第6卷，第3期

Although bone morphogenetic proteins (BMPs) initially showed effective induction of ectopic bone growth in muscle, it has since been determined that these proteins, as members of the TGF-β superfamily, play a diverse and critical array of biological roles. These roles include regulating skeletal and bone formation, angiogenesis, and development and homeostasis of multiple organ systems. Disruptions of the members of the TGF-β/BMP superfamily result in severe skeletal and extra-skeletal irregularities, suggesting high therapeutic potential from understanding this family of BMP proteins. Although it was once one of the least characterized BMPs, BMP9 has revealed itself to have the highest osteogenic potential across numerous experiments both in vitro and in vivo, with recent studies suggesting that the exceptional potency of BMP9 may result from unique signaling pathways that differentiate it from other BMPs. The effectiveness of BMP9 in inducing bone formation was recently revealed in promising experiments that demonstrated efficacy in the repair of critical sized cranial defects as well as compatibility with bone-inducing bio-implants, revealing the great translational promise of BMP9. Furthermore, emerging evidence indicates that, besides its osteogenic activity, BMP9 exerts a broad range of biological functions, including stem cell differentiation, angiogenesis, neurogenesis, tumorigenesis, and metabolism. This review aims to summarize our current understanding of BMP9 across biology and the body.

1333

After more than one hundred years of documented trials, immunotherapy has become a standard of care in the treatment of human cancer. Much of the knowledge that led to recent breakthroughs seems quite logical from today's point of view. However, what we now cite as facts were originally considered paradoxes, meaning something contrary to expectations or perceived opinion at the time. In order to make gains in the field of immunotherapy, one had to be willing to confront ideas and concepts that seemed to contradict one another, and reconcile how each could be true. This is what led to new knowledge and advances. Here, we highlight some of these paradoxes and the milestone discoveries that followed, each one critical for our understanding of immune checkpoint pathways. By outlining some of the steps that we took and the challenges that we overcame, we hope to inspire and encourage future generations of researchers to confront the paradoxes that still permeate the field.

1299

Immune checkpoint blockade therapies (ICBs) are a prominent breakthrough in cancer immunotherapy in recent years (named the 2013 "Breakthrough of the Year" by the Science magazine). Thus far, FDA-approved ICBs primarily target immune checkpoints CTLA-4, PD-1, and PD-L1. Notwithstanding their impressive long-term therapeutic benefits, their efficacy is limited to a small subset of cancer patients. In addition, ICBs induce inadvertent immune-related adverse events (irAEs) and can be costly for long-term use. To overcome these limitations, two strategies are actively being pursued: identification of predictive biomarkers for clinical response to ICBs and multi-pronged combination therapies. Biomarkers will allow clinicians to practice a precision medicine approach in ICBs (biomarker-based patient selection) such as treating triple-negative breast cancer patients that exhibit PD-L1 staining of tumor-infiltrating immune cells in ≥1% of the tumor area with nanoparticle albumin-bound (nab) epaclitaxel plus anti-PD-L1 and treating patients of MSI-H or MMR deficient unresectable or metastatic solid tumors with pembrolizumab (anti-PD-1). Importantly, the insights gained from these biomarker studies can guide rational combinatorial strategies such as CDK4/6 inhibitor/fractionated radiotherapy/HDACi in conjunction with ICBs to maximize therapeutic benefits. Further, with the rapid technological advents (e.g., ATCT-Seq), we predict more reliable biomarkers will be identified, which in turn will inspire more promising combination therapies.

1322

Arginine methylation is a common posttranslational modification that governs important cellular processes and impacts development, cell growth, proliferation, and differentiation. Arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs), which are classified as type I and type II enzymes responsible for the formation of asymmetric and symmetric dimethylarginine, respectively. PRMT5 is the main type II enzyme that catalyzes symmetric dimethylarginine of histone proteins to induce gene silencing by generating repressive histone marks, including H2AR3me2s, H3R8me2s, and H4R3me2s. PRMT5 can also methylate nonhistone proteins such as the transcription factors p53, E2F1 and p65. Modifications of these proteins by PRMT5 are involved in diverse cellular processes, including transcription, translation, DNA repair, RNA processing, and metabolism. A growing literature demonstrates that PRMT5 expression is upregulated in hematologic malignancies, including leukemia and lymphoma, where PRMT5 regulates gene expression to promote cancer cell proliferation. Targeting PRMT5 by specific inhibitors has emerged as a potential therapeutic strategy to treat these diseases.

