Does ANRIL mediate its effect through altered expression of the CDKN2A/B locus?

As ANRIL has been shown to negatively regulate p16INK4A and p15INK4B, RT-qPCR was used to determine if the knockdown of ANRIL expression also induced a change in expression of these transcripts. Following transfection of cells with Ambion E1 siRNA, there was a significant increase in p16INK4A expression (p=0.0018), however there was no change in p16INK4A expression in cells transfected with either Congrains E1 or E19 siRNAs (Figure 4.9A). Interestingly, p15INK4B expression was significantly decreased in cells transfected with each of the three ANRIL siRNAs (p=0.0045, p<0.0001 and p<0.0001), though Ambion E1 siRNA caused a smaller decrease in expression compared to either Congrains E1 and Congrains E19 siRNAs (statistical differences in expression between the three siRNAs denoted by letters in Figure 4.9B).

Expression of p14ARF, a third product of the CDKN2A/B locus was also investigated, though there is no evidence to suggest that this transcript is regulated by ANRIL. Results showed that when ANRIL expression is knocked down, there was a significant decrease in p14ARF expression when SaOS-2 cells were treated with the Congrains E1 and Congrains E19 siRNAs (p=0.0029 and p<0.0001 respectively) (Figure 4.9C).

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Figure 4.9: Expression of p16INK4A (A), p15INK4B (B) and p14ARF (C) in SaOS-2 cells compared to control when ANRIL expression has been knocked down. Graphs represent three independent transfections (n=3).

Results shown as mean ±SEM * denote comparisons to control (****p≤0.0001; ***p≤0.001; **p≤0.01; *p≤0.05) Letters denote comparisons between the three siRNA treatments.

113 4.4 Discussion

In this chapter the role of ANRIL was investigated in the osteoblast derived cell line SaOS- 2 cells. Previously, very little was known about the expression of ANRIL, which cell types it is expressed in and what its precise function is. The results in this chapter show that ANRIL is highly expressed in the osteoblast-like derived cell line SaOS-2 and primary osteoblasts, as well as human bone marrow stromal cells (hBMSCs). Transcripts containing exon 1-2, 5-6 and 18-19 were all detected. Moreover, ANRIL expression was also detected in a liposarcoma cell line (SW-872), breast cancer cell line (Hs578T) and liver cell line (HepG2), suggesting tissue wide expression patterns. Although interestingly, the relative abundance of the transcripts containing exons 1-2 and 5-6 in SW-872 cells compared to SaOS-2 cells was similar, whereas transcripts containing exons 18-19 were relatively low.

Our results show that expression of ANRIL transcripts containing exons 1-2 or 5-6 is higher in hBMSCs than those that have been differentiated into primary osteoblasts, whereas there was no significant difference in exons 18-19 expression. As ANRIL has been shown to repress p16INK4A and p15INK4B, cell cycle inhibitors, high levels of ANRIL expression in hBMSCs would be consistent with the higher self-renewal capacity of these stem cells. It has been shown across different cell types that differentiation is co-ordinated with cell cycle exit (291). In the case of ANRIL, you would potentially expect to see decreased expression in cells that are undergoing differentiation as cells exit the cell cycle in order to differentiate, which would allow for an increase in p16INK4A levels. These results do suggest that different cell types express different levels of ANRIL, which agrees with results from other research groups (238,281,284,292). Figure 4.10 shows exons present in different known ANRIL transcripts and this figure shows that exons 5 and 6 are more abundant in

Figure 4.10: Exons present in 17 different ANRIL transcripts in PBMCs. Transcripts identified using RACE

and PCR amplification. Figure replicated from Holdt et al (282).

Exons 1 2 3 4 5 6 7 7b 8 9 10 11 12 13 13b 14 15 16 17 18 19 20 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17

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ANRIL transcripts when compared to exons 18 and 19. Even though exon 1 is present in the majority of the transcripts shown in these figures, exon 2 is present in only a few of the transcripts shown. This agrees with our RT-qPCR results as we found that transcripts containing exons 5-6 were the most highly expressed and transcripts containing exons 18-19 had the lowest expression. However, even though different isoforms have been shown to be expressed in VSMCs, HeLa and PBMCs, the specific isoforms that are expressed in bone are not known.

