Ji-Su Mo

MicroRNA 375 regulates proliferation and migration of colon cancer cells by suppressing the CTGF‐EGFR signaling pathway

MicroRNA 375 (MIR375) is significantly down regulated in human colorectal cancer (CRC) tissues; we have previously identified MIR375 as a colon cancer associated microRNA (miRNA). We identified putative MIR375 target genes by comparing the mRNA microarray analysis data of MIR375‐overexpressing cells with the candidate MIR375 target genes predicted by public bioinformatic tools. We investigated that the connective tissue growth factor (CTGF) is a direct target gene of MIR375. Expression of CTGF, a ligand of epidermal growth factor receptor (EGFR), was markedly enhanced in human CRC tissues in comparison with the corresponding normal colon tissues. We demonstrated that the expression levels of molecules in EGFR signaling pathways were regulated by MIR375 in colorectal cells. Using immunohistochemistry and the xenograft of MIR375‐overexpressing colorectal cells in mice, we showed that MIR375 regulates cell growth and proliferation, angiogenesis, cell migration, cell cycle arrest, apoptosis, and necrosis in colon cells. Furthermore, results of MIR375 overexpression and cetuximab treatment indicated that the apoptosis and necrosis in colon cells were synergistically enhanced. Our results suggest that the down‐regulation of MIR375 modulates EGFR signaling pathways in human colorectal cells and tissues by increasing CTGF expression; therefore, MIR375 may have a therapeutic value in relation to human CRC.

MicroRNA 429 Regulates Mucin Gene Expression and Secretion in Murine Model of Colitis

BACKGROUND AND AIMS miRNAs are non-coding RNAs that play important roles in the pathogenesis of human diseases by regulating target gene expression in specific cells or tissues. We aimed to detect miRNAs related to ulcerative colitis [UC], identify their target molecules, and analyse the correlation between the miRNAs and their target genes in colorectal cells and dextran sulphate sodium [DSS]-induced mouse colitis. METHODS UC-associated miRNAs were identified by miRNA microarray analysis using DSS-induced colitis and normal colon tissues. The results were validated by quantitative real-time polymerase chain reaction [RT-PCR]. We identified target genes of MIR429, a colitis-associated miRNA, from our screen by comparing the mRNA microarray analysis in MIR429-overexpressed cells with predicted candidate target genes. We constructed luciferase reporter plasmids to confirm the effect of MIR429 on target gene expression. The protein expression of the target genes was measured by western blot,enzyme-linked immunosorbent assay [ELISA] analysis, or immunohistochemistry. RESULTS We identified 37 DSS-induced colitis associated miRNAs. We investigated MIR429 that is down-regulated in DSS-induced colitis, and identified 41 target genes of MIR429. We show that the myristoylated alanine-rich protein kinase C substrate [MARCKS] is a direct target of MIR429. MARCKS mRNA and protein expression levels are down-regulated by MIR429, and MIR429 regulates the expression of MARCKS and MARCKS-mediated mucin secretion in colorectal cells and DSS-induced colitis. In addition, anti-MIR429 up-regulates MARCKS expression in colorectal cell lines. CONCLUSION Our findings suggest that MIR429 modulates mucin secretion in human colorectal cells and mouse colitis tissues by up-regulating of MARCKS expression, thereby making MIR429 a candidate for anti-colitis therapy in human UC.

Identification of the polymorphisms in IFITM1 gene and their association in a Korean population with ulcerative colitis

Abstract Interferon inducible transmembrane protein (IFITM) family genes have been implicated in several cellular processes such as the homotypic cell adhesion functions of IFNs and cellular anti-proliferative activities. We previously showed that the IFITM3 single nucleotide polymorphisms (SNPs) associated with susceptibility to ulcerative colitis (UC). The present study aimed to investigate whether the polymorphisms in the IFITM1 gene are associated with susceptibility to UC. We also evaluated the expression levels in the putative functional promoter polymorphisms to determine the change of their activity. Gene expression profiles in the tissues obtained from human digestive tracts by RT-PCR, and the possible variation sites and SNPs of IFITM1 were identified by direct sequencing method. Genotype analysis in the IFITM1 SNPs was performed by high resolution melting and TaqMan probe analysis, and the haplotype frequencies of IFITM1 SNPs for multiple loci were estimated using the expectation maximization (EM) algorithm. The expression levels in the putative functional promoter polymorphisms were evaluated by performing a luciferase reporter assay. We identified two SNPs and two variation sites, g.-1920G>A (rs77537847), g.-1547delA (novel) and g.-416C>G (rs11246062) in the promoter region, and g.364delA (rs200576757) in intron 1. The genotype and allele frequencies of the g.-1920G>A polymorphism of IFITM1 gene in the UC patients were significantly different from those of the healthy controls (P =0.002 and 0.042, respectively). These results suggest that the g.-1920G>A polymorphism in IFITM1 may be associated with susceptibility to UC.

