Background: Deregulated circular RNAs (circRNAs) are associated with the development of cancer and therapy resistance. However, functional research of circRNAs mostly focus on potential miRNA or protein binding and more potential regulation of circRNA on host gene DNA in cancers are yet to be inspected. Method: We performed total RNA sequencing on clinical breast cancer samples and identified the expression patterns of circRNAs and corresponding host genes in patient blood, tumor and adjacent normal tissues. qPCR, northern blot and in situ hybridization were used to validate the dysregulation of circRNA circSMARCA5. A series of procedures including R-loop dot-blotting, DNA-RNA immunoprecipitation and mass spectrum, etc. were conducted to explore the regulation of circSMARCA5 on the transcription of exon 15 of SMARCA5. Moreover, immunofluorescence and in vivo experiments were executed to investigate the overexpression of circSMARCA5 with drug sensitivities. Results: We found that circRNAs has average higher expression over its host linear genes in peripheral blood. Compared to adjacent normal tissues, circSMARCA5 is decreased in breast cancer tissues, contrary to host gene SMARCA5. The enforced expression of circSMARCA5 induced drug sensitivity of breast cancer cell lines in vitro and in vivo. Furthermore, we demonstrated that circSMARCA5 can bind to its parent gene locus, forming an R-loop, which results in transcriptional pausing at exon 15 of SMARCA5. CircSMARCA5 expression resulted in the downregulation of SMARCA5 and the production of a truncated nonfunctional protein, and the overexpression of circSMARCA5 was sufficient to improve sensitivity to cytotoxic drugs. Conclusion: Our results revealed a new regulatory mechanism for circRNA on its host gene and provided evidence that circSMARCA5 may serve as a therapeutic target for drug-resistant breast cancer patients.

Gendicine (recombinant human p53 adenovirus), developed by Shenzhen SiBiono GeneTech Co. Ltd., was approved in 2003 by the China Food and Drug Administration (CFDA) as a first-in-class gene therapy product to treat head and neck cancer, and entered the commercial market in 2004. Gendicine is a biological therapy that is delivered via minimally invasive intratumoral injection, as well as by intracavity or intravascular infusion. The wild-type (wt) p53 protein expressed by Gendicine-transduced cells is a tumor suppressor that is activated by cellular stress, and mediates cell-cycle arrest and DNA repair, or induces apoptosis, senescence, and/or autophagy, depending upon cellular stress conditions. Based on 12 years of commercial use in >30,000 patients, and >30 published clinical studies, Gendicine has exhibited an exemplary safety record, and when combined with chemotherapy and radiotherapy has demonstrated significantly higher response rates than for standard therapies alone. In addition to head and neck cancer, Gendicine has been successfully applied to treat various other cancer types and different stages of disease. Thirteen published studies that include long-term survival data showed that Gendicine combination regimens yield progression-free survival times that are significantly longer than standard therapies alone. Although the p53 gene is mutated in >50% of all human cancers, p53 mutation status did not significantly influence efficacy outcomes and long-term survival rate for Ad-p53-treated patients. To date, Shenzhen SiBiono GeneTech has manufactured 41 batches of Gendicine in compliance with CFDA QC/QA requirements, and 169,571 vials (1.0 × 10 vector particles per vial) have been used to treat patients. No serious adverse events have been reported, except for vector-associated transient fever, which occurred in 50-60% of patients and persisted for only a few hours. The manufacturing accomplishments and clinical experience with Gendicine, as well as the understanding of its cellular mechanisms of action and implications, could provide valuable insights for the international gene therapy community and add valuable data to promote further developments and advancements in the gene therapy field.

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ObjectiveTo detect and compare the bone microstructure and osteoblast and osteoclast activity in different regions of human osteonecrotic femoral heads.MethodsOsteonecrotic femoral heads were obtained from 10 patients (6 males, 4 females; Ficat IV) undergoing total hip arthroplasty between 2011 and 2013. The samples were divided into subchondral bone, necrotic, sclerotic, and healthy regions based on micro-computed tomography (CT) images. The bone microstructure, micromechanics, and osteoblast and osteoclast activity were assessed using micro-CT, pathology, immunohistochemistry, nanoindentation, reverse transcription polymerase chain reaction (RT-PCR), tartrate-resistant acid phosphatase staining and Western blotting.Results(1) The spatial structure of the bone trabeculae differed markedly in the various regions of the osteonecrotic femoral heads. (2) The elastic modulus and hardness of the bone trabeculae in the healthy and necrotic regions did not differ significantly (P >0.05). (3) The subchondral bone and necrotic region were positive on TRAP staining, while the other regions were negative. (4) On immunohistochemical staining, RANK and RANKL staining intensities were increased significantly in the subchondral bone and necrotic region compared with the healthy region, while RUNX2 and BMP2 staining intensities were increased significantly in the sclerotic region compared with the necrotic region. (5) OPG, RANK, RANKL, RUNX2, BMP2, and BMP7 protein levels were greater in the necrotic and sclerotic region than in subchondral bone and the healthy region.ConclusionThe micromechanical properties of bone trabeculae in the necrotic region did not differ significantly from the healthy region. During the progress of osteonecrosis, the bone structure changed markedly. Osteoclast activity increased in subchondral bone and the necrotic region while osteoblast activity increased in the sclerotic region. We speculate that the altered osteoblast and osteoclast activity leads to a reduction in macroscopic mechanical strength.

