Clinical and Prognostic Significance of TZAP Expression in Cervical Cancer.

Background and Objectives: Telomeric zinc finger-associated protein (TZAP) is a telomere-associated factor that was previously called ZBTB48. This protein binds preferentially to long telomeres, competing with telomeric repeat factors 1 and 2. Genetic changes in TZAP may be associated with cancer pathogenesis; however, this relationship has not yet been elucidated for any type of cancer. In this study, we aimed to examine the clinicopathologic and prognostic value of TZAP expression in cervical cancer (CC). Materials and Methods: The data were extracted from The Cancer Genome Atlas cohorts by OncoLnc (21 cancer types, 7700 cancers). The prognostic value of TZAP for different stages of 264 CCs was examined using survival analysis. Results: The TZAP expression did not differ significantly between CC and normal matched tissues. Age, cancer stage, and viral infection were not associated with TZAP expression. Survival analysis revealed a shorter overall survival in CC patients with a lower TZAP expression (χ2 = 3.62, p = 0.057). The prognostic value of TZAP expression was greater in patients with N1 stage CC (χ2 = 5.64, p = 0.018). Conclusion: TZAP expression is a possible prognostic marker for CC, especially stage N1 CC.

N-Terminal Modification of the Tetrapeptide Arg-Leu-Tyr-Glu, a Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) Antagonist, Improves Antitumor Activity by Increasing its Stability against Serum Peptidases.

The tetrapeptide Arg-Leu-Tyr-Glu (RLYE), a vascular endothelial growth factor (VEGF) receptor-2 antagonist, has been used previously either alone or in combination with chemotherapeutic drugs for treating colorectal cancer in a mouse model. We analyzed the half-life of the peptide and found that because of degradation by aminopeptidases B and N, it had a short half-life of 1.2 hours in the serum. Therefore, to increase the stability and potency of the peptide, we designed the modified peptide, N-terminally acetylated RLYE (Ac-RLYE), which had a strongly stabilized half-life of 8.8 hours in serum compared with the original parent peptide. The IC50 value of Ac-RLYE for VEGF-A-induced endothelial cell migration decreased to approximately 37.1 pM from 89.1 pM for the parent peptide. Using a mouse xenograft tumor model, we demonstrated that Ac-RLYE was more potent than RLYE in inhibiting tumor angiogenesis and growth, improving vascular integrity and normalization through enhanced endothelial cell junctions and pericyte coverage of the tumor vasculature, and impeding the infiltration of macrophages into tumor and their polarization to the M2 phenotype. Furthermore, combined treatment of Ac-RLYE and irinotecan exhibited synergistic effects on M1-like macrophage activation and apoptosis and growth inhibition of tumor cells. These findings provide evidence that the N-terminal acetylation augments the therapeutic effect of RLYE in solid tumors via inhibition of tumor angiogenesis, improvement of tumor vessel integrity and normalization, and enhancement of the livery and efficacy of the coadministered chemotherapeutic drugs. SIGNIFICANCE STATEMENT: The results of this study demonstrate that the N-terminal acetylation of the tetrapeptide RLYE (Ac-RLYE), a novel vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitor, significantly improves its serum stability, antiangiogenic activity, and vascular normalizing potency, resulting in enhanced therapeutic effect on solid tumors. Furthermore, the combined treatment of Ac-RLYE with the chemotherapeutic drug, irinotecan, synergistically enhanced its antitumor efficacy by improving the perfusion and delivery of the drug into the tumors and stimulating the conversion of the tumor-associated macrophages to an immunostimulatory M1-like antitumor phenotype.

NF-κB-responsive miRNA-31-5p elicits endothelial dysfunction associated with preeclampsia via down-regulation of endothelial nitric-oxide synthase.

