CSCs have the characteristics of carcinogenicity, multiple differentiation and self-renewal. Some scholars believe that tumor recurrence, metastasis and resistance to radiotherapy and chemotherapy were due to the existence of tumor stem cells . The first solid tumor to prove the existence of CSCs was BC; Al-Hajj et al. proved that CD44+/CD24−/(low)–lineage cells isolated from BC tissue can form tumors in immunodeficient mice . Studies have shown that in CRC, CSCs expressing CD133+ and CD44+/CD166+ are more resistant to treatment ; CD133 is also a surface marker of pancreatic-cancer (PC) stem cells [29, 30]. CSCs rich in CD133+ and CD44+/cMet+ in PC are also more prone to drug resistance . This research on CSCs is expected to be applied to clinical treatment. Hedgehog (Hh), Wingless/Integrated (Wnt), Notch and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway inhibitors can inhibit the renewal of PC tumor stem cells; the pluripotency of stem cell factors such as Pituitary–Octamer–Unc-86 (POU) class 5 homeobox 4 (OCT4), SOX2, cellular myelocytomatosis oncogene (c-Myc) and Nanog; and tumor growth in vivo and in vitro . CD133+/CD44+ liver cancer stem cells show stem cell characteristics such as extensive proliferation, self-renewal and the ability to differentiate into cancer cells. Therefore, some researchers believe blocking CD44 signal transduction with anti-CD44 antibodies could be a potential strategy to eradicate hepatic CSCs and cure patients . Exploring the origin and markers of CSCs involved in the occurrence and growth will help us better judge the prognoses of patients with tumors and more accurately target therapies. In the early stage of this study, our group used Piwil2 to reprogram human FBs in order to establish Piwil2-iCSCs that stably expressed Piwil2-GFP. The cell biological-behavior test and tumor stem cell marker test confirmed that the reprogrammed cells were tumor-like stem cells , which could be used in an ideal in vitro CSCs model.
A piRNA is a small non-coding RNA (sncRNA) 23–31 nt long; it is characterized by common monophosphate uracil at the 5′-end and methylation modification at the 3′-end . After piRNA is combined with Piwi proteins (including Piwil2 and Piwil4), it inhibits the function of transposon via the ping-pong effect and stabilizes genes. In addition, piRNA can participate in gene regulation via DNA methylation and histone modification [12–14]. Many studies have proven that abnormally expressed piRNA is related to lung, liver, stomach, colorectal, bladder and breast cancers.
In the in vitro CSCs model constructed by reprogramming FBs with Piwil2, we speculated that the expression and functional changes of related piRNAs caused by overexpression of the Piwil2 gene might play important roles in the process of tumorigenesis, and the differentially expressed piRNA might affect tumor tissues. Therefore, the differentially expressed piRNAs could be of great value for exploring the mechanism underlying the occurrence and promotion of CSCs.
In this study, we screened 204 differentially expressed piRNAs of Piwil2-iCSC using HTS technology, and a total of 77 target genes were predicted by the miRanda algorithm. GO analysis of the selected target genes with differential expression of piRNAs revealed that the main BP regulated cell calcium ion concentration and the main MF participated in ATPase activity. After we used STRING to analyze the protein interaction network, the results showed the highest combined score of interaction between RPS8 and RPS29 (0.999) and the second-highest between RPS8 and NOP56 (0.994). MYO9A and PTPRD each had seven interacting proteins, representing the strongest interaction points. Based on these bioinformatics data, we speculated that the role of piRNA in CSCs might be related to energy metabolism. Many studies have shown that tumors and their stem cells have abnormalities in energy metabolism. As they have significantly fewer mitochondriaes than normal cells, tumors and CSCs mainly produce energy via glycolysis [27, 28]. It is speculated that piRNA regulates the energy metabolism of CSCs by targeting its target genes or by some other mechanism.
