Activation of long non-coding RNA NEAT1 leads to survival advantage of multiple myeloma cells by supporting a positive regulatory loop with DNA repair proteins

Abstract

Long non-coding RNA NEAT1 is the core structural component of the nuclear paraspeckle (PS) organelles and it has been found to be deregulated in multiple myeloma (MM) patients. Experimental evidence indicated that NEAT1 silencing negatively impacts proliferation and viability of MM cells, both in vitro and in vivo, suggesting a role in DNA damage repair (DDR). In order to elucidate the biological and molecular relevance of NEAT1 upregulation in MM disease we exploited the CRISPR/Cas9 synergistic activation mediator genome editing system to engineer the AMO-1 MM cell line and generate two clones that para-physiologically transactivate NEAT1 at different levels. NEAT1 overexpression is associated with oncogenic and prosurvival advantages in MM cells exposed to nutrient starvation or a hypoxic microenvironment, which are stressful conditions often associated with more aggressive disease phases. Furthermore, we highlighted the NEAT1 involvement in virtually all DDR processes through, at least, two different mechanisms. On one side NEAT1 positively regulates the posttranslational stabilization of essential PS proteins, which are involved in almost all DDR systems, thus increasing their availability within cells. On the other hand, NEAT1 plays a crucial role as a major regulator of a molecular axis that includes ATM and the catalytic subunit of DNA-PK kinase proteins, and their direct targets pRPA32 and pCHK2. Overall, we provided novel important insightsthe role of NEAT1 in supporting MM cells adaptation to stressful conditions by improving the maintenance of DNA integrity. Taken together, our results suggest that NEAT1, and probably PS organelles, could represent a potential therapeutic target for MM treatment.

Figure 1.

NEAT1 transactivation associates with increased paraspeckle protein expression levels. (A) Analyses of total NEAT1 and NEAT1_2 expression levels in NEAT1-transactivated AMO-1^N#5 and AMO-1^N#8 cell lines with respect to AMO-1^SCR cells, based on the real-time quantitative polymerase chain reaction (qRT-PCR) approach described in the schematic representation. NEAT1 expression was expressed as 2^-ΔΔCt relative to the scrambled (SRC) condition. *P<0.05 vs. SCR; ***P< 0.001 vs. SCR. (B) Western blot of NONO, SFPQ, and FUS in AMO-1^SAM cells. GAPDH protein expression was included for protein loading normalization. (C) Effect of NEAT1 transactivation in the presence of the protein synthesis inhibitor cycloheximide (CHX) (100 mM) on the decay of NONO and FUS protein levels in AMO-1^SCR and AMO-1^N#8 cells at indicated time points. Actin protein expression was included for protein loading normalization. The densitometric analysis of immunoreactive bands is reported with respect to SCR condition in western blotting experiments. (D) Confocal microscopy results of NEAT1 specific RNA fluorescence in situ hybridization and NONO immunofluorescence in AMO-1^SAM cells (scale bar 5µm).

Figure 2.

Fetal bovine serum starvation upregulates NEAT1 and increases paraspeckle numbers. (A) Real-time quantitative polymerase chain reaction (qRT-PCR) analyses of total NEAT1 and NEAT1_2 expression in AMO-1^SAM cells maintained for 48 hours (h) in physiological fetal bovine serum (FBS)-culturing conditions (10% FBS) and in FBS-starving medium. NEAT1 expression was expressed as 2^-ΔCt. (B) Percentage of NEAT1_2 variant contribution respect to total NEAT1 expression in AMO-1^SAM cells maintained for 48 h in 10% FBS and in FBS-starving medium. (C) Confocal microscopy results of NEAT1-specific RNA fluorescence in situ hybridization and NONO immunofluorescense in AMO-1 cells cultured for 48 h in 10% FBS, in FBS-starving medium, and upon restoring physiological 10% FBS-culturing conditions (scale bar 5µm).

Figure 3.

Hypoxia upregulates NEAT1 and increases paraspeckle numbers. (A) Realtime quantitative polymerase chain reaction (qRT-PCR) analyses of total NEAT1 and NEAT1 2 expression in AMO-1^SAM cells maintained for 48 hours (h) under normoxic and hypoxic microenvironment. NEAT1 expression was expressed as 2~^ACt. (B) Percentage contribution of NEAT1 2 respect to total NEAT1 expression in AMO-1^SAM cells maintained for 48 h under normoxic and hypoxic micro-environment. (C) Confocal microscopy results of NEAT1-specific RNA fluorescence in situ hybridization (RNA-FISH) and NONO immunofluorescence (IF) in AMO-1^SAM cells cultured for 48 h upon normoxic or hypoxic conditions (scale bar 5µm). (D) Confocal microscopy results of NEAT1-specific RNA-FISH and NONO IF in AMO-1 cells cultured for 48 h upon normoxic, hypoxic conditions, and upon restoring physiological normoxic conditions (scale bar 5µm).

Figure 4.