1359

Mesenchymal stem cells (MSCs) are ubiquitously-existing multipotent progenitors that can self-renew and differentiate into multiple lineages including osteocytes, chondrocytes, adipocytes, tenocytes and myocytes. MSCs represent one of the most commonly-used adult progenitors and serve as excellent progenitor cell models for investigating lineagespecific differentiation regulated by various cellular signaling pathways, such as bone morphogenetic proteins (BMPs). As members of TGFβ superfamily, BMPs play diverse and important roles in development and adult tissues. At least 14 BMPs have been identified in mammals. Different BMPs exert distinct but overlapping biological functions. Through a comprehensive analysis of 14 BMPs in MSCs, we demonstrated that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of MSCs. Nonetheless, a global mechanistic view of BMP signaling in regulating the proliferation and differentiation of MSCs remains to be fully elucidated. Here, we conducted a comprehensive transcriptomic profiling in the MSCs stimulated by 14 types of BMPs. Hierarchical clustering analysis classifies 14 BMPs into three subclusters: an osteo/chondrogenic/adipogenic cluster, a tenogenic cluster, and BMP3 cluster. We also demonstrate that six BMPs (e.g., BMP2, BMP3, BMP4, BMP7, BMP8, and BMP9) can induce I-Smads effectively, while BMP2, BMP3, BMP4, BMP7, and BMP11 up-regulate Smad-independent MAP kinase pathway. Furthermore, we show that many BMPs can upregulate the expression of the signal mediators of Wnt, Notch and PI3K/AKT/mTOR pathways. While the reported transcriptomic changes need to be further validated, our expression profiling represents the first-of-its-kind to interrogate a comprehensive transcriptomic landscape regulated by the 14 types of BMPs in MSCs.

1268

EZH2 is a component of the polycomb repressive complex 2 (PRC2), which is a highly conserved histone methyltransferase that methylates lysine 27 of histone 3. EZH2 mutations are associated with oncogenesis and progression of cancers. However, the relationship between the clinical outcome of patients with myeloid malignancies and EZH2 mutations is controversial. Therefore, we performed a meta-analysis of 8 studies (n=2243 patients) that evaluates the correlation between EZH2 mutations and overall survival (OS) in patients with myeloid neoplasms. EZH2 mutations were associated with significantly worse OS (hazard ratio[HR]Z 2.37, 95% confidential interval (CI), 1.48-3.79). In a word, EZH2 mutations indicate a poor prognosis for patients with myeloid neoplasms.

1355

Metabolic syndrome (MetS) is a clustering of metabolic abnormalities that is associated with increased risk of developing cardiovascular disease and type 2 diabetes. There is growing body of data showing the associations of genetic variants of the genes involved in the PI3K/AKT/mTOR pathway with diabetes and obesity. We aimed to investigate the association between MetS and its components with the genetic polymorphism in AKT1, rs1130233 (T>C). Total of 618 participants, recruited from Mashhad stroke and heart atherosclerosis disorder cohort (MASHAD study). Patients with MetS were defined by using international diabetes federation (IDF) criteria (n=326) and those without MetS (n=261) were recruited. Anthropometric and biochemical parameters were measured in all subjects. Genetic analysis for the rs1130233 polymorphism was performed, using the ABI-StepOne instruments with SDS version-2.0 software. Individuals with MetS had a significantly higher levels of BMI, waist-circumference, total cholesterol, triglyceride, high sensitivity-c reactive protein (hs-CRP) and blood-pressure, and lower concentrations of high density lipoprotein (HDL-C), compared to non-MetS individuals (P<0.05). The association between the rs1130233 and MetS was not significant. Subjects with a CC or CT genotypes had a significantly higher serum hs-CRP-level (OR: 1.5;95% CI (1.05-2.1), P=0.02). Additionally, subjects who carried the TC genotype had a higher BMI compared to the CC genotype (p value=0.045). Our findings demonstrated that AKT1, rs1130233 (T>C) polymorphism was associated with major components of MetS such as hs-CRP, and BMI, indicating further investigation in a multi-center setting to explore its value as an emerging biomarker of risk stratification marker.