Next, we chose piRNA MW557525 to further study the function of piRNAs in CSCs. Via WB, qRT-PCR, CCK-8, Transwell and FCM assays, we found that piRNA MW557525 could promote proliferation, migration and invasion and inhibit apoptosis in Piwil2-iCSCs, which suggested that it could trigger CSCs occurrence and progression. By detecting the expression of CD24, CD133, KLF4 and SOX2 via WB, we found that piRNA MW557525 could upregulate these markers of stem cell pluripotency, meaning that it could increase Piwil2-iCSCs pluripotency and decrease CSCs differentiation. Therefore, we think that piRNA MW557525 could be an important factor in the occurrence and maintenance of CSCs. Interestingly, we detected high expression of NOP56 in Piwil2-iCSC. Regarding this phenomenon, we believe that increasing the amount of Piwil2 in cells not only changes the expression of MW557525, but also regulates other genes that can target NOP56, thereby increasing the expression of NOP56 at the overall level.
Via the miRanda algorithm, we predicted NOP56 as the target gene of piRNA MW557525. NOP56 participates in the modification of rRNA precursor ribose methylation and is a component of the small-molecule nucleolar ribonucleoprotein (snoRNP) complex . Studies have found that its abnormality is related to the Myc gene mutation in Burkitt lymphoma . Some scholars have shown that NOP56 could be a promising potential therapeutic target in CRC . In our study, silencing NOP56 could promote proliferation, migration and invasion and inhibit the apoptosis of Piwil2-iCSCs. We found that upregulation of MW557525 decreased the expression of NOP56 and vice versa, and a dual LRA confirmed that NOP56 was negatively related by piRNA MW557525 and indirectly modulated by MW557525.
Many studies have shown that Piwi and piRNA regulate embryogenesis and somatic-cell development in many organisms. The ping-pong effect of piRNA and Piwi protein can consume transposable factors, thereby ensuring the stability of key genes. The piRNA pathway has been proven to influence cell stemness in primitive animals with high regenerative ability, as well as in germ lines and somatic stem cells of other animals [34, 35]. In planarians and ascidians, Piwi is an essential gene for maintaining function in pluripotent stem cells (PSCs) . In Drosophila, Piwi affects somatic-cell function through epigenetic and post-transcriptional regulation [37, 38]. Piwi/piRNA complex has also been shown to be related to brain development, memory formation and translation of neuronal damage . Meanwhile, some scholars believe that the Piwi protein family can enhance the pluripotency of mammalian cells, but it is dispensable for mouse somatic development and for reprogramming FBs into PSCs . The results of this study showed that upregulating piRNA MW557525 alone could enhance the stemness of cells. We speculate that piRNA maintains cell stemness through Piwi/piRNA complex in vitro, and that it has other ways of doing so such as direct or indirect regulation of targeted genes.
In addition to targeting related genes, piRNA has many other means of regulating the genes of tumor/cancer stem cells. Many studies have reported that piRNA is involved in tumorigenesis, most commonly by regulating the function of transposons by combining with Piwi protein to affect tumorigenesis . In addition, piRNA and Piwi/piRNA complex can also regulate tumor growth by targeting certain protein-coding genes, such as recruiting DNA methyltransferase (DNMT) to regulate DNA methylation. . Therefore, we speculate that piRNA MW557525 indirectly affects NOP56 by other means or through other elements, rather than simply targeting it directly.
This research innovatively sequenced cancer stem cells constructed artificially to obtain unknown differentially expressed piRNA.MW557525 is one of the unknown differentially expressed piRNA and its target gene is predicted to be NOP56.We conduct cell experiments to prove the function of unknown piRNA and its relationship with the predicted target gene NOP56.In conclusion, our research demonstrated that piRNA MW557525 could promote and maintain the vitality and pluripotency of Piwil2-iCSCs while NOP56 inhibited the vitality and the stemness of Piwil2-iCSCs.PiRNA MW557525 was negatively associated with NOP56 in Piwil2-iCSCs.Silencing the target gene NOP56 could also promote and maintain the vitality and pluripotency of Piwil2-iCSCs. This finding provides a novel focus, piRNA, in the exploration of CSCs’ mechanism. We next plan to research the process by which piRNA MW557525 targets NOP56, as well as MW557525’s effect on normal or cancer cells.