Slit2/Robo1的在宫颈癌细胞异常低表达与对细胞凋亡的影响

Abnormal low expression of Slit2/Robo1 in cervical cancer cells and its effect on cell apoptosis

目的研究Slit2及其受体Robo1在人宫颈癌组织中的表达及其对宫颈癌细胞凋亡的影响和潜在机制。方法分别用实时荧光定量聚合酶链式反应(real time PCR)和免疫印迹实验检测Slit2、Robo1、Bcl-2的mRNA和蛋白在20例正常宫颈上皮(NCE)、40例宫颈上皮内瘤变(CIN)、60例宫颈鳞癌(SCC)组织中的表达情况。在转染试剂Lipofectamine2000作用下,对He La细胞进行转染siRNA(沉默组)、质粒(过表达组)和单纯转染p CMV-HA质粒(p CMV-HA对照)及单纯转染无关siRNA序列的对照组(阴性对照),于特定时间后收集细胞用于后续实验。经Annexin V/PI染色后,用流式细胞术检测Robo1过表达和Slit2沉默后对细胞凋亡的影响;用免疫印迹法检测细胞凋亡指标PARP-1的剪切形式及Bcl-2的表达。结果 Slit2 mRNA在NCE、CIN、SCC中的相对表达水平分别为2. 48±0. 36,1. 78±0. 22,1. 45±0. 17,Robo1 mRNA分别为2. 69±0. 31,1. 94±0. 21,1. 48±0. 15,Bcl-2分别为1. 61±0. 21,2. 22±0. 23,2. 85±0. 28,差异均有统计学意义(均P <0. 05)。过表达组和p CMV-HA对照组的Robo1相对表达水平分别为3. 48±0. 51,1. 00±0. 14,差异有统计学意义(P <0. 05)。过表达组早期凋亡、晚期凋亡和总凋亡率分别为(7. 81±0. 80)%,(14. 22±1. 80)%和(23. 11±2. 02)%,p CMV-HA组早期凋亡、晚期凋亡和总凋亡率分别为(2. 60±0. 82)%,(7. 33±0. 81)%和(9. 91±1. 72)%,差异有统计学意义(P <0. 05)。沉默组和阴性对照组Slit2分别为1. 00±0. 11,0. 51±0. 12,差异有统计学意义(P <0. 05);阴性对照细胞早期凋亡、晚期凋亡和总凋亡率分别为(4. 91±1. 12)%,(7. 90±1. 01)%,(12. 80±1. 70)%;沉默组分别为(2. 82±1. 01)%,(5. 13±2. 10)%,(7. 92±1. 90)%,差异均有统计学意义(均P <0. 05)。结论 Slit2/Robo1在宫颈癌组织中呈异常低表达,有利于抵抗细胞凋亡,在宫颈癌发生发展中发挥重要作用。

Objective To study the expression of Slit2 and its receptor Robol in human cervical carcinoma and its effect on the apoptosis Of cer- vical cancer cells and its underlying mechanism. Methods Real - time polymerase chain reaction （PCR） and WeStern blot were used to detect the mRNA and protein of Slit2,： Robol and Bel-2 in 20 cases of normal cervical epithelium （NCE） and 40 cases of cervical intraepithelial neoplasia （CIN） , 60 cases of cervical squamous cell carcinoma （SCC） tissue expression. HeLa cells were transfected with siRNA （silencing group）, plasmid （overexpression group ） and transfected pCMV -HA plasmid （pCMV- HA control） and unrelated siRNA sequences （negative control） by the transfection reagent Lipo- fectamine2000. Cells were harvested after a specific time for subsequent experiments. After staining with Annexin V/PI, the effects of Robol overexpression and Slit2 silencing on apoptosis were detected by flow cytometry. The cleavage form of PARP - 1 and the expression of Bcl - 2 were analyzed by immunoblotting. Results The relative expression levels of Slit2 mRNA in NCE, CIN and SCC were 2. 48 ± 0. 36, 1.78 ± 0. 22, 1.45 ± 0. 17, R0bol mRNA were 2.69 ±0. 31, 1.94 _±0. 21, 1.48 ±0. 15, and Bcl -2 mRNA were 1.61 ±0. 21,2.22 ±0. 23, 2. 85 ±0. 28, all with significant difference （ all P 〈 0. 05 ）. The relative expression levels of Robol mRNA in overexpression group and pCMV - HA control group were 3.48 ±0. 51 and 1.00 ±0. 14, with significant difference （P 〈0. 05）. The early apoptosis, late apoptosis and total apoptotic rate in overexpression group were （ 7.81 ± 0. 80 ） % , （ 14. 22 ± 1.80 ） % and （23.11 ± 2. 02） %, had significant difference with those in pCMV - HA group, which were （ 2. 60 ± 0. 82 ） %, （7. 33 ± 0. 81 ） % and （9. 91 ± 1.72） % （P 〈 0. 05 ）. Slit2 in silent group and negative control group were 1.00 ± 0. 11 and 0. 51 ±0. 12, with significant difference （P 〈0. 05 ）. The early apoptosis, late apoptosis and total apoptosis rate in negative control were （4. 91 ± 1.12） %, （7.90 ± 1.01 ） %, （ 12. 80 ± 1.70） % ; had significant difference with those in silent group, which were （2.82 ±1.01）%, （5.13 ±2.10）%, （7.92±1.90）% （P〈0.05）. Conclusion Slit2/Robol is abnormally expressed in cervical cancer tissues, which is beneficial to resist apoptosis and play an important role in the development of cervical cancer.