AMO-1

Activation of NEAT1 Leading to Survival Advantage of Multiple Myeloma Cells

Long non-coding RNA NEAT1 is the core structural component of the nuclear paraspeckle (PS) organelles and it is deregulated in multiple myeloma (MM) patients. NEAT1 silencing negatively impacts the proliferation and viability of MM cells both in vitro and in vivo.

To elucidate the biological and molecular relevance of NEAT1 upregulation in MM disease, the CRISPR/Cas9 synergistic activation mediator genome editing system was exploited to engineer the AMO-1 MM cell line. The AMO-1 cell line was engineered to stably express the components of the CRISPR activation system (dCas9/ MS2-p65-HSF1. Then, AMO-1 cells were individually transduced with three sgRNA targeting the NEAT1 promoter or a scramble sgRNA. qRT-PCR of selected cells demonstrated that two of three NEAT1 targeting sgRNA induced significant NEAT1 transactivation (AMO-1^N#5 and AMO-1^N#8 cells) compared to the scramble condition (AMO-1^SCR cells) (Fig. 1).

From a biological point of view, upon FBS starvation NEAT1-activated cells showed significantly higher viability as compared to AMO-1^SCR cells (Fig. 2A). This finding is supported also by a cell cycle analysis that showed a higher percentage of cells distributed in sub-G0/G1 phase (Fig. 2B). Additionally, the presence of a significantly higher percentage of apoptotic cells (Fig. 2C) in AMO-1^SCR cells suggests also an anti-apoptotic role of NEAT1. Moreover, NEAT1 transactivation significantly increased the clonogenic potential of MM cells cultured in the same conditions (Fig. 2D).

Fig. 1 Analyses of total NEAT1 and NEAT1_2 expression levels in NEAT1-transactivated AMO cell lines. (Taiana E, et al., 2023)

Fig. 2 NEAT1 transactivation improves multiple AMO cell survival and oncogenic potential in non-physiological culturing conditions. (Taiana E, et al., 2023)

Knockdown of LAMP5 Promoting Apoptosis in MM Cells

Multiple myeloma (MM) is the second most common tumor of the hematologic system. Lysosome-associated membrane protein (LAMP) affects various cellular processes, including phagocytosis, autophagy, lipid transport, and senescence, and is involved in cancer. LAMP5 is a member of the LAMP family. The study silenced LAMP5 expression in RPMI-8226 and AMO-1 cell lines and explored the effects of LAMP5 silencing on MM cell apoptosis.

LAMP5 expression was highest in RPMI-8226 and AMO-1, followed by H929, and lowest in U266 (Fig. 3A). PMI-8226 and AMO-1 for the follow-up experiments. Four different siRNA sequences were used for cell transfection and found that si3 and si4 were the most efficient in reducing LAMP5 expression, so these two sequences were selected for the next experiments (Fig. 3B, C).

In AMO-1, the apoptosis rates were 33.05% ± 3.74%, 43.34% ± 4.27% and 44.52% ± 1.47% for siNC, si3 and si4, respectively (Fig. 4A). knockdown of LAMP5 increased the expression of the pro-apoptotic protein bax, downregulated the expression of the anti-apoptotic protein bcl-2, and upregulated the expression of cleaved caspase3 (Fig. 4B). Therefore, the study concluded that knockdown of LAMP5 gene could lead to apoptosis in MM cells.

Fig. 3 Expression of LAMP5 in MM cell lines (RPMI-8226, AMO-1, H929, and U266) and screening of siRNA for knockdown of LAMP5. (Chen Y and Ma T, 2023)

Fig. 4 Knockdown of LAMP5 in MM cell lines (RPMI-8226, AMO-1) promotes apoptosis. (Chen Y and Ma T, 2023)