Transcriptomic and morphological maturation of human astrocytes in cerebral organoids.

Cerebral organoids (CerOrgs) derived from human induced pluripotent stem cells (iPSCs) are a valuable tool to study human astrocytes and their interaction with neurons and microglia. The timeline of astrocyte development and maturation in this model is currently unknown and this limits the value and applicability of the model. Therefore, we generated CerOrgs from three healthy individuals and assessed astrocyte maturation after 5, 11, 19, and 37 weeks in culture. At these four time points, the astrocyte lineage was isolated based on the expression of integrin subunit alpha 6 (ITGA6). Based on the transcriptome of the isolated ITGA6-positive cells, astrocyte development started between 5 and 11 weeks in culture and astrocyte maturation commenced after 11 weeks in culture. After 19 weeks in culture, the ITGA6-positive astrocytes had the highest expression of human mature astrocyte genes, and the predicted functional properties were related to brain homeostasis. After 37 weeks in culture, a subpopulation of ITGA6-negative astrocytes appeared, highlighting the heterogeneity within the astrocytes. The morphology shifted from an elongated progenitor-like morphology to the typical bushy astrocyte morphology. Based on the morphological properties, predicted functional properties, and the similarities with the human mature astrocyte transcriptome, we concluded that ITGA6-positive astrocytes have developed optimally in 19-week-old CerOrgs.

Long noncoding RNA SNHG3 interacts with microRNA-502-3p to mediate ITGA6 expression in liver hepatocellular carcinoma.

SNHG3, a long noncoding RNA (lncRNA), has been linked to poor outcomes in patients with liver hepatocellular carcinoma (LIHC). In this study, we found that SNHG3 was overexpressed in LIHC and associated with poor outcomes in patients with LIHC. Functional assays, including colony formation, spheroid formation, and in vivo assays showed that SNHG3 promoted stemness of cancer stem cells (CSC) and tumor growth in vivo by interacting with microRNA-502-3p (miR-502-3p). miR-502-3p inhibitor repressed the tumor-suppressing effects of SNHG3 depletion. Finally, by RNA pull-down, dual-luciferase reporter assay, m6A methylation level detection, and m6A-IP-qPCR assays, we found that miR-502-3p targeted YTHDF3 to regulate the translation of integrin alpha-6 (ITGA6) and targeted HBXIP to inhibit the m6A modification of ITGA6 through methyltransferase-like 3 (METTL3). Our study revealed that SNHG3 controls the YTHDF3/ITGA6 and HBXIP/METTL3/ITGA6 pathways by repressing miR-502-3p expression to sustain the self-renewal properties of CSC in LIHC.

Single-cell transcriptomic sequencing data reveal aberrant DNA methylation in SMAD3 promoter region in tumor-associated fibroblasts affecting molecular mechanism of radiosensitivity in non-small cell lung cancer.

OBJECTIVE: Non-small cell lung cancer (NSCLC) often exhibits resistance to radiotherapy, posing significant treatment challenges. This study investigates the role of SMAD3 in NSCLC, focusing on its potential in influencing radiosensitivity via the ITGA6/PI3K/Akt pathway. METHODS: The study utilized gene expression data from the GEO database to identify differentially expressed genes related to radiotherapy resistance in NSCLC. Using the GSE37745 dataset, prognostic genes were identified through Cox regression and survival analysis. Functional roles of target genes were explored using Gene Set Enrichment Analysis (GSEA) and co-expression analyses. Gene promoter methylation levels were assessed using databases like UALCAN, DNMIVD, and UCSC Xena, while the TISCH database provided insights into the correlation between target genes and CAFs. Experiments included RT-qPCR, Western blot, and immunohistochemistry on NSCLC patient samples, in vitro studies on isolated CAFs cells, and in vivo nude mouse tumor models. RESULTS: Fifteen key genes associated with radiotherapy resistance in NSCLC cells were identified. SMAD3 was recognized as an independent prognostic factor for NSCLC, linked to poor patient outcomes. High expression of SMAD3 was correlated with low DNA methylation in its promoter region and was enriched in CAFs. In vitro and in vivo experiments confirmed that SMAD3 promotes radiotherapy resistance by activating the ITGA6/PI3K/Akt signaling pathway. CONCLUSION: High expression of SMAD3 in NSCLC tissues, cells, and CAFs is closely associated with poor prognosis and increased radiotherapy resistance. SMAD3 is likely to enhance radiotherapy resistance in NSCLC cells by activating the ITGA6/PI3K/Akt signaling pathway.

