516 1002-0217 2017 06-0516 - 05 3 1,2, 1, 1, 1 1. 210029 2. 244000 3 RT-PCR Western-blot RNA CCK-8 Western-blot P < 0. 05 P < 0. 001 LC3Ⅱ P < 0. 001 P62 P < 0. 001 3 R 735. 2 A DOI 10. 3969 / j. issn. 1002-0217. 2017. 06. 002 Effects of inhibiting aquaporin-3 expression on the proliferation and apoptosis of gastric cancer cells GAO Libin ZHANG Qiang CHEN Liang XU Hao Department of General Surgery The First Affiliated Hospital of Nanjing Medical University Nanjing 210029 China Abstract Objective To investigate the effects of inhibiting aquaporin-3 on the proliferation and apoptosis of gastric cancer cells and the potential mechanisms. Methods The expression level of in gastric cancer cell lines was determined by RT-PCR and Western-blot. low expression cell line was constructed by small interfering RNA. CCK-8 proliferation assay and flow cytometry were used to detect the cell proliferation and apoptosis. The autophagy level was detected by Western-blot and number of autophagosomes was measured by transmission electron microscopy. Results Significantly higher expression of was seen in the gastric cancer cells yet was markedly down-regulated following interference P < 0. 05. Increased apoptotic level P62 expression and decreased autophagy biomarker of LC3Ⅱ were also seen in the gastric cancer cells after interfering all P < 0. 001. Conclusion is highly expressed in gastric cancer cells and interfering the expression of can promote cell apoptosis by decreasing the level of autophagy and inhibiting the proliferation of gastric cancer cells. This suggests that may be potential target for treating gastric cancer. Key words aquaporin-3 autophagy proliferation apoptosis gastric cancer 4 1 2 Aquaporins AQPs 30 ku 5 3 Zhao WSW-038 2017-09-13 1978-13965208002 doctor_gaolb@ 163. com hxu@ njmu. edu. cn
517 1 1. 1 HGC27 MKN45 SGC7901 MGC803 BGC823 AGS GES-1 4 200 μl ATCC RPMI 1640 1X F12K Sigma SDS-PAGE PBS 4 d CCK-8 Tris-base Biosharp 10% CCK-8 100 μl CCK-8 LC3 P62 GAPDH 2 h 450 nm /490 Abcam Cell counting kit-8 Selleck TAKARA Faststart SYBR-green qpcr Mix TOYOBO Li- 1. 2. 5 2. 5 10 5 / - 3 pofectamine 3000 Invitrogen BCA 6 10 ml EDTA PBS Annexin V-FITC BD 1. 2 1500 r /min 3 min 1. 2. 1 10% 1% PBS RPMI 1640 37 5% CO 2 PBS Binding buffer 500 μl Fitc- 1. 2. 2 -sirna -sirna 5'-GGATATGAT- CAATGGCTTCTT-3' 5'-TTCTCCGAAC GTGTCACGT-3' 2. 5 10 5 BCA 24 h sirna 12% 5% Lipofectamine 3000 5 μl 100 μl 5 μl marker 80 V 100 μl 5 min 20 min 120 V 25min PVDF 200 μl 24 h 5% 2 h TBST 3 RNA 5 min 4 1. 2. 3 Real-time PCR RNA cdna RT-PCR β-actin β-actin-f 5'-CTTG- 1. 2. 7 10 6 CAGCTCTTCCGGAGTC-3' β-actin-r 5'-GCTCAGT- GAGCATCAGCGTG-3' -F 5'-CGAAGTGC CA- GATTGCATCATAA-3 -R 5'-TAGCCTTGTCAG 6 48 h 1. 5 ml 1500 r /min 15 min 2. 5% 4 ATAAGGAAGGA-3' SYBR 1% 1 h 5 μl 0. 3 μl 0. 3 μl cdna 1μL DEPC 3. 4 μl 10 μl JEM-1010 95 5min PCR 95 10 s 60 30 s 40 95 15 s 1. 3 SPSS 19. 0 60 30 s 95 15 s 2 -ΔΔCT x 珋 ± s 1. 2. 4 CCK8 2 10 3 96 4 nm = Annexin Ⅴ PI 5 μl 15 min 1. 2. 6 Western-blot SDS-PAGE TBST 3 2 h t
518 3 P < 0. 05 2 mrna 1A 1B Western-blot F 38. 17 24. 68 11. 29 82. 74 2. 1 RT- 31. 12 130. 1 t 4. 377 7. 865 20. 13 3. 26 PCR Western-blot 14. 69 8. 348 P < 0. 05 A RT-PCR GES1 B Western-blot GES1 * P < 0. 05 **P < 0. 01 ***P < 0. 001 1 2. 2 3 SGC7901-siRNA BGC823-siRNA RT-PCR 2A t 10. 29 12. 38 P < 0. 001 Western-blot 2B F 18. 84 4. 850 t 9. 603 9. 239 P < 0. 001 1 2C F 2. 526 2. 778 t 10. 87 38. 62 P < 0. 001 CCK-8 AQPs 6-7 2D 72h F 5 7 1. 210 1. 027 t 2. 796 3. 885 P < 0. 05 8 96 h F 13. 98 1. 197 t 6. 508 8. 357 P < 0. 01 2. 3 9 11-12 F LC3Ⅱ 1. 194 13. 57 t 29. 50 29. 46 P < 0. 001 P62 F 4. 538 1. 193 t 12. 18 28. 3 5 9 ATP 10 60 P < 0. 001 LC3 Ⅱ P62
519 14-15 Sun 16 13 A B. C. D. * P < 0. 05 **P < 0. 01 ***P < 0. 001 2
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