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This study explores the effect of COL1A2, COL6A3, and THBS2 gene silencing on proliferation, migration, invasion, and apoptosis of gastric cancer cells through the PI3K-Akt signaling pathway. The gastric cancer microarray expression data (GSE19826, GSE79973, and GSE65801) was analyzed. Gastric cancer tissues and corresponding adjacent normal tissues were extracted from patients. Positive expression rate of PI3K, Akt, and p-Akt was measured with immunohistochemistry. Two cell lines, BGC-823 and SGC-7901, were transfected and cells were grouped into blank, negative control, COL1A2-shRNA, COL6A3-shRNA, and THBS2-shRNA groups. Expressions of COL1A2, COL6A3, and THBS2 in gastric cancer cells transfected with corresponding silencing sequences were evaluated by RT-qPCR and Western blot. MTT assay, Transwell, and cell scratch tests were conducted to evaluate cell proliferation, invasion, and migration capacity, respectively. Flow cytometry was used to evaluate cell cycle distribution and apoptosis. The positive expression of PI3K, Akt, and p-Akt was higher in gastric cancer tissues compared with adjacent normal tissues, and the mRNA expression of COL1A2, COL6A3, and THBS2 was increased in gastric cancer tissues. Akt, p-Akt, and PI3K expression drastically decreased in cells transfected with COL1A2, COL6A3, and THBS2 silencing sequences. Cells transfected with COL1A2, COL6A3, and THBS2 silencing sequences exhibited promoted apoptosis but inhibited proliferation, migration, and invasion. This study demonstrates that COL1A2, COL6A3, and THBS2 gene silencing inhibits gastric cancer cell proliferation, migration, and invasion while promoting apoptosis through the PI3K-Akt signaling pathway., (© 2017 Wiley Periodicals, Inc.)

Objects: To investigate the relationship between genotype and severity of malocclusion in osteogenesis imperfecta (OI)., Setting and Sample Population: A total of 49 patients participated in this cross-sectional study (age range: 5-19 years; 28 females; diagnoses: OI type I, N = 7; OI type III, N = 11; OI type IV, N = 27; OI type V, N = 2; OI type VI, N = 2)., Materials and Methods: Sequence analysis of COL1A1/COL1A2 and other OI-related genes was compared to the Peer Assessment Rating (PAR), an index reflecting the severity of malocclusion., Results: The mutation spectrum was as follows: COL1A1, N = 22; COL1A2, N = 22, IFITM5, N = 2; SERPINF1, N = 2; no mutation detected, N = 1). Compared to patients with COL1A1 mutations, patients with COL1A2 mutations had significantly higher scores for total PAR, anterior cross-bite, anterior open bite and anteroposterior buccal occlusion. Males with COL1A2 mutations had significantly higher total PAR scores than females (median 36 vs 30, P = .047, Mann-Whitney test). Exploratory correlation between age and buccal vertical occlusion was noted in patients with COL1A2 mutations (Spearman correlation: r = .46, P = .03, power = .50). Two patients with OI type V (caused by IFITM5 mutations) had total PAR scores of 44 and 21. Both patients scored high for "segment." Patients with OI type VI (due to SERPINF1 mutations) scored similar to OI type V for "centreline." Considerable difference was observed in the total PAR score between the 2 patients with OI type VI. They had total PAR of 43 and 2., Conclusion: Type of disease-causing mutation affects the severity of malocclusion in individuals with OI., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Although over 85% of osteogenesis imperfecta (OI) cases are associated with mutations in the procollagen type I genes (COL1A1 or COL1A2), no hot spots for the mutations were associated with particular clinical phenotypes. Eight patients that were studied here, diagnosed with OI by clinical standards, are from the Polish population with no ethnic background indicated. Previously unpublished mutations were found in six out of those eight patients. Genotypes for polymorphisms (Sp1 - rs1800012 and PvuII - rs412777), linked to bone formation and metabolism were determined. Mutations were found in exons 2, 22, 50 and in introns 13 and 51 of the COL1A1 gene. In COL1A2, one mutation was identified in exon 22. Deletion type mutations in COL1A1 that resulted in OI type I had no effect on collagen type I secretion, nor on its intracellular accumulation. Also, a single base substitution in I13 (c.904-9 G>T) was associated with the OI type I. The OI type III was associated with a single base change in I51 of COL1A1, possibly causing an exon skipping. Also, a missense mutation in COL1A2 changing Gly→Cys in the central part of the triple helical domain of the collagen type I molecule caused OI type III. It affected secretion of the heterotrimeric form of procollagen type I. However, no intracellular accumulation of procollagen chains could be detected. Mutation in COL1A2 affected its incorporation into procollagen type I. The results obtained shall help in genetic counseling of OI patients and provide a rational support for making informed, life important decisions by them and their families.

