KRT23对肝癌干细胞活性的促进作用及其机制

doi:10.13481/j.1671-587X.20260415

摘要/Abstract

摘要：

目的探讨角蛋白23（KRT23）对肝癌（HCC）干细胞活性的调控作用，并阐明其作用机制。方法利用网络数据库GEPIA分析KRT23在肝癌组织和正常肝组织中的表达情况；通过Kaplan Meier Plotter生存分析比较KRT23高表达和KPT23低表达肝癌患者的生存期，评估KRT23对肝癌患者预后的影响。通过二代测序分析及京都基因与基因组百科全书（KEGG）信号通路分析发现KRT23可能参与调控的信号通路，最终选择（Wnt/β-catenin）信号通路进行机制研究。采用实时荧光定量PCR（RT-qPCR）法、Western blotting法和免疫组织化学法分别验证KRT23在肝癌细胞及肝癌组织中的表达，通过免疫荧光法明确其定位。利用已验证敲除效率的小干扰RNA（siRNA）构建KRT23低表达慢病毒载体，敲低KRT23的表达水平后，通过干细胞成球实验验证KRT23对肝癌干细胞活性的调控，并通过RT-qPCR法和Western blotting法检测相关干细胞指标的变化。采用免疫荧光法和核浆蛋白分离提取进一步验证KRT23对Wnt/β-catenin信号通路中相关指标的调节作用。结果 KRT23在肝癌组织中的表达水平较正常肝组织升高（P<0.05）。与永生化肝细胞LO2比较，肝癌细胞中KRT23表达水平升高（P<0.05）；与KPT23低表达肝癌患者比较，KRT23高表达肝癌患者预后较差（P=0.038）。二代测序和KEGG信号通路分析，KRT23可能调控干细胞活性，参与Wnt/β-catenin信号通路。免疫荧光定位，KRT23主要位于细胞胞浆。验证针对KRT23的siRNA敲除效率后，构建KRT23低表达慢病毒载体，并再次验证转染效率。KRT23表达降低后，与sh-NC组比较，sh-KRT23组干细胞球成球形成率明显降低（P<0.01），干细胞相关指标分化簇133（CD133）、分化簇44（CD44）、八聚体结合转录因子4（OCT4）和SRY-盒转录因子2（SOX2）表达水平降低（P<0.05或P<0.01）。结论 KRT23通过调控Wnt/β-catenin信号通路增强肝癌干细胞活性，其机制与Wnt/β-catenin信号通路的激活有关。

关键词: 角蛋白23, 肝肿瘤, 干细胞活性, 分化簇133, 八聚体结合转录因子4

Abstract:

