Enhancing Neuroprotection in Mouse Model of Parkinson's Disease through Protein Nanosystem Conjugation with ApoE Peptide for miR-124 Delivery.

Parkinson's disease (PD) affects millions of people's lives worldwide. The main pathogenesis of PD is dopaminergic neuron necrosis and neuroinflammation mediated by activated microglia cells. In recent years, the anti-inflammatory ability and neuroprotective effects of miR-124 in PD models were well proved, but the in vivo delivery of miR-124 remains challenging. Herein, we report a protein nanosystem modified with a brain-targeting peptide ApoE that could efficiently deliver miR-124 across the blood-brain barrier (BBB). This nanosystem showed good cell viability on brain endothelial cells and microglia cells, and administration of this nanosystem significantly decreased the neuroinflammation and dopaminergic neuron loss, as well as recovered parts of neurobehavioral deficits. This ApoE peptide-based protein nanosystem holds great promise for the delivery of RNA therapeutics to the brain and for realizing neuron protection in PD treatment.

Construction and experimental validation of a necroptosis-related lncRNA signature as a prognostic model and immune-landscape predictor for lung adenocarcinoma.

Necroptosis is a new form of cell death. Since the discovery that long non-coding RNAs can affect the proliferation of lung adenocarcinoma, much has been learned about it, yet those of necroptosis-related long non-coding RNAs (NRlncRNAs) in lung adenocarcinoma (LUAD) remain enigmatic. This study aims to explore novel biomarkers and therapeutic targets for LUAD. The LUAD data was downloaded from The Cancer Genome Atlas, and necroptosis-related genes were retrieved from published literature. Co-expression analysis, univariate Cox analysis, least absolute shrinkage and selection operator regression analysis were used to identify necroptosis-related prognostic long non-coding RNAs. A comprehensive evaluation of tumor immunity for necrosis-related features was performed, and we identified a 9-NRlncRNA signature. Kaplan-Meier and Cox regression analyses confirmed that the signature was an independent predictor of LUAD outcome in the test and train sets (all P < 0.05). The areas of 1-, 2-, and 3-year overall survival under the time-dependent receiver operating characteristics (ROC) curve (AUC) were 0.754, 0.746, and 0.720, respectively. The GSEA results showed that 9 NRlncRNAs were associated with multiple malignancy-associated and immunoregulatory pathways. Based on this model, we found that the immune status and level of response to chemotherapy and targeted therapy were significantly different in the low-risk group compared with the high-risk group. qRT-PCR assay revealed that 9 NRlncRNAs were involved in the regulation of tumor cell proliferation and may affect the expression of programmed cell death 1 (PD1) and CD28 at human immune checkpoints. Our results indicated that the novel signature involving 9 NRlncRNAs (AL031600.2, LINC01281, AP001178.1, AL157823.2, LINC01290, MED4-AS1, AC026355.2, AL606489.1, FAM83A-AS1) can predict the prognosis of LUAD and are associated with the immune response. This will provide new insights into the pathogenesis and development of therapies for LUAD.

Macrophage polarization involved the inflammation of chronic obstructive pulmonary disease by S1P/HDAC1 signaling.

Globally, chronic obstructive pulmonary disease (COPD) is the cause of high morbidity and mortality, and constitutes a huge public health burden. Previous studies have reported that inflammation is closely related to COPD, but its potential mechanism is still unclear. Since the polarization of macrophages is involved in regulating inflammation, we assume that COPD changes the polarization of macrophages. To verify this, we investigated the relationship between the expression of S1PR1, HADC1, and inflammatory macrophages in COPD patients via flow cytometry, qRT-PCR, and western blot analysis. We found that macrophages of COPD individuals differentiated into M1 phenotype, and the expression of S1PR1 increased and HDAC1 decreased. S1PR1 also inhibits the expression of HDAC1, so S1PR1/HDAC1 signal regulates the polarization of macrophages. The results of the study put forward new ideas of the pathogenesis of COPD, and also proposed the possible treatment options.

