Exosome-mediated delivery of artificial circular RNAs for gene therapy of bladder cancer.

Bladder cancer (BCa) is one of the most common malignancies affecting men. Oncogenic transcription factors function as an important regulator in the progression of human cancer. In our study, we aimed to construct artificial circular non-coding RNAs (acircRNAs) consisting of three functional units that mimic the CRISPR-Cas system and elucidate its therapeutic role in bladder cancer. Additionally, the compare of the efficiency in regulating gene expression between acircRNA and CRISPR-dCas systems was performed. We connected the cDNA sequences of TFs aptamer and constructed a circRNA. To demonstrate the platform's practicality, ß-catenin and NF-κB were chosen as functional targets, while T24 and 5637 cell lines served as test models. Real-time Quantitative PCR (qPCR), double luciferase assay and related phenotype assay were used to detect the expression of related genes and the therapeutic effect. To elucidate the functionality of acircRNAs, luciferase vectors capable of detecting ß-catenin and NF-κB expression were employed to assess the inhibitory impact of acircRNA on ß-catenin and NF-κB. Consequently, the optimal combination involving acircRNA-3 was determined. Next, qPCR assay was employed to assess the relative expression levels of target downstream genes following acircRNA treatment. The expression of c-myc and cyclin D1 were used to determine the function of ß-catenin, while Bcl-XL and TRAF1 were used to determine that of NF-κB. The acircRNAs inhibited the ß-catenin and NF-κB related signaling in BCa cells specifically. CD63-HuR fusion protein was used to loading acircRNA into exosomes. The results showed that acircRNA could inhibit the activity of the target transcription factors, and the inhibitory effect was better than that of CRIPSR-dCas9-KRAB. Furthermore, functional experiments demonstrated that the transfection of acircRNA in bladder cells resulted in decreased proliferation, enhanced apoptosis, and suppressed migration. In conclusion, our synthetic gene device exhibited anti-tumor regulatory capabilities and showed greater efficiency in tumor suppression compared to the CRISPR-dCas9-KRAB system. Therefore, our device provides a new strategy for cancer treatment and could be a useful strategy for cancer cells.

Jointly estimating bias field and reconstructing uniform MRI image by deep learning.

Bias field is one of the main artifacts that degrade the quality of magnetic resonance images. It introduces intensity inhomogeneity and affects image analysis such as segmentation. In this work, we proposed a deep learning approach to jointly estimate bias field and reconstruct uniform image. By modeling the quality degradation process as the product of a spatially varying field and a uniform image, the network was trained on 800 images with true bias fields from 12 healthy subjects. A network structure of bias field estimation and uniform image reconstruction was designed to compensate for the intensity loss. To further evaluate the benefit of bias field correction, a quantitative analysis was made on image segmentation. Experimental results show that the proposed BFCNet improves the image uniformity by 8.3% and 10.1%, the segmentation accuracy by 4.1% and 6.8% on white and grey matter in T2-weighted brain images. Moreover, BFCNet outperforms the state-of-the-art traditional methods and deep learning methods on estimating bias field and preserving image structure, and BFCNet is robust to different levels of bias field and noise.

MiR-342 attenuates lipopolysaccharide-induced acute lung injury via inhibiting MAPK1 expression.

Micro RNA (miRNA) and mitogen-activated protein kinase (MAPK) are reported as the crucial regulators of inflammatory responses in acute lung injury (ALI). This study will explore the role of the miR-342/MAPK1 axis in regulation of lipopolysaccharide (LPS)-induced ALI. We found that miR-342 was down-regulated in LPS-induced A549 cells compared with the control group with DMSO, accompanied by elevated inflammatory cytokines and apoptosis. Over-expression of miR-342 reduced LPS-induced inflammatory responses and apoptosis in LPS-stimulated A549 cells, and had a protective role in LPS-treated mice with ALI by decreasing levels of inflammatory cytokines, improving survival of mice with ALI, and ameliorating the lung permeability. Dual-luciferase reporter gene assay demonstrated that miR-342 regulated the expression of MAPK1 by directly targeting its 3' untranslated region (3'-UTR). Mechanistically, MAPK1 silencing abrogated LPS-induced inflammatory injury in A549 cells, and partially enhanced the protective effect of miR-342. Therefore, miR-342 attenuates LPS-induced ALI by targeting MAPK1 expression, thereby protecting against A549 cell injury induced by LPS and lung injury of mice with ALI.

MiR-216a alleviates LPS-induced acute lung injury via regulating JAK2/STAT3 and NF-κB signaling.

MicroRNAs (miRNAs) play an important role in the progression of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Till now, little is known about the role of miR-216a in ALI/ARDS. In this study, patients with ARDS exhibited significantly higher interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels than healthy controls (P < 0.01). However, miR-216a expression in patients with ARDS was significantly lower than healthy controls (P < 0.05), and negatively correlated with 28-day survival rate. Similar effects were observed in LPS-treated mice and A549 cells. MiR-216a over-expression reduced LPS-induced IL-1ß, IL-6 and TNF-α levels, and ameliorated lung permeability, and prolonged overall survival of ALI mice. Further, miR-216a over-expression inhibited LPS-induced apoptosis and autophagy. In addition, the janus kinase-2 (JAK2) was a direct target of miR-216a. Silencing of JAK2 partially aggravated miR-216a-inhibited inflammation injury. Besides, miR-216a obviously decreased the expressions of phosphorylated signal transducer and the activator of transcription 3 (p-STAT3), p-p56, and p-IκBα. In conclusion, miR-216a alleviates LPS-induced inflammatory injury via regulating JAK2/STAT3 and NF-κB signaling.

