A potential role of human esophageal cancer-related gene-4 in cardiovascular homeostasis.

Human esophageal cancer related gene-4 (ECRG-4) encodes a 148-aminoacid pre-pro-peptide that can be processed tissue-dependently into multiple small peptides possessing multiple functions distinct from, similar to, or opposite to the tumor suppressor function of the full-length Ecrg4. Ecrg-4 is covalently bound to the cell surface through its signal peptide, colocalized with the innate immunity complex (TLR4-CD14-MD2), and functions as a 'sentinel' molecule in the maintenance of epithelium and leukocyte homeostasis, meaning that the presence of Ecrg-4 on the cell surface signals the maintained homeostasis, whereas the loss of Ecrg-4 due to tissue injury activates pro-inflammatory and tissue proliferative responses, and the level of Ecrg-4 gradually returns to its pre-injury level upon wound healing. Interestingly, Ecrg-4 is also highly expressed in the heart and its conduction system, endothelial cells, and vascular smooth muscle cells. Accumulating evidence has shown that Ecrg-4 is involved in cardiac rate/rhythm control, the development of atrial fibrillation, doxorubicin-induced cardiotoxicity, the ischemic response of the heart and hypoxic response in the carotid body, the pathogenesis of atherosclerosis, and likely the endemic incidence of idiopathic dilated cardiomyopathy. These preliminary discoveries suggest that Ecrg-4 may function as a 'sentinel' molecule in cardiovascular system as well. Here, we briefly review the basic characteristics of ECRG-4 as a tumor suppressor gene and its regulatory functions on inflammation and apoptosis; summarize the discoveries about its distribution in cardiovascular system and involvement in the development of CVDs, and discuss its potential as a novel therapeutic target for the maintenance of cardiovascular system homeostasis.

CangFu Daotan decoction improves polycystic ovarian syndrome by downregulating FOXK1.

Objective: Polycystic ovarian syndrome (PCOS) is a prevalent gynecologic disorder, often associated with abnormal follicular development. Cangfu Daotan decoction (CFD) is a traditional Chinese medicine formula that is effective in alleviating PCOS clinically, but the specific mechanism remains unclear. Forkhead box K1 (FOXK1) is associated with cellular function. This study aimed to explore the effects of CFD and FOXK1 on PCOS.Methods: High-fat diet and letrozole were combined to establish PCOS rat models. Next, primary GCs were extracted from those PCOS rats. Then, GC cells were transfected with si-FOXK1 or oe-FOXK1. CFD-contain serum was prepared, and experiments were conducted to investigate the regulation of FOXK1 by CFD.Results: FOXK1 was highly expressed in GCs of PCOS rats. Further investigation revealed that FOXK1 overexpression resulted in inhibition of proliferation and DNA synthesis, along with promotion of apoptosis and autophagy in GCs. Additionally, it was found that FOXK1 promoted the expressions of the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway-related proteins. Interestingly, treatment with CFD reversed all the effects of FOXK1 overexpression in GCs. Conclusion: This study demonstrated that CFD exerted a protective role in PCOS by inhibiting FOXK1, which provided a research basis for the application of CFD in PCOS, and suggested that FOXK1 is a novel therapeutic target in PCOS treatment.

Downregulated lncRNA HOTAIR ameliorates polycystic ovaries syndrome via IGF-1 mediated PI3K/Akt pathway.

