Long non-coding RNA UCA1 regulated cell cycle distribution via CREB through PI3-K dependent pathway in bladder carcinoma cells
Highlights
► Microarray data showed many target genes were screened by stable knockdown of UCA1. ► Real-time PCR analysis confirmed some critical target genes. ► Western blot validated EP300 and indicated CREB was affected by UCA1 expression. ► UCA1 expression was correlated to AKT activity which influenced CREB. ► UCA1 regulated cell cycle progression through CREB via PI3K-AKT pathways.
Introduction
In the past 50 years, about 98% of the transcriptional output of the human genome was regarded as “transcriptional noise,” because these RNA molecules do not code for proteins (Tsang et al., 2007). However, with the development of high-throughput DNA sequencing and array-based technologies, various classes of lncRNAs have recently been shown to function as regulators of protein-coding gene expression in eukaryocytes. Furthermore, extensive evidence indicates that lncRNAs exert a variety of intrinsic functions and play important roles in human diseases (Taft et al., 2010).
LncRNAs have been shown to be spliced, polyadenylated, and developmentally regulated in eukaryotes, including antisense, intergenic transcripts and epigenetic regulators (Brock et al., 2009). LncRNAs are normally transcribed on the sense or antisense strand, and interplay with transcription of an adjacent gene (Mattick, 2009). Many studies strongly suggest that deregulated expression of long ncRNAs is closely correlated with the diversity of multigenetic diseases, and the increasing data emphasize their functions on the basal regulation of protein-coding genes (Ponting et al., 2009), including those crucial to normal cell development, oncogenesis during transcription and post-transcription (Perez et al., 2008). In addition, recent reports showed that some lncRNAs exhibit distinct gene expression patterns in solid tumors and leukemias (Calin et al., 2007). Moreover, more and more lncRNAs are considered as a switch to alter gene expression in different signal pathways (Gallivan, 2007, Mandal et al., 2003).
Some recent studies, including our previous work, have reported that long non-coding RNA urothelial carcinoma associated 1 (UCA1), a novel specific lncRNA displays a remarkable potential in the progression of bladder cancer (Wang et al., 2006, Xie et al., 2010). There are two of three different transcripts derived from UCA registered in the GenBank nucleotide sequence database with individual accession number(EU334869 and GU357550). EU334869 was identified from two TCC cell lines BLS-211 and BLZ-211 (cloned from one patient's sample respectively) through subtractive suppression hybridization (SSH) technique (Yang et al., 2006). Functional assay demonstrated that ectopic expression of UCA1 mRNA in BLS-211 significantly enhanced its tumorigenicity, invasive potential and drug resistance. Microarray analysis also showed several genes such as WNT6 (Peifer and Polakis, 2000), CYP1A1 (Shimada and Fujii-Kuriyama, 2004) and AURKC (Katayama et al., 2003), were associated with tumor progression, as well as apparently up- and down-regulated when UCA1 was exogenously expressed in BLS-211 cells. These findings suggested that UCA1 may regulate some aspects of the tumorigenic processes (Wang et al., 2008). Nevertheless, since it's highly possible that target genes of lncRNA differ between specific tissues and cell types, specific target genes controlled by UCA1 remained unknown. Although UCA1 can facilitate the progression of bladder cancer, little is known about the underlying molecular mechanisms.
In the present study, after knocking down the expression of lncRNA UCA1 in BLZ-211 cells by RNAi, two potential targets (p300 and CREB) are identified to be regulated by UCA1 using microarray analysis. Furthermore, the regulation mechanism was investigated in two bladder carcinoma cell lines (UCA1 positive for BLZ-211, and UCA1 negative for UMUC-2). Suppression of UCA1 expression by shRNA in BLZ-211 cells resulted in CREB down-regulation; while compensatory expression of UCA1 in UMUC-2 cells led to CREB up-regulation. The results from Western blot analysis revealed that both expression and phosphorylation of AKT and CREB are correlated to UCA1 expression. Furthermore, the activity and expression of CREB decreased, accompanied by cell arrest, when PI3K-AKT pathway was inhibited with LY294002 in BLZ-211. Therefore, we conclude that UCA1 regulates cell cycle progression through CREB via PI3K-AKT dependent signaling pathways. These results suggest that lncRNA UCA1 may serve as new diagnostic and therapeutic targets in bladder cancer (Costa, 2009).
Section snippets
shRNA plasmid construction
SiRNAs against UCA1 were designed by using the software siRNA Construct Builder (GenScript Corp., Piscataway, NJ). An extra siRNA was designed as negative control. According to standard guidelines, every individual siRNA was converted into a shRNA cassette by adding a loop (9nt) and appropriate sites for the cloning. ShRNAs were synthesized as sense and antisense oligonucleotides, annealed and subcloned into the shRNA expression vector pRNAT-U6.1/Neo (GenScript Corp., Piscataway, NJ). The
Efficiency and specificity of shRNAs in silencing UCA1 expression
In order to detect whether pRNAT-U6.1/Neo vectors could generate 19-nucleotide duplex RNAi oligonucleotides, which is able to silence UCA1 expression in BLZ-211 cells, transient transfection was performed. 48 hours after transfection, silencing capacity of shRNAs was evaluated by using RT-PCR. Among three shRNA plasmids(pRNAT-U6.1/Neo-shUCA1.1, pRNAT-U6.1/Neo-shUCA1.2, pRNAT-U6.1/Neo-shUCA1.3), pRNAT-U6.1/Neo-shUCA1.1 showed optimal effect compared to that of negative control (Fig. 1).
Microarray analysis after silencing UCA1
In order
Discussion
Although it is well-known that lncRNAs are necessary to gene transcription, most attention has been focused on microRNAs and their regulatory functions in gene expression. However, in human disease, mechanisms and functional roles of lncRNAs remain elusive at molecular level and require further investigation (Lipovich et al., 2010). In this study, we continued our efforts on revealing the regulatory role of a novel lncRNA UCA1, in order to elucidate its general function and underlying
Acknowledgments
The work was funded by grants from the National Natural Science Foundation of China (No. 30370660 and No. 81072104). We thank Dr. Zhijun Luo from Boston University school of Medicine for editing this manuscript.
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2022, International Journal of CardiologyCitation Excerpt :CHAST inhibits the expression of Plekhm1, thereby reducing autophagy and cardiomyocyte hypertrophy [13]. UCA1 is protective against hydrogen peroxide induced apoptosis in cardiac myocytes [14]. UCA1 level in AMI decreased in the early stage of the disease and increased 3 days after the onset of MI, regardless of the influence of hypertension and diabetes.