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Long non-coding RNA ROR decoys gene-specific histone methylation to promote tumorigenesis.

Fan J, Xing Y, Wen X, Jia R, Ni H, He J, Ding X, Pan H, Qian G, Ge S, Hoffman AR, Zhang H, Fan X - Genome Biol. (2015)

Bottom Line: Suppression of ROR in tumors results in silencing of TESC expression, and G9A-mediated histone H3K9 methylation in the TESC promoter is restored, which significantly reduces tumor growth and metastasis.Without ROR silencing, TESC knockdown presents consistent and significant reductions in tumor progression.Our results reveal a novel mechanism by which ROR may serve as a decoy oncoRNA that blocks binding surfaces, preventing the recruitment of histone modifying enzymes, thereby specifying a new pattern of histone modifications that promote tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, 200025, P. R. China.

ABSTRACT

Background: Long non-coding RNAs (lncRNAs) are not translated into proteins and were initially considered to be part of the 'dark matter' of the genome. Recently, it has been shown that lncRNAs play a role in the recruitment of chromatin modifying complexes and can influence gene expression. However, it is unknown if lncRNAs function in a similar way in cancer.

Results: Here, we show that the lncRNA ROR occupies and activates the TESC promoter by repelling the histone G9A methyltransferase and promoting the release of histone H3K9 methylation. Suppression of ROR in tumors results in silencing of TESC expression, and G9A-mediated histone H3K9 methylation in the TESC promoter is restored, which significantly reduces tumor growth and metastasis. Without ROR silencing, TESC knockdown presents consistent and significant reductions in tumor progression.

Conclusions: Our results reveal a novel mechanism by which ROR may serve as a decoy oncoRNA that blocks binding surfaces, preventing the recruitment of histone modifying enzymes, thereby specifying a new pattern of histone modifications that promote tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

Knockdown of ROR expression in tumors. aROR expression in different tumors and normal cells was measured by RT-PCR. ROR presented higher expression in a series of tumor cells than in two normal gastrointestinal cells (NCM460 and GES-1) and normal fibroblasts. iPSC cells were used as a positive control. GAPDH was used as the internal control. bROR knockdown by two shRNAs. EGFP was used to track the expression of ROR shRNAs in AGS and HT29 cells. Scale bars: 100 μm. c-f RT-PCR (c, d) and real-time PCR (e, f) showed ROR expression in AGS and HT29 cells. M: marker; mock: empty pGIPZ vector, *P <0.05: compared with the control
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Fig1: Knockdown of ROR expression in tumors. aROR expression in different tumors and normal cells was measured by RT-PCR. ROR presented higher expression in a series of tumor cells than in two normal gastrointestinal cells (NCM460 and GES-1) and normal fibroblasts. iPSC cells were used as a positive control. GAPDH was used as the internal control. bROR knockdown by two shRNAs. EGFP was used to track the expression of ROR shRNAs in AGS and HT29 cells. Scale bars: 100 μm. c-f RT-PCR (c, d) and real-time PCR (e, f) showed ROR expression in AGS and HT29 cells. M: marker; mock: empty pGIPZ vector, *P <0.05: compared with the control

Mentions: To investigate our hypothesis, we first examined the expression of ROR in different tumor cells. Since the over-expression of ROR has been reported in iPSCs but not in human fibroblast cells [19], we selected these two cell types as positive and negative controls of ROR expression, respectively. Moreover, we also used normal intestinal mucosal cells (NCM460) and normal gastric epithelial cells (GES-1) to serve as controls for our tests of gastrointestinal tumor cells. As expected, we found that the expression of ROR was significantly increased in a series of tumor cells (Fig. 1a, lanes 2-6), whereas all of the negative controls remained weakly expressed (Fig. 1a, lanes 7-9). Thus, to decipher the potential role of ROR in tumors, we aimed to knockdown the expression of ROR using conventional RNAi methodology. Although a validated siRNA (siROR-1) of ROR lncRNA has been demonstrated [19], we designed another three siRNAs (siROR-2, siROR-3, and siROR-4) to search for the most efficient siRNA. However, we found that siRNA-1 was the most valuable siRNA with the ability to silence the expression of ROR in AGS (gastric cancer) and HT29 (colon cancer) tumor cells (Additional file 1: Figure S1). To exclude off-target effects, we next chose two siRNAs (siROR-1 and siROR-2) for the construction of the pGIPZ ROR-shRNA plasmids. The pGIPZ ROR-shRNA vectors with an EGFP marker were then packaged into lentiviruses and transduced into human AGS and HT29 cells. We used two control cell lines: one with a mock virus carrying the empty vector and one without the virus. Using EGFP as a tracking marker, we observed green fluorescence in AGS and HT29 cells (Fig. 1b).Fig. 1


Long non-coding RNA ROR decoys gene-specific histone methylation to promote tumorigenesis.