OBJECTIVE: Polycystic ovarian syndrome (PCOS) is a common disorder that leads to infertility in reproductive-aged females. HOTAIR is highly expressed in various gynecological diseases and is associated with a poor prognosis. We aimed to explore the role of HOTAIR in PCOS. METHODS: First, PCOS rats were induced using dehydroepiandrosterone and then treated with si-HOTAIR. Next, HOTAIR mRNA expression and serum hormone levels were detected. HE staining was applied to observe estrus cycle, ovarian morphology and count the number of follicles. Apoptosis in the ovary was detected by TUNEL. Thereafter, ovarian granulosa cells (GCs) were isolated from PCOS rats, transfected with si-HOTAIR and treated with LY294002 (Akt inhibitor) or IGF-1. CCK-8 and flow cytometry assays were used to evaluate cell viability and apoptosis. IGF-1, apoptosis- and PI3K/Akt pathway-associated protein expressions in ovary and GCs were also detected. RESULTS: In in vivo experiments, si-HOTAIR decreased serum T, E2 and LH levels but increased FSH level, restored estrus cycle, ovarian morphology and inhibited apoptosis of ovary in PCOS rats. Meanwhile, in vitro assays showed that si-HOTAIR upregulated the viability but inhibited the apoptosis of PCOS GCs. Furthermore, both in vivo and in vitro assays revealed that si-HOTAIR increased Bcl-2 expression but suppressed Bax, Bad, IGF-1 expressions and PI3K, AKT phosphorylation. However, the aforementioned effects of si-HOTAIR in vitro were further enhanced by LY294002 and partially reversed by IGF-1. CONCLUSIONS: HOTAIR knockdown improved PCOS, and the mechanism may relate to IGF-1-mediated PI3K/Akt pathway, indicating HOTAIR may be a novel therapeutic target for PCOS.

Circular RNAs Involve in Immunity of Digestive Cancers From Bench to Bedside: A Review.

The immune system plays a complex role in tumor formation and development. On the one hand, immune surveillance can inhibit the growth of tumors; on the other hand, immune evasion of tumors can create conditions conducive for tumor development and growth. CircRNAs are endogenous non-coding RNAs with a covalently closed loop structure that are abundantly expressed in eukaryotic organisms. They are characterized by stable structure, rich diversity, and high evolutionary conservation. In particular, circRNAs play a vital role in the occurrence, development, and treatment of tumors through their unique functions. Recently, the incidence and mortality of digestive cancers, especially those of gastric cancer, colorectal cancer, and liver cancer, have remained high. However, the functions of circRNAs in digestive cancers immunity are less known. The relationship between circRNAs and digestive tumor immunity is systematically discussed in our paper for the first time. CircRNA can influence the immune microenvironment of gastrointestinal tumors to promote their occurrence and development by acting as a miRNA molecular sponge, interacting with proteins, and regulating selective splicing. The circRNA vaccine even provides a new idea for tumor immunotherapy. Future studies should be focused on the location, transportation, and degradation mechanisms of circRNA in living cells and the relationship between circRNA and tumor immunity. This paper provides a new idea for the diagnosis and treatment of gastrointestinal tumors.

Cardiac Expression of Esophageal Cancer-Related Gene-4 is Regulated by Sp1 and is a Potential Early Target of Doxorubicin-Induced Cardiotoxicity.

Esophageal Cancer-Related Gene 4 (Ecrg4) expressed in cardiomyocytes and the cardiac conduction system is downregulated during cardiac ischemia and atrial fibrillation. To explore whether Ecrg4 plays any role in doxorubicin (DOX)-induced cardiotoxicity. Rats and neonatal rat cardiomyocytes (NRCMs) were employed to study the effect of DOX on Ecrg4 transcription. Bioinformatics combined with promoter analysis were used to map the rat Ecrg4 promoter. ChIP assay was used to evaluate the binding of Sp1 to the Ecrg4 promoter. Transient transfection was used to study the effect of Sp1 on the expression of endogenous Ecrg4. DOX decreased endogenous Ecrg4 gene expression in the heart and cultured NRCMs. In silico analysis showed that the 5'UTR immediately upstream of the start codon ATG, harbors a putative promoter that is GC-rich, and contains CpG islands, multiple overlapping Sp1sites. Transcription is initiated mainly on the 'C' at - 15. Serial 5'-deletion combined with dual-luciferase assays showed that the rat Ecrg4 core promoter resides at - 1/- 800. Sp1 transactivated Ecrg4 gene, which was almost abolished by DOX. Furthermore, ChIP assay showed that Sp1 specifically bound to the Ecrg4 promoter was interrupted by DOX. Finally, DOX suppressed Sp1 protein expression, and restoration of Sp1 increased Ecrg4 expression that was resistant to DOX-induced Ecrg4 downregulation. Importantly, cardiomyocyte-specific loss of Ecrg4 significantly enriched the differentially expressed proteins in the signaling pathways commonly involved in DOX-induced cardiotoxicity. Our results indicate that Sp1 mediates DOX-induced suppression of Ecrg4, which may contribute indirectly to its cardiotoxicity.

