| Background and objectiveThe incidence of bladder cancer is the sixth in male solid tumors over the world,and ranking first in urogenital tumors in china.About 75%of bladder cancers are Non-Muscle Invasion Bladder Cancer(NMIBC).For these patients,transurethral resection combined with bladder infusion using chemotherapy or BCG is the gold standard treatment.The latest EAU guidelines suggested that several tumor characteristics including the number,diameter,differentiation,TNM stage,carcinoma in situ,and primary or recurrence are all risk factors associated with NMIBC recurrence,which is as high as 78%at the postoperative 5t" years.Even worse,about 20%-30%of NMIBC will progress to Muscle Invasion Bladder Cancer(MIBC),and 50%of these MIBC patients will occur distant metastases within two years after being treated by radical surgery.In terms of patients with locally advanced or advanced MIBC,gemcitabine combined with cisplatin(GC regimen)is still the standard treatment.However,the mortality rate of bladder cancer has only decreased by 1.5%over the past 15 years,which is far lower than that in breast cancer,prostate cancer,and colorectal cancer.The reasons can be attributed to the following three aspects:one,limitations of the response to GC program;two,appearance of drug resistance to tumor cells;three,absences of effective targeted drugs.It can be assured that the discovering effective targeted drugs should depended on powerful mechanisms studies on tumor progression and drug resistance,nevertheless,it is unclear so far.Long noncoding RNA usually be described as the single strand nucleotides sequence more than 200bp,although without protein coding function,lncRNA could take part in regulating gene expression at epigenetic,transcriptional,post-transcriptional level,and then influence the processes of occurrence,progression,metastasis,and even drug resistance in tumors.Taking the classic lncRNA UCA1(Urothelial Carcinoma-associated 1)for instance,a meta-analysis showed that the sensitivity of urine UCA1 to diagnose bladder cancer was 0.83(95%CI,0.77-0.88),and the specificity was 0.87(95%(CI,0.73-0.94),and the area under the receiver curve was 0.88(95%CI,0.85-0.91),which suggests that UCA1 in urine is an ideal non-invasive diagnostic marker for bladder cancer.It has potential application values in follow-up and early diagnosis for bladder cancer patient.More importantly,a series of studies found that UCA1 is significantly overexpressed in bladder cancer.Silencing UCA1 expression will significantly inhibit the proliferation,migration,and invasion,the anti-apoptotic capacity,as well as increase the sensitivity to gemcitabine for bladder cancer cells.In the mechanism,UCA1 can activate the transcription factor CREB,which could combine with the promoter region of miR-196a-5p,and then,enhancing the progress and resistance of bladder cancer;it can also increase the tolerance to chemotherapy drugs by activating the Wnt signaling pathway in bladder cancer.Recent studies reported that lncRNA might also be closely related to the stemness of bladder cancer cells.For example,lncRNA-LET expression will gradually decrease regulated by increasing TGFβ1 expression along with the extension of gemcitabine acting on bladder cancer cells,which will increase the structural stability of NF90 and eventually lead to down-regulation of miR-145 expression,enhancing stemness of bladder cancer cells and drug resistance.It is obvious that dysregulated lncRNA could play important regulatory roles in all aspects associated with tumorigenesis and development of bladder cancer.Therefore,exploring new dysregulated lncRNA molecules in bladder cancer is obviously meaningful for broadening ideas for mechanisms about bladder cancer progression and drug resistance.The competitive endogenous RNA(ceRNA)is the most common and widely recognized mechanism among the various lncRNA regulating mRNA patterns in tumors,ceRNA mechanism can be described as that some non-coding RNAs have microRNA binding sites and act as miRNA sponges,which could reverse the processes of the miRNA suppressing target genes,thereby increasing the expression level of the target gene.Some studies have found that UCA1 have a competitive binding and significantly negative relationships with miR-582-5p.Moreover,targeted gene ATG7 expression of miR-582-5p decreased significantly when UCA1 is knocked down or miR-582-5p is overexpressed,ATG7-mediated cellular autophagy is also inhibited.However,a target gene can also be regulated by more than one "ceRNA" network.In gastric,Claudin-4 is upregulated and is closely related to poor prognosis.It can enhance the proliferation,invasion,and EMT abilities of AGS,HGC-27 and SGC-7901 cancer cell lines,and such processes can be inhibited by miR-596 and miR-3620-3p overexpression.lncRNA-KRTAP5-AS1 and lncRNA-TUBB2A can play the parts of "ceRNA" to inverse the inhibition processes of the two miRNAs and enhance the carcinogenesis of Claudin-4.The two lncRNA are also considered as potential biomarkers and therapeutic targets for gastric cancer.Therefore,it is feasible to reveal the mechanisms of lncRNA regulating target genes through the perspective of "ceRNA".The purpose of this study is to discover new lncRNA molecular markers that are closely related to the occurrence and development of bladder cancer.Based on its cellular localization and distribution characteristics,we aimed to explore its roles and