Biological Function And Regulation Mechanisms Of LncRNA HAGLROS In Bladder Cancer

Objective:Bladder cancer(BC)is the most common genitourinary malignancy worldwide,with high morbidity and mortality,showing a gradually younger trend.More than 90%of BC is urothelial carcinoma.It falls into non-muscle-invasive bladder cancer(NMIBC)and muscle-invasive bladder cancer(MIBC)according to the depth of muscle infiltration.NMIBC can be treated with transurethral resection of the bladder tumour and postoperative adjuvant chemotherapy/immunotherapy.Of note,approximately 50%～70%of NMIBC will relapse,and 10%～20%will progress to MIBC or metastasize,eventually.Although MIBC or highly malignant BC is mainly treated with radical cystectomy,there are many short and long-term complications,which caused great pain to patients.Therefore,early diagnosis and treatment of BC are of great clinical significance to improve the patient life quality and prognosis.The long non-coding RNA(lncRNA)is a non-coding RNA with a length of more than 200 base pairs,which was initially ignored as "transcription noises" due to lack of protein-coding function.However,the rapid development of genomics technology highlights lncRNAs are closely related to the malignant phenotypes of various tumours,and abnormal expression of lncRNA is closely associated with increased cancer-related mortality.In recent years,growing evidences indicate that lncRNAs can affect gene expression and tumor biological function by participating in regulating transcription,gene expression and mRNA expression,which play an important role in the occurrence and progression of BC.HOXD antisense growth associated long non-coding RNA opposite strand(HAGLROS),located on chromosome 2q31.1,is a 699 bp IncRNA.Accumulating pieces of evidences indicate that HAGLROS is closely related to proliferation,invasion,apoptosis,autophagy and metabolism reprogramming,and plays an oncogenic role in cancers.However,the biological functions and mechanisms of HAGLROS in BC remain unknown.This study aimed to:1.Investigate the expression of HAGLROS in BC and its clinical significance;2.Clarify the biological effect of HAGLROS in BC;3.Explore the molecular regulatory mechanism of HAGLROS in BC;4.Provide a new direction for the diagnosis,pathogenesis and molecular targeted therapy of BC.Methods:In the first part,the InCAR database was used to predict the secondary structure of HAGLROS and evaluate its protein-coding potential and conservation.Secondly,the expression level of HAGLROS and its relationship with prognosis in BC were analyzed using the GEPIA database,the Lnc2Cancer database,the starBase database,the TCGA database and our transcriptome sequencing database.Furthermore,the expression levels of HAGLROS in BC tissues and cells were measured though real-time quantitative PCR(qRTCPCR)and in situ hybridization(ISH)assays.In the second part,firstly,the over-expressed and interfered HAGLROS lentiviral vectors were constructed.Then,the stable transfection BC cells wtih overexpressed and silencing HAGLROS vectors were cultured by using cell transfection techniques.The transfection efficiency was observed by fluorescence microscope and verified through qRT-PCR assays.Secondly,the effects of HAGLROS on the proliferation,migration,invasiveness and cell cycle of BC cells were investigated by CCK-8 assay,wound healing assay,transwell assay and flow cytometry assay.Finally,the role of HAGLROS on the growth of BC in vivo was studied via establishing tumor xenograft implantation in nude mice and immunofluorescence assay.In the third part,firstly,the nucleo-cytoplasmic separation assay and fluorescent in situ hybridization(FISH)assay were used to detect the subcellular localization of HAGLROS in BC cells.Secondly,the downstream target genes of HAGLROS were predicted using the TCGA database,the starBase database,the lncBase database and our transcriptome sequencing database.Then,qRT-PCR assay,western blot assay and immunohistochemistry(IHC)assay were used to detect the expression level of HAGLROS downstream target genes.In addition,the interaction and binding relationship between HAGLROS and its downstream target genes were explored by FISH co-localization assay and dual-luciferase reporter assay.Then,the regulatory roles between HAGLROS and its downstream target genes were explored by qRT-PCR assay and western blot assay.Finally,the effects of HAGLROS downstream genes on the proliferation,migration and invasion of BC cells were explored through rescue assays.Results:In the first part,the two secondary structures of HAGLROS were found and these two transcripts are of no protein-coding ability and highly conserved by analyzing the InCAR database analysis.The analysis result of the public databases and our transcriptome sequencing database showed HAGLROS is significantly over-expressed in BC.Moreover,qRT-PCR and ISH assays also demonstrated that HAGLROS is significantly up-regulated in BC tissues and cells,and elevated HAGLROS expression was related to higher pathologic grade and advanced clinical stage,which is significant for BC diagnosis.In the second part,the over-expressed and interfered vectors of HAGLROS were successfully constructed.The stable transfection BC cells wtih over-expressed and silencing HAGLROS vectors were successfully established and screened.Cellular function assays showed that over-expressed HAGLROS could promote the proliferation,migration and invasion of BC cells and shorten G1 phase.Knockdown HAGLROS can inhibit the proliferation,migration and invasion of BC cells,arrested in the G1 phase.Furthermore,the results of animal experiments suggested that over-expressed HAGLROS can also promote the progression of BC cells in vivo.In the third part,the results of the nucleo-cytoplasmic separation assay and FISH assay demonstrated that HAGLROS are mainly located in the cytoplasm of BC cells.The analysis results of public databases and our transcriptome sequencing database revealed that SPRR1B was significantly over-expressed in BC and miR-330-5p had common binding sites with HAGLROS and SPRR1B.Double-luciferase reporter assay confirmed that miR-330-5p shared common direct binding sites with HAGLROS and SPRR1B,and there was a competitive binding relationship among them.Western blot,IHC and qRT-PCR assays showed that SPRR1B was significantly up-regulated and miR-330-5p was obviously down-regulated in BC tissues and cells.Furthermore,HAGLROS can positively regulate SPRR1B expression,negatively regulate miR-330-5p expression,and miR-330-5p can negatively regulate SPRR1B expression in BC cells.Notably,rescue assays demonstrated that decreasing miR-330-5p could reverse the malignant phenotype inhabited by silencing HAGLROS in BC cells,and silencing SPRR1B can reverse the malignant phenotype of BC cells promoted by knockdown miR-330-5p,thereby inhibiting the cancer-promoting effect of HAGLROS.Conclusions:HAGLROS is significantly over-expressed in BC tissues and cells,and elevated HAGLROS was associated with higher pathologic grade and advanced clinical stage,which is significant for BC diagnosis;HAGLROS can promote the proliferation,migration,invasion,and affect the cell cycle in BC;HAGLROS can positively regulate the expression of SPRR1B through sponging miR-330-5p to promote the malignant phenotype of BC.