中文｜English
ISSN 1001-5256 (Print)
ISSN 2097-3497 (Online)
CN 22-1108/R
Volume 40 Issue 7
Jul.  2024
Turn off MathJax
Article Contents
Article Navigation >  Journal of Clinical Hepatology  > 2024  >  40(7): 1382-1389

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

DOI: 10.12449/JCH240716
  • 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）
  • Received Date: 2023-10-03
  • Accepted Date: 2024-01-04
  • Published Date: 2024-07-25
    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.

     

    • FullText(HTML)
  • loading
    • References(30)
  • [1]
    TSUCHIDA T, FRIEDMAN SL. Mechanisms of hepatic stellate cell activation[J]. Nat Rev Gastroenterol Hepatol, 2017, 14( 7): 397- 411. DOI: 10.1038/nrgastro.2017.38.
    [2]
    ZHANG CY, YUAN WG, HE P, et al. Liver fibrosis and hepatic stellate cells: Etiology, pathological hallmarks and therapeutic targets[J]. World J Gastroenterol, 2016, 22( 48): 10512- 10522. DOI: 10.3748/wjg.v22.i48.10512.
    [3]
    HIGASHI T, FRIEDMAN SL, HOSHIDA Y. Hepatic stellate cells as key target in liver fibrosis[J]. Adv Drug Deliv Rev, 2017, 121: 27- 42. DOI: 10.1016/j.addr.2017.05.007.
    [4]
    MARTÍ-RODRIGO A, ALEGRE F, ÁB MORAGREGA, et al. Rilpivirine attenuates liver fibrosis through selective STAT1-mediated apoptosis in hepatic stellate cells[J]. Gut, 2020, 69( 5): 920- 932. DOI: 10.1136/gutjnl-2019-318372.
    [5]
    RUPAIMOOLE R, SLACK FJ. MicroRNA therapeutics: Towards a new era for the management of cancer and other diseases[J]. Nat Rev Drug Discov, 2017, 16( 3): 203- 222. DOI: 10.1038/nrd.2016.246.
    [6]
    JIANG XP, AI WB, WAN LY, et al. The roles of microRNA families in hepatic fibrosis[J]. Cell Biosci, 2017, 7: 34. DOI: 10.1186/s13578-017-0161-7.
    [7]
    LIN HY, WANG FS, YANG YL, et al. MicroRNA-29a suppresses CD36 to ameliorate high fat diet-induced steatohepatitis and liver fibrosis in mice[J]. Cells, 2019, 8( 10): 1298. DOI: 10.3390/cells8101298.
    [8]
    SONG L, CHEN TY, ZHAO XJ, et al. Pterostilbene prevents hepatocyte epithelial-mesenchymal transition in fructose-induced liver fibrosis through suppressing miR-34a/Sirt1/p53 and TGF-β1/Smads signalling[J]. Br J Pharmacol, 2019, 176( 11): 1619- 1634. DOI: 10.1111/bph.14573.
    [9]
    LU TX, ROTHENBERG ME. MicroRNA[J]. J Allergy Clin Immunol, 2018, 141( 4): 1202- 1207. DOI: 10.1016/j.jaci.2017.08.034.
    [10]
    LOU WY, LIU JX, GAO YJ, et al. MicroRNA regulation of liver cancer stem cells[J]. Am J Cancer Res, 2018, 8( 7): 1126- 1141.
    [11]
    ROEHLEN N, CROUCHET E, BAUMERT TF. Liver fibrosis: Mechanistic concepts and therapeutic perspectives[J]. Cells, 2020, 9( 4): 875. DOI: 10.3390/cells9040875.
    [12]
    EZHILARASAN D, SOKAL E, NAJIMI M. Hepatic fibrosis: It is time to go with hepatic stellate cell-specific therapeutic targets[J]. Hepatobiliary Pancreat Dis Int, 2018, 17( 3): 192- 197. DOI: 10.1016/j.hbpd.2018.04.003.
    [13]
    LI J, CHAN MC, YU Y, et al. MiR-29b contributes to multiple types of muscle atrophy[J]. Nat Commun, 2017, 8: 15201. DOI: 10.1038/ncomms15201.
    [14]
    CAI FF, BIAN YQ, WU R, et al. Yinchenhao decoction suppresses rat liver fibrosis involved in an apoptosis regulation mechanism based on network pharmacology and transcriptomic analysis[J]. Biomed Pharmacother, 2019, 114: 108863. DOI: 10.1016/j.biopha.2019.108863.
    [15]
    HUANG YH, TIAO MM, HUANG LT, et al. Activation of miR-29a in activated hepatic stellate cells modulates its profibrogenic phenotype through inhibition of histone deacetylases 4[J]. PLoS One, 2015, 10( 8): e0136453. DOI: 10.1371/journal.pone.0136453.
    [16]
    GUO CJ, PAN Q, LI DG, et al. MiR-15b and miR-16 are implicated in activation of the rat hepatic stellate cell: An essential role for apoptosis[J]. J Hepatol, 2009, 50( 4): 766- 778. DOI: 10.1016/j.jhep.2008.11.025.
    [17]
    ZHU XQ, SHAO P, TANG YC, et al. hsa_circRNA_100533 regulates GNAS by sponging hsa_miR_933 to prevent oral squamous cell carcinoma[J]. J Cell Biochem, 2019, 120( 11): 19159- 19171. DOI: 10.1002/jcb.29245.
    [18]
    LI HZ, ZHANG YW, XU YJ, et al. MiR-933 inhibits proliferation, migration and invasion of lung cancer cell lines by regulation of KLF6 gene[J]. China Oncol, 2021, 31( 7): 581- 588. DOI: 10.19401/j.cnki.1007-3639.2021.07.004.

