MicroRNA-16 participates in the cell cycle alteration of HepG2 cells induced by MC-LR

https://doi.org/10.1016/j.ecoenv.2020.110295Get rights and content

Highlights

  • The proliferation of HepG2 cells was promoted at the low dose of MC-LR exposure.

  • miR-16 expression was suppressed in the cells following MC-LR exposure.

  • Overexpression of miR-16 suppressed proliferation of HepG2 cells induced by MC-LR.

  • miR-16 may play a vital role in HepG2 cell cycle alteration after MC-LR exposure.

Abstract

Microcystin-LR (MC-LR) is a cyclic hepatotoxin produced by cyanobacteria in freshwater, and chronic MC-LR exposure could induce human hepatitis if consumed in drinking water. In recent years, many studies have indicated that microRNAs participate in the hepatotoxicity of MC-LR. The purpose of this study was to investigate the potential function of miR-16 in the hepatocellular toxicity and cell cycle alteration induced by MC-LR in human hepatocellular carcinoma (HepG2) cells after treatment with 10 μM MC-LR. The result of flow cytometry detection showed that a low concentration of MC-LR (10 μM) failed to induce apoptosis but promoted cell cycle G1/S transition in HepG2 cells. In addition, the expression of apoptosis-related genes was suppressed after MC-LR exposure. These results confirm that MC-LR exposure at a low dose can promote the proliferation of HepG2 cells. Furthermore, we also found that microRNA-16 (miR-16) expression was suppressed in HepG2 cells following MC-LR exposure. Hence, we overexpressed miR-16 in HepG2 cells and treated them with MC-LR, and the results showed that miR-16 overexpression induced an increase in the G0/G1 phase and a decrease in the S phase cell cycle populations in HepG2 cells, suggesting that miR-16 can inhibit the cell proliferation of HepG2 cells. In conclusion, our results suggest that miR-16 may play a vital role in the cell cycle alteration of HepG2 cells after MC-LR exposure.

Introduction

Microcystins (MCs) are harmful cyclic heptapeptide toxins produced by toxigenic cyanobacteria during cyanobacterial bloom that have received more attention in recent years (Zhang et al., 2019). More than 100 types of MC isomers have been identified, but Microcystin-LR (MC-LR) is the most commonly distributed and most toxic, accounting for 46–99.8% of the total MCs in freshwater (Yang et al., 2014; Han et al., 2017; Zhou et al., 2014). The World Health Organization (WHO) proposed that the provisional value for MC-LR is 1 μg/L in drinking water (WHO, 2011). Previous studies have shown that long-term exposure to MCs can induce liver damage, kidney damage, neurotoxicity, reproductive toxicity, and gastrointestinal disorders (Kawan et al., 2019; Kleinkauf and Dohren, 1996; Schmidt et al., 2014). The main mechanism of MC-LR hepatotoxicity is the specific inhibition of the activity of protein phosphatase 1 and 2A (MacKintosh et al., 1990), which induces oxidative stress (Ma et al., 2018). lipid peroxidation (Drobac et al., 2017) and DNA damage (Li et al., 2015).

MicroRNAs (miRNAs) are noncoding RNAs 18 to 24 nt in length that widely exist in various species, such as animals, plants, and protozoa (Ranganathan et al., 2013). These miRNAs negatively regulate the target messenger RNAs (mRNAs) by acting on the 3′-UTRs of the target genes via repressing protein translation or inducing mRNA degradation, resulting in a decreased level of gene expression (Kong et al., 2014). MicroRNAs play vital roles in physiological processes, including development, cell proliferation, and apoptosis (Zhou et al., 2018). Further ectopic expression of miR-15a and miR-16 can induce apoptosis in cell lines and suppress tumorigenesis in xenograft models of leukemia and solid tumors by targeting Bcl-2 and Mcl-1 (Calin et al., 2008; Cimmino et al., 2005). Moreover, research has shown that the apparent upregulation of miR-16a can suppress cell proliferation in the liver of challenged whitefish (Brzuzan et al., 2012). Based on the results of published research, miR-16 is involved in various pathways and acts by regulating different genes in diverse cancers, such as ovarian cancer, bladder cancer, non-small-cell lung cancer, and chronic lymphoid leukemia (CLL) (Jiang et al., 2013; Ke et al., 2013).

