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. 2017 Jan 19;8(1):e2569.
doi: 10.1038/cddis.2016.438.

H19/let-7/LIN28 reciprocal negative regulatory circuit promotes breast cancer stem cell maintenance

Fei Peng  1 , Ting-Ting Li  1 , Kai-Li Wang  1 , Guo-Qing Xiao  2 , Ju-Hong Wang  3 , Hai-Dong Zhao  2 , Zhi-Jie Kang  1   4 , Wen-Jun Fan  1 , Li-Li Zhu  5 , Mei Li  3 , Bai Cui  1 , Fei-Meng Zheng  1   6 , Hong-Jiang Wang  7 , Eric W-F Lam  8 , Bo Wang  6 , Jie Xu  1 , Quentin Liu  1
Affiliations

H19/let-7/LIN28 reciprocal negative regulatory circuit promotes breast cancer stem cell maintenance

"V体育2025版" Fei Peng et al. Cell Death Dis. .

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"VSports注册入口" Abstract

Long noncoding RNA-H19 (H19), an imprinted oncofetal gene, has a central role in carcinogenesis. Hitherto, the mechanism by which H19 regulates cancer stem cells, remains elusive. Here we show that breast cancer stem cells (BCSCs) express high levels of H19, and ectopic overexpression of H19 significantly promotes breast cancer cell clonogenicity, migration and mammosphere-forming ability VSports手机版. Conversely, silencing of H19 represses these BCSC properties. In concordance, knockdown of H19 markedly inhibits tumor growth and suppresses tumorigenesis in nude mice. Mechanistically, we found that H19 functions as a competing endogenous RNA to sponge miRNA let-7, leading to an increase in expression of a let-7 target, the core pluripotency factor LIN28, which is enriched in BCSC populations and breast patient samples. Intriguingly, this gain of LIN28 expression can also feedback to reverse the H19 loss-mediated suppression of BCSC properties. Our data also reveal that LIN28 blocks mature let-7 production and, thereby, de-represses H19 expression in breast cancer cells. Appropriately, H19 and LIN28 expression exhibits strong correlations in primary breast carcinomas. Collectively, these findings reveal that lncRNA H19, miRNA let-7 and transcriptional factor LIN28 form a double-negative feedback loop, which has a critical role in the maintenance of BCSCs. Consequently, disrupting this pathway provides a novel therapeutic strategy for breast cancer. .

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Conflict of interest statement (VSports app下载)

The authors declare no conflict of interest.