Distinctive field effects of smoking and lung cancer case-control status on bronchial basal cell growth and signaling.

RATIONAL: Basal cells (BCs) are bronchial progenitor/stem cells that can regenerate injured airway that, in smokers, may undergo malignant transformation. As a model for early stages of lung carcinogenesis, we set out to characterize cytologically normal BC outgrowths from never-smokers and ever-smokers without cancers (controls), as well as from the normal epithelial "field" of ever-smokers with anatomically remote cancers, including lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) (cases). METHODS: Primary BCs were cultured and expanded from endobronchial brushings taken remote from the site of clinical or visible lesions/tumors. Donor subgroups were tested for growth, morphology, and underlying molecular features by qRT-PCR, RNAseq, flow cytometry, immunofluorescence, and immunoblot. RESULTS: (a) the BC population includes epithelial cell adhesion molecule (EpCAM) positive and negative cell subsets; (b) smoking reduced overall BC proliferation corresponding with a 2.6-fold reduction in the EpCAMpos/ITGA6 pos/CD24pos stem cell fraction; (c) LUSC donor cells demonstrated up to 2.8-fold increase in dysmorphic BCs; and (d) cells procured from LUAD patients displayed increased proliferation and S-phase cell cycle fractions. These differences corresponded with: (i) disparate NOTCH1/NOTCH2 transcript expression and altered expression of potential downstream (ii) E-cadherin (CDH1), tumor protein-63 (TP63), secretoglobin family 1a member 1 (SCGB1A1), and Hairy/enhancer-of-split related with YRPW motif 1 (HEY1); and (iii) reduced EPCAM and increased NK2 homeobox-1 (NKX2-1) mRNA expression in LUAD donor BCs. CONCLUSIONS: These and other findings demonstrate impacts of donor age, smoking, and lung cancer case-control status on BC phenotypic and molecular traits and may suggest Notch signaling pathway deregulation during early human lung cancer pathogenesis.

miR-186-5p inhibits the progression of oral squamous cell carcinoma by targeting ITGA6 to impair the activity of the PI3K/AKT pathway.

BACKGROUND: microRNAs (miRNAs) are pivotal regulators of multiple biological processes. miR-186-5p functions as a tumor suppressor in a variety of cancers and promotes the malignant proliferation of oral squamous cell carcinoma (OSCC). This study aimed to clarify the role and regulatory mechanism of miR-186-5p in OSCC. METHODS: The levels of miR-186-5p and integrin subunit alpha 6 (ITGA6) were investigated in clinical specimens and OSCC cell lines by reverse transcription-quantitative polymerase chain reaction. The effects of miR-186-5p and ITGA6 on the cell migration, proliferation, and phosphatidylinositol 3-kinase (PI3K)/serine-threonine kinase (AKT) pathway activity were evaluated by transwell assay, cell counting kit 8 assay, and western blotting, respectively. A xenograft model was used to analyze the effect of miR-186-5p on tumor growth. Bioinformatic analyses were conducted to identify the putative targets of miR-186-5p in OSCC. RESULTS: Decreased miR-186-5p expression levels were observed in OSCC tumor tissues and cell lines. The overexpression of miR-186-5p suppressed the proliferation and migration of OSCC cells, and weakened the phosphorylation of PI3K and AKT. Moreover, the overexpression of miR-186-5p in xenograft tumor models impedes tumor growth. miR-186-5p is bound to ITGA6 and negatively related to ITGA6 expression in tumor tissues. The forced expression of ITGA6 promoted OSCC cell proliferation and migration and enhanced the phosphorylation levels of PI3K and AKT, while additional miR-186-5p enrichment partly abolished these effects. CONCLUSION: miR-186-5p binds to ITGA6 to impair the activity of the PI3K/AKT signaling pathway, thereby blocking the development of OSCC. This study provides insight to understand the pathogenesis of OSCC.

