Despite significant improvements in the clinical treatment of PTCL, the 5-year survival rate for peripheral T-cell lymphoma remains low. In the context of the development of cancer therapy, epigenetics has received more and more attention. Abnormal epigenetic disorders, including DNA methylation, histone modification, and chromatin remodeling, play a key role in tumorigenesis[31–33]. Histone deacetylases (HDAC) and histone acetyltransferases (HATS) participate in epigenetic regulation of genes by controlling the acetylation status of lysine residues in the histone tail[34]. Histone acetyltransferases acetylate histone tails to form "open" chromatin structures that promote gene transcription execution; In contrast, histone deacetylases control the deacetylation of histone tails, keeping chromatin in a "closed" state and causing silencing of expression of related genes[35]. Under normal circumstances, there is a dynamic balance between HDAC and HATs, and the disruption of the balance is closely related to the occurrence and development of tumors. Histone deacetylases regulate the deacetylation of histones and certain non-histone proteins, including activation of tumor-priming related transcription factors and post-translational modification of key proteins including tumor suppressor genes [36, 37], and have become potential anti-cancer targets due to their close correlation with tumor cell proliferation, apoptosis, differentiation, migration and metastasis[38].
Currently, several molecules have been developed as HDAC inhibitors (HDACi) for the treatment of diseases such as cancer. In 2001, VPA was recognized as an HDACi and is currently considered a PAN inhibitor. Studies have found that VPA can exert its anticancer effects by changing the level of histone acetylation in tumor cells. Meanwhile, the expression level of anti-apoptotic protein survivin is down-regulated to cancel its anti-apoptotic effect and play a role in inducing apoptosis of tumor cells[39, 40]. Moreover, VPA can significantly down-regulate the expression of epithelial markers E-cadherin (E-cadherin) and zone-1 (ZO-1), and up-regulate the expression of mesenchymal markers Vimentin and N-cadherin in colorectal cancer cells[41]. It is suggested that VPA can trigger Epithelial-Mesenchymal Transition (EMT) in colorectal cancer cells. Therefore, in this experiment, we first examined the effects of VPA on the proliferation and apoptosis of PTCL cells. Our results demonstrated that VPA could significantly inhibit the proliferation and promote the apoptosis of A3 cells compared with the control group. Meantime, VPA could promote the expression of miR-3196 in a dose-dependent manner. Similarly, our results revealed that overexpression of miR-3196 inhibited proliferation and promoted apoptosis in A3 cells. Similar to previous studies, VPA and miR-3196 may have significant anticancer effects.
Mitochondria are important energy sources in the cell, and their morphology is a dynamic change process in the cellular process. Mitochondria are extremely vulnerable to the attack of reactive oxygen species, resulting in the destruction of the homeostasis of mitochondrial division and polymerization, and ultimately apoptosis[42, 43]. Studies have shown that histone deacetylase inhibitors can inhibit the formation of reactive oxygen species in tumor cells, promote the activation of caspase-8, caspase-9, caspase-3 and PARP, and ultimately lead to cell apoptosis[44]. We found that VPA and miR-3196 mimics significantly increased ROS, glucose and lactate content, and decreased ATP content in cells. Our results may reveal that VPA promotes apoptosis through the mitochondrial pathway.
The PI3K/AKT axis is the most frequently activated signaling pathway in human cancer and is often implicated in resistance to anticancer therapies[45]. Dysfunction of components of this pathway such as hyperactivity of PI3K, loss of function of PTEN, and gain-of-function of AKT, are notorious drivers of treatment resistance and disease progression in cancer[46]. A previous study demonstrated that PIK3 pathway has been shown to be highly activated in PTCL samples, high PIK3α expression was significantly associated with poor survival, even after adjustment for age, International Prognostic Index (IPI) score and anthracycline-based chemotherapy in first line[47]. Moreover, PTCL patients with high p-AKT expression showed aggressive clinical courses with significantly worse OS and PFS and a poor chemotherapy response rate[48]. Our results showed that VPA and miRNA-3196 significantly inhibited the activity of PI3K/AKT pathway in A3 cells. Moreover, VPA and miRNA-3196 had a significant synergistic effect to inhibit the activation of PI3K/Akt signaling pathway. In a word, VPA may inhibit the proliferation, and promote apoptosis, and regulate mitochondrial function, which may be achieved by regulating the miR-3196/PI3K/AKT pathway.
To further investigate the mechanism of miR-3196 promoting apoptosis of PTCL cells, we used bioinformatics methods to predict and verify the target genes of miR-3196. In this study, through GEO database and biological online software analysis such as miRTarBase and starbase2.0, and then luciferase, qRT-PCR and Western blot analysis, TWIK associated acid-sensitive Potassium channel subfamily K member 3 (TASK-1, also known as KCNK3) was confirmed to be a potential target gene of miR-3196. KCNK3 is a subtype of two-pore potassium channel. KCNK3 is extremely sensitive to various extracellular signal stimuli, resulting in changes in its expression and activity, such as PH, hypoxia, and intracellular signaling pathways[49]. Study found that the expression of KCNK3 in lung cancer is significantly increased, and overexpression of KCNK3 can promote the differentiation of A549 cells into tumor stem cells and promote the resistance of cells to gefitinib; meanwhile, overexpression of KCNK3 can promote the expression of CD133, OCT-4 and Nanog proteins, and promote EMT[50]. Down-regulation of KCNK3 inhibited EMT and reversed gefitinib sensitivity in lung cancer. Similarly, after siRNA interfered with the expression of KCNK3, apoptosis of A549 cells increased and cell proliferation decreased[51]. Inhibition of TASK-1 can significantly inhibit the proliferation of MCF-7 cells, and TASK-1 is required in MCF-7 cancer cell lines[52].
In this study, inhibition of KCNK3 expression significantly inhibited the proliferation and promoted their apoptosis of A3 cells. In combination with VPA, it further promoted the apoptosis and inhibited the proliferation of A3 cells. Interestingly, our results revealed that si-KCNK3 significantly inhibited the activity of PI3K/Akt pathway. A previous study demonstrated that the change of KCNK3 expression is closely related to the activation of PI3K/Akt pathway[53]. Moreover, an inhibitor of PI3-kinase could occlude the channel regulation function of TASK, revealing a correlation of TASK with PI3K[54]. At the same time, KCNK3 acted as the target gene of miR-3196, and VPA significantly promoted the expression of miR-3196. It is suggested that VPA regulates the proliferation and apoptosis of A3 cells is closely related to miR-3196/KCNK3/PI3K/Akt signaling axis.
In conclusion, our results showed that VPA could inhibit the proliferation and promote the apoptosis of A3 cells, and further studies showed that VPA might inhibit the expression and activation of PI3K/Akt signaling pathway by promoting the expression of miR-3196, and finally inhibit the proliferation and promote the apoptosis of PTCL cell. Through GEO database and biological online software analysis miRTarBase and starbase2.0, and then luciferase, qRT-PCR and Western blot analysis, KCNK3 was found to be a potential target gene of miR-3196. Furthermore, we speculated that by promoting the expression of miR-3196, VPA inhibits the expression of KCNK3, thus inhibiting the activation of PI3K/Akt pathway, and finally promoting the apoptosis and inhibiting the proliferation of PTCL cell. Our findings will facilitate the application of VPA in PTCL.