The Association of the GABRP Polymorphisms with Systemic Lupus Erythematosus

Gamma-aminobutyric acid receptor subunit pi (GABRP) is involved in inhibitory synaptic transmission in the central nervous system. This gene encodes multisubunit chloride channels and is also expressed in numerous nonneuronal tissues such as the uterus and the ovaries. This study was aimed to validate whether the polymorphisms in the GABRP gene are associated with the susceptibility to systematic lupus erythematosus (SLE). The genotype frequencies of the rs929763, rs732157, and rs3805455 of the GABRP gene in SLE patients were significantly different from those of the control group (P < 0.0001, P = 0.05 and 0.002, resp.). Additional analysis showed that the genotype of the rs929763 and rs3805455 of the GABRP gene were also significantly associated with female SLE patients (P < 0.0001, P = 0.005, resp.). Two haplotype frequencies including a major haplotype of GABRP SNPs were more significantly different between the SLE patients and the healthy controls (P = 0.038 and 4.2E − 24, resp.). These results suggest that the polymorphisms in the GABRP gene might be associated with the susceptibility to SLE and the haplotype of GABRP SNPs is useful genetic marker for SLE.

MicroRNA 196B Regulates HOXA5, HOXB6 and GLTP Expression Levels in Colorectal Cancer Cells

MiRNAs are non-coding RNAs that play important roles in the pathogenesis of human diseases by regulating target gene expression in specific cells or tissues. Previously we identified colorectal cancer (CRC) associated MIR196B, which was specifically up-regulated in CRC cells and tissue. We also identified 18 putative MIR196B target genes by comparing the mRNAs down-regulated in MIR196B-overexpressed cells with MIR196B target genes predicted by public bioinformatics tools. In this study, we verified the association between MIR196B and three genes, HOXA5, HOXB6 and GLTP. HOXA5, HOXB6 and GLTP transcripts were directly down-regulated by MIR196B. The mRNA and proteins levels of HOXA5, HOXB6 and GLTP were also down-regulated in CRC cells by the up-regulated MIR196B. GLTP protein expression was decreased in CRC tissues compared to adjacent non-tumor tissues. These results suggest that HOXA5, HOXB6, and GLTP were direct target genes of MIR196B in CRC cells, and that the up-regulated MIR196B in CRC tissue regulates the expression levels of HOXA5, HOXB6, and GLTP during colorectal carcinogenesis.

MicroRNA 429 regulates the expression of CHMP5 in the inflammatory colitis and colorectal cancer cells

Objective and designMicroRNAs (miRNAs) play an important role in the pathogenesis of human diseases by regulating the expression of target genes in specific cells or tissues. In this study, we analyzed the association between the MIR429 and its target gene, charged multivesicular body protein 5 (CHMP5), in human colon cancer cells and in a DSS-induced colitis mouse model.Materials and methodsA luciferase reporter system was used to confirm the effect of MIR429 on CHMP5 expression. Protein or mRNA expression of the target gene and associated molecules were measured by Western blot or quantitative RT-PCR (qRT-PCR), respectively. Flow cytometry was used to compare cell viability or cell cycle progression.ResultsCHMP5 mRNA and protein expression was directly down-regulated by MIR429. We found that MIR429 inhibited colon cancer cell growth and cell cycle progression, and CHMP5 was overexpressed in the DSS-induced colitis mouse model and human ulcerative colitis (UC) tissues.ConclusionsOur findings show that CHMP5 is a direct target of MIR429 in human colon cancer cell lines and suggest that CHMP5 up-regulation as a result of reduced MIR429 expression in DSS-induced mice colitis tissues and human UC tissues may restrict apoptosis and promote cell proliferation.

Significant association of the GABRP polymorphisms with ulcerative colitis patients

The γ-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the central nervous system. The π subunit receptor of GABA (GABRP) is a transmembrane multi-subunit chloride channel that is expressed in the CNS and several other non-neuronal tissues. The exact function of this gene in non-neuronal tissues is not clearly identified. Our previous customized pilot SNPs chip screens analysis indicated that this gene had strong susceptibility to ulcerative colitis (UC). This study was aimed to validate whether the polymorphisms in the GABRP gene are associated with the susceptibility to UC. The genotype and allele frequencies of the rs929763 rs1063310, rs3805455, rs382819 and rs732157 of the GABRP gene were significantly associated with UC patients. Two haplotype frequencies including a major haplotype of GABRP SNPs were more significantly different between the UC patients and the healthy controls. These results suggest that the polymorphisms in the GABRP gene might be associated with the susceptibility to UC, and the haplotype of GABRP SNPs are useful genetic marker for UC.