CircCRIM1 suppresses osteosarcoma progression via sponging miR146a-5p and targeting NUMB.

CircCRIM1 (hsa_circ_0002346) is a circular RNA derived from gene CRIM1 (the cysteine rich transmembrane BMP regulator 1 circRNAs) by back-splicing. Recent studies have suggested the diverse function of CircCRIM1 in the tumorigenesis of multiple malignancies, including osteosarcoma (OS). Here, we investigated the role and mechanism of circCRIM1 during OS progression. Differentially expressed circRNAs (including circCRIM1) in OS and human osteoblast (hFOB1.19) cell lines were selected by searching the circRNA expression microarray dataset of GSE96964. The expression levels of circCRIM1 and its sponging miRNAs and target genes were examined by RT-qPCR. The effects of circCRIM1 on the proliferation, migration, and invasion of OS cells were investigated by in vitro gain of function experiments. The in vivo function of circCRIM1 on OS was evaluated by measuring the subcutaneous and in situ tumor growth in nude mice. In addition, dual-luciferase reporter assay and in situ hybridization (FISH) were performed to explore the underlying mechanisms of circCRIM1 and its sponging miRNAs and target genes in OS. CircCRIM1 is downregulated in human OS cell lines and predominantly presents in the cytoplasm as demonstrated by RT-qPCR and FISH assays. Overexpression of circCRIM1 suppressed the migration, invasion, proliferation of OS cells in vitro and OS tumor growth in vivo. Mechanistically, we identified miR146a-5p as a sponge miRNA of circCRIM1 through bioinformatic prediction and confirmed their interaction and colocalization via reporter gene assay and FISH analysis. This interaction leads to increase expression of the downstream target gene NUMB, which will cause inhibition of the Notch signal pathway. We further demonstrated that miR146a-5p overexpression could reverse the antitumor effect induced by circCRIM1 in OS cells. Our results support that circCRIM1 acts as a tumor suppressor in OS by sponging miR146a-5p and its downstream target NUMB.

Stat5a promotes Col10a1 gene expression during chondrocyte hypertrophic differentiation.

OBJECTIVES: Multiple transcription factors (TFs) have previously been shown to control hypertrophic chondrocyte-specific mouse type X collagen gene (Col10a1) expression via interaction with Col10a1 promoters. This study aims to investigate the role and mechanism of the potential binding factor signal transduction and transcription activator 5a (Stat5a) of Col10a1 cis-enhancer, in controlling Col10a1 gene expression and chondrocyte hypertrophic differentiation. METHODS: The potential Col10a1 regulator was predicted by the transcription factor affinity prediction (TRAP) analysis of the 150-bp Col10a1 cis enhancer. Stat5a was screened and verified by qRT-PCR, western blot and IHC analyses. Transfection of Stat5a siRNA or expression plasmid into MCT and ATDC5 cells was performed to either knockdown or over-express Stat5a and to investigate the influence of Stat5a on Col10a1 gene expression during the chondrocyte hypertrophy. Dual-luciferase reporter assay was performed to explore the mechanism of Stat5a affecting Col10a1 transcription. Alcian blue, alkaline phosphatase, and alizarin red staining, as well as qRT-PCR analyses of related marker genes were performed to investigate the effect and possible mechanism of Stat5a on chondrocyte differentiation. RESULTS: The potential binding factor of Col10a1 cis-enhancer Stat5a and Col10a1 were both highly expressed and positively correlated within hypertrophic chondrocytes in vitro and in situ. Knockdown of Stat5a reduced Col10a1 expression, while overexpression of Stat5a enhanced Col10a1 expression in hypertrophic chondrocytes, suggesting Stat5a as a positive Col10a1 regulator. Mechanistically, Stat5a was shown to potentiate the reporter activity mediated by Col10a1 promoter/enhancer. In addition, Stat5a increased the intensity of alkaline phosphatase staining of ATDC5 cells and the expression of relevant hypertrophic marker genes including Runx2, which was consistent with the expression of Stat5a and Col10a1. CONCLUSIONS: Our results support that Stat5a promoted Col10a1 expression and chondrocyte hypertrophic differentiation, possibly via interaction with the 150-bp Col10a1 cis-enhancer.

