KLF13上调对骨肉瘤细胞侵袭、迁移及p-AKT表达的影响

潘寒松; 郭昌军; 梁一民; 梁剑伟; 李祥

doi:10.16766/j.cnki.issn.1674-4152.002228

KLF13上调对骨肉瘤细胞侵袭、迁移及p-AKT表达的影响

doi: 10.16766/j.cnki.issn.1674-4152.002228

台州市第一人民医院骨科，浙江台州 318020

基金项目:

浙江省基础公益研究计划项目 LY20H070006

台州市社会发展类一般项目 1701KY48

详细信息

通讯作者:
李祥，E-mail：lx112326@126.com

中图分类号: R738.1 R730.231
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- 文章访问数: 168
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- PDF下载量: 1
- 被引次数: 0
出版历程
- 收稿日期: 2020-06-18
- 网络出版日期: 2022-03-02

Effects of the up-regulation of KLF13 on invasion, migration and p-AKT expression of osteosarcoma cells

Department of Orthopaedics, Taizhou First People's Hospital, Taizhou, Zhejiang 318020, China

摘要

摘要: 目的研究Kruppel样因子13(KLF13)对骨肉瘤细胞(MG-63)侵袭、迁移的影响及该过程中p-AKT蛋白表达变化。方法将MG-63细胞按照随机数字表法分为对照组、NC组和LV-KLF13-OE组；显微镜观察KLF13慢病毒颗粒转染情况；实时荧光定量PCR(qRT-PCR)检测KLF13 mRNA表达情况；蛋白免疫印迹法检测KLF13、丝氨酸/苏氨酸蛋白激酶B(AKT)及磷酸化水平、E-钙黏蛋白(E-cadherin)和膜棕榈糖基化蛋白2(MPP-2)的蛋白表达情况；细胞计数试剂盒8(CCK-8)法检测细胞增殖情况；划痕实验检测迁移能力；小室法(Transwell)检测侵袭能力。结果 MG-63细胞转染效率>75%；与对照组和NC组相比，LV-KLF13-OE组细胞KLF13 mRNA和蛋白、E-cadherin蛋白表达升高(F=1 544.143、588.000、235.391，均P＜0.001)，MPP-2蛋白表达水平降低(F=260.053，P＜0.001)；LV-KLF13-OE组细胞划痕愈合率[(20.07±8.18)%]、侵袭数量[(89.29±15.04)个]、p-AKT蛋白表达(0.21±0.02)水平低于对照组[(71.61±6.02)%、(221.33±20.09)个、1.12±0.12]和NC组[(74.93±7.21)%、(212.69±25.47)个、1.09±0.08，F=109.781、76.815、226.896, 均P＜0.001]。结论 KLF13过表达可能通过下调p-AKT水平，抑制人骨肉瘤细胞迁移和侵袭。
- Kruppel样因子13 /
- 骨肉瘤细胞 /
- 增殖 /
- 迁移 /
- 侵袭 /
- 丝氨酸/苏氨酸蛋白激酶B
Abstract: Objective To study the effects of Kruppel-like factor 13 (KLF13) on the invasion and migration of osteosarcoma cells (MG-63) and changes in p-AKT protein expression during the process. Methods MG-63 cells were divided into control group, NC group and LV-KLF13-OE group according to random number table method. The transfection of KLF13 recombinant lentivirus particles was observed under a microscope. The expression levels of KLF13 mRNA were detected by real-time fluorescent quantitative PCR. In addition, the protein expression levels of KLF13, serine/threonine protein kinase B (AKT) and its phosphorylation, E-cadherin and membrane palmitoylated protein 2 (MPP-2) were detected by western blot. Proliferation was detected by the Cell Counting Kit-8 (CCK-8), the migration ability was detected by a scratch test, and invasiveness was detected by the transwell method. Results The transfection efficiency of MG-63 cells was over 75%. Compared with those in the control group and NC group, the expression levels of KLF13 mRNA and protein and E-cadherin protein in the LV-KLF13-OE group were significantly higher (F=1 544.143, 588.000, 235.391, all P < 0.001), and the expression levels of MPP-2 protein were significantly lower (F=260.053, P < 0.001). The scratch healing rate [(20.07±8.18) %], number of invasions (89.29±15.04) and p-AKT protein expression (0.21±0.02) levels in the LV-KLF13-OE group were significantly lower than those in the control group [(71.61±6.02)%, 221.33±20.09 and 1.12±0.12] and NC group [(74.93±7.21)%, 212.69±25.47 and 1.09±0.08; F=109.781, 76.815, 226.896, all P < 0.001]. Conclusion KLF13 overexpression may inhibit the migration and invasion of human osteosarcoma cells by down-regulating p-AKT expression.
- Kruppel-like factor 13 /
- Osteosarcoma cells /
- Proliferation /
- Migration /
- Invasion /
- Serine/threonine protein kinase B

