Overexpression of Long Non-coding RNA 4933425B07Rik Causes Urinary Malformations in Mice

Abstract

Congenital anomalies of the kidney and urinary tract (CAKUT) is a common birth defect and is the leading cause of end-stage renal disease in children. The etiology of CAKUT is complex and includes mainly genetic and environmental factors. However, these factors cannot fully explain the etiological mechanism of CAKUT. Recently, participation of long non-coding RNAs (lncRNAs) in the development of the circulatory and nervous systems was demonstrated; however, the role of lncRNAs in the development of the kidney and urinary tract system is unclear. In this study, we used the piggyBac (PB) transposon-based mutagenesis to construct a mouse with lncRNA 4933425B07Rik (Rik) PB insertion (RikPB/PB) and detected overexpression of Rik and a variety of developmental abnormalities in the urinary system after PB insertion, mainly including renal hypo/dysplasia. The number of ureteric bud (UB) branches in the RikPB/PB embryonic kidney was significantly decreased in embryonic kidney culture. Only bone morphogenetic protein 4 (Bmp4), a key molecule regulating UB branching, is significantly downregulated in RikPB/PB embryonic kidney, while the expression levels of other molecules involved in the regulation of UB branching were not significantly different according to the RNA-sequencing (RNA-seq) data, and the results were verified by quantitative real-time polymerase chain reaction (RT-PCR) and immunofluorescence assays. Besides, the expression of pSmad1/5/8, a downstream molecule of BMP4 signaling, decreased by immunofluorescence. These findings suggest that abnormal expression of Rik may cause a reduction in the UB branches by reducing the expression levels of the UB branching-related molecule Bmp4, thus leading to the development of CAKUT.

Overexpression of long noncoding RNA 4933425B07Rik leads to renal hypoplasia by inactivating the Wnt/β-catenin signaling pathway

“…Construction of lncRNA Rik overexpressing cell lines, RNA-seq and GO/KEGG pathway analysis Previous studies found that newborn and embryonic Rik PB/PB ;Hoxb7 mouse kidneys showed various CAKUT phenotypes, of which the most common was hypoplasia (82.4%). In addition, the expression level of Rik was increased at all stages of embryonic renal development in Rik PB/PB ;Hoxb7 mice, suggesting that Rik was involved in mouse metanephric renal development, and when Rik was overexpressed, metanephric renal development was abnormal [19] . Therefore, we constructed a Rik-overexpressing lentivirus according to the transcript sequence of RACE and infected the renal development-related cell lines MPTC and MK3.…”

“…To determine the actual transcription sequence of Rik in the mouse model in this study, we rst performed a RACE assay. Previous studies have shown that the expression level of Rik in the embryonic kidney tissue of Rik +/+ ;Hoxb7 mice is low at all stages of development [19] , leading to failure of the RACE test. While observing the renal phenotype of Rik PB/PB ;Hoxb7 mice, we also noticed that the sperm number of Rik PB/PB ;Hoxb7 male mice decreased signi cantly, suggesting that the Rik PB/PB ;Hoxb7 homozygous male mice were not sterile, but their spermatogenic ability was affected (Supplemental Fig.…”

“…Blast comparison with 1926 bp in the NCBI database showed that lncRNA Rik mainly matched the exon 5 fragment (data came from https://blast.ncbi.nlm.nih.gov/Blast.cgi). Previous studies explored the subcellular localization of Rik in MPTC cells through nuclear and cytoplasmic fractions, and the results showed that Rik was mainly located in the nucleus [19] . FISH analysis of the embryonic kidney tissue of E12.5 Rik PB/PB ;Hoxb7 mice con rmed this cytoplasm distribution (Supplemental Fig.…”

“…Rik PB/+ mice on the FVB/NJ genetic background were generated by using PB transposon-based insertional mutagenesis targeted to intron 3 of Rik [19] . Rik PB/+ mice were mated with Hoxb7/myr-Venus mice to obtain Rik PB insertion mice speci cally expressing uorescent protein in the UB epithelium (Rik PB/+ ;Hoxb7/myr-Venus abbreviated as Rik PB/+ ;Hoxb7).…”

“…In our previous study, lncRNA 4933425B07Rik (Rik) was overexpressed in Rik PB/PB ;Hoxb7 mice, and the neonatal kidneys showed multiple CAKUT phenotypes, including duplicated kidneys, hydronephrosis, kidney hypoplasia, kidney dysplasia, and vesicoureteral re ux, the most common of which was kidney hypoplasia. In vitro embryonic kidney culture showed that the number of UB tips was reduced; therefore, lncRNA Rik appears to participate in renal development, and its overexpression leads to abnormal renal development [19] . Therefore, in this study, we constructed lncRNA Rik-overexpressing cell models and a lncRNA Rik knockout cell model in vitro and used the Rik PB/PB ;Hoxb7 mouse model to explore the molecular mechanism of kidney hypoplasia induced by Rik overexpression.…”