ncRNAs-mediated high expression of TICRR promotes tumor cell proliferation and migration and is correlated with poor prognosis and tumor immune infiltration of hepatocellular carcinoma.

TICRR is a regulatory factor of DNA replication with ToPBP1 interaction. At present, the underlying function and mechanisms of TICRR remain unclear in LIHC. Our objective was to assess the function and prognosis of TICRR in LIHC. We conducted a differential expression analysis, GO/KEGG, and GSEA enrichment analysis of TICRR in LIHC. We also carried out the gene frequency and SCNA of TICRR. We found that TICRR could serve as an independent prognostic marker in LIHC by univariate and multivariate analysis. In addition, we observed that TICRR was related to immune infiltration, and TICRR had positive correlation with PD1/PD-L1 and CTLA-4 in LIHC. The hsa-miR-126-3p/IPO9-AS1 may be the candidate ncRNAs to regulate the expression of TICRR. The high rate of SCNV of TICRR might have critical effect on the function of CTL cells in LIHC. We further demonstrate through a series of experiments that TICRR facilitated the proliferation and metastasis of liver cancer cells in vitro. Altogether, TICRR might be a potential biomarker and therapeutic target in LIHC.

Glycerol monolaurate ameliorates DSS-induced acute colitis by inhibiting infiltration of Th17, neutrophils, macrophages and altering the gut microbiota.

Background and aims: Inflammatory bowel disease (IBD) places a heavy medical burden on countries and families due to repeated and prolonged attacks, and the incidence and prevalence of IBD are increasing worldwide. Therefore, finding an effective treatment is a matter of great urgency. Glycerol monolaurate (GML), which has a twelve-carbon chain, is a compound naturally found in human breast milk. Some studies have shown that GML has antibacterial and anti-inflammatory effects. However, the specific mechanism of action remains unclear. Methods: Acute colitis was established in mice using 3% DSS, and glycerol monolaurate (500 mg·kg-1) was administered for two weeks. QPCR and western blotting were performed to examine the inflammatory status. Mice described were subjected to flow cytometry analysis for immune cell activation. Results: GML treated alleviated macroscopic symptoms such as shortened colons, increased spleen weight, and caused weight loss in mice with DSS-induced colitis. In addition, GML decreased the expression of pro-inflammatory factors (NF-α, IL-1ß and IL-1α) and increased the expression of anti-inflammatory factors (IL-10 and TGF-ß). GML inhibited the activation of the MAPK and NF-κB signalling pathways, improved tissue damage, and increased the expression of intestinal tight junction proteins. In addition, LPMCs extracted from intestinal tissue via flow cytometry showed that GML treatment led to a decrease of Th17 cells, Neutrophils and Macrophages. 16S rDNA sequencing showed that GML increased the abundance of commensal bacterium such as Akkermansia and Lactobacillus murinus. Conclusions: We showed that oral administration of GML ameliorated DSS-induced colitis by inhibiting infiltration of Th17 cells, Neutrophils, and Macrophages, protecting the intestinal mucosal barrier and altered the abundance of commensal bacterium. This study provides new insights into the biological function and therapeutic potential of GML in the treatment of IBD.

Mindin serves as a tumour suppressor gene during colon cancer progression through MAPK/ERK signalling pathway in mice.

Mindin is important in broad spectrum of immune responses. On the other hand, we previously reported that mindin attenuated human colon cancer development by blocking angiogenesis through Egr-1-mediated regulation. However, the mice original mindin directly suppressed the syngenic colorectal cancer (CRC) growth in our recent study and we aimed to further define the role of mindin during CRC development in mice. We established the mouse syngeneic CRC CMT93 and CT26 WT cell lines with stable mindin knock-down or overexpression. These cells were also subcutaneously injected into C57BL/6 and BALB/c mice as well as established a colitis-associated colorectal cancer (CAC) mouse model treated with lentiviral-based overexpression and knocked-down of mindin. Furthermore, we generated mindin knockout mice using a CRISPR-Cas9 system with CAC model. Our data showed that overexpression of mindin suppressed cell proliferation in both of CMT93 and CT26 WT colon cancer cell lines, while the silencing of mindin promoted in vitro cell proliferation via the ERK and c-Fos pathways and cell cycle control. Moreover, the overexpression of mindin significantly suppressed in vivo tumour growth in both the subcutaneous transplantation and the AOM/DSS-induced CAC models. Consistently, the silencing of mindin reversed these in vivo observations. Expectedly, the tumour growth was promoted in the CAC model on mindin-deficient mice. Thus, mindin plays a direct tumour suppressive function during colon cancer progression and suggesting that mindin might be exploited as a therapeutic target for CRC.

