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The Wnt–b-catenin pathway represses let-7 microRNAexpression through transactivation of Lin28 toaugment breast cancer stem cell expansion

Wang-Yu Cai1,*, Tong-Zhen Wei1,*, Qi-Cong Luo2,*, Qiu-Wan Wu2, Qing-Feng Liu1, Meng Yang1, Guo-Dong Ye1,Jia-Fa Wu1, Yuan-Yuan Chen1, Guang-Bin Sun1, Yun-Jia Liu1, Wen-Xiu Zhao3, Zhi-Ming Zhang2,` and Bo-An Li1,`

1State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China2The First Affiliated Hospital of Xiamen University, Xiamen, Fujian, China3Zhongshan Hospital of Xiamen University, Xiamen, Fujian, China

*These authors contributed equally to this work`Author for correspondence (bali@xmu.edu.cn; zhangzhiming164@yahoo.cn)

Accepted 27 March 2013Journal of Cell Science 126, 2877–2889� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.123810

SummaryWnt signalling through b-catenin and the lymphoid-enhancing factor 1/T-cell factor (LEF1/TCF) family of transcription factors

maintains stem cell properties in both normal and malignant tissues; however, the underlying molecular pathway involved in this processhas not been completely defined. Using a microRNA microarray screening assay, we identified let-7 miRNAs as downstream targets ofthe Wnt–b-catenin pathway. Expression studies indicated that the Wnt–b-catenin pathway suppresses mature let-7 miRNAs but not the

primary transcripts, which suggests a post-transcriptional regulation of repression. Furthermore, we identified Lin28, a negative let-7biogenesis regulator, as a novel direct downstream target of the Wnt–b-catenin pathway. Loss of function of Lin28 impairs Wnt–b-catenin-pathway-mediated let-7 inhibition and breast cancer stem cell expansion; enforced expression of let-7 blocks the Wnt–b-cateninpathway-stimulated breast cancer stem cell phenotype. Finally, we demonstrated that the Wnt–b-catenin pathway induces Lin28

upregulation and let-7 downregulation in both cancer samples and mouse tumour models. Moreover, the delivery of a modified lin28siRNA or a let-7a agomir into the premalignant mammary tissues of MMTV-wnt-1 mice resulted in a complete rescue of the stem cellphenotype driven by the Wnt–b-catenin pathway. These findings highlight a pivotal role for Lin28/let-7 in Wnt–b-catenin-pathway-

mediated cellular phenotypes. Thus, the Wnt–b-catenin pathway, Lin28 and let-7 miRNAs, three of the most crucial stem cell regulators,connect in one signal cascade.

Key words: Breast cancer, Let-7, Lin28, Stem cell, Wnt–b-catenin pathway

IntroductionMany pathways are implicated in the regulation of stem cell self-

renewal and differentiation. These pathways include Hedgehog,

Notch and Wnt (Liu et al., 2010; Merchant and Matsui, 2010;

Takebe et al., 2011; Wend et al., 2010), within which the Wnt–b-

catenin pathway is crucial in both normal mammary development

and tumorigenesis (Roarty and Rosen, 2010). b-catenin plays a

central role as a transcriptional activator in the Wnt–b-catenin

pathway (Logan and Nusse, 2004). In the absence of upstream

signalling stimulation, cytoplasmic b-catenin is phosphorylated

by glycogen synthase kinase-3b (GSK-3b) in a complex that

includes Axin and the tumour suppressor adenomatous polyposis

coli (APC) and is targeted for ubiquitin-mediated proteasomal

degradation. Stimulation by Wnt ligands leads to the inhibition

of phosphorylation and degradation of b-catenin, which

subsequently enters the nucleus and binds to the LEF1/TCF

family of transcription factors. In turn, the b-catenin–LEF1/TCF

complex activates the expression of a cohort of target genes that

impact cellular functions (Amit et al., 2002; Liu et al., 2002). As

the Wnt–b-catenin pathway can regulate mammary stem cell

self-renewal and differentiation, the aberrant activation of this

pathway in human breast cancer may indicate a key role for it in

cancer stem cells. Despite the fact that the Wnt–b-catenin

pathway is a key regulator of breast cancer stem cells, the

downstream transcriptional cascade in this process remains

largely unknown.

MicroRNAs (miRNAs) play critical roles in many biological

processes, including cancer processes, by directly inhibiting the

expression of target mRNAs through a variety of molecular

mechanisms (Bartel, 2009; Ventura and Jacks, 2009). Also,

miRNAs undergo aberrant regulation during carcinogenesis and

can act as either oncogenes or tumour suppressors (Lu et al., 2005).

Let-7 is an important miRNA family consisting of 12 members

with expression that is frequently downregulated in a number of

human cancers (Calin et al., 2004; Johnson et al., 2005; Kumar

et al., 2008). Moreover, let-7 is also the key regulator of breast

cancer stem cell self-renewal and differentiation (Yu et al., 2007).

