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中华细胞与干细胞杂志(电子版) ›› 2022, Vol. 12 ›› Issue (01) : 19 -25. doi: 10.3877/cma.j.issn.2095-1221.2022.01.004

论著

circ_0000267靶向miR-198对急性淋巴细胞白血病细胞KOCL44增殖和凋亡的影响
孙文思1, 于皎皎1, 林杉,1   
  1. 1. 266000 青岛市市立医院儿科
  • 收稿日期:2021-10-28 出版日期:2022-02-01
  • 通信作者: 林杉

Role of circ_0000267 targeting miR-198 in the proliferation and apoptosis of acute lymphoblastic leukemia cells KOCL44

Wensi Sun1, Jiaojiao Yu1, Shan Lin,1   

  1. 1. Peds Qingdao Municipal Hospital, Qingdao 266000, China
  • Received:2021-10-28 Published:2022-02-01
  • Corresponding author: Shan Lin
引用本文:

孙文思, 于皎皎, 林杉. circ_0000267靶向miR-198对急性淋巴细胞白血病细胞KOCL44增殖和凋亡的影响[J/OL]. 中华细胞与干细胞杂志(电子版), 2022, 12(01): 19-25.

Wensi Sun, Jiaojiao Yu, Shan Lin. Role of circ_0000267 targeting miR-198 in the proliferation and apoptosis of acute lymphoblastic leukemia cells KOCL44[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(01): 19-25.

目的

探讨circ_0000267对急性淋巴细胞白血病(ALL)KOCL44细胞增殖和凋亡的影响及其作用机制。

方法

选择ALL细胞株KOCL44为研究对象,分别将小干扰RNA (siRNA)阴性对照(si-NC)、circ_0000267 siRNA (si-circ_0000267)、微小RNA (miRNA)阴性对照(miR-NC)、miR-198模拟物(miR-198)、circ_0000267 siRNA+miRNA抑制剂阴性对照(si-circ_0000267+anti-miR-NC)和circ_0000267 siRNA+miR-198抑制剂(si-circ_0000267+anti-miR-198)转染细胞,48 h后通过RT-qPCR检测细胞circ_0000267和miR-198相对表达水平,采用CCK-8法检测KOCL44细胞的增殖水平,流式细胞术实验检测KOCL44细胞的凋亡水平,Western blot检测KOCL44细胞Ki-67、Bcl-2和Bax蛋白表达水平,通过双荧光素酶报告实验验证circ_0000267和miR-198靶向关系。两组间比较采用独立样本t检验。

结果

与健康志愿者比较,ALL患者circ_0000267表达水平(1.00±0.06比3.19±0.21)上调,miR-198表达水平(1.00±0.07比0.41±0.03)下调,差异有统计学意义(P < 0.05)。敲低circ_0000267或者过表达miR-198可抑制KOCL44细胞增殖(0.68±0.05比0.32±0.02、0.69±0.06比0.39±0.03)、Ki-67 (0.84±0.06比0.37±0.03、0.85±0.06比0.45±0.04)和Bcl-2蛋白表达(0.63±0.05比0.22±0.02、0.65±0.04比0.29±0.02),促进细胞凋亡[(6.53±0.51)﹪比(24.29±2.06)﹪、(7.38±0.57)﹪比(20.03±1.66)﹪]和Bax蛋白表达(0.31±0.03比0.77±0.04、0.30±0.02比0.71±0.04),差异有统计学意义(P < 0.05)。双荧光素酶报告实验验证circ_0000267可以靶向miR-198表达,干扰miR-198表达可以逆转抑制circ_0000267表达对KOCL44细胞的增殖和凋亡的作用,差异有统计学意义(P < 0.05)。

结论

circ_0000267通过调控miR-198抑制ALL细胞增殖,并促进凋亡,为临床治疗ALL提供新的依据。

Objective

To study the role of circ_0000267 in the proliferation and apoptosis of acute lymphoblastic leukemia (ALL) and its mechanism.

