microRNA-933对LX-2细胞凋亡和增殖的影响及其分子机制

海龙; 马丽娜; 雒夏; 丁向春

doi:10.12449/JCH240716

microRNA-933对LX-2细胞凋亡和增殖的影响及其分子机制

DOI: 10.12449/JCH240716

海龙^{1, 2},
马丽娜^{1, 2},
雒夏^{1, 2},
丁向春^{1, 2, , ,}

1.
宁夏医科大学总医院感染疾病科，银川 750004
2.
宁夏医科大学临床医学院，银川 750004

基金项目:

宁夏医科大学总医院创新项目 (2023AAC03584)

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：海龙、丁向春负责课题设计，资料分析，撰写论文；海龙、马丽娜、雒夏参与收集数据，修改论文；丁向春负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
丁向春， 13619511768@163.com （ORCID： 0000-0003-0283-9419）

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出版历程
- 收稿日期: 2023-10-03
- 录用日期: 2024-01-04
- 出版日期: 2024-07-25

Effect of miRNA-933 on the apoptosis and proliferation of LX-2 cells and its molecular mechanism

Long HAI^{1, 2},
Lina MA^{1, 2},
Xia LUO^{1, 2},
Xiangchun DING^{1, 2
, ,
,}

1.
Department of Infectious Diseases，General Hospital of Ningxia Medical University，Yinchuan 750004，China
2.
School of Clinical Medicine，Ningxia Medical University，Yinchuan 750004，China

Research funding:

The Innovation Project of Ningxia Medical University General Hospital (2023AAC03584)

More Information

Corresponding author: DING Xiangchun， 13619511768@163.com （ORCID： 0000-0003-0283-9419）

摘要

摘要: 目的探讨microRNA-933（miRNA-933）对人肝星状细胞系LX-2细胞凋亡和增殖的调控作用及其机制。方法首先以人肝组织为研究对象，利用基因芯片技术，检测肝硬化与慢性乙型肝炎肝组织相较于正常肝组织中差异表达的基因，并从中筛选差异表达显著的miRNA，从而确定研究对象为miRNA-933。进而以人肝星状细胞系LX-2为研究对象，利用miRNA-933分子模拟剂（miRNA-933 mimic）与抑制剂（miRNA-933 siRNA），构建LX-2过表达与敲减模型，并以转染mimic-NC（过表达）或siRNA-NC（敲减）的细胞作为阴性对照。使用实时荧光定量PCR（qPCR）和Western Blot技术，检测miRNA-933与活化标志蛋白的表达水平；随后采用细胞增殖实验、流式细胞术等技术，检测miRNA-933对细胞凋亡、增殖和活化的影响，并探讨其机制。计量资料两组间比较采用成组t检验，多组间比较采用单因素方差分析，并经过Bonferroni校正。结果基于基因芯片检测结果，最终筛选出18个显著差异表达的miRNA，其中miRNA-933表达显著下调（P<0.05）。miRNA-933 mimic与siRNA转染LX-2细胞后，结果显示，与阴性对照组相比，miRNA-933 siRNA显著下调miRNA-933的表达（P=0.000 7），而miRNA-933 mimic显著上调miRNA-933的表达（P=0.000 3）；Western Blot和qPCR检测结果表明，miRNA-933 siRNA显著上调胶原蛋白Ⅰ（Collagen Ⅰ）和α-平滑肌肌动蛋白（α-SMA）的表达（P值均<0.001），而miRNA-933 mimic显著抑制Collagen Ⅰ和α-SMA的表达（P值均<0.05）。流式细胞术检测结果显示，与阴性对照组相比，miRNA-933 siRNA显著下调LX-2细胞凋亡率（P=0.031 9），miRNA-933 mimic显著上调LX-2细胞凋亡率（P=0.005 5）；Western Blot检测结果表明，与阴性对照组相比，miRNA-933 siRNA可以抑制LX-2细胞中胱天蛋白酶3（Caspase-3）和多聚ADP核糖聚合酶1（PARP-1）的表达（P值分别为0.006 7、0.003 0），上调B淋巴细胞瘤-2（Bcl-2）的表达（P=0.002 0），而miRNA-933 mimic可显著上调Caspase-3（P=0.011 8）和PARP-1（P=0.049 5）的表达，下调Bcl-2的表达（P=0.002 1）。细胞增殖实验结果显示，与阴性对照组相比，miRNA-933 siRNA可促进LX-2细胞增殖（P=0.011 5）；相反miRNA-933 mimic可抑制LX-2细胞增殖（P=0.001 2）。Western Blot和qPCR检测显示，miRNA-933 siRNA显著抑制Kruppel样因子6（KLF6）的表达，进而下调活化转录因子4（ATF4）、活化转录因子3（ATF3）、C/EBP同源蛋白（CHOP）蛋白的表达，而miRNA-933 mimic会促进以上蛋白的表达（P值均<0.05）。结论 miRNA-933可能通过促进LX-2细胞中KLF6/ATF4/ATF3/CHOP/Bcl-2信号轴的激活，进而促进细胞凋亡，抑制细胞的活化和增殖。
- 肝纤维化 /
- 微RNAs /
- 肝星状细胞
Abstract: Objective To investigate the regulatory effect of miRNA-933 on the apoptosis and proliferation of human hepatic stellate cell line LX-2 and its mechanism. Methods Firstly， with human liver tissue for research， gene microarray technology was used to detect the differentially expressed genes in liver tissue between liver cirrhosis/chronic hepatitis B tissue and normal liver tissue， among which the significantly differentially expressed miRNAs were identified， and thus miRNA-933 was determined as the research object. Then， with the human hepatic stellate cell line LX-2 for research， miRNA-933 mimic and inhibitor （miRNA-933 siRNA） were used to construct the LX-2 models of overexpression and knockdown， and the cells transfected with mimic-NC （overexpression） or siRNA-NC （knockdown） were established as the negative control group. Quantitative real-time PCR and Western blot were used to measure the expression levels of miRNA-933 and activation biomarkers； techniques such as cell proliferation assay and flow cytometry were used to investigate the effect and mechanism of miRNA-933 on cell apoptosis， proliferation， and activation. The independent-samples t test was used for comparison of continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and Bonferroni correction was also performed. Results A total of 18 significantly differentially expressed miRNAs were obtained based on the results of gene microarray， among which miRNA-933 was significantly downregulated （P<0.05）. After LX-2 cells were transfected with miRNA-933 mimic or siRNA， compared with the negative control group， miRNA-933 siRNA significantly downregulated the expression of miRNA-933 （P=0.000 7）， while miRNA-933 mimic significantly upregulated the expression of miRNA-933 （P=0.000 3）. Western blot and quantitative real-time PCR showed that miRNA-933 siRNA significantly upregulated the expression of collagen I and α-SMA （P<0.001）， while miRNA-933 mimic significantly inhibited the expression of collagen I and α-SMA （P<0.05）. Flow cytometry showed that compared with the negative control group， miRNA-933 siRNA significantly downregulated the apoptosis rate of LX-2 cells （P=0.031 9）， and miRNA-933 mimic significantly upregulated the apoptosis rate of LX-2 cells （P=0.005 5）. Western blot showed that compared with the negative control group， miRNA-933 siRNA could inhibit the expression of Caspase-3 （P=0.006 7） and poly（ADP-ribose） polymerase-1 （PARP-1）（P=0.003 0） and upregulate the expression of B-cell lymphoma-2 （Bcl-2） in LX-2 cells （P=0.002 0）， while miRNA-933 mimic could significantly upregulate the expression of Caspase-3 （P=0.011 8） and PARP-1 （P=0.049 5） and downregulated the expression of Bcl-2 （P=0.002 1）. Cell proliferation assay showed that compared with the negative control group， miRNA-933 siRNA could promote the proliferation of LX-2 cells （P=0.011 5）， while on the contrary， miRNA-933 mimic could inhibit the proliferation of LX-2 cells （P=0.001 2）. Western blot and quantitative real-time PCR showed that miRNA-933 siRNA significantly inhibited the expression of Kruppel-like factor 6 （KLF6） and downregulated the expression of activating transcription factor 4 （ATF4）， activating transcription factor 3 （ATF3）， and C/EBP homologous protein （CHOP）， while miRNA-933 mimic promoted the expression of the above proteins （all P<0.05）. Conclusion This study shows that miRNA-933 may promote cell apoptosis and inhibit cell activation and proliferation by promoting the activation of the KLF6/ATF4/ATF3/CHOP/Bcl-2 signal axis in LX-2 cells.
- Hepatic Fibrosis /
- MicroRNAs /
- Hepatic Stellate Cells

