Loss-of-function and certain missense mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) significantly decrease or increase bone mass, respectively. These human skeletal phenotypes have been recapitulated in mice harboring Lrp5 knockout and knock-in mutations. We hypothesized that measuring mRNA expression in diaphyseal bone from mice with Lrp5 wild-type (Lrp5(+/+) ), knockout (Lrp5(-/-) ), and high bone mass (HBM)-causing (Lrp5(p.A214V/+) ) knock-in alleles could identify genes and pathways that regulate or are regulated by LRP5 activity. We performed RNA-seq on pairs of tibial diaphyseal bones from four 16-week-old mice with each of the aforementioned genotypes. We then evaluated different methods for controlling for contaminating nonskeletal tissue (ie, blood, bone marrow, and skeletal muscle) in our data. These methods included predigestion of diaphyseal bone with collagenase and separate transcriptional profiling of blood, skeletal muscle, and bone marrow. We found that collagenase digestion reduced contamination, but also altered gene expression in the remaining cells. In contrast, in silico filtering of the diaphyseal bone RNA-seq data for highly expressed blood, skeletal muscle, and bone marrow transcripts significantly increased the correlation between RNA-seq data from an animal's right and left tibias and from animals with the same Lrp5 genotype. We conclude that reliable and reproducible RNA-seq data can be obtained from mouse diaphyseal bone and that lack of LRP5 has a more pronounced effect on gene expression than the HBM-causing LRP5 missense mutation. We identified 84 differentially expressed protein-coding transcripts between LRP5 "sufficient" (ie, Lrp5(+/+) and Lrp5(p.A214V/+) ) and "insufficient" (Lrp5(-/-) ) diaphyseal bone, and far fewer differentially expressed genes between Lrp5(p.A214V/+) and Lrp5(+/+) diaphyseal bone.

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An RNA‐seq protocol to identify mRNA expression changes in mouse diaphyseal bone: Applications in mice with bone property altering Lrp5 mutations

The murine double minute 2 (MDM2) oncogene exerts major oncogenic activities in human cancers; it is not only the best-documented negative regulator of the p53 tumor suppressor, but also exerts p53-independent activities. There is an increasing interest in developing MDM2-based targeted therapies. Several classes of MDM2 inhibitors have been evaluated in preclinical models, with a few entering clinical trials, mainly for cancer therapy. However, noncarcinogenic roles for MDM2 have also been identified, demonstrating that MDM2 is involved in many chronic diseases and conditions such as inflammation and autoimmune diseases, dementia and neurodegenerative diseases, heart failure and cardiovascular diseases, nephropathy, diabetes, obesity, and sterility. MDM2 inhibitors have been shown to have promising therapeutic efficacy for treating inflammation and other nonmalignant diseases in preclinical evaluations. Therefore, targeting MDM2 may represent a promising approach for treating and preventing these nonmalignant diseases. In addition, a better understanding of how MDM2 works in nonmalignant diseases may provide new biomarkers for their diagnosis, prognostic prediction, and monitoring of therapeutic outcome. In this review article, we pay special attention to the recent findings related to the roles of MDM2 in the pathogenesis of several nonmalignant diseases, the therapeutic potential of its downregulation or inhibition, and its use as a biomarker.

Targeting MDM2 for novel molecular therapy: Beyond oncology

NEU3 inhibitory effect of naringin suppresses cancer cell growth by attenuation of EGFR signaling through GM3 ganglioside accumulation.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by synovial inflammation. Fibroblast‑like synoviocytes (FLS) serve a vital role in the initiation and perpetuation of the immune response in patients with RA. The present study aimed to investigate the potential role of microRNA (miR)‑34a‑3p in the pathogenesis of RA. FLS were collected from patients with RA and osteoarthritis (OA). The miR‑34a‑3p mimics and inhibitor vectors were constructed and transfected into RAFLS using Lipofectamine® 2000. Cell proliferation was determined by Cell Counting kit‑8 assay and cell cycle progression was analyzed by flow cytometry. In addition, the expression levels of cell cycle control genes, matrix metalloproteinase (MMP)‑1 and MMP‑9, and pro‑inflammatory cytokines were detected by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. The potential targets of miR‑34a‑3p were predicted by TargetScan and MiRWalk; the target genes were further verified using a luciferase reporter assay. The expression levels of miR‑34a‑3p were generally lower in RAFLS compared with in OAFLS. miR‑34a‑3p overexpression significantly inhibited the proliferation of FLS (P<0.01) by suppressing the expression levels of cyclin‑dependent kinase 2, cell division cycle 25A and cyclin D1 (P<0.01), and arresting FLS cell cycle progression at the G1 phase. Furthermore, the expression levels of MMP‑1 and 9 were markedly decreased, as were the mRNA and protein expression levels of pro‑inflammatory cytokines (tumor necrosis factor α and interleukin 6; P<0.01). Murine double minute 4 (MDM4) was predicted and verified as a potential target gene of miR‑34a‑3p; the 547‑554 nt position of the MDM4 3'‑untranslated region harbored one potential binding site for miR‑204‑3p. The results of the present study indicated that miR‑34a‑3p may be considered a promising therapeutic target for RA through inhibiting FLS proliferation and suppressing the production of pro‑inflammatory cytokines and MMPs.

/pdf/microrna-34a-3p-inhibits-proliferation-of-rheumatoid-iwej4m142x.pdf

MicroRNA‑34a‑3p inhibits proliferation of rheumatoid arthritis fibroblast‑like synoviocytes

Paeoniflorin is an active ingredient derived from Paeonia, which has an anti-inflammatory effect. However, the potential role and basis of paeoniflorin in rheumatoid arthritis (RA) are indistinct. Cell viability, cycle distribution, migration, and invasion were evaluated via Cell Counting Kit-8 (CCK-8), flow cytometry, and transwell assays. The contents of inflammatory cytokines were examined using enzyme-linked immunosorbent assay (ELISA). RNA expression levels were determined via qRT-PCR and western blot. The targeting relationship between miR-671-5p and circ-FAM120A (hsa_circ_0003972) or murine double minute 4 (MDM4) was validated via dual-luciferase reporter assay. Paeoniflorin restrained proliferation, migration, invasion, and inflammation and accelerated cell cycle arrest in RA fibroblast-like synoviocytes (RA-FLSs). Circ-FAM120A was boosted in RA synovial tissues and RA-FLSs. Circ-FAM120A upregulation, miR-671-5p knockdown, or MDM4 augmentation reversed the repressive effect of paeoniflorin on RA-FLS progression. Moreover, paeoniflorin attenuated RA-FLS progression by regulating the circ-FAM120A/miR-671-5p/MDM4 axis. Paeoniflorin inhibited RA-FLS proliferation, mobility, and inflammation and triggered cell cycle arrest via mediating the circ-FAM120A/miR-671-5p/MDM4 pathway.

Hongwei Liu

Papers

MDM4 overexpression contributes to synoviocyte proliferation in patients with rheumatoid arthritis.