Methods

siRNA Negative control (si-NC) , circ_0000267 siRNA (si-circ_0000267) , miRNA negative control (miR-NC) , miR-198 mimics (miR-198) , si-circ_0000267+anti-miR-NC and si-circ_0000267+anti-miR-198 were transfected into the acute lymphocytic leukemia cell line KOCL44. The cells were collected 48 h after transfection. The expressions of circ_0000267 and miR-198 were detected by RT-qPCR; CCK-8 method was used to detect the proliferation of KOCL44 cells; flow cytometry was used to detect the apoptosis of KOCL44 cells; Western blot was used to detect the protein expressions of Ki-67, Bcl-2 and Bax; and double luciferase reporting assay was used to verify the relationship between circ_0000267 and miR-198. The comparison between the two groups was analyzed by independent sample t test.

Results

Compared with healthy volunteers, the expression of circ_000026 (1.00±0.06 vs 3.19±0.21) was up-regulated in ALL patients, and the expression of miR-198 (1.00±0.07 vs 0.41±0.03) was down-regulated, and the difference was statistically significant (P < 0.05) . Knockdown of circ_0000267 or overexpression of miR-198 can significantly inhibit the proliferation of KOCL44 cells (0.68±0.05 vs 0.32±0.02, 0.69±0.06 vs 0.39±0.03) , the protein expression of Ki-67 (0.84±0.06 vs 0.37±0.03, 0.85±0.06 vs 0.45±0.04) and Bcl-2 (0.63±0.05 vs 0.22±0.02, 0.65±0.04 vs 0.29±0.02) , and increase the rate of apoptosis ([6.53±0.51]﹪vs [24.29±2.06]﹪, [7.38±0.57]﹪ vs [20.03±1.66]﹪) and the protein expression of Bax (0.31±0.03 vs 0.77±0.04, 0.30±0.02 vs 0.71±0.04) . All the differences were statistically significant (P < 0.05) . The dual luciferase report experiment verified that circ_0000267 can target miR-198, and interference with the expression of miR-198 reversed the effect of inhibiting the expression of circ_0000267 on the proliferation and apoptosis of KOCL44 cells, and the difference was also statistically significant (P < 0.05) .

Conclusions

circ_0000267 inhibits all cell proliferation and promotes apoptosis by regulating miR-198, providing a new basis for clinical treatment of all.

表1 引物序列信息
图1 circ_0000267和miR-198在ALL患者中的相关性分析
表2 circ_0000267和miR-198在急性淋巴细胞白血病患者和健康志愿者单核细胞中的表达( ± s
图2 制circ_0000267表达对急性淋巴细胞白血病细胞KOCL44凋亡的影响注:a ~ b图分别为流式细胞术检测si-NC和si-circ_0000267的急性淋巴细胞白血病细胞KOCL44的凋亡率;c图为si-NC和si-circ_0000267细胞凋亡率比较;与si-NC组比较,aP < 0.05
图3 抑制circ_0000267表达对急性淋巴细胞白血病细胞KOCL44增殖和凋亡相关蛋白表达的影响注:a图为Western blot检测Ki-67、Bcl-2和Bax的蛋白表达;b图为si-NC和si-circ_0000267的Ki-67、Bcl-2和Bax的蛋白相对表达量比较;与si-NC组比较,aP < 0.05
表3 抑制circ_0000267表达对急性淋巴细胞白血病细胞KOCL44增殖的影响( ± s
图4 circ_0000267的序列中含有与miR-198互补的核苷酸序列
表4 双荧光素酶报告实验( ± s
图5 miR-198过表达对急性淋巴细胞白血病细胞KOCL44增殖和凋亡的影响注:a ~ b图分别为流式细胞术检测转染miR-NC和miR-198的KOCL44细胞凋亡率;c图为Western blot检测Ki-67、Bcl-2和Bax的蛋白表达
图6 miR-198过表达对急性淋巴细胞白血病细胞KOCL44增殖和凋亡的影响注:a ~ b图分别为流式细胞术检测转染si-circ_0000267+anti-miR-NC和si-circ_0000267+anti-miR-198的KOCL44细胞凋亡率;c图为Western blot检测Ki-67、Bcl-2和Bax的蛋白表达
表5 miR-198过表达对急性淋巴细胞白血病细胞KOCL44增殖和凋亡的影响( ± s
表6 干扰miR-198表达可以逆转抑制circ_0000267表达对急性淋巴细胞白血病细胞KOCL44增殖和凋亡的作用( ± s
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