Inflammatory cytokines, including tumor necrosis factor-α (TNFα), were elevated in patients with cardiovascular diseases and are also considered as crucial factors in the pathogenesis of preeclampsia; however, the underlying pathogenic mechanism has not been clearly elucidated. This study provides novel evidence that TNFα leads to endothelial dysfunction associated with hypertension and vascular remodeling in preeclampsia through down-regulation of endothelial nitric-oxide synthase (eNOS) by NF-κB-dependent biogenesis of microRNA (miR)-31-5p, which targets eNOS mRNA. In this study, we found that miR-31-5p was up-regulated in sera from patients with preeclampsia and in human endothelial cells treated with TNFα. TNFα-mediated induction of miR-31-5p was blocked by an NF-κB inhibitor and NF-κB p65 knockdown but not by mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase inhibitors, indicating that NF-κB is essential for biogenesis of miR-31-5p. The treatment of human endothelial cells with TNFα or miR-31-5p mimics decreased endothelial nitric-oxide synthase (eNOS) mRNA stability without affecting eNOS promoter activity, resulting in inhibition of eNOS expression and NO/cGMP production through blocking of the functional activity of the eNOS mRNA 3'-UTR. Moreover, TNFα and miR-31-5p mimic evoked endothelial dysfunction associated with defects in angiogenesis, trophoblastic invasion, and vasorelaxation in an ex vivo cultured model of human placental arterial vessels, which are typical features of preeclampsia. These results suggest that NF-κB-responsive miR-31-5p elicits endothelial dysfunction, hypertension, and vascular remodeling via post-transcriptional down-regulation of eNOS and is a molecular risk factor in the pathogenesis and development of preeclampsia.

TNF-α elicits phenotypic and functional alterations of vascular smooth muscle cells by miR-155-5p-dependent down-regulation of cGMP-dependent kinase 1.

cGMP-dependent protein kinase 1 (PKG1) plays an important role in nitric oxide (NO)/cGMP-mediated maintenance of vascular smooth muscle cell (VSMC) phenotype and vasorelaxation. Inflammatory cytokines, including tumor necrosis factor-α (TNFα), have long been understood to mediate several inflammatory vascular diseases. However, the underlying mechanism of TNFα-dependent inflammatory vascular disease is unclear. Here, we found that TNFα treatment decreased PKG1 expression in cultured VSMCs, which correlated with NF-κB-dependent biogenesis of miR-155-5p that targeted the 3'-UTR of PKG1 mRNA. TNFα induced VSMC phenotypic switching from a contractile to a synthetic state through the down-regulation of VSMC marker genes, suppression of actin polymerization, alteration of cell morphology, and elevation of cell proliferation and migration. All of these events were blocked by treatment with an inhibitor of miR-155-5p or PKG1, whereas transfection with miR-155-5p mimic or PKG1 siRNA promoted phenotypic modulation, similar to the response to TNFα. In addition, TNFα-induced miR-155-5p inhibited the vasorelaxant response of de-endothelialized mouse aortic vessels to 8-Br-cGMP by suppressing phosphorylation of myosin phosphatase and myosin light chain, both of which are downstream signal modulators of PKG1. Moreover, TNFα-induced VSMC phenotypic alteration and vasodilatory dysfunction were blocked by NF-κB inhibition. These results suggest that TNFα impairs NO/cGMP-mediated maintenance of the VSMC contractile phenotype and vascular relaxation by down-regulating PKG1 through NF-κB-dependent biogenesis of miR-155-5p. Thus, the NF-κB/miR-155-5p/PKG1 axis may be crucial in the pathogenesis of inflammatory vascular diseases, such as atherosclerotic intimal hyperplasia and preeclamptic hypertension.

REDD-1 aggravates endotoxin-induced inflammation via atypical NF-κB activation.