Cangfudaotan Decoction Alleviates Insulin Resistance and Improves Follicular Development in Rats with Polycystic Ovary Syndrome via IGF-1-PI3K/Akt-Bax/Bcl-2 Pathway.

Polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disorder prevalent in females of reproductive age; insulin resistance (IR) is the major pathogenic driver. Pharmacology is a basic option for PCOS therapy; traditional Chinese medicine (TCM), as a significant part of complementary and alternative medicine, has a long history in the clinical management of PCOS. Cangfudaotan decoction (CFD) has been used clinically for gynaecological diseases especially PCOS. In this study, first, chemical components in CFD were clarified using UPLC-Q/TOF-MS analysis. Then, an animal model of PCOS was established, granular cells were also isolated from the rats with PCOS, and CFD was administrated at different dosages in PCOS rats and granular cells, to investigate the therapeutic effect and mechanisms of CFD for PCOS treatment. The result showed that CFD treatment is effective in PCOS rats and granulosa cells. CFD was able to improve IR, restore the serum hormone levels, inhibit the inflammatory cytokines in PCOS rat, and alleviate ovary morphological injury and apoptosis in PCOS rats. In granulosa cells of PCOS, the result showed that the cell viability was improved, and cell apoptosis was inhibited after CFD administration. Further experiments suggested that CDF improves IR, follicular development, cell apoptosis, and inflammatory microenvironment, and this was associated to the regulation of IGF-1-PI3K/Akt-Bax/Bcl-2 pathway-mediated gene expression. Given that CFD sufficiently suppresses insulin resistance and improves follicular development in this study, exploring these mechanisms might help to optimize the therapeutic treatment of CFD in PCOS patients.

Straightened cervical lordosis causes stress concentration: a finite element model study.

In this study, we propose a finite element analysis of the complete cervical spine with straightened and normal physiological curvature by using a specially designed modelling system. An accurate finite element model is established to recommend plausible approaches to treatment of cervical spondylosis through the finite element analysis results. There are few reports of biomechanics influence of the straightened cervical curve. It is difficult to measure internal responses of cervical spine directly. However, the finite element method has been reported to have the capability to quantify both external and internal responses to mechanical loading, such as the strain and stress distribution of spinal components. We choose a subject with a straightened cervical spine from whom to collect the CT scan data, which formed the basis of the finite element analysis. By using a specially designed modelling system, a high quality finite element model of the complete cervical spine with straightened curvature was generated, which was then mapped to reconstruct a normal physiological curvature model by a volumetric mesh deformation method based on discrete differential properties. Then, the same boundary conditions were applied to do a comparison. The result demonstrated that the active movement range of straightened cervical spine decreased by 24-33 %, but the stress increased by 5-95 %. The stress was concentrated at the facet joint cartilage, uncovertebral joint and the disk. The results suggest that cervical lordosis may have a direct impact on cervical spondylosis treatment. These results may be useful for clinical treatment of cervical spondylosis with straightened curvature.

RET proto-oncogene genetic screening of families with multiple endocrine neoplasia type 2 optimizes diagnostic and clinical management in China.