Effect of different iodine levels on the DNA methylation of PRKAA2, ITGA6, THEM4 and PRL genes in PI3K-AKT signaling pathway and population-based validation from autoimmune thyroiditis patients.

PURPOSE: Autoimmune thyroiditis (AIT) is one of the most common autoimmune endocrine diseases. The currently recognized causes are genetic susceptibility, environmental factors and immune disorders. It is important to clarify the pathogenesis for the prevention, diagnosis, treatment of AIT and scientific iodine supplementation. This study analyzed the DNA methylation levels of PRKAA2, ITGA6, PRL and THEM4 genes related to PI3K-AKT signaling pathway, compared the DNA methylation levels between cases and controls from different water iodine levels in Shandong Province of China, and evaluated the contribution of PI3K-AKT signaling pathway-related genes in AIT. METHODS: A total of 176 adult AIT patients were included from three different water iodine areas, and 176 healthy controls were included according to gender, age and BMI. According to the results of the Illumina Methylation 850 K BeadChip in our previous research, the significant methylation differences of genes on the PI3K-AKT signaling pathway related to AIT were determined. The MethylTarget™ assay was used to detect the methylation levels of the target genes, and real-time PCR experiments were used to verify the mRNA expression levels. RESULTS: Compared with the control group, PRKAA2_3 and 15 CpG sites were hyper-methylated. ITGA6 gene and 2 CpG sites were hypo-methylated in AIT cases. The mRNA expression of ITGA6 gene was negatively correlated with the DNA methylation levels of ITGA6 gene and 2 CpG sites. Compared with cases and controls in areas with different water iodine levels, methylation differences were mainly in PRKAA2 and ITGA6 genes. The methylation levels of PRKAA2_1 and PRKAA2_3 were positively correlated with age. The methylation levels of PRL and THEM4 genes were negatively correlated with age. The methylation level of PRKAA2_3 was positively correlated with FT4. CONCLUSION: In summary, we identified aberrant DNA methylation levels of PRKAA2 and ITGA6 genes related to PI3K-AKT signaling pathway in the blood of AIT patients. Both iodine supplementation after long-term iodine deficiency and iodine excess can affect the DNA methylation levels of PRKAA2 and ITGA6 genes, and the former affects more obviously. In ITGA6 gene, this aberrant epigenetic modification is associated with the increased mRNA expression.

The transcriptional repressor BCL11A promotes breast cancer metastasis.

The phenotypes of each breast cancer subtype are defined by their transcriptomes. However, the transcription factors that regulate differential patterns of gene expression that contribute to specific disease outcomes are not well understood. Here, using gene silencing and overexpression approaches, RNA-Seq, and splicing analysis, we report that the transcription factor B-cell leukemia/lymphoma 11A (BCL11A) is highly expressed in triple-negative breast cancer (TNBC) and drives metastatic disease. Moreover, BCL11A promotes cancer cell invasion by suppressing the expression of muscleblind-like splicing regulator 1 (MBNL1), a splicing regulator that suppresses metastasis. This ultimately increases the levels of an alternatively spliced isoform of integrin-α6 (ITGA6), which is associated with worse patient outcomes. These results suggest that BCL11A sustains TNBC cell invasion and metastatic growth by repressing MBNL1-directed splicing of ITGA6 Our findings also indicate that BCL11A lies at the interface of transcription and splicing and promotes aggressive TNBC phenotypes.

PRR34-AS1 sponges miR-498 to facilitate TOMM20 and ITGA6 mediated tumor progression in HCC.

