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中华细胞与干细胞杂志(电子版) ›› 2024, Vol. 14 ›› Issue (01) : 19 -26. doi: 10.3877/cma.j.issn.2095-1221.2024.01.003

所属专题: 文献

论著

miR-126-3p靶向PIK3R1促进卵巢癌细胞增殖、迁移和侵袭
卫伟1, 王一娜2, 孔祥3,()   
  1. 1. 225000 扬州,扬州大学医学院;224000 盐城,江苏省盐城市第三人民医院妇产科
    2. 224000 盐城,江苏省盐城市第三人民医院妇产科
    3. 225001 扬州,江苏省扬州市苏北人民医院妇产科
  • 收稿日期:2023-12-10 出版日期:2024-02-01
  • 通信作者: 孔祥

miR-126-3p promotes the proliferation, migration and invasion of ovarian cancer cells by targeting PIK3R1

wei Wei1, Yina Wang2, Xiang Kong3,()   

  1. 1. Medical College of Yangzhou University, Yangzhou 225000, China; Department of Gynaecology and Obstetrics, Yancheng Third People's Hospital, Yancheng 224000, China
    2. Department of Gynaecology and Obstetrics, Yancheng Third People's Hospital, Yancheng 224000, China
    3. Department of Gynaecology and Obstetrics, Yangzhou Subei People’s Hospital, Yangzhou 225001, China
  • Received:2023-12-10 Published:2024-02-01
  • Corresponding author: Xiang Kong
引用本文:

卫伟, 王一娜, 孔祥. miR-126-3p靶向PIK3R1促进卵巢癌细胞增殖、迁移和侵袭[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(01): 19-26.

wei Wei, Yina Wang, Xiang Kong. miR-126-3p promotes the proliferation, migration and invasion of ovarian cancer cells by targeting PIK3R1[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2024, 14(01): 19-26.

目的

研究miR-126-3p/PIK3R1轴对卵巢癌细胞增殖、迁移和侵袭的影响。

方法

将miR-126-3p mimic,miR-126-3pinhibitor和PIK3R1质粒转染入SKOV3、A2780卵巢癌细胞中。采用CCK-8、划痕和Transwell实验评估细胞增殖、迁移。Western blot和qPCR检测PIK3R1、miR-126-3p表达水平,双荧光素酶测定miR-126-3p靶基因PIK3R1。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。

结果

与正常卵巢上皮细胞系IOSE80相比,卵巢癌细胞系SKOV3、A2780、HO-8910中miR-126-3p的表达水平(2.30 ± 0.18、1.86 ± 0.11、1.26 ± 0.12比1.00 ± 0.07)上调(P均< 0.05);抑制miR-126-3p表达可抑制SKOV3、A2780细胞增殖[SKOV3细胞:72 h(0.94 ± 0.13)比(1.14 ± 0.08),A2780细胞:72 h (0.83 ± 0.09)比(1.11 ± 0.12)]、迁移[(23.00 ± 6.08)%比(37.67 ± 6.43)%、(25.67 ± 4.04)%比(40.00 ± 6.58)%]、侵袭[(97.00 ± 17.35)比(134.33 ± 13.32)个、(97.00 ± 7.00)比(127.00 ± 9.17)个](P均< 0.05);双荧光素酶报告实验验证miR-126-3p靶向作用于SKOV3、A2780细胞中的PIK3R1基因;过表达miR-126-3p后SKOV3、A2780细胞中PIK3R1蛋白表达下调[(0.50 ± 0.08)比(1.01 ± 0.04)、(0.54 ± 0.03)比(1.00 ± 0.03)](P均< 0.001)。与miR-126-3p mimic相比,miR-126-3p mimic+ pPIK3R1中SKOV3、A2780细胞增殖[SKOV3细胞:72 h (1.04 ± 0.14)比(1.55 ± 0.12),A2780细胞:72 h (0.87 ± 0.09)比(1.32 ± 0.11)]、迁移能力[(27.00 ± 2.00)%比(34.00 ± 2.00)%、(24.67 ± 3.22)%比(33.00 ± 4.00)%]、侵袭能力[(60.67 ± 7.64)比(135.00 ± 6.00)个、(63.33 ± 10.02)比(125.67 ± 9.87)个]下降,PIK3R1蛋白表达量[(1.92 ± 0.16)比(1.00 ± 0.11)、(3.15 ± 0.06)比(0.99 ± 0.12)]升高(P均< 0.05)。

结论

miR-126-3p靶向PIK3R1促进卵巢癌细胞增殖、迁移和侵袭。

Objective

To investigate the role of miR-126-3p/PIK3R1 axis proliferation, migration and invasion cells in ovarian cancer.

Methods

miR-126-3p mimic, miR-126-3p inhibitor and PIK3R1 plasmid were transfected into SKOV3 and A2780 ovarian cancer cells. Cell proliferation/migration was assessed by CCK-8, scratch and Transwell assays. Western blot and qPCR were used to detect the expression levels of PIK3R1 and miR-126-3p. A Dual-luciferase reporter assay was used to detect the target gene of miR-126-3p, PIK3R1. An independent samle t-test was used to compare between two groups, a one-way analysis of variance was used to compare among multiple groups and an LSD-t test was used for pairwise comparison among various groups.