The pattern-recognition molecule mindin binds integrin Mac-1 to promote macrophage phagocytosis via Syk activation and NF-κB p65 translocation.

Mindin has a broad spectrum of roles in the innate immune system, including in macrophage migration, antigen phagocytosis and cytokine production. Mindin functions as a pattern-recognition molecule for microbial pathogens. However, the underlying mechanisms of mindin-mediated phagocytosis and its exact membrane receptors are not well established. Herein, we generated mindin-deficient mice using the CRISPR-Cas9 system and show that peritoneal macrophages from mindin-deficient mice were severely defective in their ability to phagocytize E  coli. Phagocytosis was enhanced when E  coli or fluorescent particles were pre-incubated with mindin, indicating that mindin binds directly to bacteria or non-pathogen particles and promotes phagocytosis. We defined that 131 I-labelled mindin binds with integrin Mac-1 (CD11b/CD18), the F-spondin (FS)-fragment of mindin binds with the αM -I domain of Mac-1 and that mindin serves as a novel ligand of Mac-1. Blockade of the αM -I domain of Mac-1 using either a neutralizing antibody or si-Mac-1 efficiently blocked mindin-induced phagocytosis. Furthermore, mindin activated the Syk and MAPK signalling pathways and promoted NF-κB entry into the nucleus. Our data indicate that mindin binds with the integrin Mac-1 to promote macrophage phagocytosis through Syk activation and NF-κB p65 translocation, suggesting that the mindin/Mac-1 axis plays a critical role during innate immune responses.

Mutation screening of the GJA7 (Cx45) gene in a large international series of probands with nonsyndromic hearing impairment.

Direct evidence of the critical physiological role of connexins (Cxs) has come through the associations of several human diseases with pathogenic mutations in specific Cx genes. Currently, mutations in genes coding for five Cx proteins (Cx26, Cx30, Cx31, Cx32, and Cx43) have been shown to cause sensorineural hearing loss. Cx45 is another gap junction protein, coded by the GJA7 gene. To investigate the possible contribution of GJA7 mutations to deafness, we sequenced the GJA7 gene in 341 unrelated probands with nonsyndromic hearing loss from Turkey, South Africa, United Kingdom, United States, and China. Three nucleotide variants not affecting the amino acid sequence, c.213C>T, c.906C>T, and c.912G>T, and one missense change, c.889C>A (p.D297N), were found. None of the identified changes appeared to be pathogenic. Our data suggest that GJA7 alterations have no or low genetic relevance in nonsyndromic hearing loss in these populations.

Analysis of subcellular localization of Myo7a, Pcdh15 and Sans in Ush1c knockout mice.

Usher syndrome (USH) is the most frequent cause of combined deaf-blindness in man. An important finding from mouse models and molecular studies is that the USH proteins are integrated into a protein network that regulates inner ear morphogenesis. To understand further the function of harmonin in the pathogenesis of USH1, we have generated a targeted null mutation Ush1c mouse model. Here, we examine the effects of null mutation of the Ush1c gene on subcellular localization of Myo7a, Pcdh15 and Sans in the inner ear. Morphology and proteins distributions were analysed in cochlear sections and whole mount preparations from Ush1c(-/-) and Ush1c(-/+) controls mice. We observed the same distribution of Myo7a throughout the cytoplasm in knockout and control mice. However, we detected Pcdh15 at the base of stereocilia and in the cuticular plate in cochlear hair cells from Ush1c(+/-) controls, whereas in the knockout Ush1c(-/-) mice, Pcdh15 staining was concentrated in the apical region of the outer hair cells and no defined staining was detected at the base of stereocilia nor in the cuticular plate. We showed localization of Sans in the stereocilia of controls mouse cochlear hair cells. However, in cochleae from Ush1c(-/-) mice, strong Sans signals were detected towards the base of stereocilia close to their insertion point into the cuticular plate. Our data indicate that the disassembly of the USH1 network caused by absence of harmonin may have led to the mis-localization of the Protocadherin 15 and Sans proteins in the cochlear hair cells of Ush1c(-/-) knockout mice.

