子宫颈癌患者血清中miR-101的表达及其通过Rac1调控子宫颈癌细胞丝状伪足的形成

Clinical significance of serum miR-101 expression in cervical cancer patients and its regulation of cervical cancer cell filopodia formation through Rac1

目的探讨子宫颈癌患者血清中miR-101的表达及其临床意义,探究miR-101通过调控靶基因Rac1在子宫颈癌细胞丝状伪足形成中的作用机制。方法采用qRT-PCR法检测18例子宫颈癌患者和20例健康体检者血清中miR-101的表达量。采用qRT-PCR法检测正常对照组、NC mimic组、miR-101 mimic组子宫颈癌SiHa细胞内miR-101的表达水平,并验证其转染效率。采用双荧光素酶报告基因实验验证miR-101与Rac1的靶向关系,并通过Western blot、qRT-PCR和免疫荧光实验检测Rac1 mRNA和蛋白表达水平。miR-101 mimic与Rac1 mimic共转染至SiHa细胞后电镜下观察丝状伪足的形成,罗丹明标记的鬼笔环肽染色观察F-actin的表达。结果miR-101在子宫颈癌血清(0.54±0.06)和细胞(0.93±0.03)中低表达(P<0.01),并与子宫颈癌分化程度有关(Z=2.974,P=0.003)。miR-101靶向负调控Rac1的表达(P<0.05),即上调miR-101表达后Rac1 mRNA和蛋白表达水平均降低(0.40±0.01、0.22±0.03)(P<0.05)。上调miR-101表达后SiHa细胞丝状伪足数减少(39±3)(P<0.01),且F-actin蛋白表达明显降低,过表达Rac1可部分逆转miR-101对丝状伪足形成(57±3)(P<0.01)及F-actin蛋白的抑制作用。结论子宫颈癌患者血清中miR-101低表达与其分化程度呈负相关,miR-101靶向调控Rac1抑制子宫颈癌细胞丝状伪足的形成。

Purpose To investigate the expression of miR-101 in the serum of cervical cancer patients,and to explore the mechanism of of miR-101 in the formation of filopodia in cervical cancer cells by regulating Ras related C3 botulinum toxin substrate(Rac1).Methods The expression level of serum miR-101 in 18 cases of cervical cancer patients and 20 cases of healthy people were detetected by qRT-PCR.The expression levels of miR-101 in SiHa cells in normal control group,NC mimic group and miR-101 mimic group were detected by qRT PCR and their transfection efficiency was verified.The targeting relationship between miR-101 and Rac1 was detected by double luciferase reporter gene experiment.The gene and protein expression levels of Rac1 were observed by Western blot,qRT-PCR and immunofluorescence assay.After miR-101 mimic and Rac1 mimic were cotransfected into SiHa cells,the formation of filopodia was observed by scanning electron microscopy,and the filopodia was stained with rhodamine labeled phalloidin to observe the expression of F-actin.Results The expression of miR-101 was low in the cells and serum of cervical cancer(0.54±0.06,0.93±0.03)(P<0.01),and was related to the degree of differentiation of cervical cancer(Z=2.974,P=0.003).After upregulation of miR-101 expression,the expression levels of Rac1 mRNA and protein decreased(0.40±0.01,0.22±0.03)(P<0.05).After upregulation of miR-101 expression,filopodia numbers(39±3)(P<0.01),and F-actin expression of cervical cancer cells were decreased,however,the upregulation of Rac1 can reverse the inhibiting rule of overexpressed miR-101 on filopodia formation(57±3)(P<0.01)and the expression of F-actin.Conclusion miR-101 is low expressed in cervical cancer serum and cells,and is related to differentiation degree of cervical cancer.Targeted regulation of Rac1 by miR-101 inhibits the formation of filopodia in cervical cancer cells.