Journal: Nature Communications

Article Title: Tumour mutations in long noncoding RNAs enhance cell fitness

doi: 10.1038/s41467-023-39160-7

Figure Lengend Snippet: a Plasmid-transfected cells were measured at indicated timepoints. Statistical significance was estimated by two-sided Student’s t test based on n = 3 independent replicates. Mean value +/− SD is plotted. Replicates were performed at different times (experimental replicates). b Overexpression of LOHAN1&2 in HN5 cells. RNA levels were measured by qRT-PCR; n = 3 (experimental replicates performed at different times). c Results of colony formation assay in HN5 cells. Data are presented as mean values −/+ SD of the percent of well area covered from 18 culture wells. Statistical significance was estimated using one-way ANOVA. Replicates were performed at the same time (technical replicates). d The genomic locus of the lncRNA LOLI1 . Also shown are SNVs from PCAWG and HMF cohorts. The SNVs included in the mutated plasmid are indicated in the grey boxes. e ASOs were transfected to knock down LOLI1 expression and f RNA levels measured in HuH7 cells. Statistical significance was estimated using one-sided Student’s t test; n = 3 (experimental replicates). g ASO-transfected cells were measured at indicated timepoints; n = 3. Statistical significance was estimated by linear regression model on log2 value (experimental replicates). h CRISPRa targeting LOLI1 . On the right, qRT-PCR measurements of LOLI1 with indicated sgRNAs; n = 3 (experimental replicates). i The effect of CRISPRa on HeLa cells’ viability; n = 6 (experimental replicates). Statistical significance was estimated by one-sided paired t test at 48 h. j Plasmids expressing spliced LOLI1 sequence, in wild-type (WT) or mutated form (Mut) were transfected into HuH7 cells. RNA levels were measured by qRT-PCR; n = 3 (experimental replicates). k Populations of plasmid-transfected cells were measured at indicated timepoints. Statistical significance was estimated by one-sided Student’s t test based; n = 3 (experimental replicates). l LOLI1 overexpression in immortalised human hepatocytes (IHH). RNA levels were measured by qRT-PCR; n = 3 (experimental replicates). m The viability of IHH was measured at indicated timepoints. Statistical significance was calculated by one-sided Student’s t test; n = 3 (experimental replicates). n Primary human hepatocytes were transduced to overexpress LOLI1 mutant transcript (left panel). Transduction was monitored by EGFP marker gene (left panel). The change in proliferation-associated cytokine was measured by qRT-PCR (right panel); n = 6 experimental replicates. n indicates the number of independent experiments. Data show the mean value +/− SD in ( a – c , f – n ). Source data are provided as a Source Data file.

Article Snippet: CRISPR activation in HeLa cells was performed as described by Sanson and colleagues . sgRNAs were designed using the GPP sgRNA Designer CRISPRa from the Broad Institute ( https://portals.broadinstitute.org/gpp/public/ ) (Supplementary Data ).

Techniques: Plasmid Preparation, Transfection, Over Expression, Quantitative RT-PCR, Colony Assay, Knockdown, Expressing, Sequencing, Mutagenesis, Transduction, Marker

Journal: Nature Communications

Article Title: Tumour mutations in long noncoding RNAs enhance cell fitness

doi: 10.1038/s41467-023-39160-7

Figure Lengend Snippet: a Experimental strategy to simulate tumour-like mutations in the NEAT1 gene by Cas9 protein. b Detailed map of the six NEAT1 target regions and 15 sgRNAs. Paired gRNAs used for the deletion of NEAT1_1 are indicated as KO- sgRNA1 and KO- sgRNA2. Previously described NEAT1 functional regions are indicated below . c Analysis of mutations created by Cas9 recruitment. The frequency, size and nature of resulting DNA mutations are plotted. d Competition assay to evaluate fitness effects of mutations. Above: Rationale for the assay. Below: Red/green ratios for indicated mutations. “Control1/2” indicate sgRNAs targeting AAVS1 region. “KO” indicates paired sgRNAs designed to delete NEAT1_1. N = 4 experiments were performed, and statistical significance was estimated by linear regression model on log2 values. The mean value −/+ SD is plotted. Replicates were performed at different times (experimental replicates). e Upper panel: Set-up of mini CRISPR fitness screen. HeLa cells are infected with lentivirus-carrying mixtures of sgRNAs. The sgRNA sequences are amplified and sequenced at defined timepoints. Lower panel: Abundances of displayed sgRNAs, normalised to the Control2 negative control. Statistical significance was estimated by linear regression model; n = 4 (experimental replicates). The mean value with SD is represented. f HCT116 cells were cultured as spheroids and their population measured. Data show the mean value −/+ SD of n = 4 (experimental replicates). Statistical significance was estimated using Student’s one-sided t test. g As for ( d ), but with non-transformed MRC5 lung fibroblast cells at timepoint Day 14. Statistical significance was estimated by one-sided Student’s t test. Data show the mean value -/+ SD of n = 3 (experimental replicates) . h MRC5 cells were seeded in soft agar, and the area of colonies was calculated. The mean and SD of n = 3 experiments is shown (experimental replicates). i NEAT1 mutations in Reg2 enhances cell growth in NSG mice. HeLa cells were mutated and then implanted subcutaneously. Resulting tumour weight is shown at 4 weeks post-transplantation. Statistical significance was estimated by one-sided Student’s t test based on n = 9 animals. Experiments were pooled from two groups of animals studied at different times. Data show the mean value -/+ SD. j The survival time of 184 lymphoid cancer patients from PCAWG is displayed. Patients were stratified according to whether they have ≥1 SNVs in the NEAT1 gene. Two-sided Gehan Breslow rank test with confidence interval style set to dotted lines (95% CI). Source data are provided as a Source Data file.

Article Snippet: CRISPR activation in HeLa cells was performed as described by Sanson and colleagues . sgRNAs were designed using the GPP sgRNA Designer CRISPRa from the Broad Institute ( https://portals.broadinstitute.org/gpp/public/ ) (Supplementary Data ).

Techniques: Functional Assay, Competitive Binding Assay, CRISPR, Infection, Amplification, Negative Control, Cell Culture, Transformation Assay, Transplantation Assay