The genetic bases for non-syndromic hearing loss among Chinese.

Deafness is an etiologically heterogeneous trait with many known genetic, environmental causes or a combination thereof. The identification of more than 120 independent genes for deafness has provided profound new insights into the pathophysiology of hearing. However, recent findings indicate that a large proportion of both syndromic and non-syndromic forms of deafness in the Chinese population are caused by defects in a small number of genes. Studies of the genetic epidemiology and molecular genetic features revealed that there is a clear relevance of genes causing deafness in Chinese deaf patients as well as a unique spectrum of common and rare deafness gene mutations in the Chinese population. This review is focused on the genetic aspects of non-syndromic and mitochondrial deafness, in which unique molecular genetic features of hearing impairment have been identified in the Chinese population. The current China population is approximately 1.3 billion. It is estimated that 30,000 infants are born with congenital sensorineural hearing loss each year. Better understanding of the genetic causes of deafness in the Chinese population is important for accurate genetics counseling and early diagnosis for timely intervention and treatment options.

Digenic inheritance of non-syndromic deafness caused by mutations at the gap junction proteins Cx26 and Cx31.

Mutations in the genes coding for connexin 26 (Cx26) and connexin 31 (Cx31) cause non-syndromic deafness. Here, we provide evidence that mutations at these two connexin genes can interact to cause hearing loss in digenic heterozygotes in humans. We have screened 108 GJB2 heterozygous Chinese patients for mutations in GJB3 by sequencing. We have excluded the possibility that mutations in exon 1 of GJB2 and the deletion of GJB6 are the second mutant allele in these Chinese heterozygous probands. Two different GJB3 mutations (N166S and A194T) occurring in compound heterozygosity with the 235delC and 299delAT of GJB2 were identified in three unrelated families (235delC/N166S, 235delC/A194T and 299delAT/A194T). Neither of these mutations in Cx31 was detected in DNA from 200 unrelated Chinese controls. Direct physical interaction of Cx26 with Cx31 is supported by data showing that Cx26 and Cx31 have overlapping expression patterns in the cochlea. In addition, by coimmunoprecipitation of mouse cochlear membrane proteins, we identified the presence of heteromeric Cx26/Cx31 connexons. Furthermore, by cotransfection of mCherry-tagged Cx26 and GFP-tagged Cx31 in human embryonic kidney (HEK)-293 cells, we demonstrated that the two connexins were able to co-assemble in vitro in the same junction plaque. Together, our data indicate that a genetic interaction between these two connexin genes can lead to hearing loss.

Refinement of the DFNA41 locus and candidate genes analysis.

We previously mapped the 41rst gene locus (DFNA41) for autosomal dominant hearing loss on chromosome 12q24-qter in a large multi-generational Chinese family. We determined that DFNA41 is located in a 15 cM region, proximal to the marker D12S1609. A maximum two point LOD score of 6.56 at theta = 0.0 was obtained with marker D12S343. In the current study, screening of eight candidate genes within the DFNA41 interval did not reveal the mutation causing deafness in this family. Eight highly informative single nucleotide polymorphisms (SNPs) in the region of D12S343 were selected for linkage and association study. Because the pedigree studied here is a large family with many founders, we applied the transmission/disequilibrium (TDT) test. To account for the dependence of small families and the relatively small sample size, simulations were performed to obtain P-values. For three nearby SNPs spanning a 7 kb interval, we found significant evidence of linkage and association. The highest Z score of linkage and association of 3.6 (P < or = 0.0001) was obtained for SNP rs1566667. Haplotype analysis revealed that affected individuals were heterozygous for one core SNP (rs1027560-rs1027557-rs1566667-rs1463865-rs2078105) CAGTC haplotype, confirming location and autosomal dominant inheritance of the DFNA41 locus. Examination of pairwise LD calculation identified a major haplotype block defined by the four most centromeric SNPs. This study represents a significant refinement of the DFNA41 locus and should facilitate positional cloning of the disease gene.

Characterization of Usher syndrome type I gene mutations in an Usher syndrome patient population.

