Distinct molecular profiles drive multifaceted characteristics of colorectal cancer metastatic seeds.

Metastasis of primary tumors remains a challenge for early diagnosis and prevention. The cellular properties and molecular drivers of metastatically competent clones within primary tumors remain unclear. Here, we generated 10-16 single cell-derived lines from each of three colorectal cancer (CRC) tumors to identify and characterize metastatic seeds. We found that intrinsic factors conferred clones with distinct metastatic potential and cellular communication capabilities, determining organ-specific metastasis. Poorly differentiated or highly metastatic clones, rather than drug-resistant clones, exhibited poor clinical prognostic impact. Personalized genetic alterations, instead of mutation burden, determined the occurrence of metastatic potential during clonal evolution. Additionally, we developed a gene signature for capturing metastatic potential of primary CRC tumors and demonstrated a strategy for identifying metastatic drivers using isogenic clones with distinct metastatic potential in primary tumors. This study provides insight into the origin and mechanisms of metastasis and will help develop potential anti-metastatic therapeutic targets for CRC patients.

SEVtras delineates small extracellular vesicles at droplet resolution from single-cell transcriptomes.

Small extracellular vesicles (sEVs) are emerging as pivotal players in a wide range of physiological and pathological processes. However, a pressing challenge has been the lack of high-throughput techniques capable of unraveling the intricate heterogeneity of sEVs and decoding the underlying cellular behaviors governing sEV secretion. Here we leverage droplet-based single-cell RNA sequencing (scRNA-seq) and introduce an algorithm, SEVtras, to identify sEV-containing droplets and estimate the sEV secretion activity (ESAI) of individual cells. Through extensive validations on both simulated and real datasets, we demonstrate SEVtras' efficacy in capturing sEV-containing droplets and characterizing the secretion activity of specific cell types. By applying SEVtras to four tumor scRNA-seq datasets, we further illustrate that the ESAI can serve as a potent indicator of tumor progression, particularly in the early stages. With the increasing importance and availability of scRNA-seq datasets, SEVtras holds promise in offering valuable extracellular insights into the cell heterogeneity.

circAtlas 3.0: a gateway to 3 million curated vertebrate circular RNAs based on a standardized nomenclature scheme.

Recent studies have demonstrated the important regulatory role of circRNAs, but an in-depth understanding of the comprehensive landscape of circRNAs across various species still remains unexplored. The current circRNA databases are often species-restricted or based on outdated datasets. To address this challenge, we have developed the circAtlas 3.0 database, which contains a rich collection of 2674 circRNA sequencing datasets, curated to delineate the landscape of circRNAs within 33 distinct tissues spanning 10 vertebrate species. Notably, circAtlas 3.0 represents a substantial advancement over its precursor, circAtlas 2.0, with the number of cataloged circRNAs escalating from 1 007 087 to 3 179 560, with 2 527 528 of them being reconstructed into full-length isoforms. circAtlas 3.0 also introduces several notable enhancements, including: (i) integration of both Illumina and Nanopore sequencing datasets to detect circRNAs of extended lengths; (ii) employment of a standardized nomenclature scheme for circRNAs, providing information of the host gene and full-length circular exons; (iii) inclusion of clinical cancer samples to explore the biological function of circRNAs within the context of cancer and (iv) links to other useful resources to enable user-friendly analysis of target circRNAs. The updated circAtlas 3.0 provides an important platform for exploring the evolution and biological implications of vertebrate circRNAs, and is freely available at http://circatlas.biols.ac.cn and https://ngdc.cncb.ac.cn/circatlas.

[Characteristics of genetic variants in 134 patients with Acute myeloid leukemia].