mechanisms in bladder cancer progression and drug resistance,ultimately,to provide available theoretical basis for solving drug resistance and accurate treatment.Methods1.Based on the data from High throughput sequencing on whole-transcriptomes for 10 pairs of bladder cancer tissues and normal tissues adjacent to the cancer,using five kinds of statistical methods including Aov,DESeq2,StudentT,Wilcox,edgeR,we screen candidate lncRNAs with significantly differential expression between the two groups with reference to FDR value,P value,log2FoldChange.2.The full-length sequence of ln387265,one of the candidates,was amplified using 5’RACE and 3’RACE test.Next,PhyloCSF,ORF-finder,and CPAT databases were adopted to verify the non-protein coding characteristics of ln387265.3.FISH test was used to detect the location of ln38276 in bladder cancer cell lines.4.Furtherly,the relative proportion of ln387265 distribution on bladder cancer cells was explored by Nuclear Separation Experiment,which would provide ideas for subsequent mechanism research.5.RT-qPCR was used to identify the expression level of ln382765 in bladder cancer tissues and adjacent normal tissues;The correlation analyses between ln387265 expression level and clinical pathological characteristics were conducted;ln387265 expression levels on different bladder cancer cell lines and normal urothelial cell lines were detected by RT-qPCR.6.To construct lentivirus-mediated ln3827265-shRNA bladder cancer cell lines and ln387265 overexpression bladder cancer cell lines.7.To explore the change on biological behaviors including proliferation,migration,invasion and apoptosis ability of bladder cancer cells when knock down or overexpressing ln387265 in vitro experiments8.In vivo,to inject the stably transfected cell lines under the skin of nude mice.To observe and record the growth curves of the subcutaneous tumors in nude mice in different treatment groups,and to compare the growth differences between the groups.To detect the changes of indicators related to apoptosis and proliferation by IHC.10.To examine the expression changes of ln382765 and drug resistance-associated genes in bladder cancer cell lines after being treated with gemcitabine on gradient time.11.To detect and compare the drug sensitivity(IC50)differences of the different stably transfected cell lines treated by gemcitabine.12.To detect the protein expression changes of genes related to apoptotic and drug resistance of the different stably transfected cell lines.13.To predict the target genes and competitively binding miRNAs of ln387265 using correlation analysis,RegRNA2.0 and TargetScan database.14.To verify the competitively binding relationships among target genes,predicted miRNAs and ln387265 using luciferase reporter genes and RNA pulldown experiments.15.To examine the expression changes of predicted miRNAs and target genes when adjusting the ln387265 expression on different bladder cancer cells by RT-qPCR.16.To construct co-transfection environment associated with ln387265 and predicted miRNAs expression using stably transfected cell lines,miRNA minic and miRNA inhibitor,then,to observe the changes of proliferation,migration and invasion.ability under various conditions.17.To detect the transcriptome changes when overexpressing ln387265 in bladder cancer cells using high-throughput sequencing technology,and then,to.screen the tumor-related regulatory pathways occurring significant changes through GO and KEGG enrichment analysis.1 8.To validate the changes of the pathway-related proteins in overexpressed transfected cells and negative control cells by WB.19.To validate the changes of the pathway-related proteins at the subcutaneous tumor tissues in nude mice by IHC.20.To draw the schematic diagram about regulatory mechanism of ln387265 regulating occurrence and progression of bladder cancer.Results1.High-throughput sequencing identified multiple lncRNA exhibiting significantly different expression between tumor group and adjacent normal group.In addition,according the evidences derived from different statistical algorithms,advanced analysis results,and screening criteria of FDR value<0.5,P value<0.5,log2FoldChange>2,we finally selected ln3827265 as research target.2.No homologous amino acid residues were found after RACE amplification,ORF-finder and NCBI blast being conducted in sequence,which confirmed that ln3 87265 does not have protein-coding function.3.FISH experiments found that ln387265 was mostly localized at the cytoplasm in bladder cancer cells.4.It was furtherly confirmed that the distribution ratio of ln382765 in the cytoplasm was significantly higher than that of the nucleus through nuclear separation experiments,which was consistent with the FISH results,indicating that ln387265 belonged to the cytoplasmic lncRNA.5.The expression level of ln382765 in bladder cancer tumor tissues was higher than that in normal tissues adjacent to the cancer,and ln387265 in high-grade tissues was higher than that in low-grade tissues,and ln387265 in the recurrence cases was higher than that in the primary cases.There is significantly positive correlation between ln387265 expression and tumor grade,stage and recurrence history.ln387265 expression level in T24 and T921 bladder cancer cell lines were significantly higher than that in J82 bladder cancer cells,however,the latter still over that in normal urothelial carcinoma cell line SVHUC.6.The