Usher syndrome type I (USH1), the most severe form of this syndrome, is characterized by profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. At least seven USH1 loci, USH1A-G, have been mapped to the chromosome regions 14q32, 11q13.5, 11p15, 10q21-q22, 21q21, 10q21-q22, and 17q24-25, respectively. Mutations in five genes, including MYO7A, USH1C, CDH23, PCDH15 and SANS, have been shown to be the cause of Usher syndrome type 1B, type 1C, type 1D, type 1F and type 1G, respectively. In the present study, we carried out a systematic mutation screening of these genes in USH1 patients from USA and from UK. We identified a total of 27 different mutations; of these, 19 are novel, including nine missense, two nonsense, four deletions, one insertion and three splicing defects. Approximatelly 35-39% of the observed mutations involved the USH1B and USH1D genes, followed by 11% for USH1F and 7% for USH1C in non-Acadian alleles and 7% for USH1G. Two of the 12 MYO7A mutations, R666X and IVS40-1G > T accounted for 38% of the mutations at that locus. A 193delC mutation accounted for 26% of CDH23 (USH1D) mutations, confirming its high frequency. The most common PCDH15 (USH1F) mutation in this study, 5601-5603delAAC, accounts for 33% of mutant alleles. Interestingly, a novel SANS mutation, W38X, was observed only in the USA cohort. The present study suggests that mutations in MYO7A and CDH23 are the two major components of causes for USH1, while PCDH15, USH1C, and SANS are less frequent causes.

Digenic inheritance of deafness caused by mutations in genes encoding cadherin 23 and protocadherin 15 in mice and humans.

Mutations in genes coding for cadherin 23 and protocadherin 15 cause deafness in both mice and humans. Here, we provide evidence that mutations at these two cadherin loci can interact to cause hearing loss in digenic heterozygotes of both species. Using a classical genetic approach, we generated mice that were heterozygous for both Cdh23 and Pcdh15 mutations on a uniform C57BL/6J background. Significant levels of hearing loss were detected in these mice when compared to age-matched single heterozygous animals or normal controls. Cytoarchitectural defects in the cochlea of digenic heterozygotes, including degeneration of the stereocilia and a base-apex loss of hair cells and spiral ganglion cells, were consistent with the observed age-related hearing loss of these mice beginning with the high frequencies. In humans, we also have obtained evidence for a digenic inheritance of a USH1 phenotype in three unrelated families with mutations in CDH23 and PCDH15. Altogether, our data indicate that CDH23 and PCDH15 play an essential long-term role in maintaining the normal organization of the stereocilia bundle.

Evidence of a founder effect for the 235delC mutation of GJB2 (connexin 26) in east Asians.

Mutations in the GJB2 gene encoding connexin 26 (Cx26) are a major cause of autosomal recessive and sporadic cases of congenital deafness in most populations. The 235delC mutation of GJB2 is the most frequent known mutation in some east Asian populations, with a carrier frequency of approximately 1%. In order to study the origin of 235delC among east Asians, we analyzed single-nucleotide polymorphisms (SNPs) within the coding region of GJB2 and flanking the 235delC mutation. We observed significant linkage disequilibrium between 235delC and five linked polymorphic markers, suggesting that 235delC arose from a common founder. The detection of 235delC only in east Asians, but not in Caucasians, and the small chromosomal interval of the shared haplotype suggest that 235delC is an ancient mutation that arose after the divergence of Mongoloids and Caucasians. Similarly, the finding that this mutation appears on a single haplotype provides no support for the possibility that recurrent mutation is the explanation for the high frequency of the allele.

Prestin, a cochlear motor protein, is defective in non-syndromic hearing loss.

