Genome editing HLA alleles for a pilot immunocompatible hESC line in a Chinese hESC bank for cell therapies.

Robust allogeneic immune reactions after transplantation impede the translational pace of human embryonic stem cells (hESCs)-based therapies. Selective genetic editing of human leucocyte antigen (HLA) molecules has been proposed to generate hESCs with immunocompatibility, which, however, has not been specifically designed for the Chinese population yet. Herein, we explored the possibility of customizing immunocompatible hESCs based on Chinese HLA typing characteristics. We generated an immunocompatible hESC line by disrupting HLA-B, HLA-C, and CIITA genes while retaining HLA-A*11:01 (HLA-A*11:01-retained, HLA-A11R ), which covers ~21% of the Chinese population. The immunocompatibility of HLA-A11R hESCs was verified by in vitro co-culture and confirmed in humanized mice with established human immunity. Moreover, we precisely knocked an inducible caspase-9 suicide cassette into HLA-A11R hESCs (iC9-HLA-A11R ) to promote safety. Compared with wide-type hESCs, HLA-A11R hESC-derived endothelial cells elicited much weaker immune responses to human HLA-A11+ T cells, while maintaining HLA-I molecule-mediated inhibitory signals to natural killer (NK) cells. Additionally, iC9-HLA-A11R hESCs could be induced to undergo apoptosis efficiently by AP1903. Both cell lines displayed genomic integrity and low risks of off-target effects. In conclusion, we customized a pilot immunocompatible hESC cell line based on Chinese HLA typing characteristics with safety insurance. This approach provides a basis for establishment of a universal HLA-AR bank of hESCs covering broad populations worldwide and may speed up the clinical application of hESC-based therapies.

MiR-186 bidirectionally regulates cisplatin sensitivity of ovarian cancer cells via suppressing targets PIK3R3 and PTEN and upregulating APAF1 expression.

Ovarian cancer is a highly lethal malignancy in the female reproductive system. Platinum drugs, represented by cisplatin, are the first-line chemotherapeutic agents for treatment of various malignancies including ovarian cancer, but drug resistance leads to chemotherapy failure. MicroRNAs emerged as promising molecules in reversal of cisplatin resistance. MiR-186 was reported to be downregulated in the cisplatin-resistant ovarian cell lines and miR-186 expression increased cisplatin sensitivity. However, we found the bidirectional regulatory effects of miR-186 on cisplatin sensitivity for the first time that overexpression of miR-186 at low concentration increased the cisplatin sensitivity of ovarian cancer cells A2780/DDP, while high concentration of miR-186 decreased the cisplatin sensitivity. The survival assay in other types of cancer cell lines verified the bidirectional regulatory function of miR-186 on cisplatin sensitivity in dose and cell type dependent manners. MiR-186 suppressed the protein levels of PTEN and PIK3R3 dose-dependently, which are opposite regulatory molecules of the oncogenic AKT pathway. MiR-186 also enhanced the protein levels of apoptotic gene APAF1 dose-dependently. We proposed the final effects of PTEN and APAF1 outweighed PIK3R3 when miR-186 at low concentration so as to increase the cisplatin sensitivity of ovarian cancer cells, while the final effects of PIK3R3 outweighed PTEN and APAF1 when miR-186 at high concentration so as to decrease the cisplatin sensitivity. We concluded the outcome of regulation of these opposite functional molecules contributed to the bidirectional regulatory effects of miR-186 in ovarian cancer cisplatin sensitivity. It deserves more attentions when developing therapeutic strategies based on the bidirectional functional miRNAs.

The renoprotective effect of curcumin against cisplatin-induced acute kidney injury in mice: involvement of miR-181a/PTEN axis.

