Rps6ka2 enhances iMSC chondrogenic differentiation to attenuate knee osteoarthritis through articular cartilage regeneration in mice.

Articular cartilage (AC) is most susceptible to degeneration in knee osteoarthritis (OA); however, the existing treatments for OA do not target the core link of the pathogenesis-"decreased tissue cell function activity and extracellular matrix (ECM) metabolic disorders" for effective intervention. iMSC hold lower heterogeneity and great promise in biological research and clinical applications. Rps6ka2 may play an important role in the iMSC to treat OA. In this study, the CRISPR/Cas9 gene editing Rps6ka2-/- iMSC were obtained. Effect of Rps6ka2 on iMSC proliferation and chondrogenic differentiation was evaluated in vitro. An OA model was constructed in mice by surgical destabilization of medial meniscus (DMM). The Rps6ka2-/- iMSC and iMSC were injected into the articular cavity twice-weekly for 8 weeks. In vitro experiments showed that Rps6ka2 could promote iMSC proliferation and chondrogenic differentiation. In vivo results further confirmed that Rps6ka2 could improve iMSC viability to promote ECM production to attenuate OA in mice.

Generation of Monoclonal iPSC Lines with Stable Cas9 Expression and High Cas9 Activity.

CRISPR/Cas9-mediated gene editing has been rapidly and widely applied in many organisms for delicate genetic manipulation, including human-induced pluripotent stem cells (iPSCs). Gene editing in human iPSCs is promising for genetics and biomedical research due to that gene-edited iPSC still possesses the potential to be differentiated into any cell lineages. In many cases, the generation of Cas9 expressing cell lines is a prerequisite toward performing successful editing of multiplex genes of interest. Here, we describe a simple, effective method to generate stable Cas9 expressing human iPSCs with high Cas9 activity. In this method, stable Cas9 expressing monoclonal human iPSC lines were generated through lentiviral transduction of Cas9 cassette, followed by blasticidin selection and subcloning with low seeding density. After colonies isolation and expansion, a BFP-GFP reporter assay was applied to validate the Cas9 activities of multiple monoclonal lines by flow cytometry (FACS). These Cas9 expressing human iPSCs generated by our method are single cell-derived monoclonal lines with homogenous population and Cas9 activity of up to 99%.

Upregulation of miR­598 promotes cell proliferation and cell cycle progression in human colorectal carcinoma by suppressing INPP5E expression.

Colorectal cancer (CRC) is one of the most common types of cancer worldwide. Recently, microRNAs (miRs) have been considered as novel therapeutic targets for the treatment of cancer. miR­598 is a poorly investigated miR. The underlying mechanism of miR­598 in CRC cells remains to be elucidated. In the present study, miR­598 was demonstrated to be significantly upregulated in CRC tissue by analyzing data from The Cancer Genome Atlas and the Gene Expression Omnibus. The results of a polymerase chain reaction demonstrated that miR­598 expression was significantly upregulated in CRC tissues and cells. Gain of function and loss of function assays demonstrated that miR­598 significantly promoted cell proliferation and cell cycle progression. miR­598 was demonstrated to modulate cell functions by regulating 72 kDa inositol polyphosphate­5­phosphatase (INPP5E). In addition, knockdown of INPP5E counteracted the growth arrest caused by an miR­598­inhibitor. In conclusion, the present study demonstrated that miR­598 contributed to cell proliferation and cell cycle progression in CRC by targeting INPP5E.