Patients and control samples
The study was based on information retrieved between June 2018 and June 2019 and was approved by the Ethics Committee (2019-k-368). UPJO was diagnosed based on some examinations, such as ultrasound, intravenous pyelography and diuretic renogram. Surgical indications followed EUA guidelines. Intrinsic stenosis was confirmed during the operation. UPJO caused by other pathogenies, such as vessel and ureteral polyp, were excluded. Altogether, there were 20 stenotic ureter specimens from children with UPJOand 10 control ureter specimens from children with Wilm’s tumor. The children with Wilm’s tumor were not given pre-operation radiotherapy or chemotherapy and the specimens were confirmed by histology to be unaffected. The UPJO specimens were obtained strictly. First, after cutting off the specimen, the "stenotic" portion was split longitudinally and the obviously narrow ureter found. Second, the inconspicuous narrowing was removed at both ends. Lastly, each specimen was cut into at least three pieces lengthwise and immediately stored at -80℃ preparing for immunofluorescence, WB and PCR respectively. In the control samples, 5-millimeter long pieces of ureteropelvic junction were cut off and the same process applied as for the specimens with in UPJO.
Immunofluorescence of P1 and P2
Formalin-fixed and paraffin-embedded tissues were stained by immunohistochemistry to observe the expression of P1 and P2. Immunofluorescence was performed on 4 μm paraffin sections. Dako serum-free protein block was used to block, primary and secondary incubation buffers. Whole-mount immunofluorescence was performed as usual. The antibodies used were: P1 (primary antibodies: goat, lot: ab39266; fluorescent secondary antibodies: CY3 labeled rabbit anti-goat IgG) and P2 (primary antibodies: rabbit, lot: ab238338; fluorescent secondary antibodies: 488 Labeled goat anti- rabbit IgG) (Abcam company, 1:100). Each patient’s tissue was divided into three pieces, and one piece from each was evaluated by two investigators with a confocal microscope (Nikon Ci-S, Tokyo, Japan). This was then used to split up the light spectrum and make a quantitative analysis of ﬂuorescence intensity using Inform software (PerkinElmer). Five photomicrographs (× 200) were randomly selected to count the ﬂuorescence intensity, and the means of ﬂuorescence intensity were calculated as the ratio of integral optical density.
Western blot of Ptch1 and Ptch2
Pre-cooled RIPA protein extraction reagents (50 mMTris-HCl(pH 7.4), 150 mM NaCl, 1% np-40, 0.1% SDS) were added to a protease inhibitor cocktail (Roche) (phosphorylated protein needed to be added to phosphatase inhibitor at the same time) (according to the volume ratio of 50:1).The tissue was cut into pieces, and 50mg of tissue was added to 500ul lysate. The tissue was centrifuged at 13000rpm for 20min. The supernatant was removed and the tissue stored at -80 degrees for Western blot. The separation gel was prepared according to the molecular weight of the target protein. Electrophoresis, incubation of primary and secondary antibodies and film exposure were then performed. The primary antibodies were P1 (goat, 1:300) and P2 (rabbit, 1:1000), and the secondary antibodies were goat anti rabbit IgG and goat anti mous IgG (1:10000). Band density was quantified by digital densitometry using the Gel Image system ver.4.00 (Tanon, China). Band intensity was normalized to that of β-actin.
RNA isolation and RT-PCR
The ABI 7500 fluorescence quantitative PCR instrument was used, and ΔΔCt method was used for relative quantitative analysis of the data. Quantitative real-time PCR was accomplished with SYBR Premix Ex Taq (TaKaRa) on LightCycler-GmbH D-68298 (Roche Molecular Biochemicals) under the following conditions: 95℃ for 10 s, 45 cycles of 95℃ for 5 s, 58℃ for 20 s; 65℃ for 15 s. A dissociation procedure was performed to generate a melting curve for confirmation of amplification specificity.Primer sequences were as follows: P1 forward, 5’-CGCTCTGGAGCAGATTTCCA-3’, reverse, 5’-CTCGTCCTCCAACTTCCACC3; P2 forward 5’- GCCGCCAGAGGTGATACAG-3’, reverse, 5’- GTGTGTCTGATGAGGGGGTG-3’. Beta actin 5’-ACAGAGCCTCGCCTTTGCC-3’, reverse, 5’-GATATCATCATCCATGGTGAGCTGG-3’. The relative changes in expression levels of P1 and P2 were normalised against the levels of GAPDH gene expression in each sample (ΔΔCt method). They were carried out in triplicate for each sample and primer.
Statistical software SPSS 22.0 was used to analyse data. All normally distributed data were presented as mean ± SD. Significance of differences was evaluated using a two-sample t test. P value < 0.05 was considered to be statistically significant.