整联蛋白α5β1通过介导Gli1表达影响基底细胞癌的生长与放疗敏感性

Integrinα5β1 affects the growth and radiosensitivity of basal cell carcinoma by mediating the expression of Gli1

目的探讨整联蛋白α5β1对基底细胞癌(BCC)生长和放疗敏感性的影响及分子机制。方法采用免疫组织化学染色检测皮肤BCC组织及正常皮肤组织中α5β1的表达差异。将A431细胞随机分为对照A组(细胞正常培养)、pLEX-MCS组(采用含pLEX-MCS的慢病毒转染细胞)、pLEX-α5β1组(采用含pLEX-α5β1的慢病毒转染细胞)、pLEX-α5β1+Gli家族锌指蛋白1(Gli1)抑制剂组(采用含pLEX-α5β1的慢病毒转染细胞,并添加100μmol/L Gli1抑制剂GANT61处理细胞)。采用实时荧光定量PCR和蛋白质免疫印迹法检测细胞内α5β1与Gli1的表达情况;采用MTT法和5-乙炔基-2′-脱氧尿苷(EdU)染色检测细胞增殖能力;通过Hoechst 33258染色观察细胞凋亡形态;采用Transwell实验检测细胞的迁移与侵袭能力。再将A431细胞随机分为对照B组(细胞正常培养)、4 Gy组(采用4 Gy X射线照射细胞)、pLEX-α5β1+4 Gy组(采用含pLEX-α5β1的慢病毒转染细胞,再用4 Gy X射线照射细胞),采用MTT法检测细胞增殖活性,流式细胞术检测细胞凋亡情况。结果α5β1在皮肤BCC组织中的阳性表达率高于正常皮肤组织(P<0.05)。与对照A组和pLEX-MCS组比较,pLEX-α5β1组细胞中α5β1和Gli1 mRNA和蛋白表达水平均升高,细胞存活率升高,EdU阳性细胞率升高,迁移细胞数与侵袭细胞数均增加,差异有统计学意义(P<0.05)。与pLEX-α5β1组比较,pLEX-α5β1+Gli1抑制剂组细胞中Gli1 mRNA和蛋白表达水平均下降,细胞存活率降低,EdU阳性细胞率降低,迁移细胞数与侵袭细胞数也均减少,差异有统计学意义(P<0.05)。在处理后培养24、48和72 h时,与对照B组比较,4 Gy组细胞增殖活性明显降低(P<0.05);pLEX-α5β1+4 Gy组细胞增殖活性较4 Gy组明显升高(P<0.05)。4 Gy组细胞凋亡率较对照B组明显升高(P<0.05);pLEX-α5β1+4 Gy组细胞凋亡率较4 Gy组明显降低(P<0.05)。结论α5β1在BCC组织中高表达,能够促进BCC细胞增殖、迁移及侵袭,抑制细胞凋亡,并降低细胞的放疗敏感性,该作用可能与调控Gli1表达有关。

Objective To explore the effect of integrinα5β1 on the growth and radiosensitivity of basal cell carcinoma(BCC)and its molecular mechanism.Methods Immunohistochemical staining was used to detect the expression ofα5β1 in skin BCC and normal skin tissues.A431 cells were randomly divided into control group A(cells with normal culture),pLEX-MCS group(transfected cells with pLEX-MCS lentivirus),pLEX-α5β1 group(transfected cells with pLEX-α5β1 lentivirus),pLEX-α5β1+Gli family zinc finger protein 1(Gli1)inhibitor group(cells were transfected with pLEX-α5β1 lentivirus and treated with 100μmol/L Gli1 inhibitor GANT61).The expression ofα5β1 and Gli1 in cells were detected by real-time fluorescent quantitative PCR and Western blot.MTT assay and 5-ethynyl-2′-deoxyuridine(EdU)staining were used to detect cell proliferation ability.Apoptotic morphology was observed by Hoechst 33258 staining.Transwell assay was used to detect the migration and invasion ability of the cells.Then A431 cells were randomly divided into control group B(cells with normal culture),4 Gy group(cells were irradiated with 4 Gy X-ray),pLEX-α5β1+4 Gy group(cells were transfected with pLEX-α5β1 lentivirus and irradiated with 4 Gy X-ray).MTT assay was used to detect cell proliferation activity,cell apoptosis was detected by flow cytometry.Results The positive expression rate ofα5β1 in skin BCC tissues was higher than that in normal skin tissues(P<0.05).Compared with control group A and pLEX-MCS group,the mRNA and protein expression levels ofα5β1 and Gli1 in pLEX-α5β1 group were increased,the cell survival rate was increased,the rate of EdU positive cells was increased,and the number of migrating cells and invading cells were increased,and the differences were statistically significant(P<0.05).Compared with the pLEX-α5β1 group,the expression levels of Gli1 mRNA and protein in the pLEX-α5β1+Gli1 inhibitor group were decreased,the cell survival rate was decreased,the rate of EdU positive cells was decreased,the number of migrating cells and invading cells were also decreased,and the differences were statistically significant(P<0.05).At 24,48 and 72 h of culture after treatment,the proliferation activity of cells in 4 Gy group was significantly lower than that in control group B(P<0.05),the proliferation activity of cells in pLEX-α5β1+4 Gy group was significantly higher than that in 4 Gy group(P<0.05).The apoptosis rate of cells in 4 Gy group was significantly higher than that in control group B(P<0.05).The apoptosis rate of cells in pLEX-α5β1+4 Gy group was significantly lower than that in 4 Gy group(P<0.05).Conclusionα5β1 is highly expressed in BCC tissues,which can promote the proliferation,migration and invasion of BCC cells,inhibit cell apoptosis,and reduce the radiosensitivity of cells,which may be related to the regulation of Gli1 expression.