Regulated in development and DNA damage responses 1 (REDD-1), an inhibitor of mammalian target of rapamycin (mTOR), is induced by various cell stressors, including LPS, a major player in the pathogenesis of endotoxemic shock. However, the pathologic role of REDD-1 in endotoxemia is largely unknown. We found that LPS increased REDD-1 expression, nuclear transcription factor-κB (NF-κB) activation, and inflammation and that these responses were suppressed by REDD-1 knockdown and in REDD-1+/- macrophages. REDD-1 overexpression stimulated NF-κB-dependent inflammation without additional LPS stimulation. REDD-1-induced NF-κB activation was independent of 2 classic IKK-dependent NF-κB pathways and the mTOR signaling pathway; however, REDD-1, particularly its C-terminal region (178-229), interacted with and sequestered IκBα, to elicit atypical NF-κB activation during the delayed and persistent phases of inflammation after stimulation. Moreover, REDD-1 knockdown mitigated vascular inflammation and permeability in endotoxemic mice, resulting in decreases in immune cell infiltration, systemic inflammation, caspase-3 activation, apoptosis, and consequent mortality. We further confirmed the inflammatory and cytotoxic effects of REDD-1 in endotoxemic REDD-1+/- mice. Our data support the likelihood that REDD-1 exacerbates endotoxemic inflammation via atypical NF-κB activation by sequestering IκBα.-Lee, D.-K., Kim, J.-H., Kim, J., Choi, S., Park, M., Park, W., Kim, S., Lee, K.-S., Kim, T., Jung, J., Choi, Y. K., Ha, K.-S., Won, M.-H., Billiar, T. R., Kwon, Y.-G., Kim, Y.-M. REDD-1 aggravates endotoxin-induced inflammation via atypical NF-κB activation.

A Simple Method to Locate the Master Knot of Henry Using the Correlation between the Flexor Tendon Length Parameter and the Foot Length.

The precise location of the Master Knot of Henry (MKH) has important clinical significance, but its anatomical definition has not been agreed upon. The purpose of this study is to present a linear regression equation for predicting length variables based on foot length, by evaluating the correlation of length variables related to flexor hallucis longus (FHL) and flexor digitorum longus (FDL), with respect to the location of the MKH. A total of 95 limbs were dissected from 48 adult cadavers, and were fixed in formalin. Measurements were made for the length parameter, with reference to the landmark. The relevance between length variables was analyzed through simple correlation analysis and linear regression analysis. The foot length was 213.69 ± 17.53 mm, MKH-great toe distal phalanx was 140.16 ± 14.69 mm, MKH-FHL insertion was 124.55 ± 13.46 mm, MKH-little toe distal phalanx was 121.79 ± 13.41 mm, MKH-FDL little toe insertion was 109.07 ± 14.16 mm, and the FHL-FDL angle was 33.15 ± 5.39. The correlation coefficient between all the length variables for foot length showed a high positive correlation. We derived a regression equation that can predict the length of each variable. This regression formula is considered to be highly useful because it can estimate the positional relationship of the MKH relatively simply.

Human plasminogen-derived N-acetyl-Arg-Leu-Tyr-Glu antagonizes VEGFR-2 to prevent blood-retinal barrier breakdown in diabetic mice.

Targeting the vascular endothelial growth factor (VEGF)/its receptor-2 (VEGFR-2) system has become a mainstay of treatment for many human diseases, including retinal diseases. We examined the therapeutic effect of recently developed N-acetylated Arg-Leu-Tyr-Glu (Ac-RLYE), a human plasminogen kringle-5 domain-derived VEGFR-2 antagonists, on the pathogenesis of diabetic retinopathy. Ac-RLYE inhibited VEGF-A-mediated VEGFR-2 activation and endothelial nitric oxide synthase (eNOS)-derived NO production in the retinas of diabetic mice. In addition, Ac-RLYE prevented the disruption of adherens and tight junctions and vascular leakage by inhibiting S-nitrosylation of ß-catenin and tyrosine nitration of p190RhoGAP in the retinal vasculature of diabetic mice. Peptide treatment preserved the pericyte coverage of retinal capillaries by upregulating angiopoietin-2. These results suggest that Ac-RLYE potentially prevents blood-retinal barrier breakdown and vascular leakage by antagonizing VEGFR-2; Ac-RLYE can be used as a potential therapeutic drug for the treatment of diabetic retinopathy.

