宫颈癌组织Irisin表达临床意义研究

Expression of Irisin in cervical cancer tissues and its clinical significance

目的鸢尾素(Irisin)是新发现的一种蛋白因子,表达于多种肿瘤,但在宫颈鳞癌血清及组织中鲜见报道。本研究旨在探讨Irisin在宫颈鳞癌患者血清和组织中的表达及其与临床病理参数的相关性,并通过体外实验进一步验证Irisin对宫颈鳞癌细胞的作用。方法应用酶联免疫吸附试验法(enzyme-linked immunosorbent assay,ELISA)检测2015-07-01-2017-05-20青岛市立医院120名健康志愿者和102例宫颈鳞癌患者血清中Irisin表达;蛋白质印迹法和实时定量聚合酶链反应(real-time PCR,RT-PCR)法检测50例正常宫颈组织(对照组)和48例宫颈鳞癌组织(肿瘤组)中Irisin蛋白和Irisin mRNA表达,分析Irisin mRNA与宫颈鳞癌临床病理参数的关系;通过CCK-8和划痕实验探讨Irisin对宫颈鳞癌细胞系Hela和Siha的作用。结果 ELISA法检测结果显示,宫颈鳞癌患者血清中Irisin水平为(7.39±0.78)μg/mL,明显高于健康志愿者的(5.31±1.83)μg/mL,t=14.97,P<0.05,且临床分期越晚,Irisin表达水平越高;蛋白质印迹法检测结果显示,肿瘤组组织中Irisin蛋白相对表达量为(1.715±0.359),高于对照组组织的1,t=34.48,P=0.000 8;RT-PCR结果显示,在正常宫颈组织中相对表达量为1,宫颈鳞癌组织为3.29±0.724,t=25.49,P<0.000 1,且组织中Irisin mRNA表达水平与宫颈鳞癌的临床分期及病理分级有关,P<0.05;CCK-8法检测结果显示,Hela细胞中,0.625、1.25、2.5、5和10nmol/L组分别与对照组比较,差异有统计学意义,F=3.089,P<0.05;Siha细胞中,0.625、1.25、2.5、5和10nmol/L组分别与对照组比较,差异有统计学意义,F=7.198,P<0.001;且随着时间的延长,Irisin的促增殖能力越明显。划痕实验结果显示,在12h时,Hela细胞2.5nmol/L组的愈合率为(41.19±1.41)%,高于对照组的(8.55±0.48)%,t=66.87,P<0.000 1;Siha细胞2.5nmol/L组的愈合率为(18.75±1.05)%,高于对照组的(6.34±0.34)%,t=28.95,P<0.000 1;在24h时,Hela细胞2.5nmol/L组的愈合率为(73.95±4.53),高于对照组的(37.71±2.15)%,t=32.44,P<0.000 1;Siha细胞2.5nmol/L组的愈合率为(37.71±2.15)%,高于对照组的(6.80±0.49)%,t=40.82,P<0.000 1,差异均有统计学意义。结论宫颈鳞癌患者血清和组织中Irisin表达水平明显高于健康志愿者,且Irisin mRNA表达水平与宫颈鳞癌的临床分期及病理分级有关,其过表达预示宫颈鳞癌预后不良;且Irisin能促进宫颈鳞癌细胞系Hela和Siha细胞的增殖和迁移。

OBJECTIVE To investigate the expression of Irisin in serum and tissue of patients with cervical squa mous cell carcinoma and its correlations with clinicopathological parameters, and further confirm the effects of Irisin on cervical squamous cell carcinoma in vitro. METHODS The expressions of Irisin in serum from 120 healthy volunteers and 102 patients with cervical cancer were detected by ELISA. The expression of Irisin protein and Irisin mRNA in 50 cases of normal cervical tissues （control group） and 48 cases of cervical squamous cell carcinoma （tumor group） were detected by Western Blot and Real-time PCR,and then analyzed the relationships between expressin oflrisin mRNA and clinicopatho- logical parameters of patients with cervical squamous cell carcinoma. All the specimens were acquired from patients treated in our department and healthy volunteers in the physical examination center of Qingdao Municipal Hospital from July 2015 to May 2017. The effects of Irisin on Hela and Siha were verified by in vitro CCK-8 and scratch experiments. RESULTS The results of ELISA showed that serum levels of Irisin in patients with cervical squamous ceil carcinoma were significantly higher than those in healthy volunteers [（7.39±0.78） μg/mL vs （5.31±1.83） μg/mL,t= 14.97,P〈 0. 051,and the higher the clinical stage, the higher the expression level of Irisin. The results of Western blotting showed that the expression of Irisin protein in tumor tissue was higher than that of the control group [（1. 715±0. 359 ） vs 1 ,t= 34.48,P= 0. 000 82. The RT-PCR results showed that in the normal cervical tissue relative expression of 1 unit, cervical squamous cell carcinoma was 3.29±0. 724 （t=25.49,P〈0. 000 1） ,and the expression levels of Irisin mRNA were posi- tively correlated with the clinical stage and pathological grade of cervical squamous cell carcinoma （P〈0.05）. The CCK-8 results showed that ： （1） compared Hela cells,0. 625,1.25,2.5,5 and 10 nmol/L group with the control group, the differ- ence was statistically significant, F= 3. 089, P〈0.05 ; （2） compared Siha cells, 0. 625,1.25,2.5,5 and 10 nmol/L group with the control group,the difference was statistically significant,F 7. 198,P〈0. 001;and with the extension of time, Irisin was more obviously to promote proliferation, F = 7.198, P〈0.001. Scratch test showed that, At 12 h, （ 1 ） the heal ing rate of Hela cells in 2.5 nmol/L group was higher than that in control group [（41.19±1.41）% vs （8.55±0.48）%, t=66.87,P〈0. 000 12. （2）The healing rate of Siha cells in 2.5 nmol/L group was higher than that in control group [ （18.75±1.05）% vs （6.34＋0.34）G, t= 28.95, P〈0. 000 11. At 24 h, （1） The healing rate of Hela cells in 2.5 nmol/L group was higher than that in control group [（73.95± 4.53）G vs （37.71 ±2. 15）G,t= 32.44,P〈0. 000 1]. （2）The healing rate of Siha cells in 2. 5 nmol/L group was higher than that in control group [（37. 71± 2. 15）G vs （6. 80± 0.49） ],t=40.82, P〈0. 000 1]. CONCLUSIONS The expression levels of Irisin in serum and tissue of patients with cervical squamous cell carcinoma were significantly higher than those of normal people. The expression levels of Irisin mR- NA were correlated significatly with the clinical stage and pathological grade of cervical squamous cell carcinoma. Irisin overexpression indicated a poor prognosis of patients with cervical squamous cell carcinoma. Additionally Irisin promoted the proliferation and migration of cervical squamous cell carcinoma cell lines Hela and Siha.