In the previous researches, borax ore and pure refined borax were all cytotoxic in cultured C3H/lOT1/2 cells, V79 Chinese hamster cells, human HeLa cells, and human foreskin fibroblasts at high (mg/ml) concentrations [23], which was similar to the range of toxic concentrations in animal feeding experiments [24]. It also showed that borax had toxicity, including acute toxicity, neurotoxicity, reproductive, and developmental toxicity. However, borax is a low toxicity, which is used in manufacturing, pesticides, fertilizers, and pharmaceuticals [25]. Currently, more and more research on the therapeutic effect of the borax, mainly including anti-tumor effect and affecting the tumor growth, development and metastasis [26, 27]. A great number of studies have concentrated on the application of borax for tumor prevention and illustrated a strong inverse correlation between borax and various types of cancer, including lung cancer, cervical cancer, prostate cancer and hepatocellular carcinoma (HCC) [2, 8, 9]. But, the underlying mechanisms of anti-tumor effect remain unidentified and borax administered altered the expression of miRNAs in HepG2 cells has been reported less.
In the present study, microarray analysis showed that the expression levels of 4 miRNAs were upregulated in HepG2 cells in the 2‑h treatment group. such as, miR-21-5p may be useful as a predictor of recurrence in young gastric cancer and breast cancer patients. This miRNA was combined with clinicopathological factors, which would allow patient prognoses to be more accurately predicted. 24‑h treatment with borax upregulated the expression of several miRNAs, including miR-125b-2-3p, which may affect the G2/M phase of the hepatocellular carcinoma cell cycle through the regulation of its target genes and provide early diagnosis and novel treatment of hepatocellular carcinoma [28]. miR-422a, acts as a tumor suppressor, inhibited cell invasion, proliferation, and migration by targeting PI3K/Akt signal pathway [29]. miR-34a-3p is an independent biomarker of recurrence and a factor to improve predictive value of cancer and treatment [30]. Furthermore, to examine the expression of altered miRNAs, we applied volcano plot and heatmaps analyses to uncover the presence of differentially expressed miRNAs in the borax treatment group.
To precision select the target genes affected by miRNAs, GO enrichment analyses showed that the processes significantly altered by borax involved biological regulation, cell junction, metabolism, and protein binding. Furthermore, KEGG pathway enrichment analysis showed that the target genes of differentially expressed miRNAs in HepG2 cells following borax treatment for 2 h major participated in MAPK signaling pathway, TGF-beta signaling pathway, NF-kappa B signaling pathway, cAMP signaling pathway, etc; MAPK signaling pathway, a mitogen-activated protein kinase pathway, which is generally involved in cell proliferation, apoptosis and differentiation [31]; TGF-β signaling (TGF-beta signaling) is often activated and not attenuated during the malignancy of hepatocellular carcinoma cells, so TGF-β could be a therapeutic target for the treatment of liver cancer [32]. NF-kappa B, an ubiquitous transcription factor in mammals, which enters into cellular nucleus and enhances gene expression by binding to κB site within promoter. NF-κB signaling is associated with cell inflammation, growth and differentiation and is aberrant activated frequently in numerous kinds of cancer including liver cancer and it has also been served as a target for treatment. [33]; cAMP signaling pathway, 3′-5′-cyclic adenosine monophosphate (cAMP), as a second messenger, is the key role in mediating many cellular responses. Furthermore, the study found that the cancer cell migration and survival was inhibited by application of cAMP to cancer cell [34].
Ras signaling pathway, FoxO signaling pathway, Cellular senescence, and Neurotrophin signaling pathway, etc; involved the 24‑h treatment group. Ras signaling pathway is activated in the majority of advanced HCC, and if it is inhibited that could effectively suppress the proliferation, migration, and invasion of HCC [35]. FoxO proteins, including FoxO1a and FoxO3a, are involved in multiple fundamental cellular activities and acting in transcriptional activities related to cell stress response, proliferation, and apoptosis, the tumor cells growth arrest and apoptosis are induced through activation of FoxO transcription factors [36]. Cellular senescence is a cell fate that triggered by oxidative stress, oncogene activation, and DNA damage and is an important tumor-suppression mechanism [37]. Neurotrophin signaling pathway, exerts a range of effects in the control of cell migration and proliferation in non-neuronal cells, including in cancer. Furthermore, neurotrophin binding to p75NTR that activates the c- JNK signaling cascade, which results in activation of p53 and expression of pro-apoptotic genes such as Bcl-2. Neurotrophin and their receptors have been investigated in human cancers and were over-expressed in ovarian, breast, liver, and pancreatic malignancies [38]. In present study, target genes of differentially expressed miRNAs induced by borax also involved alterations in those pathways, indicating that borax-mediated antitumour effect might involve changes in these pathways, specially in 24‑h borax exposed HepG2 cells. Thus, the study revealed that miRNAs might play an important role in development of liver cancer. The possibility exists that borax induced antitumor effects also involves differential expression of miRNAs based on our results.