A long non-coding RNA targets microRNA miR-34a to regulate colon cancer stem cell asymmetric division
- Cornell University, United States
- Duke University, United States
- Weill Corenll Medical College, United States
- Weill Cornell Medical College, United States
Figures
Figure 1 with 2 supplements
Characterization of Lnc34a.
(A) Schematic illustration of Lnc34a (shown in black) and miR-34a (shown in blue) gene structure. Lnc34a and miR-34a contain two exons and are transcribed in different directions. P, probe for …
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Figure 1—source data 1
Information of CRC patients.
Tissue specimens collected from the listed CRC patients were used for analyses of Lnc34a and miR-34a expression and miR-34a promoter methylation.
- https://doi.org/10.7554/eLife.14620.004
Figure 1—figure supplement 1
Identification of Lnc34a.
(A) Schematic illustration of Lnc34a and miR-34a gene structures. Primers for RT-PCR and rapid amplification of cDNA ends (RACE) were shown. (B) RT-qPCR and RACE that amplified Lnc34a. (C) RT-qPCR …
Figure 1—figure supplement 2
RNA FISH specificity and Lnc34a knockdown efficiency.
(A) Knockdown of Lnc34a abolished RNA FISH signals. (B) RT-qPCR showing Lnc34a knockdown efficiencies by shLnc34a1 and shLnc34a2. (C) RT-qPCR detection of Lnc34a level in cellular fractions from …
Figure 2 with 1 supplement
Lnc34a Asymmetry in CCSC division.
(A, B) RT-qPCR detection of lnc34a in ALDH1+ and ALDH1- populations isolated from spheres of two independent patient-derived lines, CCSC1 (A) and CCSC2 (B). Lnc34a is high in ALDH1+ (CCSC) but low …
Figure 2—figure supplement 1
CCSCs co-express ALDH1 and CD133.
(A) FACS showing ALDH1+ sphere cells are CD133+. (B) Co-immunofluorescence of ALDH1 and CD133 showing ALDH1 and CD133 are expressed in the same daughter cell during CCSC division. (C) Percentages of …
Figure 3
Lnc34a promotes CCSC self-renewal and tumor formation.
(A) Representative images of CCSC spheres with Lnc34a knockdown (shLnc34a1 and shLnc34a2), ectopic Lnc34a expression (Lnc34a), and ectopic Lnc34a/miR-34a expression. (B, C) Sphere formation during …
Figure 4 with 3 supplements
Lnc34a epigenetically silences miR-34a promoter.
(A, B) RT-qPCR of miR-34a levels in CCSC1 (A) and CCSC2 (B). (C, D) RT-qPCR of miR-34a levels in CCSC1 (C) and CCSC2 (D) spheres with Lnc34a knockdown (shLnc34a1 and shLnc34a2) or ectopic expression …
Figure 4—figure supplement 1
p53 symmetry.
(A) (A) Representative immunofluorescence images showing symmetric distribution of p53 during CCSC division. (B) Percentage of p53 division type. (C) Representative immunofluorescence images showing …
Figure 4—figure supplement 2
Lnc34a epigenetically silences miR-34a promoters in Caco-2 and HT29 cells.
(A, B) RT-qPCR of miR-34a levels in CRC lines Caco-2 (A) and HT29 (B). Ectopic Lnc34a expression suppressed miR-34a expression. (C, D) Bisulfite sequencing analysis showing ectopic Lnc34a expression …
Figure 4—figure supplement 3
Lnc34a, miR-34a, and promoter methylation levels in CRC specimens.
(A) RT-qPCR showing Lnc34a and miR-34a expression in individual CRC specimens. Levels are normalized to corresponding actin levels. (B) Bisulfite sequencing analysis of miR-34a promoter methylation …
Figure 5
Lnc34a recruits epigenetic regulators.
(A) Western blot following RNA-pull down showing Lnc34a interaction with PHB2, Dnmt3a and HDAC1 in CCSC1 (left) and CCSC2 (right) sphere cells. RNA-pull down was performed using CCSC lysates with …
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Figure 5—source data 1
Potential Lnc34a-associated proteins identified by biotinylated Lnc34a pull-down and mass spectrometry.
- https://doi.org/10.7554/eLife.14620.015
Author response image 1
Lnc34a upregulates TCF1 via suppression of miR-34a.
Western blot showing TCF1 was suppressed by ectopic miR-34a expression and upregulated by ectopic Lnc34a expression in CCSCs. Ectopic miR-34a abrogated the upregulation of Lnc34a on TCF1.
Author response image 2
Lnc34a and induction of miR-34a by p53 does not affect each other.
(A and B) RT-qPCR showing miR-34a and Lnc34a expression level in HCT116 (A) and CCSC1 (B) cells with p53 ectopic expression.