OBJECTIVE: To analyze the characteristics of genetic variants in 134 patients diagnosed with Acute myeloid leukemia (AML). METHODS: Clinical data of the 134 patients with AML (non-acute promyelocytic leukemia) initially diagnosed at the 940th Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army from June 2017 to June 2022 were retrospectively analyzed. Potential variants of AML-related genes were detected by next-generation sequencing, and the frequency of variants was analyzed by using SPSS v26.0 software, and likelihood ratio χ2 test and Fisher exact test were used for data analysis. RESULTS: The patients had included 72 males and 62 females, with a gender ratio of 1.7 : 1 and a median age of 51 years (9 ~ 86 years old). One hundred twenty patients (76.1%) had harbored at least one genetic variant, including 26 (19.4%) having a single variant, 27 (20.1%) having two variants, and 49 (36.6%) having >= 3 variants. 32 (23.9%) had no detectable variants. Genetic variants detected in over 10% of the 134 patients had included NPM1 (n = 24, 17.91%), FLT3-ITD (n = 21, 15.67%), DNMT3A (n = 20, 14.93%), CEBPA (single variant; n = 14, 10.45%), TET2 (n = 14, 10.45%), and NRAS (n = 14, 10.45%). The patients were also divided into low risk, intermediate risk and high risk groups based on their chromosomal karyotypes. The mutational rates for genes in different groups have varied, with 19 patients from the low risk group harboring variants of NRAS (n = 4, 21.05%), KRAS (n = 4, 21.05%), and KIT (n = 2, 10.53%); and 96 patients from the intermediate risk group harboring variants of NPM1 (n = 24, 25.00%), FLT3-ITD (n = 20, 20.83%), DNMT3A (n = 18, 18.75%), CEBPA (n = 12, 12.50%), and TET2 genes (n = 12, 12.50%). The mutational frequencies for the 19 patients from the high risk group were ASXL1 (n = 7, 21.05%), NRAS (n = 3, 15.97%), TP53 (n = 3, 15.79%), and EZH2 (n = 2, 10.53%). A significant difference was found in the frequencies of KIT, NPM1, FLT3-ITD, DNMT3A, and ASXL1 gene variants among the low-risk, medium-risk, and high-risk groups. CONCLUSION: AML patients have a high frequency for genetic variants, with 76.1% harboring at least one variant. The frequency of genetic variants have varied among patients with different chromosomal karyotypes, and there are apparent dominant variants. KIT, NPM1, FLT3-ITD, DNMT3A, and ASXL1 may be used as prognostic factors for evaluating their prognosis.

STEEL enables high-resolution delineation of spatiotemporal transcriptomic data.

Advances in spatial transcriptomics enlarge the use of single cell technologies to unveil the expression landscape of the tissues with valuable spatial context. Here, we propose an unsupervised and manifold learning-based algorithm, Spatial Transcriptome based cEll typE cLustering (STEEL), which identifies domains from spatial transcriptome by clustering beads exhibiting both highly similar gene expression profiles and close spatial distance in the manner of graphs. Comprehensive evaluation of STEEL on spatial transcriptomic datasets from 10X Visium platform demonstrates that it not only achieves a high resolution to characterize fine structures of mouse brain but also enables the integration of multiple tissue slides individually analyzed into a larger one. STEEL outperforms previous methods to effectively distinguish different cell types/domains of various tissues on Slide-seq datasets, featuring in higher bead density but lower transcript detection efficiency. Application of STEEL on spatial transcriptomes of early-stage mouse embryos (E9.5-E12.5) successfully delineates a progressive development landscape of tissues from ectoderm, mesoderm and endoderm layers, and further profiles dynamic changes on cell differentiation in heart and other organs. With the advancement of spatial transcriptome technologies, our method will have great applicability on domain identification and gene expression atlas reconstruction.

Gut microbiota dysbiosis associated with different types of demyelinating optic neuritis in patients.

