MicroRNAs in Kidney Development And Disease Ⅲ

MiRNAs in Wilms tumor 

Wilms tumor, or nephroblastoma, is the most common childhood renal cancer, with an incidence of 1 in 10,000 children in North America (111). It is primarily a sporadic disease, although familial forms occur in approximately 1%–2% of cases (112, 113). Wilms tumors arise from aberrant nephrogenesis, where pluripotent embryonic renal precursors fail to differentiate and persist abnormally into postnatal life (111, 114, 115). These tumors histologically resemble developing kidneys with a disrupted morphology (116), and mutations in genes involved in fetal nephrogenesis, including WT1 (117–119), CTNNB1 (120), SIX1/2 (121, 122), and TP53 (123, 124), are associated with approximately 40% of Wilms tumors (125). Recent studies using whole-genome and whole-exome sequencing of Wilms tumors identified novel mutations in cancer risk genes (REST, CHEK2, PALB2) (126, 127), genes encoding proteins that mediate histone modifications during nephrogenesis (BCOR, MAP3K4) (126), and miRNA-processing genes (121, 122, 128, 129).

HOW LONG DOES IT TAKE FOR CISTANCHE TO WORK FOR KIDNEY DISEASE PATIENTS?

Among the miRNA-processing genes, mutations in DROSHA, DGCR8, DICER1, TARBP2, and XPO5 (encodes exportin 5) have been reported in treatment-naive and neoadjuvant chemotherapy-treated Wilms tumors (Figure 3) (121, 122, 128, 129). About 33% of Wilms tumors examined exhibit deleterious mutations in genes of the miRNA-processing pathway (128). A recurrent hotspot mutation (E1147K) in a metal-binding (Mg2+) residue of the RNase IIIb domain of DROSHA, which appears to be unique to Wilms tumor, abolishes the catalytic activity of this domain, resulting in incomplete cleavage of pri-miRNAs and reduced miRNA maturation (128). Somatic hotspot mutations affecting the RNase IIIb domain of DICER1 impair processing of 5p miRNAs (those derived from the 5′-arm of the pre-miRNA hairpin) (129) and are often found as "second hit" mutations that act in tandem with DICER1 germline mutations to induce Wilms tumorigenesis in DICER1 syndrome (a disorder that increases susceptibility to a variety of tumors) (130–132). It remains unclear why impaired DICER1 function in Wilms tumors results in persistent and aberrant nephrogenesis, unlike the loss of Dicer1 in mouse nephron progenitors, which causes increased apoptosis and premature cessation of nephrogenesis (72–75). Some potential possibilities include that the gene dosage activity might be crucial in determining cell survival or that mutations in the RNase IIIb domain might affect the specificity of miRNA binding by DICER1.

Mutations in miRNA-processing genes are associated with the downregulation of important miRNAs, including members of the miR-200 (121) and the let-7 families (Figure 3) (121, 129). Let-7 miRNAs and the RNA-binding protein Lin28 function in concert to control the timing of cessation of murine nephrogenesis, possibly via regulation of the growth-promoting gene Igf2 (133). Overexpression of Lin28 during kidney development causes expansion of nephrogenic progenitors, by inhibiting their final wave of differentiation, which culminates in neoplastic transformation that is highly reminiscent of the human Wilms tumor (134). Increased DNA copy number of LIN28B and DNA copy loss of let-7a are seen in 25% and 46% of human Wilms tumor samples, respectively (126). In line with these observations, germline mutations in the human DISL3L2 gene, which encodes an exoribonuclease responsible for degrading preprocessed forms of let-7, cause Perlman syndrome and predisposition to Wilms tumor (135, 136). Perlman syndrome is a congenital overgrowth syndrome characterized by macrosomia, polyhydramnios, facial dysmorphology, renal dysplasia, and nephroblastomatosis (a precursor lesion for Wilms tumor) (137). Among infants with Perlman syndrome who survive past the neonatal period, 64% develop Wilms tumor (138). Interestingly, complete or partial DISL3L2 deletions were found in about 30% of sporadic Wilms tumors examined (136).

