miR-630对三阴性乳腺癌细胞侵袭的影响及其机制研究

发布时间：2018-12-18 19:06

【摘要】：目的研究miR-630在人体三阴性乳腺癌组织中的表达情况,探讨miR-630是否通过下调Sox4影响三阴性乳腺癌细胞系MDA-MB-231细胞的侵袭。方法收集157例行乳腺癌根治术患者癌组织标本及癌旁正常乳腺组织,其中三阴性乳腺癌36例,通过实时荧光定量PCR(RT-PCR)检测各组(正常乳腺组、非三阴性乳腺癌组、三阴性乳腺癌组)组织中与乳腺癌相关的miR-630、miR-21、miR-195、miR-134、miR-200a、miR-381、miR-1228的表达情况;蛋白免疫印迹试验(Western blot)检测3组标本中细胞侵袭相关蛋白COL1A1、COL1A5、MMP-2、MMP-9的表达情况;进一步在细胞实验中,通过转染miR-630mimic或空质粒后,再转染过表达Sox4的质粒或空质粒至离体培养的细胞中,Western blot检测细胞中COL1A1、COL1A5、MMP-2、MMP-9、Sox4的表达情况,划痕实验和Transwell法观察细胞迁移侵袭情况,利用荧光素酶实验验证Sox4是miR-630的靶基因。结果相比于正常乳腺组,miR-630、miR-21在三阴性乳腺癌组织中的表达降低(P0.01);miR-195、miR-134、miR-200a、miR-381、miR-1228的表达在3组间差异无统计学意义(P0.05);相比于正常乳腺组及非三阴性乳腺癌组,三阴性乳腺癌组织中COL1A1、COL1A5、MMP-2、MMP-9、Sox4的表达增多(P0.05)。在细胞实验中,相比于空质粒组,转染miR-630mimic后,MDA-MB-231细胞中COL1A1、COL1A5、MMP-2、MMP-9、Sox4的表达减少(P0.05),MDA-MB-231细胞的迁移侵袭能力减弱(P0.01);荧光素酶实验结果显示,miR-630能够显著降低Sox4-3′-UTR质粒的荧光素活性(P0.05);相比于过表达miR-630+空质粒组,转染过表达miR-630+过表达Sox4组,MDA-MB-231细胞中COL1A1、COL1A5、MMP-2和MMP-9表达增加(P0.05),细胞的迁移侵袭能力增强(P0.01)。结论在三阴性乳腺癌组织中,miR-630低表达;miR-630可以通过下调Sox4从而抑制MDA-MB-231的迁移和侵袭。
[Abstract]:Objective to investigate the expression of miR-630 in human triple-negative breast cancer tissues and whether miR-630 can affect the invasion of triple-negative breast cancer cell line MDA-MB-231 by down-regulation of Sox4. Methods 157 patients with breast cancer underwent radical mastectomy and normal breast tissues adjacent to breast cancer were collected. Among them, 36 cases of tri-negative breast cancer were detected by real-time fluorescence quantitative PCR (RT-PCR) in each group (normal mammary gland group and non-tri-negative breast cancer group). The expression of miR-630,miR-21,miR-195,miR-134,miR-200a,miR-381,miR-1228 related to breast cancer in tri-negative breast cancer group; The expression of cell invasion-associated protein (COL1A1,COL1A5,MMP-2,MMP-9) in three groups was detected by Western blot (Western blot). Further in the cell experiment, the expression of COL1A1,COL1A5,MMP-2,MMP-9,Sox4 was detected by transfection of miR-630mimic or empty plasmid, and then transfection of plasmid or empty plasmid expressing Sox4 into the cells cultured in vitro with, Western blot. The migration and invasion of cells were observed by scratch test and Transwell method. Luciferase assay was used to verify that Sox4 was the target gene of miR-630. Results the expression of miR-630,miR-21 in tri-negative breast cancer was significantly lower than that in normal breast cancer (P0.01). There was no significant difference in the expression of miR-195,miR-134,miR-200a,miR-381,miR-1228 among the three groups (P0.05). Compared with normal breast cancer group and non-tri-negative breast cancer group, the expression of COL1A1,COL1A5,MMP-2,MMP-9,Sox4 in tri-negative breast cancer tissues increased (P0.05). In the cell experiment, compared with the empty plasmid group, the expression of COL1A1,COL1A5,MMP-2,MMP-9,Sox4 in MDA-MB-231 cells decreased after transfection with miR-630mimic (P0.05), and the migration and invasion ability of MDA-MB-231 cells decreased (P0.01). Luciferase assay showed that miR-630 could significantly reduce the fluorescein activity of Sox4-3'-UTR plasmid (P0.05). Compared with the empty plasmid group of overexpression of miR-630, the expression of COL1A1,COL1A5,MMP-2 and MMP-9 in MDA-MB-231 cells increased (P0.05), and the ability of migration and invasion of MDA-MB-231 cells increased (P0.01). Conclusion the expression of miR-630 is low in tri-negative breast cancer, and miR-630 can inhibit the migration and invasion of MDA-MB-231 by down-regulating Sox4.
【作者单位】：河北大学附属医院肿瘤内科;河北省放化疗机制与规程研究重点实验室;河北大学附属医院中心实验室;河北大学附属医院中西医肿瘤科;河北大学附属医院病理科;
【分类号】：R737.9

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