BACKGROUND: Demyelinating optic neuritis (DON) causes rapid vision loss in young and middle-aged people. The limited efficacy of treatment and the toxic side effects of drugs significantly affect the quality of life of patients with DON. Therefore, DON pathogenesis has always been a research hotspot in terms of prevention and treatment. Studies have suggested that gut microbiota imbalances may be involved in autoimmune disease development via the modulation of multiple inflammatory cytokines and anti-inflammatory metabolites. Therefore, this study aims to explore gut microbiota differences between healthy controls (HCs) and patients with DON. METHODS: A total of 54 patients with DON and 41 HCs were recruited. Fecal and blood samples were collected before and after intravenous methylprednisolone pulse (IVMP) treatment. The Shannon index, gut microbiota structure, and differential bacteria were evaluated and compared. RESULTS: The Shannon diversity index was decreased in patients with DON (p < 0.001) but was higher after IVMP treatment (p < 0.05). In patients with DON, Blautia, Escherichia-Shigella, and Ruminococcus showed higher abundances, whereas Bacteroides, Faecalibacterium, Roseburia, Parabacteroides, Romboutsia, and Alistipes showed lower abundances compared to that in the HCs. After IVMP treatment, the Shannon index of the myelin oligodendrocyte glycoprotein-immunoglobulin G (+) (MOG-IgG (+)) and both aquaporin-4 (AQP4)-IgG (-) and MOG-IgG (-) groups increased (p < 0.05). Bacteroides was negatively correlated with interleukin (IL)-21, IL-17E, and tumor necrosis factor-α levels (p < 0.05, r = -0.54; p < 0.05, r= -0.50; p < 0.05, r =-0.55, respectively). Escherichia was positively correlated with macrophage inflammatory protein-3α (p < 0.05, r = 0.51). Alistipes was negatively correlated with soluble CD40 ligand (p < 0.05, r = -0.52). CONCLUSION: The gut microbiota differed significantly between patients with DON and HCs; however, IVMP treatment may restore gut microbiota diversity and structure in patients with DON. Moreover, gut microbiota changes may play a role in DON pathogenesis.

A novel peptide protects against diet-induced obesity by suppressing appetite and modulating the gut microbiota.

OBJECTIVE: The obesity epidemic and its metabolic complications continue to be a major global public health threat with limited effective treatments, especially drugs that can be taken orally. Peptides are a promising class of molecules that have gained increased interest for their applications in medicine and biotechnology. In this study, we focused on looking for peptides that can be administrated orally to treat obesity and exploring its mechanisms. DESIGN: Here, a 9-amino-acid peptide named D3 was designed and administered orally to germ-free (GF) mice and wild-type (WT) mice, rats and macaques. The effects of D3 on body weight and other basal metabolic parameters were evaluated. The effects of D3 on gut microbiota were evaluated using 16S rRNA amplicon sequencing. To identify and confirm the mechanisms of D3, transcriptome analysis of ileum and molecular approaches on three animal models were performed. RESULTS: A significant body weight reduction was observed both in WT (12%) and GF (9%) mice treated with D3. D3 ameliorated leptin resistance and upregulated the expression of uroguanylin (UGN), which suppresses appetite via the UGN-GUCY2C endocrine axis. Similar effects were also found in diet-induced obese rat and macaque models. Furthermore, the abundance of intestinal Akkermansia muciniphila increased about 100 times through the IFNγ-Irgm1 axis after D3 treatment, which may further inhibit fat absorption by downregulating Cd36. CONCLUSION: Our results indicated that D3 is a novel drug candidate for counteracting diet-induced obesity as a non-toxic and bioactive peptide. Targeting the UGN-GUCY2C endocrine axis may represent a therapeutic strategy for the treatment of obesity.

Transcriptional memory of gene expression across generations participates in transgenerational plasticity of field pennycress in response to cadmium stress.

