Mingliang Chen

A small interfering CD147-targeting RNA inhibited the proliferation, invasiveness, and metastatic activity of malignant melanoma

CD147 plays a critical role in the invasive and metastatic activity of malignant melanoma cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases and vascular endothelial growth factor. We developed a system that blocks CD147 in the human malignant melanoma cell line, A375, using RNA interference. By transfecting melanoma cells with the small interfering RNA (siRNA) that targets human CD147, we were able to establish two stable clones in which CD147 expression was significantly down-regulated. This resulted in the decreased proliferation and invasion of A375 cells in vitro. CD147 siRNA also down-regulated the expression of vascular endothelial growth factor in these cells and reduced the migration of vascular endothelial cells. The reduction in the CD147 level suppressed the size of s.c. tumors and the microvessel density in an A375 s.c. nude mouse xenograft model. In addition, the in vivo metastatic potential of A375 cells transfected with CD147 siRNA was suppressed in a nude mouse model of pulmonary metastasis.

CD147 mediates chemoresistance in breast cancer via ABCG2 by affecting its cellular localization and dimerization.

CD147 and ABCG2 both have been reported to mediate Multidrug resistance (MDR) in breast cancer. Recent study demonstrates that CD147 could form a complex with ABCG2 on the cell membrane in primary effusion lymphoma. However, whether these two molecules regulate each other in breast cancer and result in MDR is not clear. We established four MCF-7 cell lines transfected with CD147 and/or ABCG2 and found that CD147 could increase the expression and dimerization of ABCG2, affect its cellular localization and regulate its drug transporter function. The findings derived from cells were confirmed subsequently in clinic samples of chemotherapy-sensitive/resistant breast cancer.

Effect of ghrelin on angiotensin II induced human umbilicus vein endothelial cell oxidative stress and endothelial cell injury.

OBJECTIVE To determine the effect of ghrelin on protecting the human umbilical vein endothelial cells (HUVEC) from injury by angiotensin II (Ang II) in vitro. METHODS (1) HUVEC was incubated for 24 h with AngII whose final concentration in the medium varied from 10⁻⁹ to 10⁻⁶ mol/L or pretreated with 10⁻⁹ to 10⁻⁶ mol/L ghrelin for 2 h before incubation for 24 h with Ang II whose final concentration in the medium was 10⁻⁶ mol/L. HUVECs were harvested to measure the cell vitality and cell apoptosis. The cell vitality was determined by MTT and cell apoptosis rates were measured by Annexin V-FITC apoptosis detection kit. (2) HUVECs were incubated for 3, 6, 12, or 24 h with 10⁻⁹, 10⁻⁸, 10⁻⁷, or 10⁻⁶ mol/L Ang II, respectively. Before HUVECs were incubated with 10⁻⁶ mol/L Ang II for 24 h, ghrelin (10⁻⁹, 10⁻⁸, 10⁻⁷, and 10⁻⁶ mol/L) was used to pretreat the cells for 2 h. Growth hormone secregogue receptor 1a blocker [D-Lys³]GHRP-6 was added to the cells which were incubated for 24 h with 10⁻⁶ mol/L Ang II and pretreated with 10⁻⁶ mol/L ghrelin for 2 h. Cell reactive oxygen species were measured by dichlorofluorescin (DCF) fluorescence probe method. (3) HUVECs were incubated for 24 h with 10⁻⁹, 10⁻⁸, 10⁻⁷, or 10⁻⁶ mol/L Ang II and ghrelin, respectively,and then were incubated with 10⁻⁶ mol/L of Ang II for 3, 6, 12, or 24 h. Furthermore, HUVECs were pretreated with 10⁻⁹, 10⁻⁸, 10⁻⁷, or 10⁻⁶ mol/L ghrelin for 30 min,1 h, or 2 h, and then were incubated with the inhibitor of mitogen-activated protein kinase /extracellular regulated kinase (MAPK/ERK1/2),PD98059, the inhibitor of phosphoinositide-3-kinase/serine threonine kinase( PI3K/Akt)wortmannin, and [D-Lys³]GHRP-6 for 24 h. NO production was compared among groups. HUVECs were pretreated with ghrelin, PD98059, wortmannin, and [D-Lys³]GHRP-6 for 2 h and co-cultured with 10⁻⁶ mol/L Ang II for 24 h, or pretreated with ghrelin plus PD98059, wortmannin, and [D-Lys³]GHRP-6 before incubation with Ang II for 24 h. NO was measured in the endothelial cell supernatants by Griess method. (4) HUVECs were cultivated with blank or Ang II with or without pretreatment with ghrelin or both ghrelin and wortmannin. The protein expression of eNOS and phospho-protein expression of Akt were measured by Western blot analysis. RESULTS Ang II injuried the endothelial cell vitality,increased the cell apoptosis rates in HUVECs, and decreased NO production in HUVEC supernatants,whereas ghrelin protected HUVECs from Ang II injury. Ghrelin decreased the reactive oxygen species in HUVECs induced by Ang II. The effect could be attenuated by [D-Lys³]GHRP-6 pretreatment; PD98059 alleviated Ang II inhibition of NO production in HUVEC supernatants. Wortmannin and [D-Lys³]GHRP-6 could abolish protection of ghrelin from reducing NO production in HUVEC supernatants. Ang II reduced the expression of eNOS,but ghrelin increased eNOS expression. Wortmannin could cancel this effect of ghrelin. Ghrelin increased p-Akt expression and reached the peak in 10 and 20 min. CONCLUSION Ghrelin may protect HUVECs from Ang II induced injury, which is related to decreasing oxidative stress, increasing the protein expression of eNOS, and activating PI3K/Akt signal pathway through GHSR1a receptor.