Progerin, a truncated unprocessed lamin A protein, causes tissue aging and degeneration. In this study we explored the role of progerin in the pathogenesis of intervertebral disc degeneration (IDD). We also examined the effect of sulforaphane (SFN) on progerin accumulation and mitochondrial dysfunction in IDD. Methods : The role of progerin in IDD was explored using human nucleus pulposus (NP) tissues, rat NP cells, and Lmna G609G knock-in mice. Immunostaining, X-ray imaging, and Western blotting were performed to assess the phenotypes of intervertebral discs. Alterations in senescence and apoptosis were evaluated by SA-β-galactosidase, immunofluorescence, flow cytometry, and TUNEL assays. Mitochondrial function was investigated by JC-1 staining, transmission electron microscopy, and determination of the level of ATP and the activities of mitochondrial enzymes. Results : The progerin level was elevated in degenerated human NP tissues. Lmna G609G/G609G mice displayed IDD, as evidenced by increased matrix metalloproteinase-13 expression and decreased collagen II and aggrecan expression and disc height. Furthermore, progerin overexpression in rat NP cells induced mitochondrial dysfunction (decreased ATP synthesis, mitochondrial membrane potential, and activities of mitochondrial complex enzymes), morphologic abnormalities, and disrupted mitochondrial dynamic (abnormal expression of proteins involved in fission and fusion), resulting in apoptosis and senescence. SFN ameliorated the progerin-induced aging defects and mitochondrial dysfunction in NP cells and IDD in Lmna G609G/G609G mice. Conclusions : Progerin is involved in the pathogenesis of IDD. Also, SFN alleviates progerin‑induced IDD, which is associated with amelioration of aging defects and mitochondrial dysfunction. Thus, SFN shows promise for the treatment of IDD.

Intervertebral disc degeneration (IDD) has been generally accepted as the major cause of low back pain (LBP), which causes an enormous socioeconomic burden. Previous studies demonstrated that the apoptosis of nucleus pulposus (NP) cells and the dyshomeostasis of extracellular matrix (ECM) contributed to the pathogenesis of IDD, and effective therapies were still lacking. Quercetin, a natural flavonoid possessing a specific effect of autophagy stimulation and SIRT1 activation, showed some protective effect on a series of degenerative diseases. Based on previous studies, we hypothesized that quercetin might have therapeutic effects on IDD by inhibiting the apoptosis of NP cells and dyshomeostasis of ECM via the SIRT1-autophagy pathway. In this study, we revealed that quercetin treatment inhibited the apoptosis of NP cells and ECM degeneration induced by oxidative stress. We also found that quercetin promoted the expression of SIRT1 and autophagy in NP cells in a dose-dependent manner. Autophagy inhibitor 3-methyladenine (3-MA) reversed the protective effect of quercetin on apoptosis and ECM degeneration. Moreover, SIRT1 enzymatic activity inhibitor EX-527, suppressed quercetin-induced autophagy and the protective effect on NP cells, indicating that quercetin protected NP cells against apoptosis and prevented ECM degeneration via SIRT1-autophagy pathway. In vivo, quercetin was also demonstrated to alleviate the progression of IDD in rats. Taken together, our results suggest that quercetin prevents IDD by promoting SIRT1-dependent autophagy, indicating one novel and effective therapeutic method for IDD.

Circular RNA (circRNA) is a group of RNA families generated by RNA circularization, which was discovered ubiquitously across different cancers. However, the internal structure of circRNA is difficult to determine due to alternative splicing that occurs in its exons and introns. Furthermore, cancer-specific alternative splicing of circRNA is less likely to be identified. Here, we proposed a de novo algorithm, CircSplice, that could identify internal alternative splicing in circRNA and compare differential circRNA splicing events between different conditions ( http://gb.whu.edu.cn/CircSplice or https://github.com/GeneFeng/CircSplice ). By applying CircSplice in clear cell renal cell carcinoma and bladder cancer, we detected 4498 and 2977 circRNA alternative splicing (circ-AS) events in the two datasets respectively and confirmed the expression of circ-AS events by RT-PCR. We further inspected the distributions and patterns of circ-AS in cancer and adjacent normal tissues. To further understand the potential functions of cancer-specific circ-AS, we classified those events into tumor suppressors and oncogenes and performed pathway enrichment analysis. This study is the first comprehensive view of cancer-specific circRNA alternative splicing, which could contribute significantly to regulation and functional research of circRNAs in cancers. Electronic supplementary material The online version of this article (10.1186/s12943-019-0996-0) contains supplementary material, which is available to authorized users.

BackgroundBone marrow is an important source of stem cells, which can promote bone fracture healing.MethodsWe investigated the optimal time to inject bone marrow mesenchymal stem cells (BMSCs) in a C57 murine unilateral, transverse, femur fracture model. BMSCs transfected with red fluorescent protein (RFP-BMSCs) were injected via the tail vein on day 1, 7, or 14 post-fracture. AMD3100 (inhibitor of stromal cell-derived factor 1 [SDF-1]) was also injected before RFP-BMSCs in one group for comparison; a control group received saline injections. RFP-BMSC migration and fracture healing were evaluated by in vivo fluorescence assay. Micro-CT was performed and mechanical testing and histological analysis. Chemokine levels were evaluated by quantitative real-time PCR and western blotting.ResultsFollowing injection on day 7 post-fracture, RFP-BMSCs more frequently homed to the fracture site and remained for a longer duration. Bone volume and bone mineral density were increased when BMSCs were injected on day 7 post-fracture (P < 0.05). The mechanical properties of fractured femurs were improved following day-7 BMSC injection. Histology confirmed that BMSC injection improved the formation of new bones.ConclusionsChemokines that induce BMSC migration were highly expressed, and protein levels of osteogenesis-related factors were increased. Seven days after fracture may be the optimal time for injection of BMSCs to promote fracture healing. Additionally, the SDF-1/CXCR4 pathway may play an important role in fracture healing following BMSC injection.