MiR-375 Modulates HK2 via SP1 to Impact Proliferation and Glycolysis in Hepatocellular Carcinoma
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摘要:
目的 探讨肝癌中己糖激酶2(HK2)异常表达以及肝癌细胞增殖和糖酵解的调控机制。 方法 收集2017年1月—2019年12月41例在武汉市中心医院手术切除的肝癌及癌旁组织病理样本。利用免疫组化法(IHC)评估HK2和特异性蛋白1(SP1)的表达。在肝癌细胞系HepG2中敲低HK2和SP1、过表达miR-375，使用CCK-8、葡萄糖、乳酸浓度测定检测细胞增殖和糖酵解能力，利用实时荧光定量PCR和Western blotting实验验证三者之间的调控关系。利用基因表达数据库(GEO)中的肝癌数据集分析三者之间的相关性。应用荧光素酶报告基因实验验证miR-375和SP1之间的直接相互作用。 结果 IHC显示，SP1和HK2在癌旁组织中的高表达比例分别为39.0%(16/41)和26.8%(11/41)，而在肝癌组织中则升至58.5%(24/41)和53.7%(22/41)。SP1和HK2的表达评分呈正相关关系(r=0.611，P < 0.001)。下调HK2和SP1以及上调miR-375表达抑制肝癌细胞的增殖和糖酵解能力，而上调miR-375和下调SP1抑制HK2的表达。生物信息学分析显示, HK2表达与miR-375呈负相关关系(r=-0.584, P < 0.001), 与SP1呈正相关关系(r=0.297, P=0.005)。miR-375可直接靶向抑制SP1在mRNA和蛋白质的表达。荧光素酶报告基因证实miR-375与SP1存在直接相互作用。 结论 miR-375通过靶向SP1调控HK2影响肝癌的增殖和糖酵解。 Abstract:Objective To investigate the aberrant expression of hexokinase 2 (HK2) in hepatocellular carcinoma (HCC) and the regulatory mechanisms of cell proliferation and glycolysis in HCC. Methods A total of 41 cases of HCC and paracancerous tissues that underwent surgical resection at the Central Hospital of Wuhan between January 2017 and December 2019 were collected for pathological examination. The expression of HK2 and SP1 was evaluated through immunohistochemical staining. In the HepG2 cell line, HK2 and SP1 were knocked down, and miR-375 was overexpressed. The assessment of cell proliferation and glycolytic capacity was conducted through the utilization of CCK-8, glucose, and lactate concentration assays. The regulatory relationships among HK2, SP1, and miR-375 were validated through the implementation of real-time quantitative PCR (RT-qPCR) and Western blotting techniques. The correlation among these factors was analyzed through the utilization of the gene expression omnibus (GEO) HCC dataset. The direct interaction between miR-375 and SP1 was confirmed through the utilization of a luciferase reporter assay. Results IHC revealed that the high expression rates of SP1 and HK2 in paracancerous tissues were 39.0% (16/41) and 26.8% (11/41), respectively. In HCC tissues, these rates increased to 58.5% (24/41) and 53.7% (22/41) respectively. A positive correlation was observed between SP1 and HK2 expression scores (r=0.611, P < 0.001). The downregulation of HK2 and SP1, and the upregulation of miR-375, resulted in the inhibition of HCC cell proliferation and glycolysis. The upregulation of miR-375 and downregulation of SP1 resulted in the suppression of HK2 expression. Bioinformatics analysis revealed a negative correlation between HK2 and miR-375 expression (r=-0.584, P < 0.001) and a positive correlation between HK2 and SP1 expression (r=0.297, P=0.005). Additionally, miR-375 was identified as a direct target of SP1, exhibiting inhibitory effects at both mRNA and protein levels. The luciferase reporter assay provided confirmation of the direct interaction between miR-375 and SP1. Conclusion The miR-375 has been demonstrated to regulate HK2 by targeting the transcription factor SP1, thereby influencing the proliferation and glycolysis of HCC. -