Prestin, a membrane protein that is highly and almost exclusively expressed in the outer hair cells (OHCs) of the cochlea, is a motor protein which senses membrane potential and drives rapid length changes in OHCs. Surprisingly, prestin is a member of a gene family, solute carrier (SLC) family 26, that encodes anion transporters and related proteins. Of nine known human genes in this family, three (SLC26A2, SLC26A3 and SLC26A4) are associated with different human hereditary diseases. The restricted expression of prestin in OHCs, and its proposed function as a mechanical amplifier, make it a strong candidate gene for human deafness. Here we report the cloning and characterization of four splicing isoforms for the human prestin gene (SLC26A5a, b, c and d). SLC26A5a is the predominant form of prestin whereas the others showed limited distribution associated with certain developmental stages. Based on the functional importance of prestin we screened for possible mutations involving the prestin gene in a group of deaf probands. We have identified a 5'-UTR splice acceptor mutation (IVS2-2A>G) in exon 3 of the prestin gene, which is responsible for recessive non-syndromic deafness in two unrelated families. In addition, a high frequency of heterozygosity for the same mutation was observed in these subjects, suggesting the possibility of semi-dominant influence of the mutation in causing hearing loss. Finally, the observation of this mutation only in the Caucasian probands indicated an association with a specific ethnic background. This study thereby reveals an essential function of prestin in human auditory processing.

The prevalence of connexin 26 ( GJB2) mutations in the Chinese population.

Mutations in GJB2, encoding gap junction beta 2 protein (connexin 26), are responsible for the commonest form of non-syndromic recessive deafness in many populations. It has been reported recently that the most common 35delG mutation in GJB2 is exceptionally low in Japanese and Korean populations, but another deletion, 235delC, is relatively frequent. Since the Chinese constitute approximately one fifth of the global population, the frequency of GJB2 mutations in the population has important implications for understanding worldwide causes of genetic deafness. To determine whether GJB2 mutations are an important cause of deafness in Chinese, we conducted mutation screening for GJB2 in 118 deaf Chinese probands, including 60 from simplex and 58 from multiplex families with non-syndromic deafness, and 150 normal hearing Chinese controls. Four mutations, including 235delC, 299-300delAT, V37I, and 35delG, were found in the patients. Thirty-nine percent of the probands had a GJB2mutation. Of the 118 probands, 19 carried two definitely pathogenic mutations: three among the 58 multiplex cases (5.2%) and 16 among the 60 simplex cases (26.7%). Twenty-seven probands (22.9%) were found to carry only single GJB2 mutations. None of them had mutations in exon 1 of GJB2 and or the 342-kb deletion of GJB6. The 235delC mutation was the most prevalent mutation (20.3% of alleles), accounting for 81% of the pathologic alleles in multiplex cases and 67% in simplex cases. Analysis of the affected haplotypes in the patients with the homozygous 235delC mutation yielded evidence for a single origin of the mutation. The carrier frequency of the 235delC mutation in control subjects with normal hearing was 1.3%. The 35delG mutation was only noted as a heterozygous change in two simplex cases (1.2% of alleles). These results indicated that mutations in GJB2 are a major cause of inherited and sporadic congenital deafness in the Chinese population. The 235delC mutation, rather than 35delG, is the most common mutation found in the Chinese deaf population. Our data support the view that specific combinations of GJB2 mutation exist in different populations.

Mutations in the alternatively spliced exons of USH1C cause non-syndromic recessive deafness.

We have recently shown that USH1C underlies Usher syndrome type 1c (USH1C), an USH1 subtype characterized by profound deafness, retinitis pigmentosa, and vestibular dysfunction. USH1C encodes a PDZ-domain-containing protein, harmonin. Eight different Ush1c transcripts were identified in the mouse inner ear. Moreover, transcripts containing seven alternatively spliced exons (A-F, G/G) were found to be expressed in the inner ear, but not in the eye. These findings suggested that mutations involving USH1C might also be the cause of DFNB18, a form of non-syndromic deafness, which maps to a chromosomal region that includes USH1C. We screened 32 Chinese multiplex families with non-syndromic recessive deafness for USH1C mutations. In one family, congenital profound deafness without RP was associated with a C to G transversion in the alternatively spliced exon D, predicting an arginine to proline substitution at codon 608 in the proline-, serine- and threonine-rich region of harmonin. We also screened 320 deaf probands from other ethnic background and found three who were heterozygous for changes in the alternately spliced exons including Gly431Val in exon B, Arg620Leu and Arg636Cys in exon D. None of these mutations were detected in DNA from 200 control subjects with normal hearing including 110 Chinese. We also screened 121 non-Acadian probands with type 1 Usher syndrome. None carried any mutations in these exons of USH1C. Our findings show that USH1C mutations can also cause non-syndromic deafness and that some harmonin isoforms are specifically required for inner ear function.