Vol. 35 No Journal of South-Central University for Nationalities Nat. Sci. Edition Jun TNFα. . MDSCs

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1 35 2 Vol 35 No Journal of South-Central University for Nationalities Nat Sci Edition Jun 2016 ASICs TNF-α * ASICs TNFα PCR ph 7 3 ph 5 5 ASICs TNF-α TNF-α ASICs TNF-α ASICs 1 PcTx1 TNF-α ASICs 3 APETX2 TNF-α ASICS 1 TNF-α PCR R392 9 A Promoted TNF-α Expression of by ASICs under Acidic Tumor Microenvironment Hu Xin 1 Yi Pin 1 Ming Yin 2 Tong Shenglan 3 Gong Huihong 4 1 College of PharmacySouth-Central University for NationalitiesWuhan China 2 Xianning City Spa Red Flag Road Middle School of Hubei ProvinceWuhan China 3 Clinical Laboratory the First People's Hospital of Jiangxia District in WuhanWuhan China 4 College of Biomedical EngineeringHubei University of Science and TechnologyXianning China Abstract To investigate the relationship between the TNF-α expression and ASICs in under the tumor microenvironmentreal-time PCR was performed to determine the TNF-α expression changes in mice under normal physiological ph 7 3 acidic ph 5 5 and inhibitor-added conditionrespectively The results indicated that the expression of TNF-α significantly elevated in tumor under normal physiological condition as well as acidic condition In contrastthe expression was significantly inhibited by the addition of ASICs' nonspecific inhibitors amiloridediclofenacor ASICs subunit 1 inhibitor PcTx-1 Howeverpretreatment with ibuprofen or ASIC subunits 3 inhibitors APETX2 had no effect So the ASICs subunit 1 of was likely responsible for promoting the expression of TNF- α under the acidic tumor microenvironment Keywords TNF-α real-time PCR ASICs inhibitor * ghh7322@ 126 com huxin5540@ 126 com ZY008 JYX14013

2 TAM 2 3 Percoll GE Hyclone cdna RPMI-1640 TNF-α 4 TNF-α 17 Thermo PCR kd stnf-α 26 kd tmtnfα 5 TNF-α NO ROS T Green Takara SW-CJ-2FD TNF-α CO 2 BPN-150CW Thermo IMS GTR10-1 T TNF-α PCR Blab /c 18 ~ 20 g 6 ~ 8 ph 5 8 ~ ph 7 2 ~ mm g 5mL 100 μm IL mm 0 08 LAK g 5 ml TNF- 200 μm mm α IFN-γ TGF-β IL-10 IL g DMSO 200 μm 12 ASICsH + H ml 500 nm ENaC /DEG 10 nm APETX2 5 μg APETX2 ASICs 548 μl 2 mm 13 BMM T ASICs 2 1 ASICs TNF-α ml H22 3 ~ 4 Blab /c Blab /c TNF-α 6 Blab /C H22 PCR 200 TNF-α ph r /min 10 min TNF-α ASICs 1 RNA DNA Marker RealTime PCR Master Mix SYBR ABI H22-20 PcTx1 10 μg PcTx1 100 nm 2 Blab /c /0 2 75% 5 min EP 5 ml TNF-α 10 min