BACKGROUND: Genetic screening for germline mutations in the RET proto-oncogene has been extensively exploited worldwide to optimize the diagnostic and clinical management of multiple endocrine neoplasia type 2 (MEN2) patients and their relatives. However, a distinct lag period exists not only in the recognition but also in the medical treatment of patients with MEN2. Here we present a comprehensive genetic and clinical analysis of MEN2 among Chinese families followed from 1975 to 2011. Our series comprises 36 index cases and 134 relatives from 11 independent families. METHODS: Genetic diagnosis was performed in all participants by direct sequencing all relevant RET exons. Thyroidectomy was performed in 50 patients with varying cervical neck dissection procedures. Patients with pheochromocytoma (PHEO) underwent specific surgery. Demographic, clinical profiles, mutation types, tumor histopathologic features, and follow-up records were systematically analyzed. RESULTS: The RET mutations p.C634Y (n=34), p.C634R (n=6), p.C618S (n=13), p.V292M/R67H/R982C (n=7), p.L790F (n=2), and p.C634Y/V292M/R67H/R982C (n=1) were confirmed in 31 index cases and then identified in 32 at-risk relatives (mutation carriers), with MEN2A as the most common clinical subtype. The overall penetrance of PHEO in patients with MEN2A was 46.7%. A total of 50 patients underwent thyroidectomy, and there was a significant lowering of their mean age at thyroidectomy and the tumor diameter of the mutation carriers that were detected and operated on compared with the index cases (age at first surgery: 29.3 vs. 39.3 years, p<0.05; maximum size: 1.1 vs. 3.3 cm, p<0.001). There was also a decrease in the TNM staging and the proportion of patients who underwent inappropriate initial thyroid surgery (pN1: 31.6% vs. 100%, p<0.001; inappropriate surgery: 0% vs. 29%). Meanwhile, disease-free survival (DFS) increased (DFS: 100% vs. 58.1%, p<0.05). Both medullary thyroid carcinoma-specific (n=1) and PHEO-specific (n=5) deaths were reported during the study period. CONCLUSIONS: Our results further substantiate that gene scanning of all relevant RET exons is a powerful tool in the management of MEN2 patients, especially in asymptomatic carriers, and has led to earlier diagnosis and more complete initial treatment of patients with MEN2 in China.

Two novel de novo mutations of KRT6A and KRT16 genes in two Chinese pachyonychia congenita pedigrees with fissured tongue or diffuse plantar keratoderma.

BACKGROUND: Mutations in the KRT6A or KRT16 gene cause pachyonychia congenita type 1 (PC-1), while mutations in KRT16 or KRT6C underlie focal palmoplantar keratoderma (FPPK). A new classification system of PC has been adopted based on the mutated gene. PC rarely presents the symptoms of diffuse plantar keratoderma. Mutation in the tail domain of keratins is rarely reported. PC combined with fissured tongue has never been described. OBJECTIVES: To investigate the genotype-phenotype correlations between clinical features and gene mutational sites in two unrelated southern Chinese PC pedigrees (one family presented with specific fissured tongue, the other with diffuse plantar keratoderma). MATERIALS & METHODS: The whole coding regions of the KRT6A/KRT16/KRT17/KRT6B genes were amplified and directly sequenced to detect the mutation. To confirm the effect of the IVS8-2A>C mutation in KRT6A at the mRNA level, total RNA from the plantar lesion of a patient was extracted and reverse-transcribed to cDNA for sequence analysis. RESULTS: Two novel de novo mutations, a splice acceptor site variant IVS8-2A>C (p.S487FfsX72) in KRT6A and a heterozygous substitution c.AA373_374GG (p.N125G) within exon 1 of KRT16, were found separately in the two PC families. CONCLUSION: Genotype-phenotype correlations among PC patients with codon-125 mutation in KRT16 were established, while the phenotypes caused by the IVS8-2A>C mutation in KRT6A need further studies to confirm the rare feature of fissured tongue.

Complete mitochondrial DNA sequence analysis in two southern Chinese pedigrees with Leber hereditary optic neuropathy revealed secondary mutations along with the primary mutation.

