Department of Oncology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, P.R. China.
The 5th Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai 200082, P.R. China.
Int J Mol Med. 2019 Mar;43(3):1417-1429. doi: 10.3892/ijmm.2019.4062. Epub 2019 Jan 14.
Krüppel‑like factor 4 (Klf4) is a transcriptional factor involved in the progression of hepatocellular carcinoma (HCC). However, the underlying regulatory mechanisms associated with the Klf4 gene as a tumor suppressor in HCC remain unclear. microRNAs (miRNAs or miRs) are a series of small non‑coding RNAs that serve a vital role in regulating gene expression via their influence on protein translation and the associated degradation of mRNA. The mRNA expression levels of the miRNA, miR‑9‑5p, and Klf4 were measured using reverse transcription‑quantitative polymerase chain reaction. The protein expression levels of Klf4, protein kinase B (AKT), phosphorylated (p‑)AKT, mechanistic target of rapamycin (mTOR), p‑mTOR, B cell lymphoma‑2 (Bcl‑2) and Bcl‑2‑associated X protein (Bax) were determined by western blot analysis. Dual luciferase reporter assay was used to confirm a direct interaction between miR‑9‑5p and the 3'‑untranslated region (3'‑UTR) sequence of Klf4. Cell counting kit‑8 assay, wound healing assay, Transwell migration assay and flow cytometric analysis were performed to evaluate the proliferative, migratory and apoptotic capabilities of the HCC cells. In the present study, miR‑9‑5p was revealed to be overexpressed in HCC as a novel upstream gene of Klf4. miR‑9‑5p expression was inversely associated with Klf4 expression in clinical samples. Additionally, Kaplan‑Meier analysis revealed a markedly poor prognosis of HCC in the miR‑9‑5p high‑expression group. Bioinformatics analysis revealed that miR‑9‑5p bound directly to the 3'‑UTR of Klf4, which reduced the expression levels of Klf4. The miR‑9‑5p/Klf4 axis promoted HCC proliferation and migration, and inhibited HCC apoptosis. Furthermore, miR‑9‑5p upregulated the Bcl‑2/Bax protein ratio and activated AKT/mTOR signaling. Taken together, these data demonstrated that the miR‑9‑5p/Klf4 axis was able to promote HCC progression, which may occur via regulation of the AKT signaling pathway, highlighting a potential novel target in HCC treatment.
Krüppel 样因子 4（Klf4）是参与肝细胞癌（HCC）进展的转录因子。然而，Klf4 基因作为 HCC 肿瘤抑制因子的潜在调节机制尚不清楚。微小 RNA（miRNA 或 miR）是一系列小的非编码 RNA，通过影响蛋白质翻译和相关的 mRNA 降解，在调节基因表达方面发挥着重要作用。采用逆转录-定量聚合酶链反应测定 miRNA、miR-9-5p 和 Klf4 的 mRNA 表达水平。采用 Western blot 分析测定 Klf4、蛋白激酶 B（AKT）、磷酸化（p）AKT、雷帕霉素靶蛋白（mTOR）、p-mTOR、B 细胞淋巴瘤-2（Bcl-2）和 Bcl-2 相关 X 蛋白（Bax）的蛋白表达水平。双荧光素酶报告基因检测用于证实 miR-9-5p 与 Klf4 的 3'非翻译区（3'UTR）序列之间的直接相互作用。采用细胞计数试剂盒-8 检测、划痕愈合试验、Transwell 迁移试验和流式细胞术分析评估 HCC 细胞的增殖、迁移和凋亡能力。在本研究中，miR-9-5p 作为 Klf4 的新上游基因在 HCC 中呈现过度表达。miR-9-5p 的表达与临床样本中 Klf4 的表达呈负相关。此外，Kaplan-Meier 分析显示 miR-9-5p 高表达组 HCC 的预后明显较差。生物信息学分析显示，miR-9-5p 直接结合 Klf4 的 3'UTR，降低 Klf4 的表达水平。miR-9-5p/Klf4 轴促进 HCC 增殖和迁移，抑制 HCC 凋亡。此外，miR-9-5p 上调 Bcl-2/Bax 蛋白比值并激活 AKT/mTOR 信号通路。综上所述，这些数据表明，miR-9-5p/Klf4 轴能够促进 HCC 进展，可能通过调节 AKT 信号通路发生，提示 HCC 治疗的一个潜在新靶点。