Frequent amplification of 7q21-22 genomic region is known in gastric cancer. Multiple genes including SHFM1, MCM7, and COL1A2 were reported to be the potential cancer candidate genes of this 20 Mb amplicon. This amplicon has two polycistrionic miRNA clusters and in the present study, miR-106b-25 cluster located in intron-13 of MCM7 was identified to express in gastric tumors. Among the 7q21-22 candidate genes, SHFM1 and MCM7 are expressed in intestinal type gastric tumors, whereas COL1A2 is expressed in diffuse type gastric tumors. Across gastric tumors, miR-25 was identified to co-express with MCM7 and SHFM1. On the other hand, negative correlation was observed between miR-25 and COL1A2 expression. miR-25 originating from MCM7 was found capable of selectively targeting the adjacent gene COL1A2. Silencing of miR-25 was found capable of elevating the expression of COL1A2 and inhibiting E-cadherin expression, revealing the diffuse type gastric cancer suppressive role conferred by miR-25. miR-25 was also found to suppress p53, and activate c-Src revealing its intestinal type gastric cancer associated oncogenic functions. Genome-wide expression profiling upon miR-25 silencing reveals that miR-25 is capable of suppressing 40 genes which are co-expressed with COL1A2, involved in epithelial to mesenchymal transition and angiogenesis which are the typical diffuse type gastric cancer features. The results clearly demonstrate 7q21-22 amplification, MCM7, and its intronic miR-25 are the major molecular switches involved in the complex oncogenic circuits of gastric cancer., (© 2017 Wiley Periodicals, Inc.)

Genomic Copy Number Variations (CNV) and the associated gene signatures are useful for cancer prognosis, diagnosis, and targeted therapeutics. Earlier, 7q21-22 region was reported for frequent amplification in gastric cancer and potential candidate genes were identified. An analysis of the expression pattern of the 159 genes located in this amplicon revealed the consistent elevated expression of 21 genes in gastric tumors. These genes are closely arranged within the 20 Mb region, and they showed a bimodal expression pattern. SHFM1 and 14 other genes are expressed in intestinal type gastric tumors. COL1A2 and PCOLCE genes of this region are expressed in diffuse type gastric tumors. Similarly, genome-wide expression neighbors of SHFM1 and COL1A2 also showed mutually exclusive expression pattern, and stratify intestinal and diffuse type gastric tumors. The expression of COL1A2 gene-set is associated with poor prognosis, whereas the SHFM1 gene-set is associated with better prognosis among the gastric cancer patients. Despite being physical neighbors, the SHFM1 and COL1A2 genes express differentially in the two major clinical sub-types of gastric cancer in a mutually exclusive manner. The tight gene regulations operating between these juxtaposed genes deserve investigation to understand the molecular regulatory switch defining the determinants of the gastric cancer sub-types. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Objectives: Collagen type I plays an important role in the bone matrix and is encoded by COL1A2 (collagen type I alpha 2) gene that may be a potential candidate for osteoporotic fracture. The aim of this study is to determine whether EcoRI, Del38 and PvuII polymorphisms of COL1A2 are associated with the development of osteoporosis and osteopenia in post-menopausal Polish women. Moreover, analysis of relationship between frequency of COL1A2 gene polymorphic variants and clinical parameters of bone turnover and degree of osteoporosis was performed., Material and Methods: The study group comprised of women with osteoporosis (n = 90), osteopenia (n = 56) and healthy individuals (n = 56). The EcoRI, Del38 and PvuII polymorphisms in COL1A2 gene were detected by PCR-RFLP method., Results: In women with osteoporosis the TT genotype of EcoRI polymorphism had the lowest Z-score value compared to other genotypes (p = 0.034). In case of Del28 polymorphism, there was a statistically significant correlation between lower BMI values and the DD genotype in women with osteopenia (p = 0.041). There was no statistically significant correlation between polymorphic variants of Del28 polymorphism and clinical parameters of women with osteoporosis. The analysis of PvuII polymorphism showed that in women with osteopenia the CC genotype had the lowest body weight compared to other genotypes (p = 0.039). PvuII polymorphism and clinical parameters in the group of women with osteoporosis had no statistically significant correlations., Conclusions: The analyzed COL1A2 polymorphisms seem to be related to osteoporosis development and their particular clinical parameters. Hence, the COL1A2 polymorphism may be a genetic risk factor related to the development of osteoporosis.