HTML全文

图 1 肝组织中差异表达的miRNA筛选

注： a、b，基因芯片技术检测肝硬化与慢性乙型肝炎患者肝组织中miRNA的差异表达，*P<0.05；c、d，qPCR检测健康人与肝病患者肝组织中miRNA的差异表达。

Figure 1. The verified of differential expression miRNA in liver tissues

下载: 全尺寸图片幻灯片

图 2 miRNA-933对LX-2细胞活化的影响

注： a，miRNA-933 mimic和siRNA对LX-2细胞中miRNA-933表达的影响；b、c，qPCR检测LX-2细胞中Collagen Ⅰ和α-SMA mRNA的表达情况；d～f，Western Blot检测LX-2细胞中α-SMA和Collagen Ⅰ蛋白的表达情况。

Figure 2. The effect of miRNA-933 on the activation of LX-2 cells

下载: 全尺寸图片幻灯片

图 3 miRNA-933对LX-2细胞凋亡的影响

注： a，流式细胞仪检测LX-2细胞凋亡率；b，Western Blot检测LX-2细胞凋亡相关蛋白Bcl-2、PARP-1和Caspase-3的表达情况。

Figure 3. The effect of miRNA-933 on regulating apoptosis of LX-2 cells

下载: 全尺寸图片幻灯片

图 4 miRNA-933对LX-2细胞增殖的影响

注： a，细胞集落实验检测各组细胞集落形成；b，细胞集落实验统计学分析结果；c，CCK-8实验检测细胞存活率。

Figure 4. The effect of miRNA-933 on regulating proliferation of LX-2 cells

下载: 全尺寸图片幻灯片

图 5 miRNA-933对KLF6/ATF4/ATF3/CHOP信号轴激活的影响

注： a，Western Blot检测KLF6/ATF4/ATF3/CHOP蛋白表达；b～e，Western Blot半定量分析结果。

Figure 5. The effect of miRNA-933 on the activation KLF6/ATF4/ATF3/CHOP axis

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequences

引物	序列（5′-3′）
miRNA-933	F：ACACTCCAGCTGGCTGTGTGCGCAGGG‑AGACCTC
miRNA-933	R：TGGTGTCGTGGAGTCG
U6	F：CTCGCTTCGGCAGCACA
U6	R：AACGCTTCACGAATTTGCGT
miRNA-933-RT	UGUGCGCAGGGAGACCUCUCCC
miRNA-933 mimic	CAAAGUGCCUCCCUUUAGAGUG
NC mimic	UUCUCCGAACGUGUCACGUTT
miRNA-933 siRNA	GUUUCACGGAGGGAAAUCUCAC
NC siRNA	CAGUACUUUUGUGUAGUACCAA

下载: 导出CSV

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