A recent study strengthened the significance of the miRNA regulatory network in the etiology of Wilms tumor. The authors found that pleiomorphic adenoma gene 1 (PLAG1) is one of the most consistently upregulated genes in Wilms tumors with mutations in miRNA-processing genes (125). Ectopic expression of PLAG1 in the developing mouse kidney causes neoplasia, which is accompanied by transactivation of its target gene, the Wilms tumor oncogene IGF2. miR-16 and miR-34, which are downregulated in Wilms tumors, were identified as potential regulators of PLAG1 expression (125). Table 2 summarizes other studies that have reported aberrant expression of specific miRNAs associated with the etiology of Wilms tumor. Interestingly, these miRNAs can function as oncogenes (called oncomiRs) or tumor suppressors in the setting of Wilms tumor development, depending on the nature of their targets.

MiRNAs as potential biomarkers and therapeutic agents 

Apart from their intracellular location, miRNAs are also present in significant amounts in biological fluids, including blood, plasma, urine, breast milk, and saliva (139). These circulating miRNAs are found packedaged in microparticles (exosomes, microvesicles, and apoptotic bodies) (140, 141), conjugated with AGO (142) or nucleophosmin 1 proteins (143), or loaded into HDL (144), which make them remarkably stable even under unfavorable conditions, such as boiling, extreme variations in pH, extended storage, and multiple freeze-thaw cycles (145, 146). Thus, miRNA signatures in biological fluids can reflect associations with physiological or disease conditions (147). Together, these features make circulating miRNAs attractive for use as noninvasive biomarkers for disease diagnosis and prognosis.

Circulating miRNAs can be extracted directly from unfractionated biological fluids or extracellularlar vesicle preparations using commercially available extraction kits (148) or TRIzol (149). Upon isolation, miRNAs can be stored at –70°C and remain stable for up to 1 year (148). There are several platforms available for miRNA profiling, including microarray hybridization, qPCR, and next-generation sequencing (150). Microarray and qPCR are the most frequently used methodologies to investigate the expression of known miRNAs (151). Both methods have the advantages of being simple to use, relatively quick from RNA labeling to data generation, and relatively cost-effective (152). However, they rely on the availability and accurate annotation of miRNA sequences in databases for probe and primer design (150). Although more expensive, next-generation sequencing allows for the simultaneous detection of both known and novel miRNA species and offers high sensitivity (153). Furthermore, the single-nucleotide resolution of next-generation sequencing enables the identification of isomiRs, which are mature miRNA isoforms that differ from canonical ones in length, sequence, or both (154, 155), which change the targeting specificity of the miRNA (156).

Diverse studies have investigated the potential of circulating miRNAs as biomarkers for pediatric kidney diseases (157–160). For instance, one study identified 14 miRNAs that were significantly upregulated in the serum of patients with Wilms tumor. Interestingly, a signature based on miR-100-5p and miR-130-3p expression could differentiate these patients from healthy controls with accuracy, sensitivity, and specificity (159). Although the findings from this study and many other studies have provided compelling motivation to explore the potential of circulating miRNAs as biomarkers, several hurdles in the field need to be overcome before widespread clinical application. First, there is a relative lack of consensus between studies likely due to the absence of standardized methodology for purification (161) and analysis of samples (e.g., differences in miRNA profiling platforms, refs. 162, 163; or differences in smRNA-Seq library preparation methods, ref. 164). Second, is the lack of large-sample-size studies and detailed investigations on specific diseases. Another important aspect is that the influence of confounding variables such as age, sex, and external factors (e.g., tobacco, alcohol, etc.) on miRNA profiles has not been fully explored (for an in-depth review, please refer to ref. 165).