BACKGROUND: The researches on PRR34 antisense RNA 1 (PRR34-AS1) have been limited. Both translocase of outer mitochondrial membrane 20 (TOMM20) and integrin subunit alpha 6 (ITGA6) have been proven to facilitate cancer progression. Whether TOMM20 or ITGA6 affects hepatocellular carcinoma (HCC) progression has never been investigated. Some studies showed that microRNA 498 (miR-498) can suppress HCC progression. Additionally, the influence of ceRNA network (including PRR34-AS1, miR-498, and TOMM20 or ITGA6) on HCC progression has not been inquired into yet. METHODS: The knockdown or overexpression efficiency was validated via RT-qPCR. Also, RT-qPCR was applied to detect the expression of PRR34-AS1, miR-498, TOMM20, and ITGA6. Cell proliferation in HCC was tested via EdU and colony formation assays. Transwell assays presented the migratory and invasive capabilities of HCC cells. Subcellular fractionation and FISH assays showed the subcellular localization of PRR34-AS1. RNA pull down and luciferase reporter assays were performed to explore whether miR-498 combines with PRR34-AS1, TOMM20 or ITGA6. Western blot was conducted to detect protein expression. Rescue experiments were conducted to verify the relationship among PRR34-AS1, miR-498, TOMM20, and ITGA6. RESULTS: The expressions of PRR34-AS1, TOMM20, and ITGA6 were markedly high in HCC cell lines while miR-498 was lowly expressed. PRR34-AS1, TOMM20, and ITGA6 promoted HCC progression while miR-498 suppressed cell proliferation, migration, and invasion in HCC. Furthermore, PRR34-AS1, TOMM20, and ITGA6 combined with miR-498. CONCLUSION: PRR34-AS1 facilitates HCC progression by regulating miR-498/TOMM20/ITGA6 axis.

MicroRNA-377-3p alleviates IL-1ß-caused chondrocyte apoptosis and cartilage degradation in osteoarthritis in part by downregulating ITGA6.

Increasing evidence indicates that altered expression of microRNAs (miRNAs) is associated with osteoarthritis (OA) progression. In our study, we demonstrated that miR-377-3p is underexpressed in OA-affected cartilage and IL-1ß-treated chondrocytes. Overexpression of miR-377-3p enhanced chondrocyte proliferation and restrained apoptosis and signs of cartilage matrix degradation and of an inflammatory response. Furthermore, ITGA6 was identified as a target gene of miR-377-3p. The latter was found to directly bind to the 3' untranslated region (3'UTR) of ITGA6 mRNA and downregulate ITGA6. In addition, ITGA6 expression was high in OA-affected tissues and negatively correlated with miR-77-3p expression. Overexpression of ITGA6 reversed the effects of miR-377-3p on IL-1ß-caused chondrocyte apoptosis, cartilage matrix degradation, and the inflammatory response. Moreover, bioinformatic analysis and a luciferase assay indicated that miR-377-3p expression is regulated by long noncoding RNA NEAT1, which binds to miR-377-3p and inactivates it. We showed that NEAT1 was highly expressed in OA-affected cartilage, negatively correlated with miR-377-3p levels, and positively correlated with ITGA6 levels. These findings provide information for the development of future treatments of OA, suggesting that miR-377-3p may be a therapeutic target in OA.

Exosomal miR-126 blocks the development of non-small cell lung cancer through the inhibition of ITGA6.

BACKGROUND: Exosomes, emerging mediators of intercellular communication, are reported to transfer certain non-coding RNAs, such as microRNAs (miRNAs), which play a crucial role in cancer progression. The objective of this study was to determine the function of exosomal miR-126 and provide a novel mechanism of miR-126 action in NSCLC. METHODS: The morphology of exosomes was identified by transmission electron microscope (TEM), and the exosomal surface markers were quantified by western blot. The expression of miR-126 and integrin alpha-6 (ITGA6) mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR), and ITGA6 protein expression was determined by western blot. For functional analyses, cell proliferation was assessed by colony formation assay and MTT assay. Cell cycle and cell apoptosis were monitored using flow cytometry assay. Cell migration and invasion were determined by transwell assay. ITGA6 was predicted as a target of miR-126 by bioinformatics analysis, which was verified by dual-luciferase reporter assay. The role of exosomal miR-126 in vivo was determined by Xenograft tumor models. RESULTS: NSCLC serum-derived exosomes harbored low expression of miR-126 and promoted NSCLC cell proliferation, cell cycle progression, cell migration and invasion. NSCLC serum-derived exosomes loaded with miR-126 mimic inhibits NSCLC cell proliferation, colony formation, migration and invasion but induced cell cycle arrest and apoptosis. Besides, exosomal miR-126 also blocked tumor growth in vivo. In mechanism, ITGA6 was a target of miR-126, and exosomal miR-126 weakened these NSCLC cell malignant behaviors and inhibited tumor growth by degrading the expression of ITGA6. CONCLUSION: Exosomal miR-126 blocked the progression of NSCLC through the mediation of its target gene ITGA6, and exosomal miR-126 might be used as a promising biomarker for NSCLC therapy.