Mef2a is a positive regulator of Col10a1 gene expression during chondrocyte maturation.

BACKGROUND: The type X collagen gene (Col10a1) is a signature gene of hypertrophic chondrocytes that are known as the main engine of long bone growth. Multiple transcription factors (TFs), including myocyte enhancer factor 2A (Mef2a), have previously been identified by in silico analysis as potential Col10al gene regulators. OBJECTIVES: In this study, we aimed to investigate the correlation between Mef2a and Col10a1 expression and the possible effects on chondrocyte proliferation and hypertrophic differentiation in vitro. METHODS: First, Mef2a expression in proliferating and hypertrophic chondrocytes were detected by quantitative real-time PCR (qRT-PCR) and Western blotting in two chondrocytic models, ATDC5 and MCT cells, as well as in mouse chondrocytes in situ. Transfection with Mef2a small interfering fragments or Mef2a overexpression plasmids in the above chondrocytic models were performed to determine how Mef2a knockdown or overexpression may influence Col10a1 expression. The binding between Mef2a and its putative binding site within the 150 bp Col10a1 cis-enhancer which was evaluated by the dual luciferase reporter assay. The effect of Mef2a on chondrocyte differentiation was determined by examining the chondrogenic marker gene expression by qRT-PCR and by alcian blue, alkaline phosphatase (ALP), and alizarin red staining of the ATDC5 cells stably knocked down by Mef2a. RESULTS: The expression of Mef2a in hypertrophic chondrocytes was significantly higher than that in proliferative chondrocytes in both chondrocytic models as well as in mouse chondrocytes in situ. Interference with Mef2a caused decreased Col10a1 expression, while overexpression of Mef2a upregulated Col10a1. The result of the dual luciferase reporter assay showed that Mef2a enhanced Col10a1 gene enhancer activity via its putative Mef2a binding site. For the staining of ATDC5 stable cell lines, although no significant differences were seen in ALP staining, significantly weaker alcian blue staining intensity was noticed in Mef2a knockdown stable cell lines compared to the control cells at day 21, while slightly weaker alizarin red staining was seen in the stable cell lines at days 14 and 21. Correspondingly, we detected decreased runt-related transcription factor 2 (Runx2), increased SRY-box transcription factor 9 (Sox9), as well as differential expression of other chondrogenic markers in ATDC5 stable cell lines compared with the controls. CONCLUSIONS: In conclusion, our results support that Mef2a upregulates Col10a1 expression possibly by interaction with its cis-enhancer. Altered levels of Mef2a affects the expression of chondrogenic marker genes, such as Runx2 and Sox9, but may only play an insignificant role during chondrocyte proliferation and maturation.

DLX5 promotes Col10a1 expression and chondrocyte hypertrophy and is involved in osteoarthritis progression.

Osteoarthritis (OA) has been considered non-reversible as articular cartilage wears down with limited repair capacity. Enhanced chondrocyte hypertrophy and increased type X collagen gene (COL10A1) expression have been associated with OA. Therefore, regulators controlling collagen X expression and chondrocyte hypertrophy may play a role in OA intervention. Here, we investigated how Distal-less homeobox 5 (DLX5), the distal-less homeobox family member, controls murine Col10a1 gene expression and chondrocyte hypertrophy in chondrogenic cell models and its role in a murine OA model. Through qRT-PCR and Western blot analyses, we detected significantly increased levels of COL10A1 and DLX5 in hypertrophic MCT and ATDC5 cells compared to their proliferative stage. Forced expression of Dlx5 further increases, while knockdown of Dlx5 decreases COL10A1 expression in hypertrophic MCT cells. We have performed dual-luciferase reporter and ChIP assays and demonstrated that DLX5 promotes reporter activity through direct interaction with Col10a1 cis-enhancer. We established a murine OA model and detected markedly increased COL10A1 and DLX5 in the articular cartilage and subchondral bone of the OA mice compared with the controls. Notably, forced overexpression of DLX5 in hypertrophic MCT cells up-regulates RUNX2, and adjacent DLX5 and RUNX2 binding sites have previously been found within the Col10a1 cis-enhancer. Together, our data suggest that DLX5 may cooperate with RUNX2 to control cell-specific Col10a1 expression and chondrocyte hypertrophy and is involved in OA pathogenesis.