Background: The pathogenesis and etiology of systemic sclerosis (SSc) are complex and poorly understood. To date, several studies have demonstrated that the activation of the immune system undoubtedly plays a pivotal role in SSc pathogenesis. Activated immune effector T cells contribute to the release of various pro-inflammatory cytokines and drive the SSc-specific autoantibody responses. This, and a profibrotic environment, are all-important components of abnormal active immune responses that can lead to pathological disorders of SSc. CD11a is essential to inflammatory and immune responses, regulating adhesive and co-stimulatory interactions between CD4(+) T cells and other cells. Although CD11a is overexpressed in SSc patients, the mechanisms leading to this overexpression and its consequences remain unclear. DNA methylation, a main epigenetic modification, plays an important role in the regulation of gene expression and is involved in the pathogenesis of autoimmune diseases. This work aims to investigate the effect of DNA demethylation on CD11a expression in SSc CD4(+) T cells and to determine its functional significance. CD11a expression was measured using RT-PCR and flow cytometry. Bisulfite sequencing was used to determine the methylation status of the CD11a regulatory region. CD4(+) T cells were co-cultured with antigen-presenting cells, B cells, or fibroblasts with and without anti-CD11a, and proliferation of CD4(+) T cells, IgG production by B cells, and expression levels of COL1A2 mRNA by fibroblasts were evaluated., Results: Elevated CD11a expression levels were observed in CD4(+) T cells from SSc patients; these levels were found to be positively correlated with disease activity. The methylation levels of the CD11a regulatory sequences were lower in SSc patients than in controls and inversely correlated with CD11a mRNA expression. Treatment of CD4(+) T cells with 5-azacytidine (5-azaC) decreased CD11a promoter methylation and caused CD11a overexpression. SSc CD4(+) T cells and 5-azaC-treated CD4(+) T cells showed increased proliferation of CD4(+) T cells, increased production of IgG by co-cultured B cells, and induced expression of COL1A2 mRNA by co-cultured fibroblasts. These stimulatory effects were abrogated by anti-CD11a., Conclusions: Demethylation of CD11a regulatory elements and subsequent CD11a overexpression in CD4(+) T cells may mediate immunological abnormalities and fibrotic processes in SSc.

Tennis elbow defines a condition of pain and tenderness over the lateral epicondyle of the humerus. The exact aetiology of the injury is not yet fully understood. The major constituent of tendons is type 1 collagen which is encoded by COL1A1 gene. The aim of the study was to determine whether Sp1 binding site polymorphism (SNP rs1800012; 1546G/T) within the intronic region of COL1A1 gene is associated with tennis elbow. One hundred and three tennis elbow patients and one hundred and three healthy subjects without any history of previous ligament or tendon injuries were recruited for this genetic association study. All participants were genotyped for the COL1A1 Sp1 binding site polymorphism by using PCR-RFLP method. There were no observed statistical differences in the genotype (p=0.17) or allele (p=0.11) distributions between the groups. G allele frequency in patients and controls was 82.5% and 76.21%, and T allele frequency was 17.5% and 23.79% respectively. This study has shown that there is no association between this polymorphism and tennis elbow within the population studied., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Impaired wound healing is an important clinical problem in diabetes mellitus and results in failure to completely heal diabetic foot ulcers (DFUs), which may lead to lower extremity amputations. In the present study, collagen based dressings were prepared to be applied as support for the delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. The performance of NT alone and NT-loaded collagen matrices to treat wounds in streptozotocin (STZ) diabetic induced mice was evaluated. Results showed that the prepared dressings were not-cytotoxic up to 72h after contact with macrophages (Raw 264.7) and human keratinocyte (HaCaT) cell lines. Moreover, those cells were shown to adhere to the collagen matrices without noticeable change in their morphology. NT-loaded collagen dressings induced faster healing (17% wound area reduction) in the early phases of wound healing in diabetic wounded mice. In addition, they also significantly reduced inflammatory cytokine expression namely, TNF-α (p<0.01) and IL-1β (p<0.01) and decreased the inflammatory infiltrate at day 3 post-wounding (inflammatory phase). After complete healing, metalloproteinase 9 (MMP-9) is reduced in diabetic skin (p<0.05) which significantly increased fibroblast migration and collagen (collagen type I, alpha 2 (COL1A2) and collagen type III, alpha 1 (COL3A1)) expression and deposition. These results suggest that collagen-based dressings can be an effective support for NT release into diabetic wound enhancing the healing process. Nevertheless, a more prominent scar is observed in diabetic wounds treated with collagen when compared to the treatment with NT alone., (© 2013.)

