Page 33 - Haematologica May 2022

lncRNA variants in CN-AML
D
Figure 3. Prognostic significance of long non-coding RNA variants across different cytogenetically nor- mal acute myeloid leukemia cohorts. (A) Disease- free survival, (B) overall survival and (C) event-free survival of younger adult cytogeneti-cally normal acute myeloid leukemia (CN-AML) patients in the CALGB/Alliance cohort who had the C-to-T variant of the SNHG15 long non-coding RNA (SNHG15varT) and of patients with the wild-type SNHG15 lncRNA (SNHG15wt). (D) Disease-free survival and (E) event- free survival of CN-AML patients in the AMLCG cohort with the C-to-T variant of the SNHG15 lncRNA (SNHG15varT) and of those the wild-type SNHG15 lncRNA (SNHG15wt).
E
Functional relevance of prognostic long non-coding RNA variants
We hypothesized that in addition to associations with outcome, the presence of genetic variants in lncRNA could have a functional impact and affect AML blast viability and proliferation. We focused on the lncRNA SNHG15varT, whose prognostic significance was validated in an inde- pendent cohort of AML patients. We isolated total RNA from AML cell lines and amplified both the SNHG15wt and SNHG15varT transcripts. We cloned the amplicons into pcDNA3.1 expression vectors and transfected two AML cell lines (i.e., K-562 and THP-1 cells). Expression lev- els of the SNHG15 lncRNA were similar in cells transfected with the SNHG15wt and those transfected with SNHG15varT-containing vectors, when compared with the empty vector controls (Figures 4A and B). Regarding cell
viability, ectopic overexpression of the SNHG15wt and the SNHG15varT led to a discreet but consistent decrease in cell viability across cell lines, which did not reach statistical significance (Figures 4C and D).
In order to further evaluate the effect of SNHG15 on the growth kinetics of the AML cells, we performed colorimet- ric MTT assays. Forced overexpression of the SNHG15varT had no significant effect on blast growth when compared with controls. In contrast, overexpression of the SNHG15wt led to increased proliferative capacity of the leukemic cells when compared with both controls and cells overexpressing the SNHG15varT (Figures 4E and F). These findings are in line with our prognostic observations and the inferior outcome of CN-AML patients who express SNHG15wt, compared with those who express SNHG15varT.
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