LncRNA KCNQ1OT1调节miR-145/PAK4轴对结直肠癌SW480细胞恶性发展的影响

doi:10.3969/j.issn.1006-6233.2023.02.02

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摘要 目的: 探究LncRNA KCNQ1OT1在结直肠癌SW480细胞中的作用及其潜在的分子机制。方法: miRanda与TargetScan分别分析LncRNA KCNQ1OT1与miR-145的作用靶点。通过双荧光素酶报告基因实验验证相关的分子间靶标关系。qRT-PCR实验分析KCNQ1OT1、miR-145和PAK4在结直肠癌组织、癌旁组织、SW480细胞和CCC-HIE-2细胞中的基因表达量。将SW480细胞分为siRNA NC组、KCNQ1OT1 siRNA组、PAK4 siRNA组、inhibitor NC组、miR-145 inhibitor组、pcDNA-3.1(+)+mimics NC组、pcDNA-KCNQ1OT1+mimics NC组、pcDNA-3.1(+)+miR-145 mimics组、pcDNA-KCNQ1OT1+miR-145 mimics组,分别敲低KCNQ1OT1、miR-145和PAK4表达,MTT实验分析细胞活力,细胞克隆形成实验分析细胞克隆数目,流式细胞仪检测细胞凋亡率,Western blot检测SW480细胞EMT能力。结果: 与癌旁组织相比(1.37±0.27),结直肠癌组织中LncRNA KCNQ1OT1表达上调(2.32±0.89)(t=23.84,P<0.01);与癌旁组织相比(1.21±0.09),结直肠癌组织中PAK4表达上调(2.03±0.54)(t=20.75,P<0.01);与癌旁组织相比(1.26±0.35),结直肠癌组织中miR-145表达显著下调(0.45±0.13)(t=18.76,P<0.01);与CCC-HIE-2细胞相比(1.05±0.21),SW480细胞中LncRNA KCNQ1OT1表达上调(2.09±0.27)(t=18.79,P<0.01);与CCC-HIE-2细胞相比(1.18±0.62),SW480细胞中PAK4表达上调(2.58±0.82)(t=22.17,P<0.01);与CCC-HIE-2细胞相比(1.46±0.28),SW480细胞中miR-145表达显著下调(0.58±0.23)(t=17.86,P<0.01)。LncRNA KCNQ1OT1靶向miR-145,miR-145靶向PAK4。KCNQ1OT1或PAK4表达降低后,SW480细胞活力,克隆数目与EMT能力显著降低,上调了细胞凋亡率;上调KCNQ1OT1靶向miR-145促进了SW480细胞增殖和EMT,下调了细胞凋亡率。结论: 下调LncRNA KCNQ1OT1通过miR-145/PAK4轴抑制了SW480细胞增殖和EMT,促进了癌细胞凋亡。

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关键词 ： LncRNAKCNQ1OT1, miR-145, PAK4, 结直肠癌, 增殖, 凋亡

Abstract：Objective: To explore the role of LncRNA KCNQ1OT1 in colorectal cancer SW480 cells and its potential molecular mechanism. Methods: MiRanda and TargetScan were conducted to analyze the action targets of LncRNA KCNQ1OT1 and miR-145 respectively. The intermolecular target relationship was verified by dual luciferase reporter assay. The qRT-PCR assay was used to analyze the gene expression levels of KCNQ1OT1, miR-145, and PAK4 in colorectal cancer tissues, para cancer tissues, SW480 cells, and CCC-HIE-2 cells.SW480 cells were divided into siRNA NC group, KCNQ1OT1 siRNA group, PAK4 siRNA group, inhibitor NC group, miR-145 inhibitor group, pcDNA-3.1(+)+mimics NC group, pcDNA-KCNQ1OT1+mimics NC group, pcDNA-3.1(+)+miR-145 mimics group, pcDNA-KCNQ1OT1+miR-145 mimics group, the expressions of KCNQ1OT1, miR-145 and PAK4 were knocked down, respectively. Cell viability was analyzed by MTT assay, the number of clones was analyzed by cell colony formation assay, cell apoptosis rate was detected by flow cytometry, and the EMT ability of SW480 cells was detected by Western blot. Result: Compared with para cancer tissues (1.37±0.27), LncRNA KCNQ1OT1 expression in colorectal cancer tissues was up-regulated (2.32±0.89) (t=23.84, P<0.01). The expression of PAK4 in colorectal cancer tissues was up-regulated (2.03±0.54) compared with paracellular tissues (1.21±0.09) (t=20.75, P<0.01). Compared with para cancer tissues (1.26±0.35), the expression of miR-145 in colorectal cancer tissues was significantly down-regulated (0.45±0.13) (t=18.76, P<0.01). Compared with CCC-HIE-2 cells (1.05±0.21), LncRNA KCNQ1OT1 expression in SW480 cells was up-regulated (2.09±0.27) (t=18.79, P<0.01). Compared with CCC-HIE-2 cells (1.18±0.62), PAK4 expression in SW480 cells was up-regulated (2.58±0.82) (t=22.17, P<0.01). Compared with CCC-HIE-2 cells (1.46±0.28), the expression of miR-145 in SW480 cells was significantly down-regulated (0.58±0.23) (t=17.86, P<0.01). LncRNA KCNQ1OT1 targets miR-145 and MmiR-145 targets PAK4. After KCNQ1OT1 or PAK4 expression was decreased, SW480 cell viability, clone number, and EMT ability were significantly decreased, and the apoptosis rate was up-regulated. Upregulation of KCNQ1OT1 targeting miR-145 promoted proliferation and EMT of SW480 cells and down-regulated apoptosis rate. Conclusion: Down-regulating LncRNA KCNQ1OT1 inhibited SW480 cell proliferation and EMT through the miR-145/PAK4 axis, and promoted cancer cell apoptosis

Key words： LncRNA KCNQ1OT1 miR-145 PAK4 Colorectal cancer Proliferation Apoptosis

基金资助:江苏省南通市卫生和计划生育委员会科研课题,(编号:MB2020008)

通讯作者: 孙永强

引用本文:

施文, 周志强, 蔡明, 邱建, 陈锦鹏, 何向锋, 孙永强. LncRNA KCNQ1OT1调节miR-145/PAK4轴对结直肠癌SW480细胞恶性发展的影响[J]. 河北医学, 2023, 29(2): 183-189.
SHI Wen, ZHOU Zhiqiang, CAI Ming, et al. The Effect of LncRNA KCNQ1OT1 on the Malignant Development of Colorectal Cancer SW480 Cells by Regulating the miR-145/PAK4 Axis. HeBei Med, 2023, 29(2): 183-189.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2023.02.02 或 http://www.hbyxzzs.cn/CN/Y2023/V29/I2/183

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