    李海洲, 张艳炜, 许英杰, 等. miR-933调控KLF6基因影响非小细胞肺癌的作用研究[J]. 中国癌症杂志, 2021, 31( 7): 581- 588. DOI: 10.19401/j.cnki.1007-3639.2021.07.004.
    [19]
    ZHANG YT, MA YY, XU WH, et al. Association of microRNA-933 variant with the susceptibility to gastric cancer[J]. J BUON, 2017, 22( 2): 390- 395.
    [20]
    ZHOU B, GUO H, TANG J. Long non-coding RNA TFAP2A-AS1 inhibits cell proliferation and invasion in breast cancer via miR-933/SMAD2[J]. Med Sci Monit, 2019, 25: 1242- 1253. DOI: 10.12659/MSM.912421.
    [21]
    CHENG Y, SHI WW, CUI XD, et al. Long noncoding RNA TFAP2A-AS1 suppressed hepatitis B virus replication by modulating miR-933/HDAC11[J]. Dis Markers, 2022, 2022: 7733390. DOI: 10.1155/2022/7733390.
    [22]
    ISLAM ABMMK, MOHAMMAD E, KHAN MA. Aberration of the modulatory functions of intronic microRNA hsa-miR-933 on its host gene ATF2 results in type II diabetes mellitus and neurodegenerative disease development[J]. Hum Genomics, 2020, 14( 1): 34. DOI: 10.1186/s40246-020-00285-1.
    [23]
    RANE MJ, ZHAO YG, CAI L. Krϋppel-like factors(KLFs) in renal physiology and disease[J]. EBioMedicine, 2019, 40: 743- 750. DOI: 10.1016/j.ebiom.2019.01.021.
    [24]
    SYAFRUDDIN SE, RODRIGUES P, VOJTASOVA E, et al. A KLF6-driven transcriptional network links lipid homeostasis and tumour growth in renal carcinoma[J]. Nat Commun, 2019, 10( 1): 1152. DOI: 10.1038/s41467-019-09116-x.
    [25]
    LANG UE, KOCABAYOGLU P, CHENG GZ, et al. GSK3β phosphorylation of the KLF6 tumor suppressor promotes its transactivation of p21[J]. Oncogene, 2013, 32( 38): 4557- 4564. DOI: 10.1038/onc.2012.457.
    [26]
    CAI MG, SHAO W, YU HJ, et al. Paeonol inhibits cell proliferation, migration and invasion and induces apoptosis in hepatocellular carcinoma by regulating miR-21-5p/KLF6 axis[J]. Cancer Manag Res, 2020, 12: 5931- 5943. DOI: 10.2147/CMAR.S254485.
    [27]
    ZHANG N, YAN QQ, LU L, et al. The KLF6 splice variant KLF6-SV1 promotes proliferation and invasion of non-small cell lung cancer by up-regultating PI3K-AKT signaling pathway[J]. J Cancer, 2019, 10( 22): 5324- 5331. DOI: 10.7150/jca.34212.
    [28]
    CHIAM K, RYAN NK, RICCIARDELLI C, et al. Characterization of the prostate cancer susceptibility gene KLF6 in human and mouse prostate cancers[J]. Prostate, 2013, 73( 2): 182- 193. DOI: 10.1002/pros.22554.
    [29]
    GHIASSI-NEJAD Z, HERNANDEZ-GEA V, WOODRELL C, et al. Reduced hepatic stellate cell expression of Kruppel-like factor 6 tumor suppressor isoforms amplifies fibrosis during acute and chronic rodent liver injury[J]. Hepatology, 2013, 57( 2): 786- 796. DOI: 10.1002/hep.26056.
    [30]
    TIAN F, ZHAO JZ, BU SC, et al. KLF6 induces apoptosis in human lens epithelial cells through the ATF4-ATF3-CHOP axis[J]. Drug Des Devel Ther, 2020, 14: 1041- 1055. DOI: 10.2147/DDDT.S218467.
    • Relative Articles
    • Supplements(0)
    • Cited By
  • 加载中

Catalog

    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Figures(5)  / Tables(1)

    Get Citation
    PDF
    XML

    Article Metrics

    Article views (392) PDF downloads(33) Cited by()
    Proportional views
    Related
        

    The first journal specializing in hepatobiliary and pancreatic diseases in China

    Supervisor：Ministry of Education of the People's Republic of China

    Sponsor：Jilin University

    Academic Support: Chinese Society of Hepatology，Chinese Medical Association

    Address: 461 Xinjiang Road, Changchun

    Submit：0431-88782044

    Peer review：0431-88783542

    Email：lcgdb@vip.163.com

    About Journal

    Submission Guideline

    Journal Online

    Hepatobiliary College

    Guidelines

    YesterdayIP[]YesterdayPV[]TodayIP[]TodayPV[]Online[]

    Website Design © 2020 Editorial Board of Journal of Clinical Hepatology 吉ICP备10000617号-1 Supported by: Beijing Renhe Information Technology Co. Ltd  

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return