Aberrant Expression Of SLX14A1 Gene In Bladder Cancer

Urinary bladder cancer(UBC)is the second most commonly diagnosed genitourinary malignancy.It is estimated that 76,960 new cases as well as 16,390 deaths will occur in the year of 2016.Approximately 75%of the patients are diagnosed with the non-muscle invasive transitional carcinoma by their first clinic visit after symptomatic hematuria.Nearly 70%of these cases will relapse within 5 years after the first standard transurethral resection of the bladder tumor(TURBT).Thus,it demands intensive surveillance procedures,including the long-term periodical cystoscopy screening,adjuvant intravesical chemotherapy and immunotherapy,which makes it one of the most expensive and suffering cancers worldwide.During the past decades,various molecular events have been found involved in the carcinogenesis of UBC,including the deletion in chromosome 9,point mutations of the fibroblast growth factor receptor-3(FGFR3)and alterations in tumor suppressor gene TP53 and RB1.However,the bewildering story of cancer is far more complicated than we thought.In 2011,two large-scale population based study related the SLC14A1 gene to the carcinogenesis of the urinary bladder cancer.The human solute carrier family 14,member 1(SLC14A1)gene is approximately 30kb in length and located on chromosome 18q12.1-q21.1.It encodes the Type-B urea transporter(UT-B)that facilitates rapid and passive cross-membrane movement of urea and serves as a determinant antigen of the Kidd blood group on erythrocytes.There are two human UT-B isoforms.hUT-B1(389aa,43KDa),first cloned from human bone marrow and later localized in multiple tissues;and hUT-B2(445aa,38-54KDa),which has the open reading frame from exon 3,was first isolated from bovine rumen as bovine UT-B2.Only UT-B2 mRNA was reported in the human caudate nucleus while recently in the bladder.Based on functional analysis,the renal UT-B is located in the endothelial cells of descending vasa recta(DVR),and functions to maintain the urea concentration gradient in the inner medulla.Nevertheless,in the extra-renal tissues,UT-B is believed to function as a scavenger that prevents the intracelluar urea aggregation and intoxication as observed in other studies.The urinary bladder is constantly exposed to urine urea,the concentration of which is 20-100 times higher than that of the blood.Meanwhile,UT-B is abundantly expressed at all layers of the urothelium.Severe apoptosis and DNA damage was observed in the urothelium cells of the UT-B knockout mice.In 2014,Li.et al.detected the hUT-B expression was decreased in UBC from a serial of paraffin-embedded specimens.Also accordingly,the expression level of UT-B was inversely proportional to the histological grade and clinical stage.Taken together,the suppressed expression of UT-B in the bladder may lead to the generation of bladder cancer via the toxicity of the accumulated urea.Purpose:To explore the underlying mechanisms that may impair the expression of UT-B in urinary bladder cancerMethodology:In our research,62 baldder cancer samples are invoived,which includes 10 fresh bladder cancer specimens from the TURBT surgery,2 bladder cancer cell lines,3 bladder cancer total RNA,1 bladder cancer total cDNA and 46 cases of bladder cancer paired with normal tissue array.As control,we used a customized customized Western Blot membrane of normal human tissues,1 normal bladder total cDNA,5 normal bladder cancer tissues and the normal human urothelium cell line.At mRNA level,we used RT-PCR to investigate the distribution of UT-B2 and UT-B1 mRNA in the samples mentioned above.qPCR was adopted to semi-quantify the UT-B transctipts.All PCR products were send for Sanger sequencing.Target clones were then constructed into the mammalian expression pcDNA3 vector for in vitro study.At protein level,we used Western Blot and immunohistochemistry to detect the divergent expression of UT-B in normal bladder samples and bladder cancer samples.At post-translation level,we transfected the HEK 293 cells with the UT-B1,UT-B2 and ?UT-B1 as control.Lipid raft isolation was adopted to separate the glycosylated UT-B protein in HEK 293 cells and TURBT samples.The structure of the glycoprotein was analyzed by lectin precipitation and PNGase F treatment.The cell surface expression of UT-B was detected by biotinylation assay.The function of UT-B 1,UT-B2 and ?UT-B1was tested by water permeability experiment using the Xenopus oocytes.Results:1.Both UT-B2 and UT-B1 mRNA exist in normal bladder tissues.The UT-B protein in normal human bladder demonstrated as a 37-50KDa thick band.2.In bladder cancer samples,UT-B2 mRNA was only identified 56.2%cases with a faint signal.UT-B1 mRNA,however,was identified 87.5%cases,71.4%of which are bearing a 24-nt deletion with the exon 4.This novel UT-B1 variant was named ?UT-B1.The UT-B protein in bladder cancer samples showed a 38 KDa band,and the expression of which was down-regulated.Meanwhile,this down-regulation was inversely related to the tumor grade as we have observed in the tissue micro-array.In 46 cases,the average staining score of UT-B protein was 1.61 ± 0.53 in low grade cancer(n=25),and 0.52 ±0.43 high grade cancer(n=21).3.In biotinylation assay,?UT-B1 seems more likely to be aggregated on the cell membrane in HEK 293 cells.Even though the ?UT-B1 was apt to aggregate on the cell membrane of HEK 293,its water transportation capacity was significantly lower than that of the UT-B1 and UT-B2,according to the Xenopus oocytes data.4.1n HEK 293 cells,the lipid raft derived glycosyated UT-B2 showed a 38-50 KDa smear band,which can be deglycosylated to a 38 KDa single band.On the other hand,however,both UT-B1 and ?UT-B1 demonstrated as a 38 KDa single band that can be deglycosylated to 28 KDa.The glycan structure detected by the lectin pull-down assay indicated that UT-B2,UT-B1 and ?UT-B1 in HEK 293 cells are comprised of N-acetylglucosamine and sialic acid.However,the UT-B protein isolated from bladder cancer samples demonstrated as hypo-sialylated,which is independent of the nucleotide deletion.Conclusion:Both UT-B2 and UT-B1 mRNA are expressed in the normal bladder tissue.However,the transcription of UT-B2 was strikingly suppressed in bladder cancer samples.UT-B1 mRNA was expressed in most of the bladder cancer samples,approximately 70%of which are bearing a 24-nt deletion.At protein level,the expression of UT-B protein was down-regulated in bladder cancer samples.This down-regulation was inversely related to the tumor grade.Also,the UT-B protein in bladder cancer samples demonstrated as a hypo-sialylated glyco-protein,which is independent of the 24-nt deletion.In summary,the abberent experession of SLC14A1 gene in bladder cancer is demonstrated as a 24-nt deletion in UT-B1 mRNA,as well as a hypo-sialylation of UT-B1 protein.The truncated form of UT-B1 has a stronger ability to aggregate on the cell membrane of HEK 293,yet weaker capacity in water transport,compared with wild-type UT-B1 and UT-B2.The tumor specific nucleotide deletion and hypo-sialylation of UT-B1 indicates SLC14A1gene may serve as a novel biomarker in bladder cancer.