"VSports注册入口" Figures

Figure 1
Figure 1
LncRNA-H19 expression is assessed in clinical breast cancer specimens and cancer cell lines. (a) Analysis of H19 expression in breast cancerous tissues and adjacent normal tissues (n=20). The relative H19 mRNA level was normalized to ACTB. The statistical differences were analyzed using the paired t-test. (b) In situ analysis with a DIG-labeled H19 probe in breast cancerous tissue and adjacent normal tissues. The scale bar represents 100 μm. (c) Kaplan–Meier survival analysis was performed to investigate the implication of H19 level on patient overall survival (n=20). (d) Mice harboring BLG-Cre; Brca1F22–24/F22–24; p53 +/− were established by two rounds of pregnancy and then tumors developed were collected (n=3). H19 expression was further verified in tumor and mammary tissues by RT-qPCR assay. (e) The in situ expression of H19 RNA (red) was detected by FISH assay. The red fluorescent signal is from the H19 RNA probe, and the blue fluorescent signal is from nuclear DNA counterstained with DAPI. The scale bar represents 20 μm. (fand g) Detections of H19 expression in the ALDH1-positive (ALDH1+) subpopulation (f) and side population (SP) cells (g) are showed by dot plots by FACS and the relative expression levels in MDA-MB-231 cells. Data are represented as mean±S.D. **P<0.01 and ***P<0.001, n=3
Figure 2
Figure 2
LncRNA-H19 is involved in the maintenance of CSC characteristics in breast cancer cells. (a) Ectopic expression of H19 mRNA expression was confirmed by RT-qPCR after lentivirus infection in MDA-MB-231 cells. (b) Ectopic overexpression of H19 enhanced clonogenic growth in vitro. Representative images of colonies were presented (left) and colony numbers were counted after culture for 12 days (right). Data are represented as mean±S.D. ***P<0.001, n=3. (c) Cell migration was analyzed in H19 overexpression and control groups. Stained images of invaded cells are presented (left) and migrated numbers are mean±S.D. (n=3), ***P<0.001 (right). (d) Mammosphere formation was increased by H19 overexpression. Representative images were presented (left), and the size and numbers of mammospheres were counted (right). Data were shown as mean±S.D. from three independent experiments, **P<0.01 and ***P<0.001, respectively. (e) The interfering efficiency of the lentivirus encoding H19-targeting shRNAs (shH19) were confirmed by RT-qPCR, compared with negative control lentivirus (NTC). (f–h) Knockdown of H19 reduced breast cancer cells clonogenicity (f), migration (g) and mammosphere-forming ability (h). Data were shown as mean±S.D. from three independent experiments, **P<0.01 and ***P<0.001, respectively
Figure 3
Figure 3
Endogenous LncRNA-H19 is required for CSCs maintenance in vivo. (a) H19 depletion attenuated xenograft tumor growth after the first tumor transplantation. MDA-MB-231 cells were infected with lentivirus shH19-3 or NTC; cells were subcutaneously injected into nude mice, respectively. Representative images of subcutaneous tumors taken 6 weeks post inoculation (left) and the tumor growth curves were presented (right). Data are presented as the mean ± S.D. (n = 5; *P<0.05, **P<0.01 and ***P<0.001). (b) H19 knockdown significantly repressed in vivo tumor formation after second serial tumor transplantation. Shown were representative images of subcutaneous tumors collected at end point (left) and summary of tumor xenografts formation of different groups in nude mice (right). (c) The knockdown efficiency of H19 in the second tumor xenografts were detected by RT-qRCR. Error bars represent mean±S.D. of triplicates. (d–f) The cancer cells isolated from H19 knockdown tumor xenografts displayed the reduced clonogenicity (d), migration (e) and mammosphere-forming (f) abilities. Data were shown as mean±S.D. from three independent experiments, **P<0.01 and ***P<0.001, respectively
Figure 4
Figure 4