CircNRIP1 acts as a sponge of miR-1200 to suppress osteosarcoma progression via upregulation of MIA2.

Circular RNAs (circRNAs), a class of non-coding RNAs, play an essential role in embryo development and carcinogenesis, circNRIP1 was recently identified to promote development of multiple human cancers. This study investigated the role of circNRIP1 in osteosarcoma (OS) cells and the potential mechanisms relating to the sponging miRNAs and their target genes. OS cell lines and normal human osteoblasts were grown for qRT-PCR analysis of circNRIP1 expression and functions of circNRIP1 expression in OS cell proliferation, migration, and invasion in vitro. Bioinformatics analysis was then performed to predict the sponge miRNA of circNRIP1 and the target gene, which was confirmed by using the dual-luciferase reporter assay. The in vivo functions of circNRIP1 was evaluated in OS cell xenograft models, while levels of relevant marker genes were examined using immunohistochemistry. CircNRIP1 was mainly localized in OS cell cytoplasm and significantly lower in OS cell lines than in normal human osteoblasts. CircNRIP1 overexpression significantly inhibited OS cell proliferation, migration, and invasion in vitro. miR-1200 was predicted as the sponge miRNA of circNRIP1 and directly interacted with circNRIP1 confirmed by the dual-luciferase reporter assay. Moreover, miR-1200 overexpression significantly alleviated the inhibitory effect of circNRIP1 on OS cells. A protein-coding gene MIA2 was identified as the miR-1200 targeting gene and reversely associated with miR-1200 expression in OS cells. Increase in MIA2 expression in a murine OS cell xenograft model was associated with circNRIP1 expression in inhibition of OS cell xenograft growth in vivo. These data support the circNRIP1 OS-suppressive role by sponge of miR-1200 expression and in turn to upregulate MIA2 expression.

Integrated analyses of an RNA binding protein-based signature related to tumor immune microenvironment and candidate drugs in osteosarcoma.

OBJECTIVE: Osteosarcoma is the most frequent primary bone malignancy, associated with frequent recurrence and lung metastasis. RNA-binding proteins (RBPs) are pivotal in regulating several aspects of cancer biology. Nonetheless, interaction between RBPs and the osteosarcoma immune microenvironment is poorly understood. We investigated whether RBPs can predict prognosis and immunotherapy response in osteosarcoma patients. METHODS: We constructed an RBP-related prognostic signature (RRPS) by univariate coupled with multivariate analyses and verified the independent prognostic efficacy of the signature. Single-sample Gene Set Enrichment Analysis (ssGSEA) along with ESTIMATE analysis were carried out to investigate the variations in immune characteristics between subgroups with various RRPS-scores. Furthermore, we investigatedpossible small molecule drugs using the connectivity map database and validated the expression of hub RBPs by qRT-PCR. RESULTS: The RRPS, consisting of seven hub RBPs, was an independent prognostic factor compared to traditional clinical features. The RRPS could distinguish immune functions, immune score, stromal score, tumor purity and tumor infiltration by immune cells in different osteosarcoma subjects. Additionally, patients with high RRPS-scores had lower expression of immune checkpoint genes than patients with low RRPS-scores. We finally identified six small molecule drugs that may improve prognosis in osteosarcoma patients and substantiated notable differences in the contents of these RBPs. CONCLUSION: We evaluated the prognostic value and clinical application of an RBPs-based prognostic signature and identified promising biomarkers to predict immune cell infiltration and immunotherapy response in osteosarcoma.