AIM: To investigate mitochondrial factors associated with Leber hereditary optic neuropathy (LHON) through complete sequencing and analysis of the mitochondrial genome of Chinese patients with this disease. METHODS: Two unrelated southern Chinese families with LHON and 10 matched healthy controls were recruited, and their entire mitochondrial DNA (mtDNA) was amplified and sequenced with the universal M13 primer. Then DNA sequence analysis and variation identification were perfomed by DNAssist and Chromas 2 software and compared with authoritative databases such as Mitomap. RESULTS: Mutational analysis of mtDNA in these two Chinese pedigrees revealed one common LHON-associated mutation, G11778A (Arg→His), in the MT-ND4 gene. In addition, there were two secondary mutations in Pedigree 1: C3497T (Ala→Val), and C3571T (Leu→Phe) in the MT-ND1 gene, which have not been reported; and two secondary mutations occurred in Pedigree 2: A10398G (Thr→Ala) in the MT-ND3 gene, and T14502C (Ile→Val) in the MT-ND6 gene. Three polymorphisms, A73G, G94A and A263G in the mtDNA control region, were also found. CONCLUSION: Our study confirmed that the known MT-ND4*G11778A mutation is the most significant cause of LHON. The C3497T and C3571T mutations in Pedigree 1 were also both at hot-spots of MT-ND1; they may affect the respiratory chain in coordination with the primary mutation G11778A. In Pedigree 2, the two secondary mutations A10398G of MT-ND3 and T14502C of MT-ND6 may influence mitochondrial respiratory complex I, leading to the mitochondrial respiratory chain dysfunction which results in optic atrophy together with G11778A. Therefore, not only the common primary LHON mutation is responsible for the visual atrophy, but other secondary mtDNA mutations should also be considered when giving genetic counseling.

A novel mutation within the 2B rod domain of keratin 9 in a Chinese pedigree with epidermolytic palmoplantar keratoderma combined with knuckle pads and camptodactyly.

Knuckle pads and camptodactyly are overlapping symptoms associated with many genetic and environmental factors. To the best of our knowledge, all reported cases of epidermolytic palmoplantar keratoderma (EPPK) with knuckle pads have been without accompanying camptodactyly. We here report a novel KRT9 mutation-EPPK family with combined knuckle pads and camptodactyly. All the EPPK-affected individuals in this southern Chinese pedigree suffered severe diffuse palmar and plantar hyperkeratosis including hyperhidrosis and cuticle splitting: 3 females presented EPPK only, 8 adult males had notably severe knuckle pads and camptodactyly as well as EPPK, and one 6-year-old boy manifested EPPK with knuckle pads. Haplotype analysis excluded the known candidate loci for camptodactyly and/or knuckle pad-like phenotypes on chromosomes 13q12, 3q11.2-q13.12, 1q24-q25, 4p16.3 and 16q11.1-q22, while only the markers D17S1787 and D17S579 flanking KRT9 showed co-segregation with EPPK. Then a novel c.T1373C (p.L458P) mutation within the sixth exon of KRT9 was validated, and this mutation presented a more severe pathogenicity than the previously reported p.L458F. We speculated that KRT9 plays a complicated role in the genesis of EPPK with knuckle pads and camptodactyly, which needs to be further investigated.

RET germline mutations identified by exome sequencing in a Chinese multiple endocrine neoplasia type 2A/familial medullary thyroid carcinoma family.