Background: Nephrotoxicity, especially acute kidney injury (AKI), is the main dose-limiting toxicity of cisplatin. Although recent studies showed that curcumin prevented cisplatin-induced AKI effectively, further studies to understand the mechanism are required.Methods: We established an AKI mouse model. Male C57BL/6 mice were assigned to three groups: saline group (control), cisplatin group (CP), and curcumin + cisplatin group (CP + Cur). The CP group received a single intraperitoneal (i.p.) injection of cisplatin, while the control group received saline. The CP + Cur group received i.p. curcumin three days before cisplatin injection and curcumin administered for another three days until the day before euthanization. Renal injury was assessed by serological and histological analysis. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the phosphatase and tensin homolog (PTEN), and microRNA (miR)-181a expression in the renal tissues. Bioinformatics prediction and western blotting methods validated the targets of miR-181a in vitro.Results: Curcumin treatment alleviated cisplatin-induced nephrotoxicity as validated by the blood urea nitrogen (BUN) values, and histological analysis of kidneys. At the molecular level, curcumin treatment decreased miR-181a expression level, which was induced by cisplatin and restored the in vivo expression of PTEN, which was suppressed by cisplatin. We verified the direct regulation of PTEN by miR-181a in cultured human embryonic kidney 293T cells.Conclusions: We showed the involvement of miR-181a/PTEN axis in the renoprotective effect of curcumin against cisplatin-induced AKI, and provide new evidence on the ability of curcumin to alleviate cisplatin-induced nephrotoxicity.

The Dual Role of miR-186 in Cancers: Oncomir Battling With Tumor Suppressor miRNA.

MicroRNAs (miRNAs) are a class of small non-coding RNAs which regulate gene expression at post-transcriptional level. Alterations of miR-186 expression were demonstrated in numerous cancers, shown to play a vital role in oncogenesis, invasion, metastasis, apoptosis, and drug resistance. MiR-186 was documented as a tumor suppressor miRNA in the majority of studies, while conflicting reports verified miR-186 as an oncomir. The contradictory role in cancers may impede the application of miR-186, as well as other dual-functional miRNAs, as a diagnostic and therapeutic target. This review emphasizes the alterations and functions of miR-186 in cancers and discusses the mechanisms behind the contradictory findings. Among these, target abundance and dose-dependent effects of miR-186 are highlighted. The paper aims to review the challenges involved in developing diagnostic and therapeutic strategies for cancer treatment based on dual-functional miRNAs.

Effects of miR-145 on the inhibition of chondrocyte proliferation and fibrosis by targeting TNFRSF11B in human osteoarthritis.

Osteoarthritis (OA) is a common cause of functional deterioration in older adults, and altered chondrogenesis is the most common pathophysiological process involved in the development of OA. MicroRNA­145 (miR­145) has been shown to regulate chondrocyte homeostasis. However, the function of miR­145 in OA remains to be elucidated. In the present study, the expression levels of miR­145 were examined in cartilage specimens from 25 patients with knee OA using reverse transcription­quantitative polymerase chain reaction analysis. The effects of miR­145 on the proliferation and fibrosis of the C­20/A4 and CH8 cell lines were also investigated using 3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide and western blot assays in vitro. The results revealed that the expression of miR-145 was decreased in the OA cartilage tissues, compared with normal cartilage tissues. The overexpression of miR­145 by transfection of cells with miR­145 mimics significantly inhibited C­20/A4 and CH8 cell proliferation and fibrosis. Furthermore, tumor necrosis factor receptor superfamily, member 11b (TNFRSF11B) was identified as a direct target of miR­145 in chondrocytes, which was confirmed using a dual­luciferase reporter assay. The expression level of TNFRSF11B was markedly upregulated in the patients with OA, and the ectopic expression of miR­145 was capable of suppressing the expression of TNFRSF11B. In addition, the knock down of TNFRSF11B using specific small interfering RNA also inhibited the proliferation and fibrosis of C­20/A4 and CH8 cells in vitro. These data provide the first evidence, to the best of our knowledge, to suggest the critical function of miR­145 in regulating the expression of TNFRSF11B, which may have important implications on the regulation of chondrocyte proliferation and fibrosis in OA.