PIWI-interacting RNA (piRNA): a narrative review of its biogenesis, function, and emerging role in lung cancer

piRNAs save the germ cells from mutations by TE. The mechanism of piRNA biogenesis in Drosophila and many other species is itself a process of degrading TE at the post-transcriptional level. Mutations in Aub and Ago3 lead to an elevated level of transposons in the germ cells [78]. As discussed earlier for the ping-pong pathway, a piRNA targets a transposon with opposite orientation and degrades it into a new piRNA. Together these processes serve to slice TE and amplify piRNA. The transposon-containing regions of the DNA generate piRNAs, which in turn upon maturation, recruit Piwi and other proteins to silence the TE region [79, 80]. Other species, such as mice and zebrafish, also show a similar function for their PIWI family proteins. Knocking down of Piwi orthologs increased the abundance of transposons in these species [81].

Piwi and Aub conduct position effect variegation in which a stretch of euchromatin is converted to heterochromatin to variable extents among cells within the same tissue [82]. Piwi interacts with heterochromatin protein 1a (HP1a) to promote heterochromatin formation [83]. It is shown by studies in Drosophila that Piwi helps in loading the H3K9Me3 mark on DNA and converts stretches of DNA into heterochromatin. So, the loss of Piwi makes TE available for pol II [84]. In mice, Mili and Miwi2 (mouse orthologs of PIWI) together promote retrotransposon silencing by CpG DNA methylation in male germ-line cells and piRNA alone can also regulate DNA methylation in these germ cells [81, 85].

PIWI proteins have stem cell maintaining properties for which their role in germline development and maintenance is well observed. Knockdown studies have shown anomalies in development. For instance, in Drosophila both male and female piwi protein-coding gene mutants fail to form primordial germ cells and renew germline stem cells, which leads to sterility [67]. Aub protein-coding gene mutation leads to compromised PTGS and DNA damage accumulation also resulting in sterility [86]. Females with mutant ago3 lay fewer eggs and most of the time are sterile [87]. Similar results were also obtained from studies on mice. PIWI orthologs, Mili, Miwi2, and Miwi mutant mice show defects in PTGS and spermatogenesis [88]. Zebrafish PIWI-like proteins Zili and Ziwi mutations decrease the number of germ cells and increase apoptosis of germ cells respectively [89]. Mutations in other piRNA biogenesis proteins such as Tudor, Vret, and Tej also result in DNA damage and defects in development [74, 79]. piRNA clusters from the chromosomes of Drosophila germ cells play a crucial role in maintaining the integrity of the telomeric regions. Loss of piRNAs in germ cells results in decreased levels of HP1a, Rhino and H3K9Me3 association with the telomeric region and disputed nuclear positioning of the telomere [90]. In silkworms and other insects, PIWI has a role in sex determination. Loss of Bmsiwi (insect PIWI) and histone methyltransferase BmAsh2 causes female-to-male sexual reversal [91].

piRNAs can be formed from the 3′UTR region of many protein-coding genes such as vas, traffic jam, and nanos and control the mRNA turnover and protein level expression of these genes. Drosophila flies with piwi mutant flies produce an excess of these gene products, which accumulate in the cell eventually damaging the DNA [70, 92, 93]. Mouse PIWIs interact with eIF4e, which forms a cluster of proteins to control translation [94].

Although very little evidence has been found so far, studies are suggesting a greater somatic function of the PIWI/piRNA pathway. In the early stages of embryogenesis, Drosophila piwi is needed for chromatin structure maintenance and cell cycle progression [95]. piwi is also found on chromosomes of the salivary gland that mediate epigenetic regulation [96]. Loss of piwi proteins in Drosophila intestinal stem cells impairs the gut regenerative capacity of these flies [97]. In Aplysia, piRNA-mediated DNA methylation is needed for neuronal plasticity [98]. In various arthropods, the PIWI-piRNA pathway may perform as an antiviral defense mechanism in mosquitoes and silkworms [99]. In a rat model of diabetes, activities of pancreatic β-cells may be regulated by piRNAs. β-Cells express thousands of piRNAs and their expression changed when rat Piwi proteins were downregulated, resulting in defective insulin secretion [100]. Besides all these major and minor functions of the PIWI–piRNA pathway in various animals, aberrant expression in human cancers has been reported (Table 1), which will be discussed in detail in the following sections.

Induced expression of PIWIL2 in A549 cells results in increased cell proliferation by elevated expression of CDK2 and cyclin A, both in vitro and in vivo. Similar results are obtained from H460 cells in vitro. By contrast, RNAi-mediated depletion of the protein results in cell cycle arrest (G2/M) and increased apoptosis [101]. An increase in PIWIL1 activity induces cell proliferation in A549 cells and decreases in H1299 cell proliferation when knocked down and increases the colony-forming capacity of tumor cells [63, 102].