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide. The discovery of non-invasive biomarker candidates for diagnosis and prognosis is important for the management of CRC. In this study, we performed proteomic profiling of serum from patients with different stages of CRC using a 2D-LC-MS/MS based approach combined with the APEX quantitative method. 917 proteins were identified and 93 were differentially expressed in normal and three patient groups (stages I, II and III). These proteins were predominantly involved in cell adhesion, immune response, coagulation process and metabolism. Importantly, we found collagen I dynamically changed from stages I to IV, with maximum expression in stage II, as detected in serum by MS analysis. Expression of collagen I was also validated in tumor tissues from the same group of CRC patients by real-time PCR and western blotting. Furthermore, we demonstrate that serum levels of collagen I degradation telopeptide (CTx) are correlated with staging and poor disease-free survival of CRC patients by ELISA analysis. These results suggest (1) serum proteomics may reflect biological changes in colorectal tumor tissues and (2) altered collagen I expression may be an early event in CRC tumorigenesis and CTx may provide additional information for prognosis of CRC., Biological Significance: In this work, we performed a systematic characterization of serum proteomic alterations in colorectal cancer (CRC) with different stages using a LC-MS based approach combined with the APEX quantitative method, attempting to gain overview of relevant proteins in tumorigenesis and discover non-invasive CRC-derived markers. We found a significant up-regulation of collagen I based on the proteomics data, and confirmed its expression in tissue and serum of the same group of patients. In addition, we also demonstrated that serum levels of collagen I degradation telopeptide (CTx) are correlated with the staging and poor survival rate of CRC. Those findings imply that alternation of collagen I might be an early event during tumorigenesis of CRC, and might contribute to the metastasis of CRC under the degradation regulated by some specific proteases. This work provides evidence for the clinical application of serum proteomics, and would aid the understanding of the role of the ECM in the clinical progression of CRC., (© 2013.)

We investigated whether the fibrogenic actions of acetaldehyde, the immediate oxidation product of ethanol, are mediated via Wingless (WNT) and/or β-catenin pathways in human hepatic stellate cells (HSC). First, we show that both β-catenin small inhibitory RNA and a dominant negative-MYC expression vector markedly down-regulated the expressions of fibrogenic genes in freshly isolated HSC. We further show that acetaldehyde up-regulated platelet-derived growth factor receptor beta mRNA and protein expressions ranging from 4.0- to 7.2-fold (P<0.001). Acetaldehyde induced MYC and collagen type-1 alpha-2 mRNA and protein expressions were WNT independent because DKK1, an antagonist of the canonical WNT/β-catenin pathway, completely failed to block these inductions. Acetaldehyde increased phospho-glycogen synthase kinase-3 beta (GSK3B) protein by 31% (P<0.01), whereas phospho-β-catenin protein decreased by 50% (P ≤ 0.01). Significantly, in contrast to 43% (P<0.01) inhibition of β-catenin nuclear translocation in nucleoredoxin (NXN)-overexpressed HSC, acetaldehyde profoundly stimulated β-catenin nuclear translocation by 51%, (P<0.01). Acetaldehyde also increased the cellular reactive oxygen species level 2-fold (P<0.001) with a concomitant 2-fold (P<0.001) increase in 4-hydroxynonenal adducts. Conversely, there was a 44% decrease (P<0.001) in glutathione levels with a concomitant 76% (P<0.001) decrease in the level of NXN/ disheveled (DVL) complex. Based on these findings, we conclude that actions of acetaldehyde are mediated by a mechanism that inactivates NXN by releasing DVL, leading to the inactivation of GSK3B, and thereby blocks β-catenin phosphorylation and degradation. Thus, the stabilized β-catenin translocates to the nucleus where it up-regulates the fibrogenic pathway genes. This novel mechanism of action of acetaldehyde has the potential for therapeutic interventions in liver fibrosis induced by alcohol., (Published by Elsevier Inc.)

Osteogenesis imperfecta (OI) is generally caused by a dominant mutation in Collagen I, encoded by the genes COL1A1 and COL1A2. To date there is no satisfactory therapy for OI, but inactivation of the mutant allele through small interfering RNAs (siRNA) is a promising approach, as siRNAs targeting each allele of a polymorphism could be used for allele-specific silencing irrespective of the location of the actual mutations. In this study we examined the allele dependent effects of several tiled siRNAs targeting a region surrounding an exonic COL1A2 T/C polymorphism (rs1800222) in heterozygous primary human bone cells. Relative abundances of COL1A2 alleles were determined by cDNA sequencing and overall COL1A2 abundance was analyzed by quantitative PCR. One of the siRNAs decreased overall COL1A2 abundance by 71% of which 75% was due to silencing of the targeted T-allele. In conclusion, allele-preferential silencing of Collagen type I genes may be a future therapeutic approach for OI.