Long noncoding RNA opa-interacting protein 5 antisense transcript 1 promotes proliferation and invasion through elevating integrin α6 expression by sponging miR-143-3p in cervical cancer.

An increasing number of studies have shown that long noncoding RNAs (lncRNAs) play important roles in cervical cancer (CC) progression. However, the roles and underlying mechanisms of lncRNA opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) involved in the CC remain unclear. In the current study, we found that lncRNA OIP5-AS1 was upregulated in CC tissues and cell lines. High OIP5-AS1 expression was significantly correlated with advanced International Federation of Gynecology and Obstetrics (FIGO) stage, lymph node metastasis, and poor overall survival of patients with CC. Using in vitro function assays, we showed that OIP5-AS1 suppression significantly decreased the proliferation, colony formation, and invasion ability of CC cells. Moreover, we revealed that OIP5-AS1 could act as a competing endogenous RNA of miR-143-3p to regulate the ITGA6 expression. Rescue assays showed that miR-143-3p inhibitors or ITGA6 overexpression could reverse the inhibitory effects of OIP5-AS1 suppression on the proliferation and invasion in CC cells. In addition, OIP5-AS1 suppression reduced tumor growth in vivo. In conclusion, we demonstrated that OIP5-AS1 promoted proliferation and invasion of CC cells via increasing the ITGA6 expression by sponging miR-143-3p, which might be an effective therapeutic target for the treatment of patients with CC.

MicroRNA-143-3p inhibits colorectal cancer metastases by targeting ITGA6 and ASAP3.

MicroRNAs, which regulate mRNAs, operate through a variety of signaling pathways to participate in the development of colorectal cancer (CRC). In this study, we found that microRNA (miR)-143-3p expression was significantly lower in both CRC and liver metastatic CRC tissues from liver compared with normal colonic tissues. Functional assays showed that miR-143-3p inhibited CRC cell invasion and migration in vitro. Using a bioinformatics approach, we identified miR-143-3p target mRNAs. Among the candidate targets, only the expression of integrin alpha 6 (ITGA6) and ArfGAP with the SH3 domain and ankyrin repeat and PH domain 3 (ASAP3) were significantly reduced by miR-143-3p mimics as examined by western blot, and the metastasis potential of CRC cells was attenuated by endogenous ITGA6 and ASAP3 knockdown, determined by migration and invasion assays. Both ITGA6 and ASAP3 were upregulated in CRC tissues compared to normal tissues. Analysis of the relationship between clinicopathological features and ITGA6/ASAP3 protein expression in 200 patients with CRC showed a significant difference in positive ITGA6 expression between the early stage (I + II) and the advanced stage (III + IV), and ASAP3 expression levels positively correlated with metastasis in the lymph nodes. These results indicate that miR-143-3p acts as an anti-oncogene by downregulating ITGA6/ASAP3 protein expression and could offer new insight into potential therapeutic targets for CRC.

Upregulation of lncRNA ADAMTS9-AS2 Promotes Salivary Adenoid Cystic Carcinoma Metastasis via PI3K/Akt and MEK/Erk Signaling.

Neurotropic infiltrative growth and distant metastasis are the main causes of death in salivary adenoid cystic carcinoma (SACC) patients. Long noncoding RNAs (lncRNAs) are involved in many human neoplasms, however, their potential roles in SACC are unclear. In our study, we found that ADAM metallopeptidase with thrombospondin type 1 motif, 9 (ADAMTS9) antisense RNA 2 (ADAMTS9-AS2) was significantly upregulated in SACC patients with metastasis and SACC-lung metastasis (LM) cells. Moreover, ADAMTS9-AS2 expression was closely associated with the prognosis and distant metastasis in SACC patients. Next, we found that c-myc could specifically bind to the promoter of ADAMTS9-AS2 and activated its transcription. Knockdown of ADAMTS9-AS2 significantly inhibited migration and invasion of SACC cells in vitro and distant lung metastasis in vivo. Furthermore, ADAMTS9-AS2, which mainly expressed in the cytoplasm, shared microRNA (miRNA) response elements with Integrin α6 (ITGA6). Overexpression of ADAMTS9-AS2 competitively bound to miR-143-3p that inhibited ITGA6 from miRNA-mediated degradation, and thus it activated the activity of PI3K/Akt and MEK/Erk signaling and facilitated SACC metastasis. In summary, ADAMTS9-AS2 promotes migration and invasion in SACC by competing with miR-143-3p. This sheds a new insight into the regulation mechanism of ADAMTS9-AS2, and it provides a possible application for the SACC treatment.