Radiosensitizing effects of Sestrin2 in PC3 prostate cancer cells.

OBJECTIVES: The stress-responsive genes of Sestrin family are recognized as new tumor suppressor genes in breast carcinoma, however, the function of Sestrin family in human prostate cancer is not clear. Ionizing radiation (IR) is known to induce Sestrin gene expression in breast cancer cells. However, the response of Sestrin to IR has not been reported in PC3 prostate cancer cells. MATERIALS AND METHODS: Sestrin2 expression in prostate cancer cell lines (PC3, LNCaP clone FGC, and DU145) was detected by Western blot and real-time PCR. Cell counting kit (CCK-8) was used to detect cellular proliferation. The radiosensitivity of PC3 cells was detected by clonogenic assay. RESULTS: Sestrin2 expression in prostate cancer cell lines (PC3, LNCaP clone FGC, and DU145) is low. In vitro assays indicated that over-expressing Sestrin2 in human prostate cancer PC3 inhibited tumor proliferation. In addition, elevated Sestrin2 expression sensitized PC3 cells to IR. CONCLUSION: We determined Sestrin2 may function as a tumor suppressor through repressing proliferation, mediating sensitization to IR in PC3 cells.

MiRNA-200a induce cell apoptosis in renal cell carcinoma by directly targeting SIRT1.

Accumulating evidence indicates that microRNAs are implicated in tumor initiation and progression through negatively regulating oncogenes or tumor suppressor genes. In the present study, we report that the expression of miR-200a was significantly lower in renal cell carcinoma (RCC) specimens and RCC cell lines. Restoration of miR-200a suppressed cell growth, arrested cell cycle progression, and promoted cell apoptosis in RCC cell lines. We next used qRT-PCR array technology to identify Sirtuin 1 (SIRT1) as one of the downregulated proteins during miR-200a overexpression in 786-O cells. Following a further assay by luciferase reporter system, SIRT1 was validated as a direct target of miR-200a. Moreover, siRNA-mediated knockdown of SIRT1 could partially phenocopy the effects of miR-200a overexpression. In contrast, overexpression of truncated SIRT1 (without an endogenous 3'-UTR) could rescue the effect of miR-200a overexpression on 786-O cells, which suggested that SIRT1 3'-UTR is targeted by miR-200a specifically. These observations provide further evidence for a critical tumor-suppressive role of the miR-200a in RCC in addition to identifying a novel regulatory mechanism, which may contribute to SIRT1 upregulation in RCC.

[Correlation of oxidative stress with sperm DNA integrity and semen parameters in infertile men with varicocele].

OBJECTIVE: To investigate the relationship of oxidative stress with DNA integrity and semen parameters in infertile men with varicocele (VC). METHODS: This prospective study included 98 infertile males with VC. According to the levels of reactive oxygen species (ROS) in the semen, we divided the patients into a high ROS group (n=44) and a low ROS group (n=54), determined the sperm DNA fragmentation index (DFI), motility and morphology, and analyzed their correlation with ROS in the two groups of patients. RESULTS: Compared with the patients of the low ROS group, those of the high ROS group showed a significantly higher DFI (27.38±8.10 vs 34.49±6.05, P=0.039) and a higher concentration of seminal leukocytes (ï¼»0.65±0.15ï¼½×106/ml vs ï¼»0.86±0.41ï¼½×106/ml, P=0.022), but lower sperm motility (ï¼»36.16±22.83ï¼½% vs ï¼»18.22±25.21ï¼½%, P=0.017), percentage of progressively motile sperm (ï¼»23.34±11.53ï¼½% vs ï¼»16.34±9.22ï¼½%, P=0.041), sperm curvilinear velocity (ï¼»27.03±6.21ï¼½ vs ï¼»20.62±4.38ï¼½ µm/s, P=0.013), and sperm linearity (ï¼»29.75±8.24ï¼½% vs ï¼»18.30±7.93ï¼½%, P=0.024). Spearman correlation analysis indicated that the ROS level was correlated positively with the concentration of seminal leukocytes (r=0.41, P<0.01) and DFI (r=0.21, P=0.006), but negatively with sperm curvilinear velocity (r=－0.24, P=0.017), linearity (r=－0.24, P=0.021), motility (r=－0.31, P=0.002), and the percentage of progressively motile sperm (r=－0.41, P=0.012). Additionally, the sperm DFI manifested a significant negative correlation with sperm motility (r=－0.29, P<0.01) and the percentage of progressively motile sperm (r=－0.34, P<0.01). CONCLUSIONS: The level of seminal ROS is positively correlated with the sperm DFI in infertile men with varicocele, and both the ROS level and DNA integrity are associated with semen parameters.