Transgenerational plasticity (TGP) occurs when maternal environments influence the expression of traits in offspring, and in some cases may increase fitness of offspring and have evolutionary significance. However, little is known about the extent of maternal environment influence on gene expression of offspring, and its relationship with trait variations across generations. In this study, we examined TGP in the traits and gene expression of field pennycress (Thlaspi arvense) in response to cadmium (Cd) stress. In the first generation, along with the increase of soil Cd concentration, the total biomass, individual height, and number of seeds significantly decreased, whereas time to flowering, superoxide dismutase (SOD) activity, and content of reduced glutathione significantly increased. Among these traits, only SOD activity showed a significant effect of TGP; the offspring of Cd-treated individuals maintained high SOD activity in the absence of Cd stress. According to the results of RNA sequencing and bioinformatic analysis, 10,028 transcripts were identified as Cd-responsive genes. Among them, only 401 were identified as transcriptional memory genes (TMGs) that maintained the same expression pattern under normal conditions in the second generation as in Cd-treated parents in the first generation. These genes mainly participated in Cd tolerance-related processes such as response to oxidative stress, cell wall biogenesis, and the abscisic acid signaling pathways. The results of weighted correlation network analysis showed that modules correlated with SOD activity recruited more TMGs than modules correlated with other traits. The SOD-coding gene CSD2 was found in one of the modules correlated with SOD activity. Furthermore, several TMGs co-expressed with CSD2 were hub genes that were highly connected to other nodes and critical to the network's topology; therefore, recruitment of TMGs in offspring was potentially related to TGP. These findings indicated that, across generations, transcriptional memory of gene expression played an important role in TGP. Moreover, these results provided new insights into the trait evolution processes mediated by phenotypic plasticity.

Colorectal Cancer Patient-Derived 2D and 3D Models Efficiently Recapitulate Inter- and Intratumoral Heterogeneity.

Pre-existing drug resistance and tumorigenicity of cancer cells are highly correlated with therapeutic failure and tumor growth. However, current cancer models are limited in their application to the study of intratumor functional heterogeneity in personalized oncology. Here, an innovative two-dimensional (2D) and three-dimensional (3D) model for patient-derived cancer cells (PDCCs) and air-liquid interface (ALI) organotypic culture is established from colorectal cancer (CRC). The PDCCs recapitulate the genomic landscape of their parental tumors with high efficiency, high proliferation rate, and long-term stability, while corresponding ALI organotypic cultures retain histological architecture of their original tumors. Interestingly, both 2D and 3D models maintain the transcriptomic profile of the corresponding primary tumors and display the same trend in response to 5-Fluoruracil, regardless of their difference in gene expression profiles. Furthermore, single-cell-derived clones() are efficiently established and pre-existing drug-resistant clones and highly tumorigenic clones within individual CRC tumors are identified. It is found that tumorigenic cancer cells do not necessarily possess the stem cells characteristics in gene expression. This study provides valuable platform and resource for exploring the molecular mechanisms underlying the pre-existing drug resistance and tumorigenicity in cancer cells, as well as for developing therapeutic targets specifically for pre-existing drug-resistant or highly tumorigenic clones.

Exploring the cellular landscape of circular RNAs using full-length single-cell RNA sequencing.

Previous studies have demonstrated the highly specific expression of circular RNAs (circRNAs) in different tissues and organisms, but the cellular architecture of circRNA has never been fully characterized. Here, we present a collection of 171 full-length single-cell RNA-seq datasets to explore the cellular landscape of circRNAs in human and mouse tissues. Through large-scale integrative analysis, we identify a total of 139,643 human and 214,747 mouse circRNAs in these scRNA-seq libraries. We validate the detected circRNAs with the integration of 11 bulk RNA-seq based resources, where 216,602 high-confidence circRNAs are uniquely detected in the single-cell cohort. We reveal the cell-type-specific expression pattern of circRNAs in brain samples, developing embryos, and breast tumors. We identify the uniquely expressed circRNAs in different cell types and validate their performance in tumor-infiltrating immune cell composition deconvolution. This study expands our knowledge of circRNA expression to the single-cell level and provides a useful resource for exploring circRNAs at this unprecedented resolution.

Unfavourable intrauterine environment contributes to abnormal gut microbiome and metabolome in twins.

