Prostate cancer small extracellular vesicles participate in androgen-independent transformation of prostate cancer by transferring let-7a-5p.

Objectives: Androgen deprivation therapy (ADT) is a standard treatment for advanced prostate cancer (PCa). However, after 2-3 years ADT treatment, prostate cancer inevitably transits from androgen-dependent PCa (ADPC) to androgen-independent PCa (AIPC), which has a poor prognosis owing to its unclear mechanism and lack of effective therapeutic targets. Small extracellular vesicles (sEVs) play a vital role in the development of cancer. However, the role of PCa sEVs in the transformation of AIPC remains poorly understood. Materials and methods: Two different cell models were employed and compared. sEVs from ADPC cells (LNCaP) and AIPC cells (LNCaP-AI + F cells) were isolated and characterized. After co-culture of LNCaP-AI + F sEVs with LNCaP cells and of LNCaP sEVs with LNCaP-AI + F cells, androgen-independent transformation was determined respectively. Mechanically, small RNA sequencing was performed. Androgen-independent transformation was examined by the upregulation and downregulation of miRNA and downstream pathways were analyzed. Results: LNCaP-AI + F sEVs promoted the androgen-independent transformation of LNCaP cells. Interestingly, LNCaP sEVs exhibited a capacity to reverse the process.Let-7a-5p transfer was demonstrated. Furthermore, let-7a-5p overexpression promotes the androgen-independent transformation and let-7a-5p down-regulation reverses the process. Androgen receptor (AR) and PI3K/Akt pathways were identified and demonstrated by both let-7a-5p regulation and PCa sEVs coculture. Conclusions: PCa sEVs are intimately involved in the regulation of androgen-independent transformation of prostate cancer by transferring the key sEVs molecular let-7a-5p and then activating the AR and PI3K/Akt signaling pathways. Our results provide new perspectives for the development of sEVs and sEVs molecular targeted treatment approaches for AIPC patients.

Loading of "cocktail siRNAs" into extracellular vesicles via TAT-DRBD peptide for the treatment of castration-resistant prostate cancer.

Extracellular vesicles (EVs) are cell-derived, membranous nanoparticles that mediate intercellular communication by transferring biomolecules between cells. As natural vehicles, EVs may exhibit higher delivery efficiency, lower immunogenicity, and better compatibility than existing RNA carriers. A major limitation of their therapeutic use is the shortage of efficient, robust, and scalable methods to load siRNA of interest. Here, we report a novel strategy using polycationic membrane-penetrating peptide TAT to encapsulate siRNAs into EVs. Three TAT peptides were co-expressed with DRBD as 3TD fusion protein. The sequence-independent binding of DRBD facilitates multiplex genes targeting of mixed siRNAs. Functional assays for siRNA-mediated gene silencing of CRPC were performed after engineered EVs treatment. EVs were isolated using differential centrifugation from WPMY-1 cell culture medium. The increase of merged yellow fluorescence in the engineered EVs showed by TIRFM and the decrease in zeta potential absolute values certified the co-localization of siRNA with EVs, which indicated that siRNA had been successfully delivered into WPMY-1 EVs. qRT-PCR analysis revealed that the mRNA level of FLOH1, NKX3, and DHRS7 was dramatically decreased when cells were treated with engineered EVs loaded with siRNAs mixtures relative to the level of untreated cells. Western and flow cytometry results indicate that delivery of siRNA mixtures by engineered EVs can effectively downregulate AR expression and induce LNCaP-AI cell apoptosis. The uptake efficiency of the EVs and the significantly downregulated expression of three genes suggested the potential of TAT as efficient siRNA carriers by keeping the function of the cargoes.

Exosomes derived from osteogenic tumor activate osteoclast differentiation and concurrently inhibit osteogenesis by transferring COL1A1-targeting miRNA-92a-1-5p.

In patients with prostate cancer (PCa), bone lesions appear osteoblastic in radiographs; however, pathological fractures frequently occur in PCa patients, and bone resorption is observed in all metastatic lesions under histopathologic assessment. The mechanisms that balance the activities of osteoblasts and osteoclasts in PCa patients remain unclear. We unexpectedly discovered that PCa exosomes are critical mediators in the regulation of bone homeostasis that results in osteoclastic lesions and thereby promotes tumor growth in bone. We evaluated how exosomes derived from osteoblastic, osteoclastic, and mixed PCa cell lines affect osteoblast and osteoclast differentiation, revealing that all three types of PCa exosomes promoted osteoclastogenesis in vitro and induced osteolysis in vivo. Mechanistically, microRNAs (miRNAs) delivered by PCa exosomes were found to play several key roles in bone homeostasis. Among the delivered miRNAs, miR-92a-1-5p, the most abundant miRNA, downregulated type I collagen expression by directly targeting COL1A1, and thus promoting osteoclast differentiation and inhibiting osteoblastogenesis. Furthermore, PCa exosomes also markedly reduced type I collagen expression in vivo. Our findings not only offer a novel perspective on tumor bone metastasis, where-contrary to our initial hypothesis-exosomes derived from an osteoblastic tumor induce osteoclast differentiation, but also suggest potential therapeutic targets for PCa bone metastasis.