1298

Chronic Hepatitis C Viral (HCV) infection is a leading health problem worldwide and resulted in fibrotic scar formation, and finally liver-cirrhosis. Although contemporary therapies can partially reverse this destructive process, the rehabilitation is too slow and unsuitable for all chronic infections. The current study elucidates the mechanism of disease progression from early (F1) to moderate (F2, F3), and to severe fibrosis (F4)/cirrhosis in HCV genotype 3a infected patients to find out new candidates as potential disease progression markers and antiviral therapeutic agents. A total of 550 genes were found differentially regulated in the four fibrosis stages and grouped in 22 classes according to their biological functions. Gene set enrichment (GSEA) and Ingenuity pathway analysis (IPA) were used to identify the regulation of crucial biological functions and pathways involved in HCV progression. HCV differentially regulated the expression of genes involved in apoptosis, cell structure, signal transduction, proliferation, metabolism, cytokine signaling, immune response, cell adhesion and maintenance, and post translational modifications by pathway analysis. There was an increasing trend of proliferative and cell growth related genes and shutting down of immune response as the disease progress mild to moderate to advanced stage cirrhosis. The myriad of changes in gene expression showed more chances of developing liver cancer in patients infected with HCV genotype 3a in a systematic manner. The identified gene set can act as disease markers for prediction, whether the fibrosis lead to cirrhosis and its association with end stage liver disease development.

1259

Liver cancer is the sixth most commonly diagnosed cancer and the fourth leading cause of cancer death worldwide. Western medicine and therapies are the primary treatment strategies of hepatocellular carcinoma (HCC), but the general prognosis for HCC patients is still dismal. Under these circumstances, HCC prevention is particularly important. Traditional Chinese medicine (TCM) encompasses a wealth of documented therapeutic resources, and "preventative treatment" is the principle of TCM. In China, TCM has been used for HCC prevention for thousands of years, and has also been demonstrated to be effective for the treatment of HCC in modern China. However, the TCM theory for prevention and treatment of HCC is more widely accepted in China than abroad. In this review, we first summarize the herbs and ancient formulas with therapeutic effects on HCC. We also review the research status of TCM in modern medicine as well as the current obstacles in its development. Finally, we discuss the future of TCM in the context of precision and integrated medicine. After reviewing the literature, we believe that TCM, through ancient development, is an advanced method of cancer treatment with positive curative effects, despite its surrounding controversy. Furthermore, precise analyses and systematic research methods provides novel approaches to modernize TCM for the future.

1867

Despite advances in screening and treatment, colon cancer remains one of the leading causes of cancer-related death. Finding novel and useful drug treatment targets is also an urgent need for clinical applications. Tetrandrine (Tet) is extracted from the Chinese medicinal herbal medicine, which is a well-known calcium blocker with a variety of pharmacological activities, including anti-cancer. In this study, we recruited cell viability assay, flow cytometry analysis, cloning formation to con firm that Tet can inhibit the proliferation of SW620 cells, and induce apoptosis. Mechanically, we confirmed that Tet up-regulates the mRNA and protein level of BMP9 in SW620 cells. Over-expression BMP9 enhances the anticancer effects of Tet in SW620 cells, but these effects can be partly reversed by silencing BMP9. Also, Tet reduces phosphorylation of Aktl/2/3 in SW620 cells, which could be elevated by overexpressed BMP9 and impaired by silencing BMP9. Furthermore, we demonstrated that Tet reduces phosphorylated PTEN, which can be promoted by overexpressed BMP9, analogously also be attenuated through silencing BMP9. Finally, we introduced a xenograft tumor model to investigate the anti-proliferative effect of Tet, further to explore the effects of BMP9 and PTEN in SW620 cells. Our findings suggested that the anti-cancer activity of Tet in SW620 cells may be mediated partly by up-regulating BMP9, followed by inactivation PI3K/Akt through up-regulating PTEN at least.