MiR-127-3p inhibits cell growth and invasiveness by targeting ITGA6 in human osteosarcoma.

Osteosarcoma (OS) is one of the most universal malignant bone tumors that occur mostly in children and adolescents. This study aimed to investigate the roles of miR-127-3p and integrin subunit-α 6 (ITGA6) in OS proliferation, apoptosis, invasion and migration, and to explore the possible molecular mechanism and target relationship. By conducting quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, the microRNA (miRNA) and protein expressions of miR-127-3p and ITGA6 in both tissues and cells were determined. The expression of apoptosis and migration related were also detected by western blot. The target relationship between miR-127-3p and ITGA6 was predicted by TargetScan and verified by dual-luciferase reporter assay. The biological functions of miR-127-3p and ITGA6 in OS were investigated by following experiments: cell counting kit 8 (CCK-8) and colony formation assays to inspect cell proliferation, flow cytometry, and caspase 3 activity assay to examine apoptosis, and transwell and wound healing assays to analyze invasion and migration. Significant down-regulation of miR-127-3p and up-regulation of ITGA6 was found out in OS tissues and cells. ITGA6 was proved to be the downstream target gene of miR-127-3p and functioned as a tumor promotor in OS, while miR-127-3p restrained deterioration of OS by suppressing cell viability, reducing migration and invasion, and promoting apoptosis. MiR-127-3p suppressed proliferation, invasion, and migration while stimulated apoptosis of OS cells through knocking down ITGA6. © 2018 IUBMB Life, 70(5):411-419, 2018.

RFX1 maintains testis cord integrity by regulating the expression of Itga6 in male mouse embryos.

Formation and maintenance of testis cords during embryogenesis are essential for establishing testicular structure and function in adults. At least five genes (Wt1, Dhh, Sox8/Sox9, and Dax1) appear to be required for the maintenance of testis cord integrity in mice. Here, we report that RFX1 is specifically expressed in fetal Sertoli cells. Mouse embryos conditionally deficient in Rfx1 (Rfx1(flox/flox) , Amh-Cre) possessed disrupted testis cords, as the basal lamina lining was fragmented or completely absent in some areas of the testes. Spermatogenesis was blocked, leading to complete infertility. Expression of integrin alpha-6 was significantly decreased in Rfx1-deficient testes compared to control testes; indeed, luciferase and chromatin immunoprecipitation assays indicated that RFX1 directly activates transcription of Itga6 (the gene coding for integrin alpha-6). Taken together, RFX1 transcriptionally targets Itga6 in Sertoli cells, thereby, helping maintain the integrity of the basal lamina during testis cord development. Mol. Reprod. Dev. 83: 606-614, 2016. © 2016 Wiley Periodicals, Inc.

Intracellular modifiers of integrin alpha 6p production in aggressive prostate and breast cancer cell lines.

Cancer metastasis is a multi-step process in which tumor cells gain the ability to invade beyond the primary tumor and colonize distant sites. The mechanisms regulating the metastatic process confer changes to cell adhesion receptors including the integrin family of receptors. Our group previously discovered that the α6 integrin (ITGA6/CD49f) is post translationally modified by urokinase plasminogen activator (uPA) and its receptor, urokinase plasminogen activator receptor (uPAR), to form the variant ITGA6p. This variant of ITGA6 is a cleaved form of the receptor that lacks the ligand-binding domain. Although it is established that the uPA/uPAR axis drives ITGA6 cleavage, the mechanisms regulating cleavage have not been defined. Intracellular integrin dependent "inside-out" signaling is a major regulator of integrin function and the uPA/uPAR axis. We hypothesized that intracellular signaling molecules play a role in formation of ITGA6p to promote cell migration during cancer metastasis. In order to test our hypothesis, DU145 and PC3B1 prostate cancer and MDA-MB-231 breast cancer cell lines were treated with small interfering RNA targeting actin and the intracellular signaling regulators focal adhesion kinase (FAK), integrin linked kinase (ILK), and paxillin. The results demonstrated that inhibition of actin, FAK, and ILK expression resulted in significantly increased uPAR expression and ITGA6p production. Inhibition of actin increased ITGA6p, although inhibition of paxillin did not affect ITGA6p formation. Taken together, these results suggest that FAK and ILK dependent "inside-out" signaling, and actin dynamics regulate extracellular production of ITGA6p and the aggressive phenotype.

