DICER1 regulated let-7 expression levels in p53-induced cancer repression requires cyclin D1

Abstract

Let-7 miRNAs act as tumour suppressors by directly binding to the 3'UTRs of downstream gene products. The regulatory role of let-7 in downstream gene expression has gained much interest in the cancer research community, as it controls multiple biological functions and determines cell fates. For example, one target of the let-7 family is cyclin D1, which promotes G0/S cell cycle progression and oncogenesis, was correlated with endoribonuclease DICER1, another target of let-7. Down-regulated let-7 has been identified in many types of tumours, suggesting a feedback loop may exist between let-7 and cyclin D1. A potential player in the proposed feedback relationship is Dicer, a central regulator of miRNA expression through sequence-specific silencing. We first identified that DICER1 is the key downstream gene for cyclin D1-induced let-7 expression. In addition, we found that let-7 miRNAs expression decreased because of the p53-induced cell death response, with deregulated cyclin D1. Our results also showed that cyclin D1 is required for Nutlin-3 and TAX-induced let-7 expression in cancer repression and the cell death response. For the first time, we provide evidence that let-7 and cyclin D1 form a feedback loop in regulating therapy response of cancer cells and cancer stem cells, and importantly, that alteration of let-7 expression, mainly caused by cyclin D1, is a sensitive indicator for better chemotherapies response.

Keywords: DICER1; cancer stem cells; cell apoptosis; cyclin D1; let-7; regulatory loop.

Figure 1

The proliferation inhibition and apoptosis ratio of let-7 overexpressed breast cancer cells were caused by decreased cyclin D1. (A) Let-7 expression levels in lentiviral-infected MCF-7 and MDA-MB-231 cells. (B) Let-7 inhibited cell proliferation of breast cancer cells, among which, let-7b exhibited the strongest effect, * p < 0.01. (C) Overexpression of let-7 miRNAs corresponded to decreased cyclin D1 expression in MCF-7 cells but not in MDA-MB-231 cells. (D) Let-7b also induced more cell apoptosis in MCF-7 cells, with little effects in MDA-MB-231 cells, * p < 0.01. (E) TET-treated TRE3G-cyclin D1 MCF-7 cells possesses exogenous extra cyclin D1, and RFP-based shRNA-cyclin D1 exhibited decreased cyclin D1 expression in MCF-7 cells; Dicer1 is positively correlated with cyclin D1 level. Down-regulation of cyclin D1 decreased both the number (F) and size (G) of mammospheres. (H) Representative images of mammospheres acquired from shRNA mediated cyclin D1 knockdown [Correction added on 21 May 2015 after first online publication: TET-induced was removed from the text.] in MCF-7 cells are shown.

Figure 2

Cyclin D1 decreased let-7 expression through Dicer. (A) Immunofluorescent staining of Dicer (Green) in mammospheres from cyclin D1 knockdown MCF-7 cells, results showing that the decreased cyclin D1 achieved by shRNA-CCND1 (Red for RFP lentiviral) repressed Dicer expression level. (B) Let-7 expression was positively related with cyclin D1 expression, * p < 0.01. (C) Western blot results of Dicer siRNA-treated TRE3G-cyclin D1 cells. (D) Let-7 expression in cyclin D1-overexpressed MCF-7 cells was affected mainly by Dicer, * p < 0.01. (E) The illustration of cyclin D1 regulated Dicer-pGL3 Luc-vector. (F) Dicer promoter was regulated by cyclin D1 expression, * p < 0.01.

Figure 3

Let-7 signature variation in p53 and cyclin D1 regulated MCF-7 depend on Dicer. (A) Increased p53 by 10 μM of Nutlin-3 inhibited cyclin D1 and Dicer in MCF-7 but not MDA-MB-231 cells. (B) Let-7 was increased by overexpressed cyclin D1, and decreased in cyclin D1 knockdown cells, * p < 0.01. (C) p53 decreased endogenous cyclin D1 of TET treated TRE3G-cyclin D1-MCF-7 cells, and then decreased Dicer; however, in cyclin D1 knockdown cells, p53 failed to inhibited Dicer expression. (D) Nutlin-3 decreased let-7b expression in MCF-7 control and cyclin d1 overexpressed cells, but did not function in cyclin D1 knockdown cells, * p < 0.01. (E) Representative images of cell apoptosis of different groups are shown. (F) The knockdown of cyclin D1, increase in p53 and treatment with Taxol (TAX) all induced cell apoptosis, and Nutlin-3 sensitize cancer cells to TAX induced cell apopsosis, * p < 0.01. (G) TAX promoted cell apoptosis through regulating p53 and cyclin D1 levels, and N3 promoted this repression.

Figure 4

Nutlin-3 affected the let-7/cyclin D1 loop in breast cancer stem cells. (A) Representative images of first generation of mammospheres acquired from different groups. (B) Increased cyclin D1 increased mammosphere numbers, while both p53 and TAX treatment decreased sphere numbers, * p < 0.01. (C) Higher levels of cyclin D1 and Dicer were responsible for elevated let-7b; TAX and Nutlin-3 inhibited let-7b expression through down-regulating cyclin D1 and Dicer. (D) Relative let-7b expression level in MCF-7 of different groups, * p < 0.01. (E) Both nuclear and cytoplasmic Dicer were inhibited by increased p53 induced by Nutlin-3. (F) Let-7 inhibited DICER1 expression partially through cyclin D1 inhibition, forming let-7/cyclin D1/DICER1 negative feedback loop.