EPSTI1 polymorphisms are associated with systemic lupus erythematosus

Epithelial stromal interaction 1 (EPSTI1) is an interferon (IFN) response gene, which was originally identified as a stromal fibroblast-induced gene in breast cancer. Our previous study using a customized SNP chip found evidence of an association between EPSTI1 and susceptibility to the chronic inflammatory disease, systematic lupus erythematosus (SLE). This study aimed to validate whether polymorphisms in EPSTI1 are associated with susceptibility to SLE. We analyzed genotype and allele frequencies of SNPs at EPSTI1 using genomic DNA from 119 patients with SLE and 512 healthy controls. We found that the genotype frequencies of rs1044856 and rs1359184 in patients with SLE were significantly different from those found in the control group (P = 0.03 and P = 0.01, respectively). In addition, we found that genotype and allele frequencies of rs1359184 in female patients with SLE were significantly different from those found in female controls (P = 0.02 and P = 0.04, respectively). We identified two major haplotypes in EPSTI1 that were significantly different between patients with SLE and healthy controls (P = 0.01 and P = 0.05, respectively). Furthermore, we found that rs1359184 and rs1044856 in EPSTI1 were associated with antinuclear antibody (ANA) and erythrocyte sedimentation rate (ESR) levels in patients with SLE (P = 0.0035 and P = 0.021, respectively). Our findings indicate that polymorphisms in EPSTI1 are associated with susceptibility to SLE and that haplotypes at EPSTI1 may be useful genetic markers for SLE.

Identification of the Polymorphisms in IFITM2 and IFITM5 Genes and their Association with Ulcerative Colitis

Interferon inducible transmembrane protein (IFITM) family genes have been implicated in various cellular processes such as the homotypic cell adhesion functions of IFNs and cellular anti-proliferative activities. The present study aimed to investigate whether the polymorphisms of the IFITM2 and IFITM5 genes are associated with susceptibility to UC. We identified a total of thirteen polymorphisms (eleven SNPs and two variations) in the IFITM2 gene and twelve polymorphisms (eleven SNPs and one variation) in the IFITM5 gene, by the direct sequencing method. Genotype analysis in the IFITM2 and IFITM5 SNPs was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and Taq-Man probe analysis, and the haplotype frequencies of IFITM2 and IFITM5 SNPs for multiple loci were estimated using the expectation maximization (EM) algorithm. The genotype and allele frequencies of IFITM2 SNPs, as well as IFITM5 SNPs, in UC patients were not significantly different from those of the healthy controls. We also analyzed the combined frequencies of rs77537847 of IFITM1, rs909097 of IFITM2, and rs56069858 of IFITM5 in the UC patients and the healthy controls. Although the distribution of the major combined genotype frequency did not differ significantly between the healthy controls and the UC patients, the GGT combined frequency in the healthy controls was significantly different from that in the UC patients (P=0.002). This result suggests that the combined genotype of the IFITMs polymorphisms may be associated with a susceptibility to UC and could be a useful genetic marker for UC.

Association of Thymic Stromal Lymphopoietin Receptor (TSLPR) Polymorphisms with the Susceptibility of Rheumatoid Arthritis in a Korean Population

Human thymic stromal lymphopoietin receptor (TSLPR) might play an important role in the development of inflammatory and allergic responses. We previously identified eleven single nucleotide polymorphisms (SNPs) and two variation sites in the TSLPR gene and showed that all the SNPs of the TSLPR gene are associated with susceptibility to atopic asthma. The present study aimed to investigate whether the TSLPR gene SNPs are associated with susceptibility to rheumatoid arthritis (RA). We compared the genotype and the allele frequencies of the TSLPR SNPs in 457 RA patients and 570 healthy controls. The genotype and the allele frequencies of the TSLPR gene SNPs in the RA patients were not significantly different from the respective frequencies of the healthy controls. Additional analysis showed that the genotype and the allele frequencies of the TSLPR gene SNPs did not appear to be associated with RA in female RA patients. The TSLPR gene SNPs in the RA patients did not affect the production of rheumatoid factor (RF) and antisynthetic cyclic citrullinated peptide (CCP). Our results suggest that the TSLPR gene SNPs are not associated with susceptibility to RA in the Korean population.