Targeted m6A demethylation of ITGA6 mRNA by a multisite dCasRx-m6A editor inhibits bladder cancer development.

INTRODUCTION: N6-methyladenosine (m6A) modification contributes to the pathogenesis and development of various cancers, including bladder cancer (BCa). In particular, integrin α6 (ITGA6) promotes BCa progression by cooperatively regulating multisite m6A modification. However, the therapeutic effect of targeting ITGA6 multisite m6A modifications in BCa remains unknown. OBJECTIVES: We aim to develop a multisite dCasRx- m6A editor for assessing the effects of the multisite dCasRx-m6A editor targeted m6A demethylation of ITGA6 mRNA in BC growth and progression. METHODS: The multisite dCasRx- m6A editor was generated by cloning. m6A-methylated RNA immunoprecipitation (meRIP), luciferase reporter, a single-base T3 ligase-based qPCR-amplification, Polysome profiling and meRIP-seq experiments were performed to determine the targeting specificity of the multisite dCasRx-m6A editor. We performed cell phenotype analysis and used in vivo mouse xenograft models to assess the effects of the multisite dCasRx-m6A editor in BC growth and progression. RESULTS: We designed a targeted ITGA6 multi-locus guide (g)RNA and established a bidirectional deactivated RfxCas13d (dCasRx)-based m6A-editing platform, comprising a nucleus-localized dCasRx fused with the catalytic domains of methyltransferase-like 3 (METTL3-CD) or α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5-CD), to simultaneously manipulate the methylation of ITGA6 mRNA at four m6A sites. The results confirmed the dCasRx-m6A editor modified m6A at multiple sites in ITGA6 mRNA, with low off-target effects. Moreover, targeted m6A demethylation of ITGA6 mRNA by the multisite dCasRx-m6A editor significantly reduced BCa cell proliferation and migration in vitro and in vivo. Furthermore, the dCasRx-ALKBH5-CD and ITGA6 multi-site gRNA delivered to 5-week-old BALB/cJNju-Foxn1nu/Nju nude mice via adeno-associated viral vectors significantly inhibited BCa cell growth. CONCLUSION: Our study proposes a novel therapeutic tool for the treatment of BC by applying the multisite dCasRx-m6A editor while highlighting its potential efficacy for treating other diseases associated with abnormal m6A modifications.

RAC1-mediated integrin alpha-6 expression in E-cadherin-deficient gastric cancer cells promotes interactions with the stroma and peritoneal dissemination.

Diffuse-type gastric cancer (DGC) is a subtype of gastric cancer that is prone to peritoneal dissemination, with poor patient prognosis. Although intercellular adhesion loss between cancer cells is a major characteristic of DGCs, the mechanism underlying the alteration in cell-to-extracellular matrix (ECM) adhesion is unclear. We investigated how DGCs progress and cause peritoneal dissemination through interactions between DGC cells and the tumour microenvironment (TME). P53 knockout and KRASG12V-expressing (GAN-KP) cells and Cdh1-deleted GAN-KP (GAN-KPC) cells were orthotopically transplanted into the gastric wall to mimic peritoneal dissemination. The GAN-KPC tumour morphology was similar to that of human DGCs containing abundant stroma. RNA sequencing revealed that pathways related to Rho GTPases and integrin-ECM interactions were specifically increased in GAN-KPC cells compared with GAN-KP cells. Notably, we found that Rac Family Small GTPase 1 (RAC1) induces Integrin Subunit Alpha 6 (ITGA6) trafficking, leading to its enrichment on the GC cell membrane. Fibroblasts activate the FAK/AKT pathway in GC cells by mediating extracellular matrix (ECM)-Itga6 interactions, exacerbating the malignant phenotype. In turn, GC cells induce abnormal expression of fibroblast collagen and its transformation into cancer-associated fibroblasts (CAFs), resulting in DGC-like subtypes. These findings indicate that Cdh1 gene loss leads to abnormal expression and changes in the subcellular localization of ITGA6 through RAC1 signalling. The latter, through interactions with CAFs, allows for peritoneal dissemination.