Activation of JAK-STAT1 signal transduction pathway in lesional skin and monocytes from patients with systemic lupus erythematosus.

OBJECTIVE To study the activation of Janus protein tyrosine kinase (JAK)/signal transducer and activator of transcription 1 (STAT1) signaling pathway and its inhibitor-signal transducer and activator of transcription-1(SOCS-1) in patients with systemic lupus erythematosus. METHODS A total of 45 patients with active systemic lupus erythematosus (SLE) and 30 healthy controls were randomly selected. Western blot was performed to measure the expression of Stat1 protein and phospho-Stat1 protein (an activated form of Stat1 protein) in the monocytes after stimulation with recombinant high mobility group box1 (rHMGB1) at various time points. Expression of Stat1 protein in the skin or lesional skin was also detected. Phasic expressions of SOCS-1 mRNA in the monocytes after rHMGB1 stimulation were detected by real-time reverse transcription-polymerase chain reaction. SOCS-1 gene expression in the skin or lesional skin was also detected. RESULTS The expression level of Stat1 proteins in the monocytes from patients with SLE was higher than that from healthy controls (t=9.16, P<0.01) and positively correlated with SLE disease activity index (SLEDAI) (r=0.59, P<0.01). Expression of phospho-Stat1 in the monocytes from SLE patients was time-dependently upregulated after stimulation with rHMGB1 at various time points, while expression of SOCS-1 mRNA remained unchanged (all P>0.05). Expressions of phospho-Stat1 protein and SOCS-1 mRNA in the monocytes from healthy controls were increased transiently after stimulation with rHMGB1 (all P<0.05). Both expressions of phospho-Stat1 protein and SOCS-1 gene in the lesional skin from patients with SLE were upregulated compared with those in normal skin from healthy controls (all P<0.01). CONCLUSION There are hyperactivation of JAK-STAT1 signaling pathway and negative feedback down-regulation of SOCS-1 in patients with systemic lupus erythematosus. HMGB-1 may be partly involved in the pathogenesis of SLE by the abnormal mediating function of JAK-STAT1 signal transduction pathway.

CD147 is highly expressed on peripheral blood neutrophils from patients with psoriasis and induces neutrophil chemotaxis.

