of the Wnt/ß-catenin pathway by XAV939 negated the effect of TDP-43 overexpression. Importantly, we found that TDP-43 protein could interact with GSK3ß mRNA and regulate the level of GSK3ß protein translation. Taken together, our findings suggest that TDP-43 may activate the Wnt/ß-catenin pathway by targeting the inhibition of GSK3ß protein translation, thus inducing the proliferation and metastasis of HCC cells, which supports its potential value as a therapeutic target for the treatment of metastatic HCC.