Results

Compared with the normal ovarian epithelial cell line IOSE80, the expression levels of miR-126-3p were up-regulated in ovarian cancer cell lines SKOV3, A2780, HO-8910 [ (2.30 ± 0.18) , (1.86 ± 0.11) , (1.26 ± 0.12) vs (1.00 ± 0.07) ] (all P < 0.05) ; Inhibition of miR-126-3p expression inhibited the proliferation [SKOV3 cells : 72 h (0.94 ± 0.13) vs (1.14 ± 0.08) , A2780 cells: 72 h (0.83 ± 0.09) vs (1.11 ± 0.12) ], migration [ (23.00 ± 6.08) % vs (37.67 ± 6.43) %, (25.67 ± 4.04) % vs (40.00 ± 6.58) %], invasion of SKOV3, A2780 cells[ (97.00 ± 17.35) vs (134.33 ± 13.32) , (97.00 ± 7.00) vs (127.00 ± 9.17) cells] (all P < 0.05) . And dual luciferase report experiment verified miR-126-3p targeted PIK3R1 gene in SKOV3 and A2780 cells. After overexpression of miR-126-3p, the expression of PIK3R1 protein was down-regulated in SKOV3 and A2780 cells [ (0.50 ± 0.08) vs (1.01 ± 0.04) , (0.54 ± 0.03) vs (1.00 ± 0.03) ] (all P < 0.001) . Compared with miR-126-3p mimic, miR-126-3p mimic+pPIK3R1 inhibited the proliferation [SKOV3 cells: 72 h (1.04 ± 0.14) vs (1.55 ± 0.12) , A2780 cells: 72 h (0.87 ± 0.09) vs (1.32 ± 0.11) ], migration ability[ (27.00 ± 2.00) %vs (34.00 ± 2.00) %, (24.67 ± 3.22) %vs (33.00 ± 4.00) %], invasion ability of SKOV3 and A2780 cells [ (60.67 ± 7.64) vs (135.00 ± 6.00) , (63.33 ± 10.02) vs (125.67 ± 9.87) cells], the protein expression of PIK3R1 was increased [ (1.92 ± 0.16) vs (1.00 ± 0.11) , (3.15 ± 0.06) vs (0.99 ± 0.12) ] (all P < 0.05) .

Conclusion

miR-126-3p promotes the proliferation, migration and invasion of ovarian cancer cells by targeting PIK3R1.

表1 引物序列信息
图1 miRNA-126-3p在泛癌中的差异表达
图2 miRNA-126-3p inhibitor对卵巢癌细胞增殖能力的影响注:a图为qRT-PCR检测卵巢癌细胞中miR-126-3p的表达,与IOSE80细胞比较,aP < 0.05,与SKOV3细胞比较,bP < 0.05,与A2780细胞比较,cP < 0.05;b图为qRT-PCR检测miR-126-3p抑制剂在SKOV3和A2780细胞中的转染效率,与对照比较,aP < 0.05;c ~ d图为CCK-8检测抑制剂转染到SKOV3和A2780细胞中的增殖曲线,与对照比较,aP < 0.05,n = 3
图3 倒置显微镜下观察miRNA-126-3p inhibitor对卵巢癌细胞迁移和侵袭能力的影响(×200)注:0 h、24 h迁移结果可见,与对照比较,在转染miRNA-126-3p inhibitor后SKOV3、A2780细胞迁移率下降;结晶紫染色观察卵巢癌细胞的侵袭,可见与对照比较,在转染miRNA-126-3p inhibitor后SKOV3、A2780细胞侵袭率下降
表2 miRNA-126-3p inhibitor对卵巢癌细胞迁移和侵袭能力的影响( ± s
图4 miRNA-126-3p与PIK3R1靶向结合示意
图5 PIK3R1-WT和PIK3R1-MUT双荧光素酶报告实验注:a、b图分别为SKOV3细胞、A2780细胞,与对照比较,aP < 0.05,ns为差异无统计学意义,n = 3
图6 PIK3R1在卵巢癌中的表达量注:a图为转染miR-126-3p mimic后,检测SKOV3和A2780细胞中PIK3R1的表达水平;b图为GEPIA数据库中卵巢癌患者PIK3R1表达量;c图为qRT-PCR检测卵巢癌细胞中PIK3R1表达;与IOSE80比较,aP < 0.05,与SKOV3比较,bP < 0.05,与A2780比较,cP < 0.05,n = 3
表3 miR-126-3p mimic对卵巢癌细胞中PIK3R1蛋白表达的影响( ± s
图7 Western blot检测转染PIK3R1质粒进入细胞后PIK3R1表达
图8 CCK-8检测PIK3R1和miR-126-3p过表达后SKOV3、A2780细胞吸光度值比较注:a、b图分别为SKOV3、A2780细胞吸光度值,与对照比较,aP < 0.05,与miR-126-3p mimic比较,bP<0.05;n = 3
图9 倒置显微镜下观察PIK3R1和miR-126-3p过表达后卵巢癌细胞的迁移与侵袭(×200)注:0 h、24 h迁移结果可见,相较于miR-126-3p mimic,在转染miR-126-3p mimic+pPIK3R1后SKOV3、A2780细胞迁移率下降;结晶紫染色观察卵巢癌细胞的侵袭可见,相较于miR-126-3p mimic,在转染miR-126-3p mimic+pPIK3R1后SKOV3、A2780细胞侵袭数目下降
表4 miR-126-3p mimic和pPIK3R1对卵巢癌细胞中PIK3R1蛋白的影响( ± s
表5 miR-126-3p mimic和pPIK3R对卵巢癌细胞迁移和侵袭能力的影响( ± s
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