Objective To discuss the regulatory effect of keratin 23 （KRT23） on the stem cell activity in hepatocellular carcinoma （HCC）， and to clarify its mechanism. Methods The Gene Expression Profiling Interactive Analysis （GEPIA） database was used to analyze the expression of KRT23 in hepatocellular carcinoma tissues and normal liver tissues； Kaplan-Meier Plotter survival analysis was performed to compare the survival of hepatocellular carcinoma patients with high and low expression of KRT23， and to evaluate the impact of KRT23 on the prognosis of hepatocellular carcinoma patients. Second-generation sequencing analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） signaling pathway analysis were conducted to identify the signaling pathways potentially regulated by KRT23， and the Wnt/β-catenin signaling pathway was finally selected for mechanistic study. Real-time fluorescence quantitative PCR （RT-qPCR）， Western blotting， and immunohistochemistry methods were used to verify the expression of KRT23 in HCC cells and HCC tissue， respectively， and immunofluorescence was used to clarify its subcellular localization. A lentiviral vector for KRT23 knockdown was constructed using a small interfering RNA （siRNA） with verified knockdown efficiency. After knocking down the expression level of KRT23， the stem cell sphere formation assay was performed to verify the regulatory effect of KRT23 on hepatocellular carcinoma stem cell activity， and RT-qPCR and Western blotting methods were used to detect the changes of related stem cell markers. Immunofluorescence and nuclear-cytoplasmic protein extraction were further used to verify the regulation effect of KRT23 on stem cells related indicators in the Wnt/β-catenin signaling pathway. Results The expression level of KRT23 in hepatocellular carcinoma tissues was higher than that in normal liver tissue. Compared with immortalized hepatocyte LO2， the expression levels of KRT23 in hepatocellular carcinoma cells were increased （P<0.05）； compared with the hepatocellular carcinoma patients with low expression of KR23， the hepatocellular carcinoma patients with high expression of KRT23 had a poorer prognosis （P=0.038）. The second-generation sequencing and KEGG signaling pathway analysis results showed that KRT23 might regulate stem cell activity and participate in the Wnt/β-catenin signaling pathway. The immunofluorescence localization results showed that KRT23 was mainly located in the cytoplasm. After verification of the knockdown efficiency of siRNA targeting KRT23， a lentiviral vector for KRT23 knockdown was constructed， and the transfection efficiency was re-verified. After KRT23 expression was reduced， compared with sh-NC group， the stem cell sphere formation rate in sh-KRT23 group was significantly decreased （P<0.01）， and the expression levels of stem cell-related markers cluster of differentiation 133 （CD133）， cluster-of-differentiation 44（CD44）， pctamer-blinding transcription factor 4（OCT4）， and SRY-box transcription-factor 2（SOX2） were also decreased （P<0.05 or P<0.01）. Conclusion KRT23 enhances the stem cell activity in hepatocellular carcinoma by regulating the Wnt/β-catenin signaling pathway， and the mechanism is related to the activation of the Wnt/β-catenin signaling pathway.

Key words: Keratin 23, Liver neoplasm, Stem cell activity, Cluster of differentiation 133, Pctamer-blinding tramscription factor 4

中图分类号:

R735.7

郭丹,李雅睿,杨佳卉,张丹,卢桂芳,任牡丹,和水祥. KRT23对肝癌干细胞活性的促进作用及其机制[J]. 吉林大学学报(医学版), 2026, 52(4): 1034-1042.

Dan GUO,Yarui LI,Jiahui YANG,Dan ZHANG,Guifang LU,Mudan REN,Shuixiang HE. Promotion effect of KRT23 on hepatocellular carcinoma stem cell activity and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2026, 52(4): 1034-1042.

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Primer	Sequence
KRT23	Forward: AACTAGCACGTGGCTCCCG
	Reverse: TTTTTTTTTTTTTTTTTTTGCAGCTAGTGCGGAGTT
CD133	Forward:AGTCGGAAACTGGCAGATAGC
	Reverse:GGTAGTGTTGTACTGGGCCAAT
CD44	Forward:CTGCCGCTTTGCAGGTGTA
	Reverse:CATTGTGGGCAAGGTGCTATT
OCT4	Forward: TATGCAAAGCAGAAACCCTCGTG
	Reverse: CTGGCGCCGTTACAGAACCA
SOX2	Forward:ATGACCAGCTCGCAGACCTAC
	Reverse:TTGACCACCGAACCCATGGAG
β-actin	Forward: ATCGTGCGTGACATTAAGGAGAAG
	Reverse: AGGAAGGAAGGCTGGAAGAGTG

SiRNA	Sequence
SiKRT23#1	5'-3' GGAUGGCAGUGGAUGACUUTT
	3'-5' AAGUCAUCCACUGCCAUCCTT
SiKRT23#2	5'-3' GGACACUUGGUAUAAAGAATT
	3'-5' UUCUUUAUACCAAGUGUCCTT
SiKRT23#3	5'-3' CCAAAGAUCAAGGCCAUAATT
	3'-5' UUAUGGCCUUGAUCUUUGGTT
SiP21	5'-3' CCUCUGGCAUUAGAAUUAUTT
	3'-5' AUAAUUCUAAUGCCAGAGGTT
Negative control	5'-3' UUCUCCGAACGUGUCACGUTT
	3'-5' ACGUGACACGUUCGGAGAATT