Integrin α6-containing extracellular vesicles promote lymphatic remodelling for pre-metastatic niche formation in lymph nodes via interplay with CD151.

Heterogeneous extracellular vesicles (EVs) from various types of tumours are acknowledged for inducing the formation of pre-metastatic "niches" in draining lymph nodes (LNs) to promote lymphatic metastasis. In order to identify the specific subpopulations of EVs involved, we performed high-resolution proteomic analysis combined with nanoflow cytometry of bladder cancer (BCa) tissue-derived EVs to identify a novel subset of tumour-derived EVs that contain integrin α6 (ITGA6+EVs) and revealed the positive correlation of ITGA6+EVs with the formation of pre-metastatic niche in draining LNs and lymphatic metastasis in multicentre clinical analysis of 820-case BCa patients. BCa-derived ITGA6+EVs induced E-selectin (SELE)-marked lymphatic remodelling pre-metastatic niche and promoted metastasis in draining LNs through delivering cargo circRNA-LIPAR to lymphatic endothelial cells in vivo and in vitro. Mechanistically, LIPAR linked ITGA6 to the switch II domain of RAB5A and sustained RAB5A GTP-bound activated state, thus maintaining the production of ITGA6+EVs loaded with LIPAR through endosomal trafficking. ITGA6+EVs targeted lymphatic vessels through ITGA6-CD151 interplay and released LIPAR to induce SELE overexpression-marked lymphatic remodelling pre-metastatic niche. Importantly, we constructed engineered-ITGA6 EVs to inhibit lymphatic pre-metastatic niche, which suppressed lymphatic metastasis and prolonged survival in preclinical models. Collectively, our study uncovers the mechanism of BCa-derived ITGA6+EVs mediating pre-metastatic niche and provides an engineered-EV-based strategy against BCa lymphatic metastasis.

ZC3H13 promotes ITGA6 m6A modification for chronic obstructive pulmonary disease progression.

Chronic obstructive pulmonary disease (COPD) is potentially fatal, and as society ages, its effects on human health are predicted to deteriorate. The potential function of m6A modifications within COPD has become a hot topic recently. This study was conducted to clarify the function and related mechanisms of the m6A methylation transferase ZC3H13 in COPD. The expression of m6A-associated protease and ITGA6 in COPD tissues was assessed using GEO data, qRT-PCR, and western blot. COPD models in cells and mice were established through cigarette smoke extract (CSE) and smoke exposure. Inflammatory marker levels were measured by ELISA, apoptosis by flow cytometry, and mRNA stability with Actinomycin D assay. m6A modification levels were checked by MeRIP-PCR. HE and Masson staining evaluated lung pathology, and alveolar lavage fluid analysis included total cell count and Giemsa staining. ZC3H13 and METTL3 were differentially expressed m6A regulators in COPD, with ZC3H13 being more significantly upregulated. Further analysis revealed the ZC3H13 expression-related differentially expressed genes (DEGs) functions were enriched in the immunoinflammatory pathway, indicating ZC3H13's involvement in COPD pathogenesis through inflammation, and immune responses. Knockdown studies in cellular and mouse models demonstrated ZC3H13's role in exacerbating COPD symptoms, including inflammation, apoptosis, and EMT, and its suppression led to significant improvements. The identification of ITGA6 as a target gene further elucidated the mechanism, showing that ZC3H13 enhances ITGA6 expression and mRNA stability through m6A modification, influencing bronchial epithelial cell inflammation and fibrosis. In conclusion, targeting ZC3H13/ITGA6 could be an underlying therapeutic approach for treating COPD.