doi: 10.1053/j.gastro.2023.04.037.
Epub 2023 May 12.
Discovery and Initial Characterization of Long Intergenic Noncoding RNAs Associated With Esophageal Adenocarcinoma
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- PMID: 37182784
- PMCID: PMC10524377
- DOI: 10.1053/j.gastro.2023.04.037
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Discovery and Initial Characterization of Long Intergenic Noncoding RNAs Associated With Esophageal Adenocarcinoma
Gastroenterology.
2023 Aug.
No abstract available
Conflict of interest statement
Figures
(A)
Left: qPCR analysis demonstrating expression of lincPRKD and lincRTL predominantly in EACs in the discovery (Top) and validation (Bottom) cohorts. EAC (esophageal adenocarcinoma); NDSBE (never-dysplastic stable Barrett’s esophagus) obtained from BE patients with no history of dysplasia or cancer development upon long-term follow-up (median follow-up 9yrs); SQ (normal esophageal squamous); GAST (normal gastric cardia); and BE (Barrett’s metaplasia tissue from patients with no follow-up). Y-axes indicate log2 fold-changes (fc) in lincPRKD and lincRTL expression, normalized to the EAC sample with the lowest expression of respective lincRNAs in respective cohorts. ND (samples in which lincRNA expression was not detected by qPCR). The few normal esophageal SQ tissues showing lincRTL-positivity were each obtained from respective EAC patients. Right: Pie chart (Top) indicate the percentages of EACs that were positive for lincPRKD and/or lincRTL. PCR analysis (Bottom) of respective lincRNAs in EAC cell lines (n=3 pooled technical replicates) resolved on an agarose gel. (B) Cell fractionation analyses demonstrating the predominantly nuclear localization of lincRTL in EAC (SKGT4) cells, compared to both cytoplasmic and nuclear localization of lincPRKD (representative of n=2 biologic replicates). Expression of RPS14 (Ribosomal protein S14) and the non-coding RNA, MALAT1 (Metastasis associated lung adenocarcinoma transcript 1), were used as positive controls for cytoplasmic (Cyt) and nuclear (Nuc) cell-fractions, respectively. Y-axes of the bar graphs indicate PCR-based expression fold-changes of candidate lincRNAs in the nucleus, normalized to cytoplasmic fractions. PCR products resolved on an agarose gel were provided on the X-axes of respective bar graphs. (C)
Left: RNAscope ISH analyses demonstrating the nuclear localization of lincRTL in lincRTL-positive (Pos) EAC (SKGT4) cells (magenta dots), but absent in the lincRTL-Negative (Neg) EAC (FLO-1) cells. Peptidylprolyl Isomerase B (PPIB), showing positive signal in the cytoplasm (magenta dots), was used as a positive control for RNAscope ISH; Scale bar, 20μm. Right: Representative RNAscope-ISH images in treatment-naïve lincRTL-positive primary EAC tumor; Scale bar, 100μm. Note the strong nuclear signal (magenta dots) in EAC cells, with absence of signal in BE and normal SQ tissues. Magnified areas with respective image panels were indicated with black squared boxes. (D)
Left: qPCR analysis of candidate lincRNAs in BE lesions concurrent with EAC tumors (high-risk BE / HR-BE). Data from NDSBE and BE samples from Panel A, with no progression or concurrent dysplasia/EAC, were combined and considered as Low-risk BE (LR-BE). Note the induction of candidate lincRNAs in a significant fraction of HR-BE samples, compared to none in the LR-BE group (Fisher’s exact test). Right: RNAscope-ISH analyses of lincRTL in representative matched EAC and concurrent BE lesions; Scale bar, 150μm. Note the strong nuclear lincRTL positivity (magenta dots) in EAC and matched BE lesions from respective patients. Magnified areas with respective image panels were indicated with black squared boxes. (E) Representative IncuCyte cell proliferation (Left) and colony forming ability (Right) assays in EAC cells (n≥6 biologic replicates), following gapmer(gpr)-based knockdown of respective lincRNAs in representative lincRTL-positive (SKGT4) and lincPRKD -positive (FLO-1) EAC cells. A non-targeting random gapmer was used as control in these experiments. Two distinct gapmers per respective lincRNAs were used. Images provided at the top are PCR-based products resolved on agarose gels, indicating knockdown of respective lincRNAs with gapmers. Y-axes in cell proliferation graphs indicate fold-change (fc) in cell confluency over time (X-axes), normalized to time zero, for respective control and lincRNA knockdown groups. Y-axes in colony forming assay bar graphs indicate fold-change in colony forming ability (CFA) upon knockdown of respective lincRNAs, compared to control gapmer treated cells (n=3 biologic replicates). (F)
Top: RNAseq-based analyses showing the number of differentially-expressed (diff-exp) coding genes following knockdown of lincRTL in EAC (SKGT4) cells, or differentially-expressed between lincRTL high vs. low parental EAC cells. LAMC2 was among the list of 21 genes satisfying both the above criteria. Middle: qPCR-based confirmation of differential LAMC2 RNA expression following knockdown of lincRTL in EAC (SKGT4) cells (pooled n=3 biologic replicates) and its endogenous expression across parental EAC cells; associated Western Blot analyses of LAMC2 protein (pooled n=3 biologic replicates) are provided below. (A-F) All data are plotted as mean ± SEM, obtained from atleast three independent replicate measurements. *** (P < 0.0005), and ** (P < 0.005) indicate significant differences between the respective test versus control arms, estimated using a Student’s t-test, unless otherwise stated. Beta-2-Microglobulin (B2M) and β-actin were used as endogenous controls for RNA (qPCR) and protein (Western Blots), respectively, where appropriate.
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