On the therapeutic side, several miRNA-based drugs are currently in clinical trials but have not been granted FDA approval yet (166, 167). The main approaches for miRNA therapy involve restoration of miRNA levels using miRNA mimics, or inhibition of specific miRNAs using antagomiRs (168). One of the challenges associated with the development of miRNA-based therapeutics is the identification of miRNA candidates for each disease. Because multiple miRNAs are dysregulated in each disease, a careful analysis of patient samples in combination with in vitro and in vivo assays that address the pathophysiological mechanisms affected by the miRNAs in question should be performed to narrow down the candidate miRNAs for therapeutic intervention (168, 169). Another challenge involves the development of strategies to improve in vivo stability and site-specific drug delivery with minimal toxicity and off-target effects. RNA molecules are chemically unstable due to the presence of the 2′-hydroxyl group on the pentose ring. To provide higher stability and protection from nucleases present in serum or the endocytic compartment of cells, biotech companies have generated RNA molecules with chemical modifications (2′-O-methyl group, phosphorothioate, or locked nucleic acids) in their backbone (166). As for in vivo delivery, technologies include lipid-based (e.g., lipid nanoparticle and neutral liposome) and dendrimers conjugated to a targeting moiety, among many other strategies. Major challenges associated with miRNA delivery systems are immunotoxicity and target-specific affinity toward a disease site (170). Delivery strategies by various methods of administration (intraperitoneal, intravenous, and subcutaneous injections) or by using vectors containing kidney-specific and inducible promoters have been successfully used for selective kidney targeting and to avoid potential adverse effects in other tissues and organs (171, 172).

Table 2. List of miRNAs altered in Wilms tumor

List of miRNAs, samples/specimens analyzed, miRNA levels observed in these samples/specimens compared with appropriate controls, target gene, consequences of miRNA changes, and references. CAMKK2, calcium/calmodulin-dependent protein kinase kinase 2; CDC7, cell division cycle 7; CDH1, cadherin 1; CREB1, cAMP-response element-binding protein 1; EYA1, eyes absent homolog 1; E2F3, E2F transcription factor 3; FRS2, FGF receptor substrate 2; Glut1, glucose transporter 1; IGF1R, IGF1 receptor; Jag1, jagged1; MET, mesenchymal-epithelial transition; MKNK1, MAPK-interacting serine/threonine kinase 1; PTEN, phosphatase and tensin homolog; PUMA, p53-upregulated modulator of apoptosis; p73, tumor protein p73; SIX1, sineoculis homeobox homolog 1; TGFBR1, TGF-β receptor 1; WT, Wilms tumor.

Summary 

There has been an explosion of information regarding miRNA biogenesis, the regulation of miRNA expression, and miRNA function since the initial discovery of miRNAs in 1993 (6, 7). This has been accompanied by an ever-increasing understanding of how miRNAs function both in normal physiology and in the pathophysiology of many diseases. It has become clear that dysregulation of miRNA expression disrupts early kidney development and is implicated in the pathogenesis of developmental kidney diseases, such as CAKUT and Wilms tumor. With recent developments in the use of miRNAs as biomarkers and as novel drug targets, insights into how miRNAs regulate kidney development and disease are critical to understanding how they might be utilized in novel diagnostic and therapeutic approaches to these diseases. To fully realize these efforts, future studies identifying the function of specific miRNAs in kidney development are critical, in addition to technologies to optimize targeting small oligonucleotide therapeutics to the kidney 

Acknowledgments

JH's laboratory is supported by grants from the NIH National Institute of Diabetes and Digestive and Kidney Diseases (R01DK125015 and R01DK102843). DMC was supported by the Nephrotic Syndrome Study Network Career Development Award and the Children's Hospital of Pittsburgh Research Advisory Council Postdoctoral Fellowship. MT was supported by the University of Pittsburgh Summer Research Internship Program (R25DK119180). The figures were created with BioRender.com. Address correspondence to: Jacqueline Ho, Division of Nephrology, Department of Pediatrics, UPMC Children's Hospital of Pittsburgh, Rangos Research Center, 4401 Penn Ave., Pittsburgh, Pennsylvania 15224, USA. Phone: 412.692.9440; Email: jacqueline.ho2@chp.edu.

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