LncRNA-H19 acts as an endogenous miRNA let-7 sponge in breast cancer cells. (a) Comparison of the expression of H19 in cytoplasm and in nucleus by RT-qPCR. Data are represented as mean±S.D. ***P<0.001, n=3. (b) Shown was the representative image of the in situ location of H19 transcripts in MDA-MB-231 cells. The scale bar represents 20 μm (left) and 10 μm (right). (c) Immunoprecipitation using anti-AGO2 antibody (lane 3) or IgG (lane 2) followed by western blot analysis using a mouse monoclonal anti-AGO2 (right). Co-IP with rabbit anti-AGO2 antibody or preimmune IgG from extracts of MDA-MB-231 cells. H19 RNA levels in immunoprecipitates were determined by RT-qPCR (left). Data were shown as mean±S.D. from three independent experiments, **P<0.01. (d) The target validation using luciferase reporters; the indicated constructs were each transfected into MDA-MB-231 cells together with negative control miRNA (NC) or let-7 mimics (mlet-7) at a final concentration of 48 nM. Numbers are mean±S.D. (n=3, ***P<0.001). (e) Let-7 sensor (psiCHECK2-let-7 4x) was transfected into MDA-MB-231 cells, together with 0, 20, 40 or 80 ng of sponge plasmid wide type H19 (WT) or mutant H19 (Mut). Numbers are mean±S.D. (n=3, *P<0.05, **P<0.01). (f) Empty vector (EV) or full-length H19 (WT) was transfected into MDA-MB-231 cells. The relative H19 mRNA level was normalized to ACTB and let-7a/7b miRNA levels were normalized against those of U6B. Numbers are mean±S.D. (n=3). (g) The protein levels of let-7 targets DICER and RAS were confirmed by western blot. (h) MDA-MB-231 cells were transfected with siRNA targeting H19 (siH19) or negative control RNA (siNC), H19 expression levels and let-7a/7b miRNA levels were evaluated by RT-qPCR, and (i) the protein levels of let-7 targets were detected by western blot
Figure 5
Figure 5
H19/let-7 axis modulates LIN28 expression and CSCs maintenance. (a) The protein levels of a panel of core pluripotency factors were evaluated in H19 overexpression MDA-MB-231 (left) or SK-BR-3 (right) cells. (b) The protein level of LIN28 were confirmed by western bolt in H19 depletion cancer cells. (c) H19 shared regulatory let-7 miRNA with LIN28. The target validation was confirmed by using a luciferase reporter (psi-LIN28) transfected into MDA-MB-231 cells. Numbers are mean±S.D. (n=3, **P<0.01). (d) Psi-LIN28 was transfected into MDA-MB-231 cells, together with 0, 20, 40 and 80 ng of sponge plasmid WT H19 or Mut H19. Numbers are mean±S.D. (n=3, *P<0.05 and **P<0.01). (e) The protein levels of LIN28 were determined in the indicated groups 48 h post transfection. (f) Overexpression of LIN28 rescued lentivirus shH19-mediated reduction of self-renewal in mammosphere formation assays. Numbers are mean±S.D. (n=3, **P<0.01 and ***P<0.001). (g) Analysis of LIN28 expression in breast cancerous tissue and adjacent normal tissues (n=20). The relative LIN28 mRNA level was normalized to ACTB. The statistical differences were analyzed using the paired t-test. (h) The protein level of LIN28 in three pairs of randomly chosen clinical specimens was confirmed by western blot. (i) Spearman correlation showed positive correlations between expression of H19 and LIN28 in a statistically significant manner (n=20)
Figure 6
Figure 6
LncRNA-H19 formed a double-negative circuitry with miR-let-7 and LIN28 in breast cancer cells. (a) MDA-MB-231 cells were transfected with LIN28-overexpressing vector (LIN28) or empty vector (EV), the protein level of LIN28 was quantified by western blot 72 h post transfection. (b) The relative H19 mRNA level was analyzed by RT-qPCR when overexpressed LIN28. Numbers are mean±S.D. (n=3, ***P<0.001). (c) The protein level of LIN28 was detected 72 h after transfection of siRNAs targeting LIN28 (siLIN28) or siNC. (d) LIN28 depletion decreased expression of H19. Numbers are mean±S.D. (n=3, **P<0.01 and ***P<0.001). (e) LIN28-overexpressing vector (LIN28) or empty vector (EV) was transfected into MDA-MB-231 cells. After transfection 48 hours, let7a/7b miRNA levels were measured by RT-qPCR. Numbers are mean ± SD (n = 3, **P<0.01). (f) MDA-MB-231 cells were transfected with 48 nM control miRNA (NC), let-7 mimics (mlet-7) or let-7 inhibitors (ilet-7). RNAs were extracted 48 h later and RT-qPCR analysis performed. Numbers are mean±S.D. (n=3, **P<0.01 and ***P<0.001). (g) MDA-MB-231 cells were transfected with the indicated mixture, and the relative H19 mRNA level was detected by RT-qPCR. Numbers are mean±S.D. (n=3, *P<0.05). (hand i) Let-7a (h) or let-7b (i) miRNA levels in clinical specimens were assessed by RT-qPCR. Numbers are mean±S.D. (n=3, **P<0.01 and ***P<0.001). (j) Model for the H19/let7/LIN28 regulatory loop in the modulation of BCSCs maintenance
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