MicroRNA-638 induces apoptosis and autophagy in human liver cancer cells by targeting enhancer of zeste homolog 2 (EZH2)

https://doi.org/10.1016/j.etap.2020.103559Get rights and content

Highlights

  • MicroRNA-638 is downregulated in liver cancer tissues and cell lines.

  • Overexpression of miR-638 inhibits liver cancer cell proliferation.

  • Overexpression of miR-638 induces apoptosis and autophagy in liver cancer cells.

  • MicroRNA-638 exerts its effects by targeting enhancer of zeste homolog 2(EZH2).

Abstract

Liver cancer is of the devastating human cancers and its incidence is increasing at an alarming rate. The clinical outcomes are far from descent due to lack of efficient therapeutic targets and chemotherapeutic agents. Studies have revealed the therapeutic implications of microRNAs in the management of different human cancers. This study was designed to explore the role and therapeutic potential of miR-638 in liver cancer via modulation of zeste homolog 2 (EZH2). The results revealed significant (P < 0.05) downregulation of miR-638 in human liver cancer tissues and cell lines. Overexpression of miR-638 led to a significant (P < 0.05) decline in liver cancer cell proliferation. Nonetheless, inhibition of miR-638 could promote the proliferation of the human liver cancer cells. The DAPI and annexin V/PI staining assays revealed that miR-638 induces apoptosis in human liver cancer cells which was accompanied by enhancement of Bax and depletion of Bcl-2 expression. Furthermore, miR-638 overexpression also leads to a significant (P < 0.05) increase of autophagosomes and autolysosomes in liver cancer cells suggestive of autophagy. The induction of autophagy was further confirmed by increase and decrease in expression of LC3B-II and Beclin-1 proteins, respectively. In contrary, inhibition of miR-638 prevented both apoptosis and autophagy of the liver cancer cells. In silico analysis and the dual luciferase assay revealed EZH2 as the molecular target of miR-638 at post-transcriptional level. The qRT-PCR showed that EZH2 to be significantly (P < 0.05) upregulated in the human liver cancer tissues and cell lines. However, the expression of EZH2 was considerably suppressed upon miR-638 overexpression in SNU-423 cells. Taken together, these findings suggest the tumor-suppressive role of miR-638/EZH2 axis liver cancer and point towards the potential of miR-638 as therapeutic target in the treatment of liver cancer.

Introduction

Liver cancer is one of the devastating human cancers and ranked as the 5th most prevalent type of cancer across the globe (Akinyemiju et al., 2017). Causing significant number of deaths, it is ranked 3rd in terms of mortality and morbidity (Asrani et al., 2019). The liver cancer treatments include partial hepatectomy, liver transplantation together with procedures like ablation or embolization. Chemo/immune- and/or radio-therapies are used against the advanced stage disease (Anwanwan et al., 2020). However, the clinical outcomes of these treatments are far from descent which necessitate the unraveling of the molecular events, controlling the development and progression of liver cancer for the development of efficient treatment strategies. Of the various molecular factors which have been shown to regulate the onset and subsequent progression of of human cancers, the microRNAs (miRs) have emerged as pivotal players in controlling the tumor growth and proliferation (Sun et al., 2014). The miRs are relatively small group of regulatory RNAs which play key regulatory roles in various biological processes in human body (Zhang, 2008). The miRs affect the vital physiological processes like proliferation of eukaryotic cells, cell differentiation and apoptotic cell death pathways (Hwang and Mendell, 2006). As per the recent research findings, the dysregulation of miRs is has been linked with the development of several cancer and thus are considered as crucial biomarkers for cancer prognosis (Hata and Kashima, 2016). They have also been shown exhibit therapuetic applications in controlling the progression and angiogenesis of many cancers (Costouros et al., 2002). Several studies have revealed the regulatory role of different miRs in liver cancer progression and metastasis (Greene et al., 2013). The miR-638 has been previously shown to exert profound regulatory roles in human cancers like colorectal cancer and gastric cancer (Zhang et al., 2014; Zhao et al., 2014). Studies have also shown miR-638 downregulation in liver cancer (Shi et al., 2018). The miR-638 has been shown to regulate the cell invasion and epithelial-to-mesenchymal transition of liver cancer through post-transcriptional targeting of SOX2 (Zhang et al., 2017). Furthermore, miR-638/VEGF axis has been shown to control the liver cancer angiogenesis (Cheng et al., 2016). Nonetheless, the role of miR-638 on the growth of human of liver cancer via modulation of zeste homolog 2 (EZH2) expression is still unknown. The present study was therefore designed to investigate the role of miR-638/EZH2 axis in liver cancer growth and to unravel the underlying mechanisms.