REDD1 is a determinant of low-dose metronomic doxorubicin-elicited endothelial cell dysfunction through downregulation of VEGFR-2/3 expression.

Low-dose metronomic chemotherapy (LDMC) inhibits tumor angiogenesis and growth by targeting tumor-associated endothelial cells, but the molecular mechanism has not been fully elucidated. Here, we examined the functional role of regulated in development and DNA damage responses 1 (REDD1), an inhibitor of mammalian target of rapamycin complex 1 (mTORC1), in LDMC-mediated endothelial cell dysfunction. Low-dose doxorubicin (DOX) treatment induced REDD1 expression in cultured vascular and lymphatic endothelial cells and subsequently repressed the mRNA expression of mTORC1-dependent translation of vascular endothelial growth factor receptor (Vegfr)-2/3, resulting in the inhibition of VEGF-mediated angiogenesis and lymphangiogenesis. These regulatory effects of DOX-induced REDD1 expression were additionally confirmed by loss- and gain-of-function studies. Furthermore, LDMC with DOX significantly suppressed tumor angiogenesis, lymphangiogenesis, vascular permeability, growth, and metastasis in B16 melanoma-bearing wild-type but not Redd1-deficient mice. Altogether, our findings indicate that REDD1 is a crucial determinant of LDMC-mediated functional dysregulation of tumor vascular and lymphatic endothelial cells by translational repression of Vegfr-2/3 transcripts, supporting the potential therapeutic properties of REDD1 in highly progressive or metastatic tumors.

The Prognostic Significance of TERT Locus Polymorphism (rs36115365) in Surgically Resected Non-Small Cell Lung Cancer.

OBJECTIVE: Telomere length is an important factor for the development of non-small cell lung cancer (NSCLC), and current articles focused on telomere associated genes. We studied the clinicopathological and prognostic implications of rs36115365 polymorphism of the TERT-CLPTM1L locus in NSCLC. The association between rs36115365 and telomere length was investigated in 176 NSCLCs. METHODS: DNA was extracted from NSCLC tissues and polymorphism and telomere length were analyzed. RESULTS: The rs36115365 polymorphism showed the following frequencies according to the genotype: G/G in 81.8% of the patients, G/C in 14.2%, and C/C in 4.0%. Average telomere length in the tumor tissues were 3.06-fold longer than telomeres in paired non-tumor tissues (SD=1.87), and telomere length was not significantly different according to rs36115365 (p=0.134). The rs36115365 polymorphism did not have any relationships with clinicopathological characteristics. A poor overall survival result was found in NSCLC with C allele carriers than that with G/G allele (p=0.034). However, disease free survival rate was not different statistically (p=0.938). CONCLUSIONS: These findings suggest that rs36115365 may contribute to the progression of NSCLC.

Clinicopathological Characteristics of TZAP Expression in Colorectal Cancers.

PURPOSE: The zinc finger protein, ZBTB48, is a telomere-associated protein. It was renamed as telomeric zinc finger-associated protein (TZAP) binding to elongated telomeres. However, its expression level was not measured in cancers. PATIENTS AND METHODS: We analyzed TZAP mRNA levels in 60 colorectal cancers (CRC) and its correlation with telomere length and TERT was studied. RESULTS: TZAP mRNA in CRC was higher statistically than that in paired non-cancerous tissues (p = 0.033). Higher TZAP was found in carcinoembryonic antigen (CEA)-positive CRCs (>5 ng/mL) (p = 0.012). Shorter telomere was found in CRCs with high TZAP expression than that with low TZAP expression (p = 0.010). According to quantitative correlation analysis, TZAP has a correlation with age (r = -0.349, p = 0.007), TERT (r = 0.279, p = 0.041) and telomere length (r = -0.305, p = 0.021). TZAP expression did not harbor prognostic value in CRC. Inhibition of TZAP expression by siRNA suppresses cell growth in HT29 cells; however, it resulted in increased cell viability in HCT116 cells. TZAP inhibition induces a decrease in mRNA levels of TERT in both HT29 and HCT116 cells. TCGA data analysis showed higher expression of TZAP showed poorer overall survival in colon cancer (p = 0.001); however, it did not have a significance in rectal cancer (p = 0.951). CONCLUSION: We suggested that TZAP may be a possible biomarker for CRC.

