piR-020829 通过胰岛素样生长因子结合蛋白-3 调控肝癌增殖的机制

Mechanism of piR-020829 in regulating hepatocellular carcinoma proliferation via insulin-like growth factor-binding protein 3

摘要

目的 探讨 piR-020829 调控原发性肝癌恶性增殖的作用及相关分子机制。方法 采用实时定量 PcR 法检测肝癌和癌旁肝组织 piR-020829 表达水平的差异；采用 Kaplan-Meier 生存曲线分析 piR-020829 表达水平与肝癌患者总生存期、无进展生存期的关系；采用短发夹 RNa(short hairpin RNa， shRNa)干扰技术敲低 SNU387 细胞中 piR-020829 的表达(敲低组)，并以转染阴性对照载体的 SNU387 细胞为敲低对照组；采用 piR-020829 模拟物处理 hep3b 细胞实现过表达(piR-020829 过表达组)，并以转染模拟物阴性对照的 hep3b 细胞为过表达对照组。采用 ccK-8 法检测 piR-020829 敲低表达/ 过表达后对肝癌细胞 SNU387/ hep3b 增殖活力的影响；采用肝癌细胞裸鼠皮下移植瘤实验，将 32 只裸鼠均分为拮抗剂组(尾静脉注射 piR-020829 antagomir)、拮抗剂对照组(注射 antagomir Nc)、模拟物组(注射 piR-020829 mimic)和模拟物对照组(注射 mimic Nc)；至实验结束，观察不同处理组对裸鼠皮下成瘤瘤体质量的影响；采用克隆形成实验检测 piR-020829 敲低表达/ 过表达后对肝癌细胞 SNU387/ hep3b 体外增殖能力的影响。结果 相较于癌旁组织，piR-020829 在肝癌组织表达水平升高。生存分析显示，piR-020829 相对高表达的肝癌患者总生存时间、无进展生存时间较低表达患者缩短，提示 piR-020829 高表达肝癌患者预后差。体外实验证实，piR-020829 敲低表达后导致肝癌细胞增殖活力下降，克隆形成数目减少( P < 0. 05)；piR-020829-mimic 处理后导致肝癌细胞增殖活性升高，克隆形成数目增加。裸鼠皮下移植瘤实验证实，piR-020829 antagomir 处理后导致肝癌细胞裸鼠皮下成瘤质量低于拮抗剂对照组；piR-020829-mimic 处理后导致肝癌细胞裸鼠皮下成瘤瘤质量高于模拟物对照组。拮抗剂组裸鼠移植瘤体免疫组织化学染色显示，Ki-67 阳性率低于拮抗剂对照组(P<0.05)。piR-020829 敲低表达导致凋亡相关蛋白 b 细胞淋巴瘤-2(b-cell lymphoma-2， bcl-2)、b 细胞淋巴瘤-xl(b cell lymphoma-xl，bcl-xl)表达下调，半胱天冬酶-3(cysteine-aspartic protease 3， caspase3)表达上调(P< 0.05)；piR-020829 敲低表达导致细胞周期蛋白依赖性激酶 4(cyclin-dependent kinase 4， cdK4)、细胞周期蛋白 d1(g1/ S-specific cyclin-d1， cyclin d1)表达下调(P<0.05)；piR-020829 敲低表达导致其靶分子胰岛素样生长因子结合蛋白-3(insulin-like growth factor-binding protein 3， IgfbP3)表达上调(P<0.05)。结论 piR-020829 通过靶向抑制 IgfbP3 表达而促进肝癌细胞恶性增殖，该分子有望成为抗肝癌靶向治疗新的分子靶标。

Abstract

Objective To investigate the role of piRNA-020829 (piR-020829) in regulating malignant proliferation of hepatocellular carcinoma (HCC) and its underlying molecular mechanism. Methods Quantitative real-time PCR was used to detect the expression levels of piR-020829 in HCC tissues and adjacent non-tumor liver tissues. Kaplan-Meier survival analysis was performed to evaluate the correlation between piR-020829 expression and overall survival (OS) and progression-free survival (PFS) in HCC patients. Short hairpin RNA interference technology was employed to knockdown piR-020829 expression in SNU387 cells (knockdown group), with SNU387 cells transfected with negative control vector serving as negative control group. piR-020829 mimic was used to overexpress piR-020829 in Hep3B cells (overexpression group), with Hep3B cells transfected with mimic negative control serving as overexpression negative control group. CCK-8 assay was used to detect cell proliferation viability in SNU387/Hep3B cells after piR-020829 knockdown or overexpression. For in vivo experiments, 32 nude mice were randomly divided into 4 groups: antagomir group (tail vein injection of piR-020829 Antagomir), antagomir control group (injection of Antagomir NC), mimic group (injection of piR-020829 mimic), and mimic control group (injection of mimic NC); tumor weight was observed in each group at the end of the experiment. Colony formation assay was used to detect the in vitro proliferative capacity of SNU387/Hep3B cells after piR-020829 knockdown or overexpression. Results Compared with adjacent normal tissues, the expression level of piR-020829 was significantly elevated in hepatocellular carcinoma (HCC) tissues. Survival analysis showed that HCC patients with relatively high expression of piR-020829 had significantly shorter overall survival and progression free survival than those with low expression, indicating that high piR-020829 expression predicts a poor prognosis in HCC patients. In vitro experiments confirmed that knockdown of piR-020829 led to a significant decrease in the proliferation viability and colony formation ability of HCC cells (P < 0.05), while treatment with piR-020829 mimic increased HCC cell proliferation activity and colony formation. Nude mouse subcutaneous xenograft experiments demonstrated that treatment with piR-020829 Antagomir resulted in a significantly lower tumor weight compared with the antagomir control group, whereas treatment with piR-020829 mimic led to a significantly higher tumor weight compared with the mimic control group. Immunohistochemical staining of xenograft tumors from the antagomir group showed a significantly lower Ki-67 positive rate than that in the antagomir control group (P< 0.05). Knockdown of piR-020829 downregulated the expression of the apoptosis-related proteins B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-xl (Bcl-xl), and upregulated the expression of cysteine-aspartic protease 3 (Caspase-3) (P<0.05). Knockdown of piR-020829 also downregulated the expression of the cell cycle-related proteins CDK4 and Cyclin D1 (P < 0.05). Furthermore, mechanistic investigation revealed that knockdown of piR-020829 significantly upregulated the expression of its target molecule insulin-like growth factor-binding protein 3 (IGFBP3) (P<0.05). Conclusion piR-020829 promotes malignant proliferation of HCC cells by targeting and inhibiting IGFBP3 expression, and this molecule may serve as a novel molecular target for anti-HCC targeted therapy.

关键词

Piwi 相互作用 RNa / 胰岛素样生长因子结合蛋白-3 / 肝癌 / 增殖 / 凋亡

Key words

Piwi-interacting RNa / Insulin-like growth factor-binding protein 3 / hepatocellular carcinoma / Proliferation / apoptosis

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基金资助

山东省自然科学基金面上项目(ZR2020MH240)

国家自然科学基金青年项目(82000579)