BACKGROUND: Whole exome sequencing provides a labor-saving and direct means of genetic diagnosis of hereditary disorders in which the pathogenic gene harbors a large cohort of exons. We set out to demonstrate a suitable example of genetic diagnosis of MEN 2A/FMTC (multiple endocrine neoplasia type 2/familial medullary thyroid carcinoma) using this approach. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced the whole exome of six individuals from a large Chinese MEN2A/FMTC pedigree to identify the variants of the RET (REarranged during Transfection) protooncogene and followed this by validation. Then prophylactic or surgical thyroidectomy with modified or level VI lymph node dissection and adrenalectomy were performed for the carriers. The cases were closely followed up. Massively parallel sequencing revealed four missense mutations of RET. We unexpectedly discovered that the proband's daughter with MEN 2A-related MTC presented a novel p.C634Y/V292M/R67H/R982C compound mutation, due to the involvement of p.C634Y in the proband with MEN 2A and p.V292M/R67H/R982C in the proband's husband with FMTC. In the maternal origin, p.C634Y caused bilateral MTC in all 5 cases and bilateral pheochromocytoma in 2 of the 5; the earliest onset age was 28 years. In the paternal origin, one of the six p.V292M/R67H/R982C carriers presented bilateral MTC (70 years old), one only had bilateral C-cell hyperplasia (44 years), two had bilateral multi-nodules (46 and 48 years) and two showed no abnormality (22 and 19 years). CONCLUSIONS/SIGNIFICANCE: The results confirmed the successful clinical utility of whole exome sequencing, and our data suggested that the p.C634Y/V292M/R67H/R982C mutation of RET exhibited a more aggressive clinical phenotype than p.C634Y or p.V292M/R67H/R982C, while p.V292M/R67H/R982C presented a relatively milder pathogenicity of MTC and likely predisposed to FMTC.

A c.1363C>T (p.R455X) nonsense mutation of RB1 gene in a southern Chinese retinoblastoma pedigree.

Retinoblastoma (RB) is the most common malignant intraocular tumor in children. Fifty percent of RB patients are carriers of a predisposing germline mutation with high penetrance. RB1 has been identified as the only pathological gene. We present the rapid detection of an RB1 gene mutation in a Han pedigree of two RB patients from southern China. Total RNA was extracted from whole blood for reverse transcriptase-polymerase chain reaction (PCR) to analyze RB1 transcripts, and genomic DNA for PCR and direct sequencing to test RB1 exons. Allele-specific PCR was used to verify the mutation. The results showed that the bilaterally affected son and the unilaterally affected father were both heterozygous for the nonsense mutation c.1363C>T (p.R455X) in exon 14 of RB1. Our studies suggest the molecular basis of RB in this Chinese family and provide further evidence that codon 455 is one of the recurrent spots for mutations in RB1.

Deletion analysis of SMN1 and NAIP genes in Southern Chinese children with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a disorder characterized by degeneration of lower motor neurons and occasionally bulbar motor neurons leading to progressive limb and trunk paralysis as well as muscular atrophy. Three types of SMA are recognized depending on the age of onset, the maximum muscular activity achieved, and survivorship: SMA1, SMA2, and SMA3. The survival of motor neuron (SMN) gene has been identified as an SMA determining gene, whereas the neuronal apoptosis inhibitory protein (NAIP) gene is considered to be a modifying factor of the severity of SMA. The main objective of this study was to analyze the deletion of SMN1 and NAIP genes in southern Chinese children with SMA. Here, polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was performed to detect the deletion of both exon 7 and exon 8 of SMN1 and exon 5 of NAIP in 62 southern Chinese children with strongly suspected clinical symptoms of SMA. All the 32 SMA1 patients and 76% (13/17) of SMA2 patients showed homozygous deletions for exon 7 and exon 8, and all the 13 SMA3 patients showed single deletion of SMN1 exon 7 along with 24% (4/17) of SMA2 patients. Eleven out of 32 (34%) SMA1 patients showed NAIP deletion, and none of SMA2 and SMA3 patients was found to have NAIP deletion. The findings of homozygous deletions of exon 7 and/or exon 8 of SMN1 gene confirmed the diagnosis of SMA, and suggested that the deletion of SMN1 exon 7 is a major cause of SMA in southern Chinese children, and that the NAIP gene may be a modifying factor for disease severity of SMA1. The molecular diagnosis system based on PCR-RFLP analysis can conveniently be applied in the clinical testing, genetic counseling, prenatal diagnosis and preimplantation genetic diagnosis of SMA.