ln387265-shRNA T24 and T921 stably transfected cell lines were successfully constructed.The expression level of ln3 87265 in the knockdown groups were significantly lower than that in the negative control group.The ln387265-overexpression J82 stably transfected cell line was successfully constructed.The ln387265 expression level in overexpression group was significantly higher than that in negative control group.7.Functional experiments showed that the proliferation,colony formation,migration,and invasion capabilities in ln387265-shRNA groups were significantly reduced compared to those in NC group.Flow cytometry showed that the apoptosis rate of ln387265-shRNA groups was higher than that in NC group.8.Functional experiments showed that the proliferation,colony formation,migration,and invasion capabilities in ln3 87265-overexpression group were significantly enhanced.Flow cytometry showed that the proportion of apoptotic cells in ln387265-overexpression group was lower than that in NC group.9.Subcutaneous tumor formation in nude mice and IHC showed that the ability of tumor formation,growth,and Ki-67 protein expression in ln387265-shRNA group were lower than those in the negative control group,and the expression of apoptosis-related protein Casp3 was higher than that in NC group,which phenomenon was contrary to that in ln3 87265-overexpression group.10.The qPCR test found that the expression of ln387265 and GTSE1 in bladder cancer cells gradually increased along with the extension of time under gemcitabine treatment.11.The IC50 values of ln378265-shRNA groups were lower than that in NC groups,which indicated that the drug sensitivity to gemcitabine would increase to some extent when knocking down ln387265 expression.Moreover,the IC50 value in ln387265 overexpression group was higher than that in NC group,suggesting that the drug sensitivity to gemcitabine decline when up-regulating ln387265 expression.12.WB experiments showed that the protein expression of GTSE1 decreased in ln387265-shRNA groups,while the expression of apoptotic genes Casp3 and Casp7 increased significantly compared with those in NC group.However,such tendencies in terms of the three genes expression were opposed to those in ln387265-overexpression group.13.Correlation analysis and database information suggested that ln387265 might have several competitively binding sites with miRNA-608,and GTSE1 may be a downstream target gene of ln387265/miRNA-608.14.The luciferase reporter gene showed that ln387265 had a competitive binding relationship with miRNA-608,and miRNA-608 had a competitive binding relationship with GTSE1.15.RNA pull down test suggested that there was a binding relationship between ln387265 and GTSE1 mRNA.16.RT-qPCR test revealed that miRNA-608 expression increased and GTSE1 expression was down-regulated after knocking down ln387265 expression in T24 bladder cancer cell lines,while miRNA-608 expression was significantly up-regulated and GTSE1 expression increased after overexpressed ln387265 expression in J82 bladder cancer cell lines17.KEGG analysis from high-throughput transcriptome results showed that significantly dysregulated genes between ln387265-overexpression and NC group were enriched on the NF-kB signaling pathway.18.WB results showed that the expression of NF-Kb/p65 and downstream proliferation-related genes Cyclin D1,anti-apoptosis-related gene Survivin,tumor metastasis-related gene MMP-9 in ln387265 overexpression group were higher than those in NC group,while p53 protein expression was lower.19.IHC results suggested that the expression of NF-Kb/p65,Cyclin D1,Survivin,MMP-9,GTSE1,and p53 proteins were consistent with WB results.20.In bladder cancer,up-regulated ln387265 have a competitively binding relationship with miRNA-608,which could even inhibit the interference effect of miRNA-608 on GTSE1,thereby,to promote the expression of GTSE1.The high-expressing GTSE1 in the nucleus enhances the transfer of p53 from the nucleus to the nucleus,reducing the level of p53 protein in the nucleus.However,p53 could interact with p65,and low expression of p53 in the nucleus contributes to the activation of the NF-kB signaling pathway:p65 in the nucleus could act on the specific sequences of DNA to initiate or enhance the transcription of tumor-related genes,and therefore induce the expression of downstream genes Cyclin D1,Survivin,and MMP-9.These genes could strengthen tumor proliferation,metastasis,drug resistance,and anti-apoptosis ability,ultimately,promote progression of bladder tumors.ConclusionLong non-coding RNA n387265 is significantly overexpressed in bladder cancer tissues,and has a significant correlation with high stage,poor differentiation and recurrence of bladder cancer;As a cancer-promoting gene,ln3 87265 in bladder cancer cells play vital roles on enhancing proliferation,migration,invasion and anti-apoptotic ability,and reducing drug sensitivity to gemcitabine;In terms of regulatory mechanism,ln387265 might promote the expression of GTSE1 by competitively binding miRNA-608,then,induce the activation of the NF-kB pathway,which could enhance the progression of bladder cancer.In the present study,we revealed the roles and mechanism of ln387265 on the occurrence and progression in bladder cancer,which would provide new ideas for targeted therapy of bladder cancer,and have potential application value in clinical. |
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