Since the liver is a key organ for MC-LR toxic responses, many studies have focused on screening hepatocytes as an in vitro experimental model. However, the isolation and culture of human primary hepatocytes is difficult (Ma et al., 2002). Liver cells isolated from different human bodies have individual differences, and the biggest limitation is that they are difficult to passage and culture for long periods of time (Zhou et al., 2009). To overcome the limitations associated with human primary hepatocytes, researchers have developed several hepatocyte-based cell models for experimental studies (Jetten et al., 2013). The HepG2 cell line is derived from human hepatocellular carcinoma; the cell line has many liver-specific functions and can be passaged indefinitely under standard culture conditions (Liguori et al., 2008). Therefore, these advantages make HepG2 cells a very popular model for in vitro toxicology tests. In terms of MC-LR toxicology experiments, HepG2 cells express OATPs required for MC-LR transport (Kullak-Ublick et al., 1996). Therefore, HepG2 cells have become a very suitable liver-derived cell model for detecting and evaluating the toxic effects of MC-LR.

Dysregulated expression of miRNAs in various organs may lead to the tumorigenesis of multiple toxicant-associated diseases (Sonkoly and Pivarcsi, 2011; Hou et al., 2011). The number of existing studies indicates that miRNAs have attracted attention in environmental toxicology (Ma et al., 2019). Moreover, many research reports have demonstrated that miRNAs might play a pivotal role in cellular responses to MC-LR exposure (Ma and Li, 2017; Lema and Cunningham, 2010). Accumulated evidence indicates that miRNAs might be involved in the toxicity mechanism of toxicants; hence, they might be earlier regulators than other genomic toxicological biomarkers (Shah et al., 2011; Vacchi-Suzzi et al., 2012). Hence, the purpose of the present study was to investigate the functional regulation of miR-16 on hepatocellular toxicity and cell cycle transformation induced by MC-LR exposure in HepG2 cells.

Section snippets

Cell culture and MC-LR exposure

Human hepatocellular carcinoma (HepG2) cells were provided by Xinxiang Medical College, China. HepG2 cells were passaged by 0.25% trypsin digestion and seeded into RPMI-1640 (Hyclone, USA) complete medium (containing 10% fetal bovine serum, 100 U/L penicillin, and 100 μg/L streptomycin) in a humidified atmosphere at 37 °C with 5% CO2 during the study. The cells were passaged once every three days and then treated with 10 μM MC-LR for 24 and 48 h. MC-LR (C49H74N10O12, ≥98% purity) was purchased

Effect of MC-LR exposure on the cell viability, cell cycle, and apoptosis of HepG2 cells

The viability of HepG2 cells after exposure to MC-LR is depicted in Fig. 1 A. MC-LR (0.5 and 10 μΜ) exposure for 24 h inhibited cell proliferation, but at 48 h, MC-LR exposure promoted cell proliferation. The results showed that MC-LR could affect the proliferation of HepG2 cells. Cell cycle assessment showed that MC-LR exposure for 24 h had no effect on the cell cycle by flow cytometry (Fig. 1 B: a, b). However, compared with the effects in the control groups, cell growth and cell cycle G0/G1

Discussion

There are an increasing number of reports expressing concern about the environmental safety of cyanobacteria toxins. One reason for the concern is that the toxins harm the health of living organisms and even human beings via drinking water. Previous studies showed that MC-LR exposure could cause apoptosis and cell cycle arrest (Campos and Vasconcelos, 2010; Žegura et al., 2003). A number of epidemiological studies have shown that drinking cyanobacteria-contaminated water under laboratory

Conclusion

In conclusion, this study demonstrated that low-dose MC-LR exposure promoted the proliferation of HepG2 cells and that the expression of apoptosis-related genes was suppressed following MC-LR exposure. Furthermore, miR-16 expression was also downregulated after MC-LR exposure. Our results also indicate that overexpression of miR-16 can suppress the proliferation of HepG2 cells following MC-LR treatment by altering the expression of p53, CDK6, PTEN, and c-myc. These results suggest that the role

Declaration of competing interest

The authors declare that there is no potential conflict of interest.

Acknowledgements

This research was supported by grants from the National Science Foundation of China (Grant No. 31702349), the Key Research Project of Henan Normal University in China (Grant No. 19zx011), and the Key Research and Development Promotion Project in Henan Province (Grant No. 202102310343).

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