MicroRNA-16 participates in the cell cycle alteration of HepG2 cells induced by MC-LR
Graphical abstract
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).
References (58)
- et al.
Expression profiling in vivo demonstrates rapid changes in liver microRNA levels of whitefish (Coregonus lavaretus) following microcystin-LR exposure
Aquat. Toxicol.
(2012) - et al.
The multiple roles of PTEN in tumor suppression
Cell
(2000) - et al.
Reversible tumorigenesis by MYC in hematopoietic lineages
Mol. Cell.
(1999) - et al.
Altered expression of p53, Bcl-2 and Bax induced by microcystin-LR in vivo and in vitro
Toxicon
(2005) - et al.
Environmental chemicals and microRNAs
Mutat. Res. Mol. Mech. Mutagen.
(2011) - et al.
Recovery of reproductive function of female zebrafish from the toxic effects of microcystin-LR exposure
Aquat. Toxicol.
(2019) - et al.
Downregulation of miR-16 promotes growth and motility by targeting HDGF in non-small cell lung cancer cells
FEBS Lett.
(2013) - et al.
A kinase-independent function of CDK6 links the cell cycle to tumor angiogenesis
Canc. Cell
(2013) - et al.
Molecular and functional characterization of bile acid transport in human hepatoblastoma HepG2 cells
Hepatology
(1996) - et al.
MicroRNAs and their implications in toxicological research
Toxicol. Lett.
(2010)
Microcystin-LR promotes proliferation by activating Akt/S6K1 pathway and disordering apoptosis and cell cycle associated proteins phosphorylation in HL7702 cells
Toxicol. Lett.
Oxidative stress-mediated p53/p21WAF1/CIP1 pathway may be involved in microcystin-LR-induced cytotoxicity in HepG2 cells
Chemosphere
Fish and Shell fi sh Immunology Chronic exposure to the ionic liquid [ C 8 mim ] Br induces in fl ammation in silver carp spleen : involvement of oxidative stress-mediated p38MAPK/NF- κ B signalling and microRNAs
Fish Shellfish Immunol.
Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants
FEBS Lett.
Pseudolaric acid B^|^ndash;Induced autophagy contributes to senescence via enhancement of ROS generation and mitochondrial dysfunction in murine fibrosarcoma L929 cells
J. Pharmacol. Sci.
The multitudinous role of microRNAs in various biological systems
J. Pharm. Res.
CDK6 as a key regulator of hematopoietic and leukemic stem cell activation
Blood
MicroRNAs in inflammation and response to injuries induced by environmental pollution
Mutat. Res. Mol. Mech. Mutagen.
Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2
Toxicon
Elimination kinetics and detoxification mechanisms of microcystin-LR during UV/Chlorine process
Chemosphere
MicroRNA regulation of Toll-like receptor signaling pathways in teleost fish
Fish Shellfish Immunol.
Dual targeting of p53 and c-MYC selectively eliminates leukaemic stem cells
Nature
MiR-15a and miR-16-1 cluster functions in human leukemia
Proc. Natl. Acad. Sci. Unit. States Am.
Molecular mechanisms of microcystin toxicity in animal cells
Int. J. Mol. Sci.
MYC regulates the antitumor immune response through CD47 and PD-L1
Science 84
Changing rates for liver and lung cancers in qidong, China
Chem. Res. Toxicol.
miR-15 and miR-16 induce apoptosis by targeting BCL2
Proc. Natl. Acad. Sci. Unit. States Am.
Microcystin accumulation and potential effects on antioxidant capacity of leaves and fruits of Capsicum annuum
J. Toxicol. Environ. Health Part A
Health risk assessment of cyanobacterial (Blue-green algal) toxins in drinking water
Int. J. Environ. Res. Publ. Health
Cited by (14)
Microcystin-LR-induced autophagy via miR-282–5p/PIK3R1 pathway in Eriocheir sinensis hepatopancreas
2023, Ecotoxicology and Environmental SafetyMicrocystin-leucine arginine exhibits adverse effects on human aortic vascular smooth muscle cells in vitro
2022, Toxicology in VitroCitation Excerpt :A low concentration of MC-LR failed to induce apoptosis but promoted cell cycle G1/S transition in HepG2 cells. miR-16 may play a vital role in the cell cycle alteration of HepG2 cells after MC-LR exposure (Feng et al., 2020). S-phase is a critical period for the synthesis of DNA and histone proteins, in which histone deacetylase (HDAC) affects the binding of DNA and histones to induce cell cycle arrest (Lucchini and Sogo, 1995).
Gene expression of glutathione S-transferase alpha, glutathione S-transferase rho, glutathione peroxidase, uncoupling protein 2, cytochrome P450 1A, heat shock protein 70 in liver of Oreochromis niloticus upon exposure of microcystin-LR, microcystin-RR and toxic cyanobacteria crude
2022, Gene ReportsCitation Excerpt :Subsequent studies reported the occurrence of MCs hepatotoxicity referred to inhibition of the serine/threonine protein phosphatases (PP1 and PP2A) activities or altering the expression levels of microRNAs (Yang et al., 2018). Recently, Feng et al. confirmed that MC-LR exposure at a low dose promote the proliferation of human hepatocellular carcinoma cells (Feng et al., 2020). MCs can also induce hepatotoxicity by production of reactive oxygen species (ROS), superoxide anion, hydrogen peroxide and hydroxyl radical (Puerto et al., 2009).
Metatranscriptomic insight into the effects of antibiotic exposure on performance during anaerobic co-digestion of food waste and sludge
2022, Journal of Hazardous MaterialsCitation Excerpt :Besides, different techniques have been widely used to investigate the mechanism of functional communities, such as by culturing (Kong et al., 2018), next-generation sequencing (Wang et al., 2020a), qPCR (Tao et al., 2020) and metagenomic sequencing (Lei et al., 2019). Although functional microorganisms during AcoD have been identified by these techniques, the existence of functional genes to the change of environmental conditions could not reflect the functional activities, the metabolic functions respond to environmental change mainly reflect via the proteins, which were timely translated through mRNA (Cai et al., 2021; Feng et al., 2020). Therefore, the focus on functional expressions of related microorganisms was essential to realize how active microorganisms and related functions affecting AcoD performance.
Dissemination of antibiotic resistance under antibiotics pressure during anaerobic co-digestion of food waste and sludge: Insights of driving factors, genetic expression, and regulation mechanism
2022, Bioresource TechnologyCitation Excerpt :Therefore, studies on the variation of ARGs expression under antibiotics exposure and how antibiotics regulating ARGs variation during AcoD are very necessary. Although numerous ARGs in host-associated environments have been identified by qPCR and metagenomic sequencing approaches, these approaches provided limited information regarding the ARGs activity (at the RNA level) with strong dissemination ability for further assessing the potential risk (Feng et al., 2020; Wang et al., 2020a,b,c). In this study, qPCR and metatranscriptomic analyses were conducted to explore the effects of antibiotics with environmentally relevant concentration (5 mg/L) (Li et al., 2013; Martín et al., 2015) and higher concentrations (50 mg/L, probably exhibited noticeable effect) on ARGs dissemination during AcoD.