肝内胆管细胞癌中hsa_circ_0089153的表达及其生物学功能
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1.福建医科大学省立临床医学院,福建 福州 350001;2.福建省南平市第一医院 普通外科,福建 南平 353000;3.福建省立医院 肝胆外科,福建 福州 350001

作者简介:

陈小彬,福建医科大学省立临床医学院硕士研究生/福建省南平市第一医院副主任医师,主要从事肝胆胰疾病基础与临床方面的研究。

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福建省自然科学基金资助项目(2017J01244);福建省卫生计生委医学创新课题基金资助项目(2018-CX-8)。


Expression of hsa_circ_0089153 in intrahepatic cholangiocarcinoma and its biological function
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1.Shengli Clinical Medical College of Fujian Medical University, Fuzhou 350001, China;2.Department of General Surgery, Nanping First Hospital, Nanping, Fujian 353000, China;3.Department of Hepatobiliary Surgery, Fujian Provincial Hospital, Fuzhou 350001, China

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    摘要:

    背景与目的 肝内胆管细胞癌(ICC)是仅次于肝细胞癌的第二大肝脏恶性肿瘤,其发病隐匿、恶性度高、预后差。环状RNA(circRNA)异常表达与多种恶性肿瘤的发生发展关系密切,但circRNA与ICC的关系研究较少。因此,本研究初步探讨ICC中差异表达circRNA及其生物学功能。方法 采用福建省立医院经病理确诊的5例ICC患者标本,通过高通量circRNA芯片技术筛选出ICC及对应癌旁组织中差异表达的circRNA,进一步在长度500~1 500 bp之间选取其中变化最为明显的circRNA,并通过qRT-PCR进行验证;然后在HCCC-9810、RBE细胞系中通过慢病毒过表达该circRNA,应用CCK-8实验、划痕实验、Transwell实验和流式细胞术分析该circRNA对ICC细胞生物学功能的影响。结果 与癌旁正常组织比较,ICC中共有76个差异表达的circRNA,包括63个上调与13个下调的circRNA,其中低表达的hsa_circ_0089153变化倍数最大(FC=103.214);qRT-PCR验证结果与基因芯片结果一致。hsa_circ_0089153过表达后,ICC细胞的增殖活性明显增强[HCCC-9810:过表达组(114.816±5.138)vs.空载组(100.000±4.897),RBE:过表达组(117.235±7.079)vs.空载组(100.000±14.896)(均P<0.05)];凋亡率明显降低[HCCC-9810:过表达组(11.277±0.243)vs.空载组(16.673±0.660),RBE:过表达组(4.527±0.301)vs.空载组(6.793±0.534)(均P<0.05)];迁移能力与侵袭能力明显增强[HCCC-9810:过表达组(9.070±1.090)vs.空载组(6.206±1.197),RBE:过表达组(6.197±0.687)vs.空载组(4.198±1.170)(均P<0.05)与HCCC-9810:过表达组(0.370±0.026)vs. 空载组(0.274±0.014),RBE:过表达组(0.297±0.016)vs.空载组(0.250±0.019)(均P<0.05)]。结论 ICC中存在多种差异表达的circRNA,其中hsa_circ_0089153在ICC组织中呈明显低表达,但与多数情况不同,hsa_circ_0089153过表达对ICC的恶性生物学行为有促进作用,因此,hsa_circ_0089153在ICC中的生物学功能及其关联的调控网络仍需进一步探讨。

    Abstract:

    Background and Aims After hepatocellular carcinoma, intrahepatic cholangiocarcinoma (ICC) is the second most common malignant tumor of the liver, with an insidious onset, high malignant level, and a unfavorable prognosis. Although aberrant circRNA expression is linked to the onset and progression of several malignant tumors, the association between circRNA and ICC is rarely investigated. As a result, this study was conducted as a preliminary test to examine circRNA's differential expression in ICC and its biological function.Methods By using high-throughput circRNA chip technology, five specimens of ICC patients confirmed by pathology in Fujian Provincial Hospital were used to screen out differentially expressed circRNA in ICC and corresponding adjacent tissues, and the circRNA with the most obvious change was selected from 500-1500 bp in length and verified by qRT-PCR. The circRNA was then overexpressed in HCCC-9810 and RBE cell lines using lentivirus, and the impact of the circRNA on the biological function of ICC cells was investigated using CCK-8, scratch, Transwell, and flow cytometry.Results Compared with normal tissues adjacent to cancer, there are 76 differentially expressed circRNAs in ICC, including 63 upregulated and 13 downregulated circRNAs. The downregulated hsa_circ_0089153 had a highest fold-change (FC=103.214). The results of qRT-PCR were consistent with those of gene chip. After hsa_circ_0089153 overexpression, the proliferation activity of ICC cells significantly increased [HCCC-9810: over-expression group (114.816±5.138) vs. no-load group (100.000±4.897), RBE: over-expression group (117.235±7.079) vs. no-load group (100.000±14.896) (all P<0.05)]. The apoptosis rate significantly decreased [HCCC-9810: over-expression group (11.277±0.243) vs. no-load group (16.673±0.660) (all P<0.05)]. The migrated and invasive ability significantly increased [HCCC-9810: over-expression group (9.070±1.090) vs. no-load group (6.206±1.197), RBE: over-expression group (6.197±0.687) vs. no-load group (4.198±1.170) (all P<0.05) and HCCC-9810: over-expression group (0.370±0.026) vs. no-load group (0.274±0.014), RBE: over-expression group(0.297±0.016) vs. no-load group (0.250±0.019) (all P<0.05)].Conclusion There are a number of aberrantly expressed circRNAs in ICC, among which hsa_circ_0089153 has a noteworthy low expression. However, unlike most circumstances, overexpression of hsa_circ_0089153 can promote the malignant biological behavior of ICC cells. So, the biological role of hsa_circ_0089153 in ICC and the associated regulatory network still need to be further clarified.

    图1 qRT-PCR检测circ_0089153的表达 A:circ_0089153扩增曲线;B:circ_0089153溶解曲线;C:circ_0089153标准曲线;D:β-actin扩增曲线;E:β-actin溶解曲线;F: β-actin标准曲线Fig.1 Detection of circ_0089153 expression by qRT-PCR A: Amplification curve of circ_0089153; B: Dissolution curve of circ_0089153; C: Standard curve circ_0089153; D: Amplification curve of β-actin; E: Dissolution curve of β-actin; F: Standard curve of β-actin
    图2 荧光观察转染效果(×100)Fig.2 Fluorescence staining for analysis of transfection effect (×100)
    图3 PCR验证转染效率Fig.3 Transfection efficiency verification by PCR
    图4 CCK-8检测细胞增殖活性Fig.4 Determination of cell proliferation by CCK-8 assay
    图5 流式细胞术检测细胞凋亡Fig.5 Apoptosis analysis by flow cytometry
    图6 细胞划痕实验检测细胞迁移能力Fig.6 Cell migration ability examined by cell scratch assay
    图7 Transwell实验检测细胞侵袭能力Fig.7 Cell invasion ability measured by Transwell assay
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陈小彬,吴嘉艺,黄理铭,白燕南,唐忠,严茂林.肝内胆管细胞癌中hsa_circ_0089153的表达及其生物学功能[J].中国普通外科杂志,2022,31(2):199-207.
DOI:10.7659/j. issn.1005-6947.2022.02.008

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  • 收稿日期:2021-06-25
  • 最后修改日期:2021-08-28
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  • 在线发布日期: 2022-03-04