Dear Editor, The precise mechanisms responsible for the genesis and development of psoriasis, a chronic, relapsing inflammatory skin disease, remain unclear. Characteristic neutrophil accumulations by chemoattractants such as interleukin (IL)-8 to the stratum corneum that form Munro’s microabscesses are observed in the highly inflammatory and therapyresistant areas of psoriatic lesions. This associated inflammation-boosting loop may explain the localized ‘‘acute’’ inflammatory changes seen in patients with psoriasis. Our previous study has shown that the expression of CD147 ⁄basigin, a highly glycosylated transmembrane protein, belongs to the immunoglob-

Influence of component 5a receptor 1 (C5AR1) −1330T/G polymorphism on nonsedating H1-antihistamines therapy in Chinese patients with chronic spontaneous urticaria

BACKGROUND The nonsedating H1-antihistamines are the first-line medicines for chronic spontaneous urticaria (CSU) patients. However, not all these patients respond well to the antihistamines, and the mechanisms underlying the interindividual differences are still unclear. C5AR1 gene encodes the component 5a receptor (C5aR) protein, which has been reported to play an important role in chronic spontaneous urticaria. OBJECTIVE This study aimed to investigate whether the single nucleotide polymorphisms (SNPs) in C5AR1 are associated with CSU susceptibility and antihistamines therapeutic efficacy in Chinese CSU patients. METHODS A total of 191 CSU patients and 102 healthy controls were prospectively studied in our study. CSU patients were treated by nonsedating H1-antihistamines monotherapy for 4 weeks. The C5AR1 -1330T/G (rs11673309) genotype was determined by Sequenom Massarray. RESULTS Among these 191 CSU patients, there were 114 patients who were treated with desloratadine, 65 were treated with mizolastine, and 12 with fexofenadine. The-1330T alleles in CSU patients were significantly higher than controls (0.555 vs. 0.466, P=0.040, OR=1.429 [1.016-2.010]). The poorest response to desloratadine was observed in heterozygotes, when compared with either GG or TT homozgote (P=0.001). However, there was no significant difference in three genotypes when treated with mizolastine group (P=0.215). CONCLUSION We concluded that the C5AR1 SNP -1330T/G may serve as a useful pharmacodynamic predictor of nonsedating H1-antihistamines efficacy in CSU patients, and -1330T alleles may be taken as a risk factor for the CSU.

Brain natriuretic peptide rs198388 polymorphism and essential hypertension in Hunan Han people

OBJECTIVE To investigate the relation between brain natriuretic peptide (BNP) rs198388 polymorphism and the susceptibility of essential hypertension in Han population of Hunan. METHODS A total of 567 patients with hypertension (the hypertension group) and 555 healthy volunteers (the control group) were enrolled. Gender, age, smoking and drinking history of the 2 groups were not significantly different. Blood pressure was measured in the 2 groups. After fasting for 12 h or more, blood glucose, total cholesterol, triglycerides, high density lipoprotein cholesterol and low density lipoprotein cholesterol were measured. DNA polymorphism analysis was done by polymerase chain reaction-restriction fragment length polymorphism method, and genotype was determined by agarose gel electrophoresis. RESULTS The GG, GA, and AA genotypes were detected.The frequencies of GA and AA genotypes and A allele were significantly lower in the hypertension group (GA and AA:12.3%;A:6.9%) than those in the control group (GA and AA:18.4%; A:9.7%; P=0.009, and P=0.014, respectively). CONCLUSION BNP rs198388 polymorphism may be associated with essential hypertension in Han people in Hunan. Carrying rs198388 GA and AA genotypes and A allele may be the reason for low risk of hypertension.

