Expression of CC Chemokine Ligand 5 in Patients with Rheumatoid Arthritis and Its Correlation with Disease Activity and Medication

Objective To determine the levels of CC chemokine ligand 5 (CCL5) in serum and synovial fluid (SF) from patients with rheumatoid arthritis (RA) and their relations with disease activity and medication. Methods CCL5 in serum and SF was quantified by enzyme-linked immunosorbent assay (ELISA) in 28 RA patients and 21 osteoarthritis (OA) patients. In RA patients, the correlations of CCL5 levels in serum and SF with disease activity were analyzed. Meanwhile, the serum CCL5 levels among RA patients treated with disease-modifying antirheumatic drugs (DMARDs), Tripterygium Glucosides, and other Chinese herbs without disease-modifying effects were also compared. Results CCL5 levels in both serum and SF of RA patients were significantly higher than those of OA patients (P<0.05). Moreover, the level of CCL5 was higher in SF than that in serum of RA patients (P<0.01). Serum CCL5 level was correlated significantly with the number of swollen joints (r=0.3329, P<0.05), erythrocyte sedimentation rate (r=0.4001, P<0.05), and C reactive protein (r=0.3735, P<0.01). In addition, the level of CCL5 had a trend of lower in patients treated with DMARDs or Tripterygium Glucosides than those treated with other Chinese herbs, although the difference was not significant among those patients due to the small number of patients in each group. Conclusions In RA patients, the expression of CCL5 increases and correlates with some clinical and laboratory parameters of RA, which indicate that CCL5 plays an important role in RA and may serve as a useful marker of disease activity. DMARDs and Tripterygium Glucosides might exert their clinical effects through reducing CCL5 production in RA.

Association of gene and protein expression and genetic polymorphism of CC chemokine ligand 4 in colorectal cancer

BACKGROUND Leukocytes,such as T cells and macrophages,play an important role in tumorigenesis.CC chemokine ligand(CCL)4,which is produced by lymphocytes and macrophages,has been found to be expressed in the mucosa of the gastrointestinal tract and is a potent chemoattractant for various leukocytes.AIM To examine CCL4 expression and its genetic polymorphism rs10491121 in patients with colorectal cancer(CRC)and evaluate their prognostic significance.METHODS Luminex technology was used to determine CCL4 Levels in CRC tissue(n=98),compared with paired normal tissue,and in plasma from patients with CRC(n=103),compared with healthy controls(n=97).Included patients had undergone surgical resection for primary colorectal adenocarcinomas between 1996 and 2019 at the Department of Surgery,Ryhov County Hospital,Jönköping,Sweden.Reverse transcription quantitative PCR was used to investigate the CCL4 gene expression in CRC tissue(n=101).Paired normal tissue and TaqMan single nucleotide polymorphism assays were used for the CCL4 rs10491121 polymorphism in 610 CRC patients and 409 healthy controls.RESULTS The CCL4 protein and messenger RNA expression levels were higher in CRC tissue than in normal paired tissue(90%,P<0.001 and 45%,P<0.05,respectively).CRC tissue from patients with localized disease had 2.8-fold higher protein expression levels than that from patients with disseminated disease.Low CCL4 protein expression levels in CRC tissue were associated with a 30%lower cancer-specific survival rate in patients(P<0.01).The level of plasma CCL4 was 11%higher in CRC patients than in healthy controls(P<0.05)and was positively correlated(r=0.56,P<0.01)with the CCL4 protein level in CRC tissue.The analysis of CCL4 gene polymorphism rs10491121 showed a difference(P<0.05)between localized disease and disseminated disease in the right colon,with a dominance of allele A in localized disease.Moreover,the rate of the A allele was higher among CRC patients with mucinous cancer than among those with nonmucinous cancer.CONCLUSION The present study indicates that the CRC tissue levels of CCL4 and CCL4 gene polymorphism rs10491121,particularly in the right colon,are associated with clinical outcome in CRC patients.

Expression of CC Chemokine Ligand 20 and CC Chemokine Receptor 6 mRNA in Patients with Psoriasis Vulgaris

Summary: In order to explore the possible role of CC chemokine ligand 20 (CCL20) and its receptor CC chemokine receptor 6 (CCR6) in the pathogenesis of psoriasis, the expression levels of mRNA of them in psoriatic lesions were investigated. The skin biopsies were collected from skin lesions in 35 cases of psoriasis vulgaris and 18 normal controls. RT-PCR was used to semi-quantitatively analyze the mRNA expression of CCL20 and CCR6 in the psoriatic lesions and the normal skin tissues. The results showed that the mRNA of CCL20 and CCR6 was present in every specimen. The expression levels of CCL20 mRNA in skin lesions were 1.1397±0.0521, which were greatly higher than those in normal controls (0.8681±0.0308) (P<0.001). The expression levels of CCR6 mRNA in skin lesions were 1.1103±0.0538, significantly higher than in the controls (0.9131±0.0433, P<0.001). These findings indicate that up-regulated expression of CCL20 and CCR6 mRNA might be related to the pathogenesis of psoriasis.