Section snippets

Tissue samples

The liver cancer tissues, and the normal adjacent tissues were collected prior to chemotherapy at first affiliated hospital of Zhengzhou University from the year June 2018 to June 2019. The general characteristics of the patients are presented in Table 1. The human tissue specimens were obtained under the proper ethical guidelines and written consent was taken from the patients before obtaining the specimens. The study was approved by the Institutional ethics under approval number HZU-401HT-19.

miR-638 is downregulated in liver cancer

The expression level of miR-638 was examined in 25 clinical samples of each cancerous and normal surrounding liver tissues using qRT-PCR method. The results showed that miR-638 is significantly (P < 0.05) downregulated in liver tissues (Fig. 1A). Similarly, the miR-638 expression was also deduced in the cancer cell lines (SNU-182, SNU-423, SNU-449 and SNU-387) and in the normal human cell line (THLE-3). All four liver cancer cell lines exhibited markedly lower transcript levels of miR-638 in

Discussion

Human liver cancer causes significant number of human mortalities and morbidities worldwide (Wong et al., 2017). The overall 5-year survival rates of liver cancer are exceptionally low signifying the necessity of formulating more competent anti-cancer stratagems against this fatal malignancy (Lepage et al., 2015). Recent studies have laid tremendous focus on understanding the various molecular mechanisms regulating the proliferation of liver cancer (Aravalli et al., 2008). Among the different

Conclusion

The results of the present study revealed that miR-638 is significantly downregulated in human liver cancer. Overexpression of miR-638 exerts tumor suppressive effects via induction of apoptosis and autophagy. The study also revealed the involvement of EZH2 in the miR-638 exerted tumor suppressive effects. Taken together, the findings point towards the therapeutic potential of miR-638/EZH2 axis in the management of liver cancer.

CRediT authorship contribution statement

Hongyu Zhang: Conceptualization, Methodology, Software, Data curation, Writing - original draft, Writing - review & editing. Hongxia Liang: Data curation, Writing - original draft. Shuhuan Wu: Data curation, Writing - original draft. Yingying Zhang: Data curation, Writing - original draft. Zujiang Yu: Conceptualization, Methodology, Software, Supervision, Writing - review & editing.

Declaration of Competing Interest

The authors declare that there are no conflicts of interest

References (30)

  • R.N. Aravalli et al.

    Molecular mechanisms of hepatocellular carcinoma

    Hepatol.

    (2008)
  • S. Chang et al.

    Hypoxic reprograming of H3K27me3 and H3K4me3 at the INK 4A locus

    FEBS Lett.

    (2016)
  • J. Cheng et al.

    Downregulation of miRNA-638 promotes angiogenesis and growth of hepatocellular carcinoma by targeting VEGF

    Oncotarget

    (2016)
  • N.G. Costouros et al.

    Molecular imaging of tumor angiogenesis

    J. Cell. Biochem.

    (2002)
  • H.Y.C. Emily et al.

    BCL-2, BCL-XL sequester BH3 domain-only molecules preventing BAX-and BAK-mediated mitochondrial apoptosis

    Mol. Cell

    (2001)
  • Cited by (21)

    • Epigenetic regulation of autophagy by non-coding RNAs in gastrointestinal tumors: Biological functions and therapeutic perspectives

      2023, Pharmacological Research
      Citation Excerpt :

      miR-638 suppresses EZH2 signaling, and then, expression levels of LC3B-II and Beclin-1 enhance to mediate autophagy. Silencing miR-638 was associated with the inhibition of apoptosis and autophagy [210]. Autophagy status is closely associated with invasion and metastasis of HCC cells.

    • SASH1 knockdown suppresses TRAF6 ubiquitination to regulate hemangioma progression by mediating EZH2 degradation

      2022, Experimental Cell Research
      Citation Excerpt :

      Increasing evidences proved that targeting EZH2 was an attractive treatment for cancer. For example, EZH2 promoted autophagy and apoptosis in liver cancer cells [41]. The overexpression of EZH2 contributed to chemoresistance in ovarian cancer [42].

    • EZH2 as a new therapeutic target in brain tumors: Molecular landscape, therapeutic targeting and future prospects

      2022, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      Fig. 1 demonstrates EZH2 signaling and related molecular pathways. Increasing evidence demonstrates the role of EZH2 in cancer and its ability in regulating molecular landscapes that are responsible for tumor growth and aggressive behavior [30–35]. The EZH2 overexpression prevents apoptosis and autophagy in liver tumor to enhance progression.

    • Overview of micro-RNA

      2022, MicroRNA: From Bench to Bedside
    View all citing articles on Scopus
    View full text