环状RNA hsa_circ_0063772对口腔鳞状细胞癌细胞增殖、迁移和侵袭能力的影响

Effect of circular RNA hsa_circ_0063772 on proliferation, migration and invasion of oral squamous cell carcinoma cells

目的探讨环状RNA hsa_circ_0063772对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)细胞增殖、迁移和侵袭能力的影响。方法纳入2017年2月至2018年12月因口腔鳞状细胞癌就诊于北京大学深圳医院口腔颌面外科的患者33例,使用实时荧光定量PCR检测环状RNA hsa_circ_0063772在OSCC组织、相应癌旁组织、OSCC细胞系(SCC15、SCC25、SCC9、CAL27)及人类角质形成细胞系中的表达水平。使用慢病毒过表达OSCC细胞系SCC15和CAL27细胞系中hsa_circ_0063772的表达水平,通过细胞计数实验、划痕实验、Transwell实验、蛋白质印迹法和裸鼠成瘤实验检测环状RNA hsa_circ_0063772对OSCC细胞增殖、迁移和侵袭能力的影响。结果环状RNA hsa_circ_0063772在OSCC组织中的表达(9.38±0.34)显著低于癌旁组织(11.30±0.31)(t=5.20,P<0.001);在OSCC细胞系中的表达(SCC15∶0.12±0.01;SCC25∶0.18±0.02;SCC9∶0.21±0.01;CAL27∶0.13±0.01)显著低于人类角质形成细胞(1.02±0.02)(tSCC15=41.91,tSCC25=29.21,tSCC9=35.16,tCAL27=41.86,P<0.001)。细胞计数实验结果显示过表达组OSCC细胞的增殖活性(SCC15∶0.76±0.01;CAL27∶0.74±0.02)显著低于空载组(SCC15∶1.22±0.04;CAL27∶0.99±0.03;tSCC15=12.58;tCAL27=6.97)(P<0.05);Transwell迁移实验结果显示过表达组迁移细胞数(SCC15∶148.00±14.57;CAL27∶243.00±13.00)显著低于空载组(SCC15∶580.30±42.91;CAL27∶424.70±18.66)(P<0.01);Transwell侵袭实验结果显示过表达组(SCC15∶123.70±6.98;CAL27∶326.00±17.01)侵袭细胞数显著低于空载组(SCC15∶517.70±9.96;CAL27∶454.30±8.09)(P<0.01)。裸鼠体内成瘤实验结果显示过表达组裸鼠肿瘤体积[(306.40±16.51)mm^3]及肿瘤质量[(289.40±11.44)mg]均显著小于空载组肿瘤体积[(582.60±32.51)mm^3,t=7.58,P<0.05]及肿瘤质量[(599.60±21.27)mg,t=7.58,P<0.001]。结论与癌旁组织相比,环状RNA hsa_circ_0063772在OSCC组织中低表达;过表达hsa_circ_0063772能抑制口腔鳞状细胞癌细胞的增殖、迁移和侵袭能力。

Objective To investigate the effects of circular RNA hsa_circ_0063772 on the proliferation,migration and invasion of oral squamous cell carcinoma (OSCC) cells.Methods Thirty-three patients with oral squamous cell carcinoma who were admitted to the Department of Oral and Maxillofacial Surgery,Peking University Shenzhen Hospital from February 2017 to December 2018 were enrolled in this study.Real-time quantative polymerase chain reaction was used to detect the expression level of circular RNA hsa_circ_0063772 in OSCC and corresponding adjacent tissues,OSCC cell lines and human keratinocytes.The expression level of hsa_circ_0063772 was overexpressed in SCC15 and CAL27 cells by using lentivirus,and the effects of this gene on proliferation,migration and invasion of OSCC cells were detected by cell counting assay,scratch assay,Transwell assay,Western blotting and nude mice tumor formation assay.Results The expression of circular RNA hsa_circ_0063772 in OSCC tissues (9.38±0.34) was lower than that in adjacent tissues (11.30±0.31)(t=5.20,P<0.001),and the expression in OSCC cells (SCC15: 0.12±0.01;SCC25: 0.18±0.02;SCC9: 0.21±0.01;CAL27: 0.13±0.01) was significantly lower than that in human keratinocytes (1.02±0.02)(tSCC15=41.91,tSCC25=29.21,tSCC9=35.16,tCAL27=41.86,P<0.001).Overexpression of hsa_circ_0063772 in SCC15 and CAL27 cells can affect tumor cell proliferation,cell counting assay showed that tumor cell proliferation ability in high expression group (SCC15: 0.76±0.01;CAL27: 0.74±0.02) were lower than empty group (SCC15∶1.22±0.04;CAL27: 0.99±0.03;tSCC15=12.58,tCAL27=6.97;P<0.05).Transwell migration experiment showed the number of migrated cell in high expression group (SCC15∶148.00±14.57;CAL27: 243.00±13.00) were less than empty group (SCC15: 580.30±42.91;CAL27: 424.70±18.66,P<0.01);Transwell invasion assay showed the number of invased cell in high expression group (SCC15: 123.70±6.98;CAL27: 326.00±17.01) were less than empty group (SCC15: 517.70±9.96;CAL27: 454.30±8.09,P<0.01).The results of tumor formation in nude mice showed that the tumor volume and mass of the overexpressed group [(306.40±16.51) mm^3;(289.40±11.44) mg] were lower than that of the empty group [(582.60±32.51) mm^3,t=7.58,P<0.05;(599.60±21.27) mg,t=7.58,P<0.001].Conclusions Compared with adjacent tissues,hsa_circ_0063772 is lowly expressed in oral squamous cell carcinoma.High expression of hsa_circ_0063772 can inhibit the proliferation,migration and invasion of OSCC cells.