钙网蛋白在胆囊癌组织中的表达及对胆囊癌细胞生物学行为影响

The expression of calreticulin on gallbladder carcinoma tissues and its effect on the biologicalbehavior in gallbladder cancer cells

目的探讨钙网蛋白（calreticulinCRT）在胆囊癌中的表达及其对胆囊癌细胞GBC—SD细胞生物学行为影响。方法利用免疫组织化学及RT—qPCR检测CRT在胆囊癌组织中的表达，小干扰RNA转染技术沉默胆囊癌GBC—SD细胞CRT表达，通过Westernblotting检测转染效果，cellcountingkit．8（CCK-8）及平板克隆实验检测细胞的增殖能力；采用流式细胞技术检测细胞凋亡及细胞周期的变化；运用划痕及transwell侵袭小室检测细胞迁移能力；Westernblotting方法检测p-Akt及MMP-9蛋白表达。结果与癌旁及慢性胆囊炎组织比较，CRTmRNA在胆囊癌组织中高表达（f=5．571，P〈0．05）。siCRT-1、siCRT．2实验组24h和48h相对存活率分别为71．5％±6．3％，79．5％±2．7％；62．6％±8．8％，55．6％±2．6％。空白组，阴性对照组，siCRT-1，siCRT-2的凋亡率分别为3．0％±1．8％，4．7％±1．3％，13．6％±1．0％，20．0％±4．0％。划痕实验显示：空白组，阴性对照组，siCRT，1，siCRT-2的划痕修复率分别为（0．67±0．02），（0．58±0．02），（0．22±0．01），（0．37±0．04）。Transwell结果显示，空白组，阴性对照组，siCRT-1，siCRT-2的细胞穿膜个数分别为（302±11），（297±15），（178±10），（165±12）。与空白组及阴性对照组比较，siCRT-1，siCRT-2实验组24，48h相对存活率明显降低，凋亡上升，迁移能力下降（分别F：29．310，118．618，69．651，144．515，190．145，均P〈0．05）。沉默CRT表达后，与空白组及阴性对照组比较，p-Akt及MMP-9蛋白表达水平均下调。结论CRT在胆囊癌中高表达，CRT表达下调介导的胆囊癌生物学行为的改变可能与p-Akt／MMP-9通路相关。

Objective To explore the expression of calreticulin （CRT） in gallbladder cancer tissue and its effect on the biological behavior in gallbladder cancer GBC-SD cells. Methods Immunohistochemistry and RT-qPCR were applied to detect the expression of CRT. Small interfering RNA was transfected into gallbladder cancer GBC-SD cells and Western blotting were used to detect the expression of CRT. The proliferation was determined by using cell counting kit-8 （CCK-8） and clone assays. Flow cytometry were applied to detect the apoptosis and cell cycle. Migration was detected by wound healing and transwell assays, respectively. The expression of p-Akt and MMP-9 were detected by using Western blotting. Results Expression of CRT in gallbladder cancer tissues is higher than adjacent cancer tissues and chronic cholecystitis tissues（ t = 5.571,P 〈 0. 05）. The relative growth rate in the siCRT- 1, siCRT-2 experimental group for 24 hours,48 hourrs were 71.5% ± 6. 3% , 79. 5% ± 2.7% ;62.6% ±8.8%, 55.6% ±2. 6% , respectively. The apoptosis rate in the blank group, the negative control group, siCRT-1 and siCRT-2 group were 3.0% ± 1.8%, 4. 7% ±1.3%, 13.6% ±1.0%, 20.0% ±.0%, respectively. Wound healing assays showed that the wound closure ratio in the blank group, negative control group, siCRT-1 and siCRT-2 group were（0. 67 ± 0. 02）, （0. 58 ± 0. 02）, （0. 22 ±0. 01 ）, （0. 37 ± 0. 04）,respectively. Transwell experiments showed that the numbers of migration of GBC-SD cells in the blank group, negative control group, siCRT-1 and siCRT-2 group were （ 302 ±11 ）, （ 297 ± 15 ）, （ 178 ±10 ）, （ 165 ±-12 ）, respectively, compared with the blank group and the negative control group, the relative growth rate for 24 hours and 48 hours was significantly lower, the apoptosis rate was higher, the numbers of migration was lower （F = 29. 310, 118. 618, 69. 651, 144. 515,190. 145 ,P 〈 0. 05）. Compared with the blank group and the negative control group, the expression of p-Akt and MMP-9 decreased after down-regulating the expression of CRT. Conclusions The expression of CRT in gallbladder cancer tissue was higher. CRT downregnlation mediated changes of biological behaviors in gallbladder cancer may be associated with p-Akt/ MMP-9 signal pathway.