| BackgroundLong non-coding RNA(LncRNA)is a group of RNA that exceed 200 nucleotides in length and have no protein-coding potential.Initially LncRNA was considered a "noise" from genome transcription and did not have any biological function,but Lnc RNA has become a hot spot of cancer research in the past several years.Recent evidence from large numbers of studies shows that LncRNA presents abnormal expression in many kinds of malignant tumors and plays an important role in the regulation of multiple biological processes.However,the literatures regarding LncRNA in bladder cancer are limited,and the contributions of LncRNA to bladder cancer are remains unclear.ObjectiveTo identify the differential expression of LncRNA in bladder cancer,to clarify the effect of linc00346 on the biological function of bladder cancer cells,and to preliminarily explore the molecular mechanism of linc00346 during the biological process.Methods1.We presented the differential expression of LncRNA in 6 pairs of bladder cancer tissue samples and their corresponding normal urothelium tissue samples by using LncRNA microarray,then quantitative real-time polymerase chain reaction(qRT-PCR)was performed to validate the results of microarray.Furthermore,bioinformatics analyses such as GO and pathway analyses were used to discover the function and associated pathways of differentially expressed LncRNAs,and Gene-coexpression network based on LncRNAs and mRNAs was constructed to identify the interactions between genes and IncRNAs.2.According to the results of bioinformatics analyses and Gene-coexpression network,linc00346 was screened as the targeted LncRNA for the next step in-depth study.We determined the expression of linc00346 in 52 pairs of bladder cancer tissue samples and matched normal urothelium tissue samples,as well as in bladder cancer cell lines T24 and SW780 by qRT-PCR.3.To evaluate the biological functions of linc00346,we knockdown linc00346 respectively from bladder cancer cell lines T24 and SW780 by constructing lentiviral short hairpin RNA(shRNA).Then a series of experiments for testing biological behavior of the cells,including CCK-8 assay,colony formation assay,transwell migration and invasion assay,flow cytometry,western blot analysis,immunocytochemistry assay and subcutaneous implanted tumor formation models,were performed.4.To preliminarily explore the molecular mechanism of linc00346in the process of the occurrence and development of bladder cancer,western blotting analysis was used to detect the expression of PI3K/Akt signal pathway associated proteins(PI3K,p-PI3K,Akt,p-Akt)on each group T24 and SW780 cells.In order to identify the effect of linc00346 on PI3K/Akt signal pathway,these proteins was detected again after exposuring T24 and SW780 cells to LY294002 which could block this signal pathway observably.Results1.We determined a total of 2548 differentially expressed LncRNAs(Value of fold change>2.0)in bladder cancer tissue,including 1539 unregulated ones and 1009 downregulated ones.Furthermore,126 LncRNAs were significantly upregulated in the tumor tissues(Value of fold change>10)when 103 LncRNAs were downregulated.Linc00346 which was screened out by biointormatic analysis may play a key role in the development of bladder cancer,and we would put it as the research object in the fllowing study.2.The results of qRT-PCR showed that the expression of linc00346 was significantly upregulated in bladder cancer tissue samples,as well as the expression levels of linc00346 in T24 and SW780 were higher than that in human normal urothelium cell line HUM-CELL-0046.3.After silencing linc00346 in T24 and SW780 cell lines,the results of CCK-8 assay,colony formation assay,immunocytochemistry assay of Ki67 and subcutaneous implanted tumor formation models showed that the proliferation of tumor cells were inhibited both in vitro and in vivo.The results of flow cytometry and western blot analysis showed that knocking down linc00346 induced a G0/G1 phase arrest in cell cycle,and promoted cell apoptosis in bladder cancer cells.The results of transwell migration and invasion assay showed that the capacity of migrating and invasing were significantly decreased.4.Silencing linc00346 could down-regulate the expression of p-PI3K-p-Akt in bladder cancer cells,and inhibit the phosphorylation of PI3K/Akt pathway.We used PI3K/AKT signaling pathway inhibitors to further study the role of linc00346 in this pathway.After application of the PI3K/AKT inhibitor LY294002,we observed that the inhibitory effect on phosphorylation of PI3K and Akt was more significant.Conclusion1.A series of differentially expressed LncRNAs were identified in bladder cancer tissue,and it was likely that these LncRNAs play a key or partial role in the progression of bladder cancer.2.Linc00346 was found up-regulated in bladder cancer tissue and cells,and the preliminary study indicated that linc00346 could promote proliferation,inhibit cell apoptosis,intensify invasion and metastasis in bladder cancer cell lines.3.These data suggest that linc00346 may play an significant role in the development of bladder cancer by activating the phosphorylation of PI3K/Akt pathway. |
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