DLX5 promotes osteosarcoma progression via activation of the NOTCH signaling pathway.

The distal-less (dlx) homeobox transcription factors have been implicated roles in bone development. DLX5, in particular, was shown to play essential roles in osteoblast differentiation by targeting RUNX2, a master transcription factor for bone development. Interestingly, DLX5 has also been shown to play an oncogenic role in lung and other cancers, possibly via regulation of MYC expression. Given its dual roles in bone and cancer, this study aimed to investigate the effect of DLX5 on progression of osteosarcoma (OS), the primary bone cancer that is characterized by abnormal bone formation and osteoblast activity. Expression of DLX5 in OS cell lines was detected by quantitative real-time PCR (qRT-PCR) and western blot (WB). In vitro and in vivo assays were performed to investigate the oncogenic function of DLX5 in OS cells and xenograft models. Luciferase reporter assay was performed to determine the underlying mechanism of DLX5-mediated OS aggressiveness. The results showed that DLX5 was differentially expressed in OS cell lines, with significantly upregulated levels in HOS and MG-63 and relatively low levels in U2OS and 143B cell lines, compared with the normal bone cell line. DLX5 knockdown in HOS and MG-63 cell lines by siRNA inhibited OS cell growth and progression, and induced cell apoptosis and cell cycle changes both in vitro and in vivo. Meanwhile, DLX5 overexpression had the opposite effect on U2OS and 143B cell lines. Notably, a positive correlation between the expression patterns of NOTCH1 and DLX5 was also observed. The expression levels of NICD (NOTCH1 intracellular domain) and HES1 (classical target of NOTCH) were closely associated with DLX5 expression. Whereas knockdown of DLX5 in OS cells resulted in decreased expression of NOTCH1 and reduced cell proliferation and migration, which were rescued by overexpression of NOTCH1. We further analyzed DLX5 and NOTCH1 genes using JASPAR software and found two potential DLX5 binding sites within the NOTCH1 promoter. Dual-luciferase assay demonstrated that DLX5 specifically activates the NOTCH1 promoter and controls its expression. Taken together, our results support that DLX5 plays an oncogenic role in OS development, which can at least partially, be attributed to activation of the NOTCH signaling pathway.

Expression Profiling and Functional Analysis of Candidate Col10a1 Regulators Identified by the TRAP Program.

Hypertrophic chondrocytes and their specific marker, the type X collagen gene (Col10a1), are critical components of endochondral bone formation during skeletal development. We previously found that Runx2 is an indispensable mouse Col10a1 gene regulator and identified many other transcription factors (TFs) that potentially interact with the 150-bp Col10a1 cis-enhancer. However, the roles of these candidate TFs in Col10a1 expression and chondrocyte hypertrophy have not been elucidated. Here, we focus on 32 candidate TFs recently identified by analyzing the 150-bp Col10a1 enhancer using the transcription factor affinity prediction (TRAP) program. We found that 12 TFs (Hoxa3, Lsx, Evx2, Dlx5, S8, Pax2, Egr2, Mef2a, Barhl2, GKlf, Sox17, and Crx) were significantly upregulated and four TFs (Lhx4, Tbx5, Mef2c, and Hb9) were significantly downregulated in hypertrophic MCT cells, which show upregulation of Col10a1 expression. Most of the differential expression pattern of these TFs conformed with the results obtained from ATDC5 cell model and primary mouse chondrocytes. Notably, Tbx5 was downregulated upon Col10a1 upregulation, overexpression of Tbx5 decreased Col10a1 expression, and knock-down of Tbx5 increased Col10a1 expression in hypertrophic chondrocytes, suggesting that Tbx5 is a negative regulator of Col10a1. We further generated a stable Tbx5-overexpressing ATDC5 cell line and ColX-Tbx5 transgenic mice driven by Col10a1-specific enhancers and promoters. Tbx5 overexpression decreased Col10a1 expression in ATDC5 cells cultured as early as day 7 and in limb tissue on post-natal day 1. Slightly weaker alkaline phosphatase staining was also observed in cell culture on day 7 and in limb digits on embryonic day 17.5, indicating mildly delayed ossification. Further characterization of these candidate Col10a1 transcriptional regulators could help identify novel therapeutic targets for skeletal diseases associated with abnormal chondrocyte hypertrophy.