OBJECTIVE: Fetal growth restriction (FGR) is a devastating pregnancy complication that increases the risk of perinatal mortality and morbidity. This study aims to determine the combined and relative effects of genetic and intrauterine environments on neonatal microbial communities and to explore selective FGR-induced gut microbiota disruption, metabolic profile disturbances and possible outcomes. DESIGN: We profiled and compared the gut microbial colonisation of 150 pairs of twin neonates who were classified into four groups based on their chorionicity and discordance of fetal birth weight. Gut microbiota dysbiosis and faecal metabolic alterations were determined by 16S ribosomal RNA and metagenomic sequencing and metabolomics, and the long-term effects were explored by surveys of physical and neurocognitive development conducted after 2~3 years of follow-up. RESULTS: Adverse intrauterine environmental factors related to selective FGR dominate genetics in their effects of elevating bacterial diversity and altering the composition of early-life gut microbiota, and this effect is positively related to the severity of selective FGR in twins. The influence of genetic factors on gut microbes diminishes in the context of selective FGR. Gut microbiota dysbiosis in twin neonates with selective FGR and faecal metabolic alterations features decreased abundances of Enterococcus and Acinetobacter and downregulated methionine and cysteine levels. Correlation analysis indicates that the faecal cysteine level in early life is positively correlated with the physical and neurocognitive development of infants. CONCLUSION: Dysbiotic microbiota profiles and pronounced metabolic alterations are associated with selective FGR affected by adverse intrauterine environments, emphasising the possible effects of dysbiosis on long-term neurobehavioural development.

Clinical efficacy evaluation of 595-nm pulsed dye lasers, microwave tissue coagulation, and liquid nitrogen cryotherapy in the treatment of verruca vulgaris in children.

OBJECTIVE: To evaluate the clinical efficacy and safety of 595-nm pulsed dye laser in the treatment of verruca vulgaris in children and to compare the efficacy of this method against two other methods, microwave tissue coagulation and liquid nitrogen cryotherapy. METHODS: A total of 90 children being treated in the dermatology outpatient department of our hospital from 2019 to 2021 were selected and divided into three groups: the treatment group A (n = 30, treated with a 595-nm pulsed dye laser), treatment group B (n = 30, treated with microwave tissue coagulation), and treatment group C (n = 30, treated with liquid nitrogen cryotherapy). All the patients in the treatment group A, treatment group B, and treatment group C were treated once every two weeks, with a maximum of six treatments. RESULTS: The response rate of the treatment group A was 93.3%, which was higher than the 83.3% rate of treatment group B and the 66.7% rate of treatment group C. The average treatment times of the treatment group A (2.45 ± 1.10) were lower than group B (3.51 ± 0.98) and group C (4.63 ± 0.96). The adverse reaction rate in the treatment group A (16.7%) was significantly lower than that in treatment group B (56.7%) and treatment group C (63.3%). The differences were statistically significant (all p < 0.05). CONCLUSIONS: The 595-nm pulsed dye laser is safe and seems to be the most effective treatment for verruca vulgaris in children. Further high-level clinical trial is warranted to verify our results.

circVAMP3 Drives CAPRIN1 Phase Separation and Inhibits Hepatocellular Carcinoma by Suppressing c-Myc Translation.

Previous studies have identified the regulatory roles of circular RNAs (circRNAs) in human cancers. However, the molecular mechanisms of circRNAs in hepatocellular carcinoma (HCC) remain largely unknown. This study screens the expression profile of circRNAs in HCC and identifies circVAMP3 as a significantly downregulated circRNA in HCC tissues. HCC patients with low circVAMP3 expression present poor prognosis. circVAMP3 negatively regulates the proliferation and metastasis of HCC cells in vitro and in vivo by driving phase separation of CAPRIN1 and promoting stress granule formation in cells, which can downregulate the protein level of Myc proto-oncogene protein by inhibiting c-Myc translation. Furthermore, circVAMP3 is widely expressed in many human tissues and is downregulated in related cancers. Therefore, circVAMP3 is a potential prognostic indicator for HCC and may serve as a therapeutic target for HCC treatment.

Screening Linear and Circular RNA Transcripts from Stress Granules.

Stress granules (SGs) are cytoplasmic ribonucleoprotein assemblies formed under stress conditions and are related to various biological processes and human diseases. Previous studies have reported the regulatory role of some proteins and linear RNAs in SG assembly. However, the relationship between circular RNAs and SGs has not been discovered. Here, we screened both linear and circular RNAs in SGs using improved total RNA sequencing of purified SG cores in mammalian cells and identified circular transcripts specifically localized in SGs. Circular RNAs with higher SG-related RBP binding abilities are more likely to be enriched in SGs. Furthermore, some SG-enriched circular RNAs are differentially expressed in hepatocellular carcinoma and adjacent tissues. These results suggest the regulatory role of circular RNAs in SG formation and provide insights into the biological function of circular RNAs and SGs in hepatocellular carcinoma.