Key words:
- Hepatocellular carcinoma /
- MicroRNA /
- MiR-375 /
- Specificity protein 1 /
- Hexokinase2 /
- Glycolysis /
- Cell proliferation
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图 1 肝癌和癌旁组织中HK2和SP1的表达情况(免疫组化法，×200)
注：肝癌中HK2和SP1表达上调。
Figure 1. HK2 and SP1 expression in hepatocellular carcinoma (immunocytochemistry, ×200)
图 2 miR-375、SP1和HK2对肝癌细胞的增殖和糖酵解的影响
注：HepG2细胞转染miR-375类似物(375 mimic)、3种不同序列的SP1和HK2 siRNA及阴性对照(NC)。A为用TaqMan-PCR定量成熟miR-375，用Western blotting检测SP1、HK2的表达；B为细胞增殖情况；C为细胞培养基中乳酸产量情况；D为细胞培养基中葡萄糖消耗量情况; aP < 0.05。
Figure 2. miR-375, SP1 and HK2 affect proliferation and glycolysis in hepatocellular carcinoma cells
图 3 miR-375和SP1对HK2表达的影响
注：A为HepG2细胞转染miR-375类似物和阴性对照(NC)，qRT-PCR和Western blotting检测HK2 mRNA水平和蛋白水平。B为转染SP1 siRNA-3(表现出更好的干扰效果)和对照，用qRT-PCR和Western blotting检测HK2 mRNA水平和蛋白水平。
Figure 3. The effects of miR-375 and SP1 on HK2 expression
图 4 miR-375在肝癌细胞中的荧光素酶报告基因实验
注：A为转染miR-375类似物和阴性对照(NC)后，检测SP1 mRNA和蛋白水平。B为荧光素酶报告基因构建图，野生型(sp1-wt)或突变型(sp1-mut), 突变位点用红色标记。C为共转染sp1-wt或sp1-mut和375类似物或NC后，检测相对荧光素酶活性。
Figure 4. Luciferase reporter assay of miR-375 in hepatocellular carcinoma
表 1 人工合成寡核苷酸序列
Table 1. Synthetic oligonucleotide sequences
基因 序列 miR-375 mimic 5’-UUUGUUCGUUCGGCUCGCGUGA-3’ 3’-AAACAAGCAAGCCGAGCGCACU-5’ miR-375 NC 5’-UUUGUACUACACAAAAGUACUG-3’ 3’-AAACAUGAUGUGUUUUCAUGAC-5’ HK2_siRNA1 CTGTGAAGTTGGCCTCATT HK2_siRNA2 ACGACAGCATCATTGTTAA HK2_siRNA3 CTGGCTAACTTCATGGATA SP1_siRNA1 GCAACATCATTGCTGCTAT SP1_siRNA2 GCCAATAGCTACTCAACTA SP1_siRNA3 CTCCCAACTTACAGAACCA sp1-wt-3’UTR 5’-CCGACTAGTGAAGCACATGATCATGGGAATGATAGCCCAGAACAA AAAGAAATCTTGAAGCTTGGG-3’ 3’-GGCTGATCACTTCGTGTACTAGTACCCTTACTATCGGGTCTTGTTTT TCTTTAGAACTTCGAACCC-5’ sp1-mut-3’UTR 5’-CCGACTAGTGAAGCACATGATCATGGGAATGATTCGGCAGTTGTTT AAGAAATCTTGAAGCTTGGG-3’ 3’-GGCTGATCACTTCGTGTACTAGTACCCTTACTAAGCCGTCAACAAA TTCTTTAGAACTTCGAACCC-5’ 
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表 2 不同HK2表达情况肝癌患者临床特征比较(例)
Table 2. Comparison of clinicopathologic characteristics of hepatocellular carcinoma patients with different expression levels of HK2(cases)
项目 低表达(n=19) 高表达(n=22) P值 项目 低表达(n=19) 高表达(n=22) P值 年龄 ≤50岁 10 12 0.902 饮酒史 >50岁 9 10 是 7 10 0.577 性别 否 12 12 男性 17 22 0.209 HBV感染 女性 2 0 是 16 22 0.053 家族史 否 3 0 是 3 4 0.999 HBV-DNA阳性 否 16 18 是 4 6 0.922 甲胎蛋白水平 否 15 16 ≤20 ng/mL 9 5 0.097 肝硬化 >20 ng/mL 10 17 是 6 9 0.536 肿瘤大小 否 13 13 ≤5 cm 2 1 0.895 门脉癌栓 >5 cm 17 21 是 0 1 0.999 分化程度 否 19 21 高分化 5 0 0.010 中低分化 14 22 注：比较均采用Fisher精确检验。
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