NF-κB-dependent miR-31/155 biogenesis is essential for TNF-α-induced impairment of endothelial progenitor cell function.

Endothelial progenitor cell (EPC) dysfunction impairs vascular function and remodeling in inflammation-associated diseases, including preeclampsia. However, the underlying mechanism of this inflammation-induced dysfunction remains unclear. In the present study, we found increases in TNF-α and miR-31/155 levels and reduced numbers of circulating EPCs in patients with preeclampsia. Patient-derived mononuclear cells (MNCs) cultured in autologous serum had decreased endothelial nitric oxide synthase (eNOS) expression, nitric oxide production, and differentiation into EPCs with angiogenic potential, and these effects were inhibited by a TNF-α-neutralizing antibody and miR-31/155 inhibitors. Moreover, TNF-α treatment of normal MNCs increased miR-31/155 biogenesis, decreased eNOS expression, reduced EPC differentiation, and impaired angiogenic potential. The TNF-α-induced impairment of EPC differentiation and function was rescued by NF-κB p65 knockdown or miR-31/155 inhibitors. In addition, treatment of MNCs with synthetic miR-31/155 or an eNOS inhibitor mimicked the inhibitory effects of TNF-α on eNOS expression and EPC functions. Moreover, transplantation of EPCs that had been differentiated from TNF-α-treated MNCs decreased neovascularization and blood perfusion in ischemic mouse hindlimbs compared with those of normally differentiated EPCs. These findings suggest that NF-κB activation is required for TNF-α-induced impairment of EPC mobilization, differentiation, and function via miR-31/155 biogenesis and eNOS downregulation. Our data provide a new role for NF-κB-dependent miR-31/155 in EPC dysfunction under the pathogenic conditions of inflammation-associated vascular diseases, including preeclampsia.

The Prognostic Value of Measuring PD-L1 mRNA Expression Levels in Surgically Resected Non-Small Cell Lung Cancer.

The expression of programmed cell death 1-ligand 1 (PD-L1) by tumor cells acts as an immune-checkpoint when it interacts with its receptor on immune cells, effectively preventing the immune system from attacking the tumor. Inhibitors against PD-L1 have shown remarkable therapeutic activity in non-small cell lung cancer (NSCLC); however, the prognostic value of this potential biomarker has yet to be conclusively shown. To investigate the clinicopathological and prognostic value of PD-L1 expression, we examined PD-L1 mRNA levels in surgically resected NSCLC. We compared PD-L1 mRNA expression in tumor and adjacent normal tissue from 71 NSCLC patient samples using real-time polymerase chain reaction. To validate PD-L1 mRNA expression by this method, PD-L1 protein expression by immunohistochemistry was analyzed and their strong correlation was confirmed (r=0.728, P=0.041). PD-L1 mRNA expression was significantly increased in tumor tissue as compared to adjacent normal tissue (P<0.001). Tumor tissue on average expressed 3.72 fold higher levels of PD L1 mRNA compared to normal tissue counterparts, and higher expression levels were found in 25.4% (18/71) of NSCLC samples. PD-L1 mRNA expression levels were not associated with clinicopathological characteristics of NSCLC. However, NSCLC patients with increased PD-L1 mRNA expression have a better prognosis than patients with low levels of PD-L1 mRNA (63.49 months vs. 98.53 months, χ2=3.73, p=0.053). Disease-free survival was 82.23 months and 45.68 months in patients with and without high levels of PD-L1 expression, respectively, although this difference was not significant (χ 2= 3.23, p=0.073). These results suggest that PD-L1 mRNA expression correlates to alterations in NSCLC disease progression; therefore, further comprehensive analyses should be performed.