The hidden genomic diversity of ciliated protists revealed by single-cell genome sequencing.

BACKGROUND: Ciliated protists are a widely distributed, morphologically diverse, and genetically heterogeneous group of unicellular organisms, usually known for containing two types of nuclei: a transcribed polyploid macronucleus involved in gene expression and a silent diploid micronucleus responsible for transmission of genetic material during sexual reproduction and generation of the macronucleus. Although studies in a few species of culturable ciliated protists have revealed the highly dynamic nature of replicative and recombination events relating the micronucleus to the macronucleus, the broader understanding of the genomic diversity of ciliated protists, as well as their phylogenetic relationships and metabolic potential, has been hampered by the inability to culture numerous other species under laboratory conditions, as well as the presence of symbiotic bacteria and microalgae which provide a challenge for current sequencing technologies. Here, we optimized single-cell sequencing methods and associated data analyses, to effectively remove contamination by commensal bacteria, and generated high-quality genomes for a number of Euplotia species. RESULTS: We obtained eight high-quality Euplotia genomes by using single-cell genome sequencing techniques. The genomes have high genomic completeness, with sizes between 68 and 125 M and gene numbers between 14K and 25K. Through comparative genomic analysis, we found that there are a large number of gene expansion events in Euplotia genomes, and these expansions are closely related to the phenotypic evolution and specific environmental adaptations of individual species. We further found four distinct subgroups in the genus Euplotes, which exhibited considerable genetic distance and relative lack of conserved genomic syntenies. Comparative genomic analyses of Uronychia and its relatives revealed significant gene expansion associated with the ciliary movement machinery, which may be related to the unique and strong swimming ability. CONCLUSIONS: We employed single-cell genomics to obtain eight ciliate genomes, characterized the underestimated genomic diversity of Euplotia, and determined the divergence time of representative species in this subclass for the first time. We also further investigated the extensive duplication events associated with speciation and environmental adaptation. This study provides a unique and valuable resource for understanding the evolutionary history and genetic diversity of ciliates.

Translocation of vaginal microbiota is involved in impairment and protection of uterine health.

The vaginal and uterine microbiota play important roles in the health of the female reproductive system. However, the interactions among the microbes in these two niches and their effects on uterine health remain unclear. Here we profile the vaginal and uterine microbial samples of 145 women, and combine with deep mining of public data and animal experiments to characterize the microbial translocation in the female reproductive tract and its role in modulating uterine health. Synchronous variation and increasing convergence of the uterine and vaginal microbiome with advancing age are shown. We also find that transplanting certain strains of vaginal bacteria into the vagina of rats induces or reduces endometritis-like symptoms, and verify the damaging or protective effects of certain vaginal bacteria on endometrium. This study clarifies the interdependent relationship of vaginal bacterial translocation with uterine microecology and endometrial health, which will undoubtedly increase our understanding of female reproductive health.

miR-92b-3p Regulates Cell Cycle and Apoptosis by Targeting CDKN1C, Thereby Affecting the Sensitivity of Colorectal Cancer Cells to Chemotherapeutic Drugs.

Colorectal cancer (CRC) is the third most common malignant tumor in the world and the second leading cause of cancer death. Multidrug resistance (MDR) has become a major obstacle in the clinical treatment of CRC. The clear molecular mechanism of MDR is complex, and miRNAs play an important role in drug resistance. This study used small RNAomic screens to analyze the expression profiles of miRNAs in CRC HCT8 cell line and its chemoresistant counterpart HCT8/T cell line. It was found that miR-92b-3p was highly expressed in HCT8/T cells. Knockdown of miR-92b-3p reversed the resistance of MDR HCT8/T cells to chemotherapeutic drugs in vitro and in vivo. Paclitaxel (PTX, a chemotherapy medication) could stimulate CRC cells to up-regulate miR-92b-3p expression and conferred cellular resistance to chemotherapeutic drugs. In studies on downstream molecules, results suggested that miR-92b-3p directly targeted Cyclin Dependent Kinase Inhibitor 1C (CDKN1C, which encodes a cell cycle inhibitor p57Kip2) to inhibit its expression and regulate the sensitivity of CRC cells to chemotherapeutic drugs. Mechanism study revealed that the miR-92b-3p/CDKN1C axis exerted a regulatory effect on the sensitivity of CRC cells via the regulation of cell cycle and apoptosis. In conclusion, these findings showed that miR-92b-3p/CDKN1C was an important regulator in the development of drug resistance in CRC cells, suggesting its potential application in drug resistance prediction and treatment.