3 2 ASICs TNF-α 53 5 ml 10% FCS-RIPM min r /min 10min 2 ml 10% FCS-1640 p < 0 05 Percoll 2 ml PBS 2 ml 50% 60% 70% 100% Percoll 2900 r /min 30 min 2 80% MDSC 2 2 RNA ASICs g 5 min RNA cdna mg PCR 7 2 μl 400 μl BufferR-I 1 ml 1 0 μl cdna 0 8 μl Master Mix 10 μl 8 ~ PCR ml 150 μl BufferR- PCR II 15 ~ 30 s g 4 5 min Ct PCR ml EP 250 μl Delta-delta Ct 2 ml EP mrna = 2 n n = Ct - Ct g 4 2 min EP Ct - Ct 2 ml EP 500 ml Buffer W1A g 3 min EP TNF-α ml EP 700 μl BufferW g 3 min 1 2 ml EP g 3 min ml EP 70 μl g 4 3 min RNA 2 3 PCR Genebank TNF-α mrna NM TNF-α 700 bp 5'- ACCCTCACACTCAGATCATCTTCTC-3' 5'- 1 CAGATTGACCTCAGCGCTGAGTTG-3' β-actin 300bp 5'-TCACCCACACTGTGCCCA Fig 1 TCTACGA-3' 5'-CATCGGAACCGCTCGTTGC CAATAG-3' μl PCR 94 3 min s s s min GraphPad ASICs TNF-α ph 7 3 ph h * p < 0 05 ** p < 0 01 *** p < N7 3 normalph 7 3 T5 5 tumorph 5 5 ph TNF-α TNF-α expression in of various group under different ph or with the addition of amiloride 1 2 ph 7 3 TNF-α

4 54 35 * p < 0 05 ** p < 0 01 *** p < N7 3 normalph7 3 T7 3 normalph7 3 T5 5 tumor ph PH PcTx1 APETX2 ph TNF-α TNF-α Fig 2 TNF-α expression in of various group under different ph or with the addition of diclofenac and ibuprofen Fig 2 TNF-α expression in of various group under different ph or with the addition of PcTx1 and APETX2 TNF-α TNF-α TNF-α p < 0 01p < TNF-α ASICs TNF-α TNF-α TNF-α 3 2 ASICs 1 3 PcTx-1 RNA cdna PCR 3 1 TNF-α 3 PcTx1 TNF-α p < 0 01 APETX2 ASICs TNF-α TNF-α ASICs 1 TNF-α TNF-α ph ASIC1 ASIC3 ASIC2 ph TNF-α ASIC2 PcTx1 APETX2 ASIC1 ASIC2 PCR TNF-α 4 * p < 0 05 ** p < 0 01 *** p < N7 3 normal ph7 3 T7 3 normal ph7 3 T5 5 tumor ph5 5 tmtnf-α NO IL-10 TGF-β APETX2 TNF-α CD4 + T CD8 + T MDSC ph ph 5 5pH h ASICs PcTx1 APETX2 H + ASICs ph ASICs T DC BMM TNF-α TNF-α TNF-α ph TNF-α MDSC

5 2 ASICs TNF-α 55 ASIC1 PcTX TNF-α ASIC3 6Szlosarek P WBalkwill F R Tumour necrosis factor α APETX2 a potential target for the therapy of solid tumoursj TNF-α ASIC1 TNF-α ASIC3 ASICs TNF-α ASICs TNF-α lymphoma cellsj J Leukocyte Biol ASICs Yan DQin NZhang Het al Expression of TNF-α leader sequence renders MCF-7 tumor cells resistant to the cytotoxicity of soluble TNF-α J Breast Cancer Res Tr Johnston D ADong BHughes C C W TNF induction of jagged-1 in endothelial cells is NFκB-dependent J 1 Gene J 11Chuang M JSun K HTang S Jet al Tumor-derived tumor necrosis factor-alpha promotes progression and 2Ostrand-Rosenberg S Sinha P Myeloid-derived suppressor epithelial-mesenchymal transition in renal cell carcinoma cells linking inflammation and cancerj J Immunol cells J Cancer Sci Ostrand-Rosenberg S Myeloid-derived suppressor cells more mechanisms for inhibiting antitumor immunity J Cancer Immunol Immun TNF-α MDSC D TNF-α Lancet Oncol Tisdale M J Cancer cachexia J Langenbeck Arch Surg Zhang HYan DShi Xet al Transmembrane TNF-α mediates forward and reverse signalinginducing cell death or survival via the NF-κB pathway in Raji Burkitt 12Müller B Fischer B Kreutz W An acidic microenvironment impairs the generation of non-major histocompatibility complex-restricted killer cells J Immunology Tong JWu W NKong Xet al Acid-sensing ion channels contribute to the effect of acidosis on the function of dendritic cells J J Immunol C/ /