Further work would need to be done in order to determine which specific ANRIL transcripts are expressed in bone. This could be done by rapid amplification of cDNA ends (RACE), where reverse transcription of RNA and PCR amplification are used to determine the sequence of unknown transcripts, which can then be mapped back to its unique genomic region. An alternative method would be to use PCR, with forward and reverse primers mapping to sequences within the exons of known ANRIL transcripts.

To determine what phenotypic and functional effects ANRIL has on bone development, siRNAs were used to knock down ANRIL expression in the osteoblast-like cell line SaOS-2. Three siRNAs were used to knock down ANRIL expression, and the results show that ANRIL was successfully knocked down by all three siRNAs. The effectiveness of knockdown was different for the three siRNAs depending on the transcript examined. For example, as Congrains E19 siRNA is less effective in knocking down transcripts containing exons 1-2 compared to Congrains E1 or Ambion E1, this suggests that not every ANRIL transcript that contains exon 1 also contains exon 19. Although, as exons 18-19 expression is knocked down to the same level when cells are treated with both Congrains E1 and Congrains E19, this may additionally suggest that the majority of ANRIL transcripts that do contain exon 19 also contain exon 1. To fully understand the significance of these results, the next step would be to map the ANRIL isoforms present in these cells. Interestingly the Ambion E1 siRNA did not knock down ANRIL expression to the same extent as Congrains E1 siRNA, which suggests there is a difference between the effectiveness of the siRNAs directed against exon 1 of ANRIL, potentially due to the different sequence that is targeted within exon 1 or a difference in secondary structure of the region.

The results show that when ANRIL expression is successfully knocked down by all three siRNAs, SaOS-2 cell number decreases. This decrease could be due to different mechanisms which include cell cycle arrest, an increase in apoptosis or potentially both of these mechanisms working together. To determine if the cells were being held in G0/G1 phase or whether an increase in cell death is the cause of the decrease in cell number, samples were stained using propidium iodide and analysed using FACS. Ambion E1 siRNA induced a significant increase in the number of cells in the G0/G1 phase of the cell cycle, but no effect on G0/G1 cell number was seen

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with the other ANRIL siRNAs. Congrains E1 and E19 transfection lead to a significantly higher number of cells undergoing apoptosis compared to the ‘scrambled control’. This difference is interesting especially between the two siRNAs directed against exon 1. Ambion E1 siRNA was less effective at knocking down exons 1-2 and exons 5-6 expression than Congrains E1 siRNA and less effective at knocking down ANRIL exons 18-19 expression than either Congrains E1 or E19 siRNAs. Even though there are 6 known variants of exon 1 across the 17 sequenced ANRIL transcripts, both Ambion E1 and Congrains E1 siRNAs map to all 6 different exon 1 sequences, so this does not explain the difference seen in knock down efficiency. Whether this difference in response is then due to the severity of the knock down is not clear, however it could probably be tested by transfecting SaOS-2 cells with an increasing concentration range of ANRIL Ambion E1 siRNA. However, another possibility is that the secondary structure of the sequence of mRNA maybe be different, therefore this could affect the effectiveness of siRNA binding and its knock down efficiency.

As the Ambion E1 siRNA from Life Technologies caused an increase in the number of cells being held in G0/G1 phase, this points to ANRIL working in cis to prevent cell cycle arrest. Consistent with this, treatment with Ambion E1 siRNA did lead to an increase in p16INK4A expression, while the others did not. As ANRIL negatively regulates p16INK4A, expression of this transcript should increase when ANRIL expression decreases. This would lead to cell cycle arrest and prevention of cell cycle progression into S phase. The results shown here agree with what have previously been demonstrated in other cell types (237,243,293) and agrees with all the theories that low expression of ANRIL leads to an increase in the expression of p16INK4A and, consequently, an increase in the number of cells held in the Go/G1 phase of the cell cycle.

A decrease in ANRIL expression and increase in p16INK4A expression, in relation to bone development, could lead to premature cell cycle exit and differentiation. This could lead to an imbalance in bone formation as it has been shown in mice that premature osteoblast differentiation can lead to deregulated bone formation (294) and there is also evidence to suggest that premature cell cycle exit and early differentiation can lead to mislocalised transcription factors (295). An increase in p16INK4A expression has also been linked to replicative senescence (239), which is when cells no longer undergo proliferation (296), and this could potentially be another ‘state’ that the cells adopt upon ANRIL knockdown.