linc00941通过miR-203/CCL2轴调控食管鳞状细胞癌细胞增殖、侵袭和糖酵解

目的:探究linc00941作为ceRNA吸附miR-203上调CC-趋化因子配体2(CC chemokine ligand 2,CCL2)的表达在食管鳞癌(esophageal squamous cell carcinoma,ESCC)中的作用机制。方法:选取南阳医学高等专科学校第一附属医院58例ESCC患者的癌组织和癌旁组织,其中,男性患者33例,年龄(49.3±18.6)岁,女性患者25例,年龄(44.6±20.7)岁。qPCR法检测linc00941、miR-203、CCL2在ESCC组织和4株人ESCC细胞系(EC9706、KYSE30、ECA109和TE1)以及人正常食管上皮细胞株HET-1A细胞系中的表达。构建linc00941-wt、linc00941-mut、CCL2-wt、CCL2-mut质粒并分别与miR-203 NC或miR-203模拟物共转染到293T细胞中。双荧光素酶报告基因实验验证linc00941、miR-203、CCL2之间的相互作用。CCK-8和Transwell实验检测细胞的增殖与侵袭能力。乳酸含量检测评价细胞的糖酵解能力。流式细胞术检测细胞的凋亡情况。糖酵解抑制剂2-DG以及linc00941共同干预ESCC细胞,以进一步观察linc00941对ESCC细胞的调控作用。结果:在ESCC组织中和细胞系中linc00941、CCL2表达均上调,miR-203表达下调(均P<0.05)。linc00941与miR-203、miR-203与CCL2的相互作用在ECA109细胞中得到证实。下调linc00941能够抑制ECA109细胞的增殖、侵袭和糖酵解,并诱导细胞凋亡,该作用被miR-203抑制剂部分逆转(均P<0.05)。过表达CCL2可以部分逆转敲减linc00941对ECA109细胞增殖、侵袭、糖酵解和凋亡的影响(均P<0.05)。结论:linc00941能够吸附miR-203进而上调CCL2的表达,促进ESCC细胞的增殖、侵袭和糖酵解,诱导细胞凋亡。linc00941对ESCC细胞增殖、侵袭和凋亡的影响可能是通过调控糖酵解实现的。

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR FOR HUMAN CCL21 AND CHARACTERIZATION OF ITS CHEMOTACTIC ACTIVITY

Objective： To obtain recombinant human CCL21 with biological activity from eukary0tic expression system for further use in cancer gene therapy. Methods： A fragment of human CCL21 gene was obtained from pSK-hCCL21 plasmid digested by Xho I and BamH I, inserted into the responding sites of eukaryotic expression vector pVAX1, and then transfected into COS-7 cells by electroporation method. The expression of hCCL21 protein was detected by western blotting analysis. The in vitro chemotaxis assay was used to test the chemotactic function of the expression product to lymphocytes. Results： Human CCL21 protein was expressed by transfected COS-7 cells with recombinant plasmid containing hCCL21 gene, and was verified by western blotting. The in vitro chemotaxis assay demonstrated that human CCL21 protein had a potent chemotactic function to lymphocytes. Conclusion： Human CCL21 was successfully and transiently expressed in eukaryotic cells, which lays some foundation for the study of CCL21 gene therapy in murine tumor models.

Aconite aqueous extract inhibits the growth of hepatocellular carcinoma through CCL2-dependent enhancement of natural killer cell infiltration

Objective:Aconite is a traditional Chinese herbal medicine that has been found to inhibit the development of liver cancer;however,its exact molecular mechanisms in this process remain unclear.This study explores how aconite aqueous extract(AAE)inhibits hepatocellular carcinoma(HCC).Methods:An in vivo mouse model of subcutaneous liver cancer was established.After AAE treatment,immunohistochemistry(IHC)was used to determine the effect of AAE on natural killer(NK)cells.Subsequently,C57BL/6 mice were used to establish the subcutaneous tumor model,and a group of these mice were treated with anti-PK163 antibody to remove NK cells,which was verified by flow cytometry and IHC.The effect of AAE on the proliferation of HCC cells in vitro was determined using cell counting kit-8.The effect of AAE on chemokine production in HCC cells was measured using real-time quantitative polymerase chain reaction and an enzyme-linked immunosorbent assay.The effect of AAE on the migration of NK cells was determined using a transwell assay.Finally,the molecular mechanism was investigated using the Western blotting method.Results:We demonstrated that the ability of AAE to induce overexpression of the cytokine C–C motif chemokine ligand 2(CCL2)in HCC cells is fundamental to the infiltration of NK cells into the tumor bed.Mechanistically,we found that the upregulation of CCL2 was achieved by the activation of c-Jun Nterminal kinase but not extracellular regulated protein kinase or p38.Conclusion:Our findings suggest that AAE can be used as an effective immune adjuvant to enhance antitumor immunity by increasing NK cell infiltration into tumors,which could help to improve the efficacy of HCC treatments.