HTML全文

图 1 慢病毒转染MG-63细胞荧光表达情况

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图 2 KLF13蛋白凝胶电泳图

注：1为对照组；2为NC组；3为LV-KLF13-OE组。

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图 3 各组MG-63细胞迁移图(×100)

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图 4 各组MG-63细胞侵袭图(结晶紫染色，×40)

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图 5 p-AKT、AKT、E-cadherin、MPP-2蛋白凝胶电泳图

注：1为对照组；2为NC组；3为LV-KLF13-OE组。

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表 1 KLF13和β-actin引物序列

基因	上游引物	下游引物
KLF13	5′-CAGAGGAAGCACAAGTGCCACT-3′	5′-CGCGAACTTCTTGTTGCAGTCC-3′
β-actin	5′-AAATCGTGCGTGACATTAA-3′	5′-CTCGTCATACTCCTGCTT-3′

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表 2 转染后MG-63细胞KLF13 mRNA及蛋白表达变化情况(x±s)

组别	n	KLF13 mRNA	KLF13蛋白
对照组	6	1.12±0.01	0.21±0.01
NC组	6	1.10±0.03	0.19±0.02
LV-KLF13-OE组	6	1.71±0.02^ab	0.57±0.03^ab
F值		1 544.143	588.000
P值		＜0.001	＜0.001
注：与对照组比较，^aP＜0.05；与NC组比较，^bP＜0.05。

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表 3 转染后MG-63细胞存活率变化情况(x±s，%)

组别	n	MG-63细胞存活率
组别	n	12 h	24 h	48 h
对照组	6	101.45±1.55	105.71±1.61	103.19±1.93
NC组	6	102.67±2.04	101.89±1.97	104.74±1.49
LV-KLF13-OE组	6	86.49±1.39^ab	70.15±2.11^abc	52.93±1.58^abcd
F值		171.988	627.875	1852.565
P值		＜0.001	＜0.001	＜0.001
注：与对照组比较，^aP＜0.05；与NC组比较，^bP＜0.05；与LV-KLF13-OE组12 h比较，^cP＜0.05；与LV-KLF13-OE组24 h比较，^dP＜0.05。

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表 4 转染后MG-63细胞p-AKT、AKT、E-cadherin、MPP-2蛋白表达情况(x±s)

组别	n	p-AKT蛋白	AKT蛋白	E-cadherin蛋白	MPP-2蛋白
对照组	6	1.12±0.12	1.09±0.07	0.36±0.02	0.83±0.03
NC组	6	1.09±0.08	1.01±0.09	0.38±0.04	0.80±0.05
LV-KLF13-OE组	6	0.21±0.02^ab	0.97±0.10	0.89±0.02^ab	0.41±0.02^ab
F值		226.896	2.922	235.391	260.053
P值		＜0.001	0.085	＜0.001	＜0.001
注：与对照组比较，^aP＜0.05；与NC组比较，^bP＜0.05。

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