Autophagy inhibition and microRNA­199a­5p upregulation in paclitaxel­resistant A549/T lung cancer cells.

Multidrug resistance (MDR) is one of the major reasons for the clinical failure of cancer chemotherapy. Autophagy activation serves a crucial role in MDR. However, the specific molecular mechanism linking autophagy with MDR remains unknown. The results of the present study demonstrated that autophagy was inhibited and microRNA (miR)­199a­5p levels were upregulated in MDR model lung cancer cells (A549/T and H1299/T) compared with those in the parental cell lines. Paclitaxel (PTX) treatment increased the expression levels of miR­199a­5p in parental lung cancer cells compared with those in PTX­untreated cells, and these expression levels were negatively correlated with PTX sensitivity of the cells. miR­199a­5p knockdown in A549/T cells induced autophagy and resensitized cells to multiple chemotherapeutic drugs including PTX, taxotere, topotecan, SN38, oxaliplatin and vinorelbine. By contrast, miR­199a­5p overexpression in A549 cells suppressed autophagy and desensitized cells to these chemotherapeutic drugs. Mechanistically, the results of the present study demonstrated that miR­199a­5p blocked autophagy by activating the PI3K/Akt/mTOR signaling pathway and inhibiting the protein expression of autophagy­related 5. Furthermore, p62 protein was identified as a direct target of miR­199a­5p; miR­199a­5p bound to p62 mRNA to decrease its mRNA and protein expression levels. In conclusion, the results of the present study suggested that miR­199a­5p may contribute to MDR development in lung cancer cells by inhibiting autophagy and targeting p62. The regulatory effect of miR­199a­5p on autophagy may provide novel insights for future multidrug­resistant lung cancer chemotherapy.

Binding of RNA m6A by IGF2BP3 triggers chemoresistance of HCT8 cells via upregulation of ABCB1.

The overexpression of ATP-binding cassette transporters subfamily B member 1 (ABCB1) is known to be the primary trigger of multidrug resistance (MDR) in colorectal cancer (CRC), leading to chemotherapy failure. However, factors that regulate chemoresistance in CRC cells are largely unknown. To identify proteins involved in MDR in CRC, we used proteomics and transcriptomics approaches to analyze HCT8/T cells and parental HCT8 cells. Results showed that the expression of insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) was upregulated in HCT8/T cells, and siIGF2BP3 remarkably elevated the sensitivity of HCT8/T cells to DOX. Overexpression of IGF2BP3 promoted ABCB1 expression, and reduced the sensitivity to ABCB1 substrates. Conversely, knockdown of IGF2BP3 reduced ABCB1 expression, and increased the sensitivity to ABCB1 substrates in vitro and in vivo. This phenomeon was further confirmed by the strong association of IGF2BP3 and ABCB1 expression with DOX sensitivity. Mechanistically, IGF2BP3, as a N6-methyladenosine (m6A) reader, directly bound to the m6A-modified region of ABCB1 mRNA, thereby promoting the stability and expression of ABCB1 mRNA. Overall, the results showed that IGF2BP3 bound to the m6A modification region of ABCB1 mRNA, and conferred chemoresistance in CRC cells via upregulation of ABCB1. These findings suggest that IGF2BP3 might be a potential biomarker for predicting the development of MDR in CRC. Targeting IGF2BP3 might be an important chemotherapeutic strategy for preventing MDR development in CRC.