A novel mutation for disseminated superficial actinic porokeratosis in the MVK gene

M. TOHYAMA K. HASH IMOTO Y . KANO T. SH IOHARA K . I TO H. FU J I TA M. A IHARA H. ASADA Department of Dermatology, Nara Medical University School of Medicine, 840 Shijocho, Kashihara, Nara 634-8522, Japan Department of Dermatology, Showa University School of Medicine, Tokyo, Japan Department of Dermatology, Ehime University Graduate School of Medicine, Matsuyama, Japan Department of Dermatology, Kyorin University School of Medicine, Tokyo, Japan Department of Environmental ImmunoDermatology, Yokohama City University Graduate School of Medicine, Yokohama, Japan Correspondence: Hideo Asada. E-mail: asadah@naramed-u.ac.jp

The expression of mCTLA-4 in skin lesion inversely correlates with the severity of psoriasis

BACKGROUND Psoriasis is a chronic inflammatory disease characterized by epidermal hyperplasia and increased T cell infiltration. Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a key factor that affects T cell function and immune response. However, whether the expression of CTLA-4 affects the severity of psoriasis is still unknown. OBJECTIVE The aim of the project was to investigate the correlation between the expression of CTLA-4 and the severity of psoriasis. METHODS The plasma soluble CTLA-4 levels and membrane CTLA-4 expression were measured by enzyme-linked immunosorbent assay and immunohistochemistry analysis in mild, moderate and severe psoriasis patients, respectively. Imiquimod-induced mouse model of psoriasis was treated with CTLA-4 immunoglobulin fusion protein (CTLA-4 Ig) or anti-CTLA-4 antibody. Epidermal thickness and infiltrating CD3+ T cell counts were evaluated. RESULTS The plasma soluble CTLA-4 levels had no significant difference among mild, moderate, and severe patients (p > 0.05). However, the membrane CTLA-4 expression in skin was significantly higher in mild psoriasis patients compared to moderate and severe psoriasis patients (17652.86 ± 18095.66 vs 6901.36 ± 4400.77 vs 3970.24 ± 5509.15, p < 0.001). Furthermore, in imiquimod-induced mouse model of psoriasis, the results showed that mimicking CTLA-4 function improved the skin phenotype and reduced epidermal thickness (172.87 ± 28.25 vs 245.87 ± 36.61 μm, n = 6, p < 0.01) as well as infiltrating CD3+ T cell counts (5.09 ± 3.45 vs 13.45 ± 4.70, p < 0.01) compared to control group. However, blocking CTLA-4 function aggregated the skin phenotype including enhanced epidermal thickness and infiltrating CD3+ T cell counts compared to control group. CONCLUSION These results indicated that the expression of mCTLA-4 in skin lesion inversely correlated with the severity of psoriasis and CTLA-4 might play a critical role in the disease severity of psoriasis.

Whole Exome Sequencing in Psoriasis Patients Contributes to Studies of Acitretin Treatment Difference

Psoriasis vulgaris is an immune-mediated inflammatory skin disease. Although acitretin is a widely used synthetic retinoid for moderate to severe psoriasis, little is known about patients’ genetics in response to this drug. In this study, 179 patients were enrolled in either the discovery set (13 patients) or replication set (166 patients). The discovery set was sequenced by whole exome sequencing and sequential validation was conducted in the replication set by MassArray assays. Four SNPs (single nucleotide polymorphisms) (rs1105223T>C in CRB2, rs11086065A>G in ANKLE1, rs3821414T>C in ARHGEF3, rs1802073 T>G in SFRP4) were found to be significantly associated with acitretin response in either co-dominant or dominant models via multivariable logistic regression analysis, while CRB2 rs1105223CC (OR = 4.10, 95% CI = 1.46–11.5, p = 0.007) and ANKLE1 rs11086065AG/GG (OR = 2.76, 95% CI = 1.42–5.37, p = 0.003) were associated with no response to acitretin after 8-week treatment. Meanwhile, ARHGEF3 rs3821414CT/CC (OR = 0.25, 95% CI = 0.10–0.68, p = 0.006) and SFRP4 rs1802073GG/GT (OR = 2.40, 95% CI, 1.23–4.70, p = 0.011) were associated with a higher response rate. Four new genetic variations with potential influences on the response to acitretin were found in this study which may serve as genetic markers for acitretin in psoriasis patients.