There is now also evidence from mouse models demonstrating that premature senescence can lead to impaired osteogenic differentiation potential, with bone marrow stromal cells from 3 different mouse strains favouring adipogenic differentiation (297–299). These mice showed lower trabeculae number, decreased bone strength and reduced bone mineral density

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from a young age, further highlighting the need for proper regulation of osteogenesis. If osteoblasts became prematurely senescent, this could cause an imbalance in bone cell turnover which favoured osteoclasts and bone degradation. This would potentially lead to a lower peak bone mass and greater risk of fracture and osteoporosis in later life (114).

ANRIL siRNAs Congrains E1 and E19 induced apoptosis in the SaOS-2 cells, though no effect was seen upon treatment with Ambion E1 siRNA. There is evidence to suggest that ANRIL has a role in apoptosis pathways, though the evidence is contradictory. Holdt et al have demonstrated a pro-apoptotic effect of ANRIL knockdown in PBMCs and MonoMac cells (a monocytic cell line), shown by a significant increase in Annexin V+ cells (282), whereas Congrains et al have demonstrated an anti-apoptotic effect when VSMC are treated with an siRNA against exon 19 of ANRIL (237). This demonstrates the potential of ANRIL having cell type specific trans functions and if, as my results suggest, ANRIL has an anti-apoptotic function in osteoblasts, this could have adverse effects on bone development by decreasing bone formation and potentially leading to a lower peak bone mass (64,300,301).

Interestingly, our results show that p16INK4A expression is not changed upon knock down of ANRIL with Congrains E1 and E19 siRNAs, which suggests that the cells are not undergoing premature senescence or cell cycle arrest via p16INK4A. This agrees with results previously shown by other groups (282), though others have shown that when ANRIL expression is knocked down p16INK4A expression increases and cells become senescent (243). In this study ANRIL knock down did induce a decrease in p15INK4B expression when ANRIL expression is knocked down. As ANRIL is the antisense RNA to the CDKN2B gene, one would expect an increase in p15INK4B expression as previously demonstrated by independent groups (276,289). However, these experiments were not carried out in bone cells; therefore this may not be the case for SaOS-2 cells. As ANRIL acts as a scaffold lncRNA to recruit PRC complexes to the p15INK4B gene locus, if ANRIL expression is knocked down, this could allow for other repressive complexes to bind to this region instead, potentially acting as a compensatory mechanism due to the importance of the cell cycle pathway. Dysregulation of cell cycle control regulators may induce apoptosis or ANRIL may do this through an independent mechanism, through which it could alter other regulatory factors that would otherwise cause the upregulation of p15INK4B.

The Ambion E1 siRNA also induced a decrease in p14ARF expression. Though there is evidence to suggest that p14ARF can induce apoptosis in cells by stabilising p53, which leads to the induction of Bax expression (302), this is unlikely to be the case with SaOS-2 cells as this cell line is p53-null. However, there is evidence to suggest that a p53 homologue, p73, can induce apoptosis independently of p14ARF expression (303), and its expression has been demonstrated in SaOS-2

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cells (304). Interestingly, our results show that upon knock down of ANRIL expression with Congrains E1 and E19 siRNAs there is a decrease in the expression of p14ARF, though there is no evidence in the literature to suggest that ANRIL directly regulates the expression of this transcript. However, the first exon of the ANRIL transcript does cover the promoter region of p14ARF; therefore a decrease in ANRIL expression could allow for potential repressor complexes to bind to the promoter of this transcript and cause the decrease in expression that is observed.