Prevalence, incidence and risk factors of strabismus in a Chinese population-based cohort of preschool children: the Nanjing Eye Study.

AIMS: To evaluate the prevalence, incidence and their related risk factors of strabismus among preschool children in China. METHODS: Children born between September 2011 and August 2012 in Yuhuatai District of Nanjing were invited to participate in the Nanjing Eye Study for a comprehensive eye examination annually since 2015. The data presented in this paper were obtained from 2015 to 2017, when these children grew from the age of 3 to 5 years. Eye examinations included visual acuity, anterior segment, posterior segment, refraction, and ocular alignment and motility. Risk factors were evaluated using univariable and multivariable logistic regression models for prevalent and incident strabismus. RESULTS: In 2015, a total of 2018 children (87.7% response rate) of 2300 eligible preschoolers completed the baseline eye examination when they were 3 years old. Among the 2018 participants, 50 had strabismus (prevalence rate, 2.48%). In multivariable analysis, prevalent strabismus was independently associated with parental strabismus history (OR=11.60, p<0.001), hyperopia (OR=6.22, p<0.001), prematurity (OR=3.07, p=0.01) and astigmatism (OR=2.15, p=0.04). Among 1766 children followed up for 2 years, 63 developed strabismus (annual incidence rate, 1.78%), of whom 57 had exotropia and 6 had esotropia. In multivariable analysis, incident strabismus was significantly associated with parental strabismus history (OR=5.55, p=0.04) and prematurity (OR=3.77, p<0.001). CONCLUSIONS: In this population-based cohort study, we found a higher incidence of strabismus and a higher exotropia:esotropia ratio than previous studies in preschool children. Parental strabismus history and prematurity were associated with a higher risk for both prevalent and incident strabismus.

Exosome-Derived LncRNA TCONS_00072128 Mediated Osteogenic Differentiation and Inflammation by Caspase 8 Regulation.

Postmenopausal osteoporosis (PMOP) is a systemic metabolic bone disease in postmenopausal women. It has been known that long non-coding RNAs (lncRNAs) play a regulatory role in the progression of osteoporosis. However, the mechanism underlying the effects of exosome-derived lncRNA on regulating the occurrence and development of PMOP remains unclear. Exosomes in the serum of patients PMOP were collected and identified. RNA sequencing was performed to obtain the expression profile of exosome-derived lncRNAs in the serum of PMOP patients. RNA sequencing identified 26 differentially expressed lncRNAs from the exosomes between healthy people and PMOP patients. Among them, the expression of TCONS_00072128 was dramatically down-regulated. A co-location method was employed and searched its potential target gene caspase 8. TCONS_00072128 knockdown notably decreased the expression of caspase 8, while the osteogenic differentiation of BMSCs was also reduced. Reversely, TCONS_00072128 overexpression enhanced caspase 8 expression and osteogenic differentiation of BMSCs. Moreover, the continuous expression of caspase 8 regulated by TCONS_00072128 significantly activated inflammation pathways including NLRP3 signaling and NF-κB signaling. Simultaneously, RIPK1 which has emerged as a promising therapeutic target for the treatment of a wide range of human neurodegenerative, autoimmune, and inflammatory diseases, was also phosphorylated. The results of the present study suggested that exosome-derived lncRNA TCONS_00072128 could promote the progression of PMOP by regulating caspase 8. In addition, caspase 8 expression in BMSCs was possible to be a key regulator that balanced cell differentiation and inflammation activation.

Exosomal miR-141-3p regulates osteoblast activity to promote the osteoblastic metastasis of prostate cancer.

Exosomes from cancer cells, which contain microRNA and reach metastasis loci prior to cancer cells, stimulate the formation of a metastatic microenvironment. Previous studies have shown that exosomal miR-141-3p is associated with metastatic prostate cancer (PCa). However, the role and regulatory mechanism of miR-141-3p in the microenvironment of bone metastases require further study. In this study, we performed a series of experiments in vivo and in vitro to determine whether exosomal miR-141-3p from MDA PCa 2b cells regulates osteoblast activity to promote osteoblastic metastasis. We demonstrate that extracts obtained from cell culture supernatants contained exosomes and that miR-141-3p levels were significantly higher in MDA PCa 2b cell exosomes. Via confocal imaging, numerous MDA PCa 2b exosomes were observed to enter osteoblasts, and miR-141-3p was transferred to osteoblasts through MDA PCa 2b exosomes in vitro. Exosomal miR-141-3p from MDA PCa 2b promoted osteoblast activity and increased osteoprotegerin OPG expression. miR-141-3p suppressed the protein levels of the target gene DLC1, indicating its functional significance in activating the p38MAPK pathway. In animal experiments, exosomal miR-141-3p had bone-target specificity and promoted osteoblast activity. Mice injected with miR-141-3p-mimics exosomes developed apparent osteoblastic bone metastasis. Exosomal miR-141-3p from MDA PCa 2b cells promoted osteoblast activity and regulated the microenvironment of bone metastases, which plays an important role in the formation of bone metastases and osteogenesis damage in PCa. Clarifying the specific mechanism of bone metastasis will help generate new possibilities for the treatment of PCa.