1286

In Egypt, Sofosbuvir (SOF) in combination with Dataclasvir (DCV) is the broadly used DAAs with excellent therapeutic profile. This study is designed to explore the relation between IL28B/TLR4 genetic variants and each of the followings; HCC development post SOF/DCV treatment, progression to HCC in naïve patients and SOF/DCV therapy outcome. A total of 493 blood samples were collected (controls (n=70); HCV patients treated with SOF/DCV (n=252) of whom 65 patients developed HCC, 187 patients didn't develop HCC (125 responders, 62 relapsers); naïve HCV patients (n=171) had early (n=48), late liver fibrosis (n=21) and HCC (n=102)). Both SNPs were genotyped using a TaqMan 5′ allelic discrimination assay. At IL28B rs12979860 SNP, the C allele was significantly correlating with the response rate more than Tallele (OR 1.9, 95% CI 1.29-2.9, p=0.004), while at TLR4 rs4986791 SNP, no association was found (OR 6.5, 95% 0.57-75.28, p=0.09). Both SNPs couldn't detect the probability for HCC emergence after treatment. In naïve patients, the protective alleles were detected in their lowest frequency in HCC patients (p=0.1, for rs12979860 and, p=0.001 for rs4986791). SOF/DCV combination improved SVR rates in HCV genotype 4a infected patients regardless of IL28B genotype, with the best rates in those lacking the T allele.

1891

Vps34 (vacuolar protein-sorting 34) plays important role in autophagy and endosomal trafficking. These processes are closely associated protein ubiquitination and degradation. We have hypothesized that Vps34 ubiquitination status would also control its degradation. Here, we report that our results did not support this assumption. In cells transiently transfected with ubiquitin (UB) constructs contained different lysine residues (Ks), Vps34 ubiquitination could occur regardless of the presence of any Ks in UB. However, Vps34 protein levels were not significantly altered in cells transiently transfected with these UB mutants. We further found that Vps34 protein was altered by pharmacological manipulation of E2/E3 activity; yet this effect was not significantly affected by UB overexpression. In vivo experiments revealed that in APP/PS1 mice, an animal model of Alzheimer's disease (AD), although ubiquitination of Vps34 was significantly reduced, Vps34 protein levels remained unchanged. Vps34 indeed was subjected to proteasomal or lysosomal degradation, as prolonged treatment of proteasomal inhibitor MG132 or lysosomal inhibitor chloroquine elevated Vps34 protein levels. We conclude that unlike most of other proteins, Vps34 ubiquitination is not closely associated with its degradation.

1861

This study aimed at investigating the vitamin D status of pediatric patients with upper limb fractures and finding out the influences of age, gender, season and fracture sites. 695 patients were admitted for upper limb fractures between November 1st 2014 and October 31st 2015. 224 healthy children were included during the same period. Serum 25(OH)D was measured at the time of visit and their demographic data were recorded. Generally fracture patients had significant lower vitamin D level than the healthy children. The average serum 25(OH)D of the patients was 24.5 ng/ml, compared to 28.1 ng/ml in healthy children. Vitamin D status worsened with the increase of age and adolescents had a deficiency rate of as high as 68.6%. Gender and fracture sites had no influence on vitamin D level. This study revealed high prevalence of vitamin D deficiency and insufficiency in pediatric patients with upper limb fractures. Adolescents had such high rate of vitamin D deficiency that called for vast attention.

1923

Circular RNAs (circRNAs) are a novel class of endogenous noncoding RNAs that play important roles in gene expression regulation. This study aimed to evaluate the potential role of circRNAs in decidual tissue of patients with early recurrent miscarriage (RM). We constructed circRNA expression profiles in decidual tissue using microarray data. A total of 123 differentially expressed circRNAs, including 78 upregulated and 45 downregulated circRNAs were detected in the early RM group compared with the control group (P < 0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also revealed the enrichment of specific circRNAs. The verified circRNA-targeted miRNA-mRNA network was constructed, most of the circRNAs harbored miRNA binding sites. The network involved 3 circRNAs, 27 microRNAs and 82 mRNAs. Hsa_circRNA_103092-miR-224-PRLR network was selected to verify by qRT-PCR. These results showed that circRNAs are aberrantly expressed in the decidual tissue in early RM patients and play potential roles in the development of early RM. It gives new insights into the mechanism of recurrent miscarriage.