CDK inhibitors in cancer therapy, an overview of recent development.

Dysregulated cell division, which leads to aberrant cell proliferation, is one of the key hallmarks of cancer. Therefore, therapeutic targets that block cell division would be effective for cancer treatment. Cell division is mainly controlled by a complex composed of cyclin and cyclin dependent kinases (CDKs). To date, the CDK inhibitors (CDKIs), specifically the ones that block the enzyme activity of CDK4 and CDK6 (CDK4/6), have been approved by FDA for the treatment of metastatic hormone receptor positive breast cancer. However, due to the non-selectivity and significant toxicity, most of the first generation CDK inhibitors (so called pan-CDK inhibitors that target several CDKs), have not been approved for clinical application. Despite this, great efforts and progress have been made to enable pan-CDK inhibitors application in the clinical setting. Notably, the development of combination therapy strategies in recent years has made it possible to reduce the toxicity and side effects of pan-CDK inhibitors. Thus, as a combination therapy approach, pan-CDK inhibitors regain great potential in clinical application. In this review, we introduced the CDK family members and discussed their major functions in cell cycle controlling. Then, we summarized the research progress regarding CDK inhibitors, especially those other than CDK4/6 inhibitors. We reviewed first-generation pan-CDKIs Flavopiridol and Roscovitine, and second-generation CDKIs Dinaciclib, P276-00, AT7519, TG02, Roniciclib, RGB-286638 by focusing on their developing stages, clinical trials and targeting cancers. The specific CDKIs, which targets to increase specificity and decrease the side effects, were also discussed. These CDKIs include CDK4/6, CDK7, CDK9, and CDK12/13 inhibitors. Finally, the efficacy and discrepancy of combination therapy with CDK inhibitors and PD1/PDL1 antibodies were analyzed, which might give insights into the development of promising strategy for cancer treatment.

TAp63γ influences mouse cartilage development.

Depletion of tumor protein p63 results in severe epithelial as well as limb defects in mice, suggesting that p63 is also required for endochondral ossification during long bone development. A key stage in endochondral ossification is chondrocyte hypertrophy, which has been associated with elevated levels of the p63 variant TAp63γ. To investigate the role of TAp63γ in chondrocyte differentiation and maturation, we developed stable TAp63γ expressing ATDC5 cells. Compared to control cells, TAp63γ cells showed significant upregulation of Col10a1 after 4 and 7 days in culture. Moreover, alkaline phosphatase, Alizarin red, and Alcian blue staining were stronger in TAp63γ cells, suggesting that TAp63γ promotes chondrocyte proliferation, hypertrophic differentiation, and possibly matrix mineralization. To investigate the in vivo function of TAp63γ during endochondral bone formation, we established transgenic mice that express flag-tagged TAp63γ driven by Col10a1 regulatory elements. Skeletal staining of transgenic mice at postnatal day 1 showed accelerated ossification in long bone, tail, and digit bones compared to wild-type littermates. Furthermore, Sox9 expression was reduced and Runx2 expression was increased in the proliferative and/or hypertrophic zones of these mice. Altogether, these results suggest that TAp63γ promotes endochondral ossification and skeletal development, at least partially via controlling chondrocyte differentiation and maturation.