NEAT1 transactivation improves multiple myeloma cells survival and oncogenic potential in non-physiological culturing conditions. (A) Growth curve and viability of AMO-1^SAM cells cultured for 72 hours (h) in fetal bovine serum (FBS)-starving conditions. *P<0.05 vs. scrambled (SCR). (B) Cell cycle analysis by propidium iodide (PI) staining performed in AMO-1^SAM cells after 48 h of culture in FBS-starving conditions; specific histograms representing the percentage of cells in sub G0/G1 are also shown. *P<0.05 vs. SCR. (C) Flow cytometric analysis of apoptosis in AMO-1^SCR, AMO-1^N#5 and AMO-1^N#8 cultured for 72 h in FBS-starving conditions. (D) Colony formation assay performed on AMO-1^SAM cultured for 31 days in FBS-starving conditions; representative pictures of colonies distribution at day 31 are also shown. (E) Representative pictures of colonies formed in AMO-1^SCR, AMO-1^N#5 and AMO-1^N#8 24 days after seeding (10x magnification). (F) Werstern blot of pERK 1/2, ERK 1/2, pAKT, and AKT in AMO-1^SAM cells after 48 h of culture in FBS-starving conditions. GAPDH protein expression was included for protein loading normalization. Percentage of pERK1/2 and pAKT with respect to total ERK1/2 and AKT (both normalized for GAPDH expression) is also shown.

Figure 5.

NEAT1 transactivation upregulates proteins involved in the DNA repair process. (A) Western blot (WB) of pRPA32 and RPA32 in AMO-1^SAM cells. (B) Confocal microscopy results of pRPA32 specific immunofluorecense (IF) in AMO-1^SAM cells cultured under physiological culturing conditions (scale bar 5mm). (C) WB of pCHK2, CHK2, pCHK1, and CHK1 in AMO-1^SAM cells. (D) WB of pATR, ATR, pATM, ATM, and DNA-PKcs in AMO-1^SAM. The densitometric analysis of DNA-PKcs immunoreactive bands is reported with respect to the scrambled (SCR) condition. Furthermore, the percentage of the activated fraction of all proteins with respect to the relative total (tot) amount (both normalized for GAPDH expression) is reported. (E) Confocal microscopy results of DNA-PKcs specific IF in AMO-1^SAM cells cultured under physiological culturing conditions (scale bar 5mm).

Figure 6.

Multiple myeloma cells under stressful conditions overexpress NEAT1 and other proteins involved in the DNA repair process. (A) Confocal microscopy results of DNA-PKcs and pRPA32 specific immunofluorescence (IF) in AMO-1 cells cultured in physiological fetal bovine serum (FBS)-culturing condition (10% FBS) and in FBS-starving medium (scale bar 20µm). (B) Western blot (WB) analysis of pRPA32, RPA32, DNA-PKcs and ATM in AMO-1^SAM cells after 48 hours (h) of culture in FBS- starving condition. GAPDH protein expression was included for protein loading normalization. Percentage of pRPA32 with respect to total (tot) RPA32 (both normalized for GAPDH expression) is also shown. (C) WB analysis of DNA-PKcs in AMO-1^SAM cells after 48 h of culture in normoxic and hypoxic conditions. Actin protein expression was included for protein loading normalization. The densitometric analysis of immunoreactive bands is reported with respect to the scrambled (SCR) condition.

Figure 7.

NEAT1 silencing downregulates proteins involved in the DNA repair process. (A) Scheme of LNA-gapmeR localization on NEAT1 transcript; real-time quantitative polymerase chain reaction (qRT-PCR) analyses of NEAT1 expression levels in AMO-1 and LP1 MM cell lines upon gymnotic delivery of g#N1_E or g#N1_G LNA-gapmeR. NEAT1 expression was expressed as 2~^ACt. (B) Western blot analysis of pRPA32, RPA32, DNA-PKcs, and ATM in AMO-1 and LP1 cells after gymnotic delivery of NEAT1-targeting gapmeR (5 DM). GAPDH protein expression was included for protein loading normalization. The densitometric analysis of DNA-PKcs and ATM immunoreactive bands is reported with respect to the scrambled (SCR) condition. Furthermore, the percentage of pRPA32 with respect to total RPA32 (both normalized for GAPDH expression) is also shown. tot: total.

Figure 8.

ATM and DNA-PK inhibitions abrogate NEAT1 pro-survival advantages. (A) Optical microscopy results of May Grun-wald-Giemsa (MGG) staining obtained in AMO-1^SCR and AMO-1^N#8 cells after 3 days of culture in fetal bovine serum (FBS)-starving conditions, in the presence for the last 24 hours (h) of ATM and DNA-PK inhibitors (100x magnification). (B) Optical microscopy results of MGG staining obtained in NCI-H929 and CD138+ multiple myeloma (MM) primary cells after 3 days from the gymnotic delivery of NEAT1-targeting gapmeR (5 mM) (100x magnification). (C) Cartoon summarizing the molecular circuit driven by NEAT1 and its crucial role for MM cell survival under stressful conditions. PC: plasma cells.