Prognostic impact of telomeric repeat-containing RNA expression on long-term oncologic outcomes in colorectal cancer.

Telomeres are transcribed into long, noncoding telomeric repeat-containing RNAs (TERRA) that have been implicated in the regulation of telomerase, the enzyme that lengthens telomeres, in heterochromatin formation at telomeres, and in telomere stability. This study aimed to evaluate the correlation between TERRA expression and long-term oncologic outcomes in colorectal cancer (CRC).We evaluated 18p TERRA expression and telomere length using quantitative real-time PCR in 60 patients who underwent surgical resection for CRC between June 2008 and November 2010.Patients were grouped according to 18p TERRA expression, with 29 (48.3%) and 31 (51.7%) patients in the low and high TERRA expression groups, respectively. The median follow-up period was 80 months (range 2-103). The 18p TERRA expression was marginally significantly associated with preoperative carcinoembryonic antigen (CEA; P = .082) and was significantly associated with telomere length (P < .05). Multivariate analysis revealed that preoperative CEA (hazard ratio [HR], 2.728; 95% confidence interval [CI], 0.832-8.944, P = .098) and 18p TERRA expression (HR, 0.113; 95% CI, 0.011-1.126, P = .071) were marginally significant independent prognostic factors for overall survival (OS), whereas preoperative CEA (HR, 4.254; 95% CI, 1.394-12.985, P = .011) and 18p TERRA expression (HR, 0.108; 95% CI, 0.011-1.037, P = .054) were significant independent prognostic factors for disease-free survival (DFS). According to our prognostic model with 2 prognostic factors, the OS and DFS rate increased to 76.2% and 80.63%, respectively, in patients with high 18p TERRA expression and CEA levels ≤5 (P = .178, P = .057, respectively).18p TERRA expression was marginally significantly associated with preoperative CEA and significantly associated with telomere length, rendering it a potential prognostic factor for long-term oncologic outcomes in CRC.

Clinical and Prognostic Value of Human Mammary Tumor Virus in Korean Patients with Breast Carcinoma.

BACKGROUND: Human mammary tumor virus (HMTV) is 90-95% homologous to mouse mammary tumor virus, one of the causal agents of murine mammary tumors. Although HMTV has been frequently detected in human breast cancers, its clinical and prognostic value remains unknown. METHODS: In the present study, we analyzed HMTV infection using polymerase chain reaction (PCR) in 128 breast cancers. RESULTS: HMTV was found in 9.4% (12/128) of breast cancers and was significantly associated with breast pain (66.7% vs. 11.7%, p=0.007). It had a tendency to be detected more frequently in breast cancer patients with lower BMI<25, although this result was not statistically significant (18.8% vs. 5.4%, p=0.103). Kaplan-Meier survival analysis showed no prognostic value of HMTV in breast cancer (χ2=0.148, p=0.700). For the first time, we investigated the clinical and prognostic value of HMTV in Korean patients with breast cancer. CONCLUSION: Although our study revealed that HMTV infection does not have important clinical significance in breast cancer, the possibility remains that it may be a prominent causative agent of the disease.

Arg-Leu-Tyr-Glu Suppresses Retinal Endothelial Permeability and Choroidal Neovascularization by Inhibiting the VEGF Receptor 2 Signaling Pathway.