1945

Glioblastoma multiform (GBM) is the most lethal intracranial tumor in adults. Glioblastoma stem-like cells (GSCs) are responsible for tumorigenesis and chemotherapy resistance. BMPs are known to increase temozolomide (TMZ) response in GSCs, however, the intracellular molecular mechanism remains largely unknown. In this study, we built a GSC cell model called U87S, and performed RNA sequencing to identify differentially expressed (DE) miRNA profiles in U87S cells treated with BMP2, TMZ or combined BMP2 and TMZ respectively. Bioinformatics analysis revealed that most DE miRNAs were involved in the cancer pathways, suggesting their crucial roles in gliomagenesis. Eight miRNAs from RNA-seq were validated. Four out of these miRNAs (has-miR-199a-3p, hsa-miR-374b-5p, hsa-miR-320d, and hsa-miR-339-5p) were found significantly up-regulated in GBM tumor tissues. One of them, hsa-miR-199a-3p, was significantly correlated with the survival of GBM patients, and differentially expressed in U87S cells. Expression of hsa-miR-199a-3p was up-regulated by BMP. Overexpression of hsa-miR-199a-3p in U87S cells inhibited cell viability and enhanced the cytotoxicity of TMZ. And activation of BMP boosted the effect of hsa-miR-199a-3p on cell viability and TMZ-mediated cytotoxicity. Besides, expressions of five predicted targets of hsa-miR-199a-3p were evaluated. Four of them were differentially expressed in GBM tumors. And one of them, SLC22A18, was associated with the survival of GBM patients. In the end, a hsa-miR-199a-3p-mediated ceRNA network was constructed for the convenience of future study. Together, our data provided DE miRNA expression profiles associated with BMP2 and TMZ in GSCs, which might lead to finding out miRNA-based target therapies that specially target GSCs.

1871

Cleft lip and/or palate (CL/P) is a most common craniofacial birth defect which has multifactorial etiology. In our study, we aimed to discover the underlying etiological gene variation in a Chinese family diagnosed as non-syndromic CL/P (NSCL/P). The blood sample of the proband and her parents were detected by whole exome sequencing. The Mendelian inheritance pattern, allele frequency, variation location, function analysis and literature search were applied to filtrate and screen the mutation. Besides, the candidates were confirmed by Sanger sequencing. We meanwhile explored the conservative analysis and protein homology simulation. As a result, a start-lost mutation c.1A > GAtg/Gtg in the Frizzled-6 (FZD6) gene predicting p. Met1 was detected. The variation has not been reported before and was predicted to be harmful. The alteration caused missing of two starting amino acids that are evolutionarily conserved for FZD6 protein. Moreover, the specific structure of the mutant protein obviously changed according to the results of the homologous model. In conclusion, the results suggest c.1A > GAtg/Gtg in the FZD6 (NM_001164616) might be the genetic etiology for non-syndromic CL/P in this pedigree. Furthermore, this finding provided new etiologic information, supplementing the evidence that FZD6 is a strong potential gene for CL/P.

1808

The number and spatial configuration of the screws will affect the stability and prognosis of the fractures. In our study, we assessed the biomechanical effects of the double-head cannulated compression screw (DhCCS) and ordinary cannulated compression screw (OCCS) for the treatment of femoral neck fractures by using computer finite element analysis. The original digital imaging and communications in medicine (DICOM) data of a proximal femur were imported into Materialise's interactive medical image control system (MIMICS) software for modeling. Both DhCCS and OCCS 3D-models were obtained by using the 3D scan technique. Using the fracture model and internal fixation assembly model with an inverted triangle, two horizontal and vertical distribution were established in UG software. Next, the displacement and stress distribution were calculated in ANSYS software. The displacement value of the femoral head in the DhCCS group was smaller than that in the OCCS group, and the displacement value in the two horizontal groups was smaller than that in the vertical group. The stress distribution in the DhCCS group was concentrated on the screw rod at the fracture block and thread end, while only at the fracture block in the OCCS group. The stress in the horizontal group was more dispersed on the screws than that in the vertical group. DhCCS has reliable stability for the fixation of femoral neck fractures and applied in the clinical work and 2 horizontal fixation can be used when two screws are selected.

1961