钙调磷酸酶调节因子1.4对食管癌KYSE30细胞增殖、迁移和侵袭能力的影响

Effect of calcineurin regulatory factor 1.4 on the proliferation,migration and invasion of esophageal carcinoma KYSE30 cells

目的探讨钙调磷酸酶调节因子1.4(RCAN 1.4)对食管癌KYSE 30细胞增殖、迁移和侵袭能力的影响。方法将对数生长期HEK-293T细胞分为HEK-293T-A组和HEK-293T-B组,培养至融合70%~80%时,分别加入用慢病毒表达载体质粒[短发卡RNA(shRNA)RCAN1.4]和LentiPac混合包装质粒制备的“DNA质粒-EndoFectin”转染复合物、“空载体质粒-EndoFectin”转染复合物,转染14 h后弃去旧培养基,加入10 mL含体积分数5%胎牛血清、105 U·L^-1青霉素、100μg·L^-1链霉素的达尔伯克改良伊格尔培养基及20μL“Titer Boost”滴度增强剂继续培养,于转染48 h后收集HEK-293T-A组和HEK-293T-B组细胞培养液,离心制备“shRNA-RCAN1.4慢病毒液”和“阴性对照慢病毒液”,分别转染体外培养的食管癌KYSE30细胞,获得shRNA干扰RCAN1.4组(shRCAN1.4组)和shRNA干扰阴性对照组(shNC组)KYSE30细胞,采用Western blot法检测2组KYSE30细胞中RCAN1.4蛋白的表达;平板克隆形成实验检测2组KYSE30细胞的克隆形成能力;划痕实验检测2组KYSE30细胞的迁移能力;Transwell小室实验检测2组KYSE30细胞的侵袭能力。结果shRCAN1.4组和shNC组KYSE30细胞中RCAN1.4蛋白相对表达量分别为0.28±0.02、0.80±0.06,shRCAN 1.4组KYSE30细胞中RCAN1.4蛋白相对表达量显著低于shNC组(t=21.360,P<0.05)。shRCAN1.4组和shNC组KYSE30细胞克隆形成数分别为124.33±6.03、56.67±6.11,shRCAN1.4组KYSE30细胞克隆形成数显著多于shNC组(t=13.665,P<0.05)。细胞划痕培养24 h后,shRCAN1.4组和shNC组KYSE30细胞划痕愈合率分别为(70.07±3.11)%、(19.14±2.58)%,shRCAN1.4组KYSE30细胞划痕愈合率显著高于shNC组(t=21.831,P<0.05)。shRCAN1.4组和shNC组KYSE30细胞侵袭数分别为143.33±12.22、83.33±8.51,shRCAN1.4组KYSE30细胞侵袭数显著多于shNC组(t=7.232,P<0.05)。结论下调食管癌KYSE30细胞中RCAN1.4基因表达可显著增强细胞的增殖、迁移和侵袭能力,RCAN1.4基因可能参与了食管癌细胞的增殖、迁移和侵袭过程。

Objective To investigate the effect of regulatory factor of calcineurin 1.4 gene(RCAN1.4)on the proliferation,migration and invasion of esophageal cancer cell line of KYSE 30.Methods The HEK-293T cells in the logarithmic growth stage were divided into HEK-293T group A and HEK-293T group B.When the cells were cultured to 70%-80%fusion,the"DNA plasmid-EndoFecin"and"empty corpuscle-EndoFecin"transfection complexes prepared by lentivirus expression vector plasmid[short hairpin RNA(shRNA)-RCAN1.4]and LentiPac mixed packaging plasmids were added respectively and transfected for 14 hours.The old medium was discarded and 10 mL of Dulbeccco′s modified Eagle′s medium containing volume fraction 5%fetal bovine serum,105 U·L^-1 penicillin,100μg·L^-1 streptomycin and 20μL titer enhancer of"titer boost"were added for further culture.The cell culture medium of HEK-293T group A and HEK-293T group B were collected at 48 h after transfection and centrifuged to prepare the"shRNA-RCAN1.4 lentivirus solution"and"negative control lentivirus solution"and then they were transfected into the esophageal cancer KYSE30 cells in vitro;the KYSE30 cells of shRNA interferes with RCAN1.4(shRCAN1.4)group and shRNA interferes with negative control(shNC)group were obtained.The expression of RCAN1.4 protein in KYSE30 cells in the two groups were detected by Western blot.The colony forming ability of KYSE30 cells in the two groups was detected by plate clone fornation assay.The migration ability of KYSE30 cells in the two groups was detected by scratch test.The invasion ability of KYSE 30 cells in the two groups was detected by Transwell chamber.Results The relative expression of RCAN 1.4 protein in KYSE30 cells in the shRCAN 1.4 group and the shNC group was 0.28±0.02 and 0.80±0.06,respectively;the relative expression of RCAN 1.4 protein in KYSE30 cells in the shRCAN 1.4 group was significantly lower than that in the shNC group(t=21.360,P<0.05).The number of clone of KYSE30 cells in shRCAN 1.4 group and shNC group was 124.33±6.03 and 56.67±6.11,respectively;the number of clone of KYSE30 cells in shRCAN 1.4 group was significantsly more than that in the shNC group(t=13.665,P<0.05).The scratch healing rate of KYSE30 cells in the shRCAN1.4 group and the shNC group was(70.07±3.11)%and(19.14±2.58)%at 24 hours after scratching;the scratch healing rate of KYSE30 cells in the shRCAN1.4 group was significantly higher than that in the shNC group(t=21.831,P<0.05).The numbers of invasion cells of KYSE30 cells in the shRCAN 1.4 group and the shNC group was 143.3±12.22 and 83.33±8.51,respectively;the number of invasion cells of KYSE30 cells in the shRCAN 1.4 group was significantly higher than that in the shNC group(t=7.232,P<0.05).Conclusion Down regulating the expression of RCAN1.4 gene in KYSE30 cells can significantly enhanced the proliferation,migration and invasion of cells;RCAN 1.4 gene may be involved in the proliferation,migration and invasion of esophageal cancer cells.