Vascular endothelial growth factor (VEGF) plays a pivotal role in pathologic ocular neovascularization and vascular leakage via activation of VEGF receptor 2 (VEGFR2). This study was undertaken to evaluate the therapeutic mechanisms and effects of the tetrapeptide Arg-Leu-Tyr-Glu (RLYE), a VEGFR2 inhibitor, in the development of vascular permeability and choroidal neovascularization (CNV). In cultured human retinal microvascular endothelial cells (HRMECs), treatment with RLYE blocked VEGF-A-induced phosphorylation of VEGFR2, Akt, ERK, and endothelial nitric oxide synthase (eNOS), leading to suppression of VEGFA-mediated hyper-production of NO. Treatment with RLYE also inhibited VEGF-A-stimulated angiogenic processes (migration, proliferation, and tube formation) and the hyperpermeability of HRMECs, in addition to attenuating VEGF-A-induced angiogenesis and vascular permeability in mice. The anti-vascular permeability activity of RLYE was correlated with enhanced stability and positioning of the junction proteins VE-cadherin, ß-catenin, claudin-5, and ZO-1, critical components of the cortical actin ring structure and retinal endothelial barrier, at the boundary between HRMECs stimulated with VEGF-A. Furthermore, intravitreally injected RLYE bound to retinal microvascular endothelium and inhibited laser-induced CNV in mice. These findings suggest that RLYE has potential as a therapeutic drug for the treatment of CNV by preventing VEGFR2-mediated vascular leakage and angiogenesis.

NF-κB-responsive miR-155 induces functional impairment of vascular smooth muscle cells by downregulating soluble guanylyl cyclase.

Vascular smooth muscle cells (VSMCs) play an important role in maintaining vascular function. Inflammation-mediated VSMC dysfunction leads to atherosclerotic intimal hyperplasia and preeclamptic hypertension; however, the underlying mechanisms are not clearly understood. We analyzed the expression levels of microRNA-155 (miR-155) in cultured VSMCs, mouse vessels, and clinical specimens and then assessed its role in VSMC function. Treatment with tumor necrosis factor-α (TNF-α) elevated miR-155 biogenesis in cultured VSMCs and vessel segments, which was prevented by NF-κB inhibition. MiR-155 expression was also increased in high-fat diet-fed ApoE-/- mice and in patients with atherosclerosis and preeclampsia. The miR-155 levels were inversely correlated with soluble guanylyl cyclase ß1 (sGCß1) expression and nitric oxide (NO)-dependent cGMP production through targeting the sGCß1 transcript. TNF-α-induced miR-155 caused VSMC phenotypic switching, which was confirmed by the downregulation of VSMC-specific marker genes, suppression of cell proliferation and migration, alterations in cell morphology, and NO-induced vasorelaxation. These events were mitigated by miR-155 inhibition. Moreover, TNF-α did not cause VSMC phenotypic modulation and limit NO-induced vasodilation in aortic vessels of miR-155-/- mice. These findings suggest that NF-κB-induced miR-155 impairs the VSMC contractile phenotype and NO-mediated vasorelaxation by downregulating sGCß1 expression. These data suggest that NF-κB-responsive miR-155 is a novel negative regulator of VSMC functions by impairing the sGC/cGMP pathway, which is essential for maintaining the VSMC contractile phenotype and vasorelaxation, offering a new therapeutic target for the treatment of atherosclerosis and preeclampsia.

Loss of the Association between Telomere Length and Mitochondrial DNA Copy Number Contribute to Colorectal Carcinogenesis.

Positive association between telomere length and mitochondrial DNA (mtDNA) copy number were introduced in healthy and patients with psychiatric disorder. Based on frequent genetic changes of telomere and mitochondria in colorectal carcinomas (CRC), we studied their clinical characteristics and their association in colorectal carcinogenesis. DNA was extracted from 109 CRCs, 64 colorectal tubular adenomas (TAs), and 28 serrated polyps (SPs), and then, telomere length and mtDNA copy number were analyzed in these legions by using a real-time PCR assay. Telomere length and mtDNA copy number (mean ± S.D) in CRCs was 1.87 ± 1.52 and 1.61 ± 1.37, respectively. In TAs and SPs, relative mtDNA copy number was 0.92 ± 0.71 and 1.84 ± 1.06, respectively, shoing statistical difference (p = 0.017). However, telomere length was similar in these precancerous legions. Telomere length and mtDNA copy number did not show clinical and prognostic values in CRCs, however, positive correlation between telomere length and mitochondrial DNA copy number were found in CRC (r = 0.408, p < 0.001). However, this association was not shown in precancerous lesions (r = -0.031, p = 0.765). This result suggests that loss of co-regulation between telomeres and mitochondrial function may induce the initiation or play a role as trigger factor of colorectal carcinogenesis.