To see whether ANRIL has any osteoblast specific functions that affect phenotype, we looked at the expression of differentiation and mineralisation markers in SaOS-2 cells where ANRIL expression had been knocked down. Our results show that upon ANRIL knock down differentiation markers significantly increase, as shown by increases in both RUNX2 expression (an early stage differentiation marker) and ALP expression (a late stage differentiation marker). No significant changes were seen with regards to the mineralisation maker osteocalcin (BGLAP). These results suggest that knock down of ANRIL enhances differentiation down the osteogenic lineage, regardless of the effect on p16INK4A, all siRNAs also effected the expression of these osteogenic markers, which suggests that this change in expression could be p16INK4A independent. There is evidence to show that lncRNAs and other epigenetic mechanisms have a role to play in osteogenic differentiation. A recent paper by Zhu et al has demonstrated that the downregulation of lncRNA-ANCR (anti-differentiation ncRNA) promotes osteogenic differentiation by regulating RUNX2 expression through associations with enchancer of zeste 2 (EZH2) (214), thus demonstrating the potential of ANRIL as a mediator of osteoblast differentiation. lncRNA expression assays are also identifying new lncRNAs that have roles to play in the early differentiation of MSCs (213). However, as there was a change in p15INK4B and p14ARF expression, this could suggest that the effects seen on RUNX2 expression and ALP expression could be p15INK4B and p14ARF dependent, rather than through direct regulation by ANRIL. Though there is evidence to suggest that RUNX2 can regulate the osteoblast cell cycle.

In cells from wild type and Runx2 deficient mice, osteoblasts from Runx2 deficient mice proliferated at a higher rate than those from wild type mice. When Runx2 was reintroduced to the cells, it caused a decrease in cell proliferation, suggesting that stringent cell cycle control had been restored (305). This could explain the decrease in p14ARF and p15INK4B expression seen upon ANRIL knock down, with RUNX2 potentially acting as a compensatory mechanism in order to maintain cell cycle control, or that the increase in RUNX2 expression causes the decrease in the expression of these two inhibitors.

There is evidence demonstrating that RUNX2 can have an anti-proliferative effect in osteoblasts by causing cell cycle arrest at the G0/G1 phase of the cycle. Galindo et al have

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demonstrated that an increase in RUNX2 expression in MC3T3 cells (a murine osteoblast cell line) suppresses proliferation and causes an increase in the number of cells being held in G1 phase (306). This effect was also seen in primary murine calvarial cells. Pratap et al have also shown that MC3T3 and primary calvarial cells that do not express Runx2 (Runx2-/-) or express a protein that lacks the Runx2 COOH terminus (Runx2C/C), which is essential for the interaction of this protein with several different pathways, exhibit an increase rate of cell growth. When Runx2 was then reintroduced to the cells through use of an adenovirus, the rate of cell growth reduced to a rate that was comparable to the control cells (305).

RUNX2 has been shown to have a role in pathways that regulate cell proliferation, such as Smad and MAPK pathways (305) as well as G protein-coupled signalling pathways (307). However, it is also known to be regulated by cell signalling pathways that are involved in bone homeostasis (308), such as the BMP, Wnt and IHH pathways. If ANRIL has a role in regulating one of these pathways, when it’s expression is knocked down it could lead to an increase in RUNX2 expression, which could, in turn, lead to a reduction in cell growth and proliferation. Either of these scenarios could potentially explain the increase in RUNX2 that is seen in SaOS-2 cells that have been treated with siRNAs against ANRIL.

4.5 Summary

We sought to demonstrate that ANRIL expression is cell type and tissue type specific by looking at the expression of three different ANRIL exons across a range of cell types. Our results show that ANRIL expression does differ across cell types, with ANRIL expression being lower in cells that have undergone osteogenic differentiation when compared to undifferentiated hBMSCs. We sought to establish the functional effects of ANRIL in the osteoblast-like cell line SaOS- 2 by transfecting these cells with siRNA against ANRIL and knocking down its expression. We showed that ANRIL knock down caused a decrease in cell number, which is most likely due to an increase in pro-apoptotic pathways as p16INK4A and p15INK4B expression did not increase upon ANRIL knock down. This suggests that the cells were not becoming senescent or undergoing cell cycle arrest. Our results show that RUNX2 and ALP expression, both markers of osteogenic differentiation, significantly increase when ANRIL expression is knocked down. This suggests that ANRIL may have a role in the regulation of these two proteins.

In order to confirm our findings experiments need to be repeated in primary osteoblasts as there are limitations to using cell lines. SaOS-2 cells do not have Rb, which is a downstream repressor of E2F and is indirectly regulated by p16INK4A, or p53, both of which have roles in the cell

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cycle pathway, which is why we may have seen a decrease in p15INK4B expression and no change in p16INK4A expression.

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Chapter 5

Characterisation of ANRIL in Human