Fan J, Xing Y, Wen X, Jia R, Ni H, He J, Ding X, Pan H, Qian G, Ge S, Hoffman AR, Zhang H, Fan X - Genome Biol. (2015)

Knockdown of ROR expression in tumors. aROR expression in different tumors and normal cells was measured by RT-PCR. ROR presented higher expression in a series of tumor cells than in two normal gastrointestinal cells (NCM460 and GES-1) and normal fibroblasts. iPSC cells were used as a positive control. GAPDH was used as the internal control. bROR knockdown by two shRNAs. EGFP was used to track the expression of ROR shRNAs in AGS and HT29 cells. Scale bars: 100 μm. c-f RT-PCR (c, d) and real-time PCR (e, f) showed ROR expression in AGS and HT29 cells. M: marker; mock: empty pGIPZ vector, *P <0.05: compared with the control
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4499915&req=5

Fig1: Knockdown of ROR expression in tumors. aROR expression in different tumors and normal cells was measured by RT-PCR. ROR presented higher expression in a series of tumor cells than in two normal gastrointestinal cells (NCM460 and GES-1) and normal fibroblasts. iPSC cells were used as a positive control. GAPDH was used as the internal control. bROR knockdown by two shRNAs. EGFP was used to track the expression of ROR shRNAs in AGS and HT29 cells. Scale bars: 100 μm. c-f RT-PCR (c, d) and real-time PCR (e, f) showed ROR expression in AGS and HT29 cells. M: marker; mock: empty pGIPZ vector, *P <0.05: compared with the control
Mentions: To investigate our hypothesis, we first examined the expression of ROR in different tumor cells. Since the over-expression of ROR has been reported in iPSCs but not in human fibroblast cells [19], we selected these two cell types as positive and negative controls of ROR expression, respectively. Moreover, we also used normal intestinal mucosal cells (NCM460) and normal gastric epithelial cells (GES-1) to serve as controls for our tests of gastrointestinal tumor cells. As expected, we found that the expression of ROR was significantly increased in a series of tumor cells (Fig. 1a, lanes 2-6), whereas all of the negative controls remained weakly expressed (Fig. 1a, lanes 7-9). Thus, to decipher the potential role of ROR in tumors, we aimed to knockdown the expression of ROR using conventional RNAi methodology. Although a validated siRNA (siROR-1) of ROR lncRNA has been demonstrated [19], we designed another three siRNAs (siROR-2, siROR-3, and siROR-4) to search for the most efficient siRNA. However, we found that siRNA-1 was the most valuable siRNA with the ability to silence the expression of ROR in AGS (gastric cancer) and HT29 (colon cancer) tumor cells (Additional file 1: Figure S1). To exclude off-target effects, we next chose two siRNAs (siROR-1 and siROR-2) for the construction of the pGIPZ ROR-shRNA plasmids. The pGIPZ ROR-shRNA vectors with an EGFP marker were then packaged into lentiviruses and transduced into human AGS and HT29 cells. We used two control cell lines: one with a mock virus carrying the empty vector and one without the virus. Using EGFP as a tracking marker, we observed green fluorescence in AGS and HT29 cells (Fig. 1b).Fig. 1

Bottom Line: Suppression of ROR in tumors results in silencing of TESC expression, and G9A-mediated histone H3K9 methylation in the TESC promoter is restored, which significantly reduces tumor growth and metastasis.Without ROR silencing, TESC knockdown presents consistent and significant reductions in tumor progression.Our results reveal a novel mechanism by which ROR may serve as a decoy oncoRNA that blocks binding surfaces, preventing the recruitment of histone modifying enzymes, thereby specifying a new pattern of histone modifications that promote tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, 200025, P. R. China.

ABSTRACT

Background: Long non-coding RNAs (lncRNAs) are not translated into proteins and were initially considered to be part of the 'dark matter' of the genome. Recently, it has been shown that lncRNAs play a role in the recruitment of chromatin modifying complexes and can influence gene expression. However, it is unknown if lncRNAs function in a similar way in cancer.

Results: Here, we show that the lncRNA ROR occupies and activates the TESC promoter by repelling the histone G9A methyltransferase and promoting the release of histone H3K9 methylation. Suppression of ROR in tumors results in silencing of TESC expression, and G9A-mediated histone H3K9 methylation in the TESC promoter is restored, which significantly reduces tumor growth and metastasis. Without ROR silencing, TESC knockdown presents consistent and significant reductions in tumor progression.

Conclusions: Our results reveal a novel mechanism by which ROR may serve as a decoy oncoRNA that blocks binding surfaces, preventing the recruitment of histone modifying enzymes, thereby specifying a new pattern of histone modifications that promote tumorigenesis.

No MeSH data available.


Related in: MedlinePlus