A TERT-CLPTM1 locus polymorphism (rs401681) is associated with EGFR mutation in non-small cell lung cancer.

Telomere length is associated with lung carcinogenesis, and recent studies have focused on telomere-maintaining genes and their polymorphisms. Cancer susceptibility of the rs401681 polymorphism, located in the TERT-CLPTM1L locus, has been studied in many cancers. We examined the clinicopathological and prognostic value of rs401681 variants in lung cancer. The relationship between rs401681 variants and telomere length was analyzed in 134 non-small cell lung cancers (NSCLCs). The rs401681 polymorphism had the following genotype frequencies: C/C in 52.2% of the samples, C/T in 30.6%, and T/T in 17.2%. The T allele showed a strong correlation with EGFR mutation (p=0.037). Telomeres in the tumor samples were 3.26-fold longer, on average, than telomeres in matched normal samples (SD=0.48), and there were no differences in telomere length according to rs401681 polymorphism. Smoking was associated with telomere shortening (p=0.01). Survival analysis showed no prognostic value for rs401681 polymorphisms or telomere length in NSCLC. These results suggested that the rs401681 polymorphism contributes to lung carcinogenesis only in patients harboring an EGFR mutation. However, the polymorphism was not associated with survival; therefore, further comprehensive analysis should be performed.

Telomere shortening in non-tumorous and tumor mucosa is independently related to colorectal carcinogenesis in precancerous lesions.

Telomere shortening is associated with colorectal carcinogenesis and recent studies have focused on its characteristics in both normal mucosa and tumor tissues. To clarify the role of telomeres in colorectal carcinogenesis, we analyzed telomere shortening in normal and tumor regions of 93 colorectal precursor lesions. Telomere length was examined in 61 tubular adenomas (TAs) and 32 serrated polyps (SPs), and PIK3CA expression, KRAS mutation, BRAF mutation, and MSI were also analyzed. Telomere length was similar in normal and tumor tissues of TAs and SPs. In normal tissues of TAs, telomere shortening was associated with PIK3CA amplification (81.3% vs. 18.8%, p < 0.001), whereas it was associated with BRAF mutation in normal tissues of SPs (66.7% vs. 23.1%, p = 0.060). According to the analysis on tumor tissues, KRAS and BRAF mutations were mutually exclusive in TAs and SPs (p < 0.001), and telomere shortening was associated with mitochondrial microsatellite instability (63.6% vs. 36.4%, p = 0.030). These data suggested a pivotal role of telomere shortening in normal colorectal tissue for proceeding to TAs or SPs along with PIK3CA amplification and BRAF mutation, respectively. Moreover, telomeres in TAs may collaborate with mitochondrial instability for disease progression.

TERT-CLPTM1 locus polymorphism (rs401681) is associated with the prognosis of hepatocellular carcinoma.

Telomere length is associated with the development of hepatocellular carcinoma (HCC), and recent studies have focused on the genetic alteration or polymorphism in telomere-maintaining genes. We examined the clinicopathologic and prognostic value of rs401681 polymorphism, located in the TERT-CLPTM1L locus, in HCC. The relationship between rs401681 variants and telomere length was also analyzed in 156 HCC patients. The rs401681 polymorphism had the following genotype frequencies: C/C in 51.3% of the samples, C/T in 39.7%, and T/T in 9.0%. Telomeres in the tumor samples were 4.04-fold longer, on average, than the telomeres in matched normal samples (SD =1.32), and there were no differences in telomere length according to rs401681 polymorphism (p=0.802). Our results indicate that the rs401681 C allele was significantly associated with increased T and International Union for Cancer Control stages (p<0.01). Univariate and multivariate survival analyses showed that HCC with C allele had poorer prognosis (p<0.01). In conclusion, our findings suggest that rs401681 is a possible prognostic biomarker for HCC patients.