A positive correlation of cell proliferation with aberrant expression of piRNAs can be postulated. piR-651 is one example of such piRNAs whose expression is altered significantly in many cancers including in lung cancer patient samples and cancer cell lines such as NCIH446 and 95-D [103]. piR-651 maintains the cell population by keeping proapoptotic proteins in check [103]. Inhibition of piR-651 decreases cell proliferation and increases apoptosis in A549 and HCC827 cells. piR-651 negatively regulates proapoptotic proteins while increasing the activities of antiapoptotic proteins [104]. piR-651 helps cyclin D1 and CDK-4 overexpression and upregulates proliferation of transfected A549 cells both in vitro and in vivo [105]. The expression of RASSF1C has been shown to regulate certain piRNA expression and cancer progression. Upregulation of this oncoprotein correlated with overexpression of piR-52200 and underexpression of piR-35127 inpatient samples while piR-34871 and piR-46545 were additionally up- and downregulated respectively in the non-small-cell lung cancer (NSCLC) cell line H1299 along with the previous 2 piRNAs. In vitro, overexpressing underexpressed and knocking down overexpressed piRNAs decreases cell proliferation. Specifically, knock down of piR-52200 in A549 cells, piR-34871 in HT520 cells decreases cell proliferation significantly. By contrast, H1299 responded in most knockdown and overexpression studies. A low level of colony formation was observed in normal lung tissues after manipulation of piRNA expression [106]. piR-55490 acted as an anticancer agent in vitro and in xenograft studies. In lung cancer cell lines such as A549, H460, and H1299, piR-55490 expression was originally suppressed and upon overexpression, these cell lines showed decreased proliferation. It is postulated that piR-55490 binds to mTOR and degrades it decreasing tumor cell proliferation [107]. Two piRNAs overlapping in the 15th chromosome and sharing a common single nucleotide polymorphism, rs11639347, piR-5247, and piR-5671, increase proliferation of A549 cells [108].

Human PIWI proteins are now proven to maintain the stemness of certain cell populations when present in testis. Hiwi inhibition resulted in the loss of ALDH-1 (cancer cell marker) positive cells and decreased tumor mass in immunocompromised mice when injecting SSC^lo Alde^br stem cells isolated from an SPC-A1 cell line [109]. Overexpression of RASSF1C promotes CD133⁺ (stem cell marker) A549 cell tumor sphere formation. RASSF1C induces PIWIL1 expression, which maintains stem cell properties and regulates the wnt/β-catenin pathway. Coexpression of RASSF1C and IGFBP-5 reduces PIWIL1 expression [110].

The interplay between PIWIs and piRNAs aids more than one hallmark of cancer. Inhibition of piR-651 decreases migration of highly invasive cell lines 95-D, A549, and HCC827 cells [106, 107]. Inhibition of PIWIL1 interferes with metastatic activity in H1299 cells, while increased expression induces A549 cell migration [102].

Human case study databases like The Cancer Genome Atlas (TCGA) show a positive correlation between PIWIL1 expression and poor overall survival of patients [102]. Patient samples show similar results, increased PIWIL1 correlating to shorter time to relapse (TTR) and shorter overall survival. Whereas, decreased PIWIL4 correlated with shorter TTR and less overall survival [102]. Patients with higher piR-55490 expression have longer overall survival [107].

Studies in NSCLC and lung squamous cell carcinoma (LSCC) have revealed another class of sncRNAs, the piRNA-Like RNAs (piR-Ls). These RNAs have similar as well as distinguishing features to piRNAs. Two variants have been discovered to date, piR-L-138 and piR-L-163, and both are similar to piRNAs in length. However, 2 major differences are that, unlike piRNAs, they are expressed in adult tissues, and they bind directly to phosphorylated protein targets (p-proteins) to regulate their functional efficacy. Therefore, they are designated as protein functional effector sncRNAs (pfeRNAs). piR-L-138 expression in LSCC increases after cisplatin-based chemotherapy, which eventually leads to chemoresistance by the tumor cells. By contrast, targeting piR-L-138 in LSCC cell lines such as H157 and SKMES-1 increases apoptosis. piR-L-138 regulates p60MDM2 to control cell proliferation [111, 58]. Another study showed piR-L-163 binds to Ezrin, Radixin, and Moesin (p-ERM), which in turn increases the binding capacity of p-ERM to EBP50 and F acting. Blocking piR-L-163 induces cell growth and invasion revealing it as a negative regulator of tumor progression [112].

Cancer prognosis is related to 2 important facets namely early detection and delayed chemoresistance. In a study with 20 pairs of malignant and nonmalignant tissues, piR-hsa-211106 was downregulated in all malignant tissues as it prevented metastasis and induced apoptosis in lung cancer cells [61]. This piRNA interacts with pyruvate carboxylase, which prevents cisplatin resistivity in lung cancer cells [61].

Lin et al. [60] and Li et al. [62] demonstrated that piRNAs can also be used for diagnosis of lung cancer. Sputum from 32 lung cancer patients was used as a source of epithelial cells from the bronchus and cRNA was profiled [60]. Lung cancer patients had upregulated piR-004987 and piR-020809 expression and downregulated expression of piR-023338 and piR-011186 [60]. Li et al. [62] examined 19 lung tissues from lung cancer patients and compared their piRNA profile with noncancerous lung tissues (from different sites in the same patients). They found 10 piRNAs unregulated in cancerous tissues compared with noncancerous tissue samples (see Table 1). Of these, 2 exosomal piRNAs, namely piR-hsa-26925 and piR-hsa-5444, were found in patient sera. Exosomes are double-layered lipid extracellular vesicles containing macromolecules like nucleic acids (in this case piRNA) used for cell-to-cell communication. Thus, such exosomes identifiable from sera can act as diagnostic markers for lung cancer.