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CN 22-1108/R
Volume 40 Issue 7
Jul.  2024
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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.

     

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    • 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.
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