A large body of evidence indicates that many miRNAs can

regulate the Wnt–b-catenin pathway through the targeting of Wnt

components (Huang et al., 2010); however, little is known

regarding whether the Wnt–b-catenin pathway regulates the

expression of miRNAs, especially in cancer stem cells.

Recent studies on induced pluripotent stem cells (iPS cells)

support the concept that cancer stem cells arise through a

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reprogramming-like mechanism (Krizhanovsky and Lowe,

2009). By enforcing the expression of so-called reprogrammingfactors (OCT4, KLF4, SOX2, and Lin28 or c-MYC),differentiated somatic cells can be converted to iPS cells,

which can differentiate into any tissue type. Several of thesereprogramming factors are upregulated in human tumours and areconsidered to be putative oncogenes (Krizhanovsky and Lowe,2009). Thus, iPS cells and cancer stem cells may share similar

mechanisms that control the stem cell properties. Lin28, one ofthese reprogramming-like factors, is highly abundant inembryonic stem cells and developing cells, but in later stages,

its expression declines (Moss and Tang, 2003). Lin28 may beoverexpressed in some tumours (Viswanathan and Daley, 2010).Moreover, Lin28 has been found to be positively correlated with

the percentage of ALDH1+ tumour cells in breast cancer,suggesting that Lin28 plays an important role in the regulationof breast cancer stem cells (Yang et al., 2010). Lin28 and Lin28B

function as negative regulators of let-7 biogenesis through theirability to directly interact with the loop region of let-7 hairpins.Lin28/Lin28B accelerates the turnover of let-7 precursors andprevents both Drosha- and Dicer-mediated let-7 processing (Heo

et al., 2008; Newman et al., 2008; Piskounova et al., 2008; Rybaket al., 2008). Conversely, let-7 targets Lin28 and downregulatesits expression (Yang et al., 2010). These results suggest that a

regulatory feedback loop exists between let-7 and Lin28 (Yang etal., 2010) that is crucial in modulating the self-renewal anddifferentiation of breast cancer stem cells.

In this study, we have demonstrated that the activation of the

Wnt–b-catenin pathway suppresses mature let-7 miRNAs but notthe primary transcripts, which suggests a post-transcriptionalregulation of let-7 expression. We further identified Lin28 as a

novel direct downstream target of the Wnt–b-catenin pathway.Accordingly, loss of function of Lin28 impairs Wnt–b-catenin-pathway-mediated let-7 inhibition and breast cancer stem cell

expansion. Moreover, enforced expression of let-7 blocks Wnt–b-catenin-pathway-stimulated breast cancer stem cell expansion.Thus, the Wnt–b-catenin pathway, Lin28 and let-7 miRNAs,

three of the most crucial stem cell regulators, connect in onesignal cascade.

ResultsPost-transcriptional repression of let-7 family miRNAs byWnt–b-catenin pathway

Studies have shown that miRNAs play pivotal roles in controllingthe maintenance and function of stem cells. We sought to identify

the downstream target miRNAs of the Wnt–b-catenin pathway byscreening the ZR-75-30 breast cancer cell line using a miRNAmicroarray system. Specifically, miRNA expression profileswere examined in high-Wnt and low-Wnt states with stable

overexpression of b-catenin and GFP. All miRNAs showing a1.5-fold or greater upregulation or downregulation in the high-Wnt state were chosen for further analysis (supplementary

material Table S1). Of these miRNAs, we found that sevenmembers of the let-7 miRNA family were downregulatedby ,2.5-fold in b-catenin stable overexpression cells, and

quantitative real-time PCR (qPCR) results confirmed thechanges (Fig. 1A). To verify the expression changes in morebreast cancer cells and more Wnt stimulations, MDA-MB-231

and T-47D cells were also used. Consistent with the results in b-catenin activated ZR-75-30 cells, the expression levels of let-7a,let-7f and let-7g in these cells were dramatically decreased when

the cells were treated with LiCl, a GSK3b inhibitor, as well as bystably overexpressing of HA–Wnt1 (Fig. 1B; supplementary

material Fig. S1A). Conversely, knockdown of b-cateninexpression resulted in the induction of let-7a and let-7f in bothZR-75-30 and MDA-MB-231 cells (Fig. 1C; supplementarymaterial Fig. S1B). These results suggest that let-7 miRNAs

are downstream target miRNAs of the Wnt–b-catenin pathway inbreast cancer cells.

To investigate the mechanism through which the Wnt–b-

catenin pathway regulates let-7 miRNAs, we used qPCR analysisto examine further the abundance of their primary transcripts inZR-75-30 cells. Unexpectedly, we found that the expression of

the let-7a/let-7f/let-7d and let-7g primary transcripts was notrepressed in b-catenin stable overexpression cells (Fig. 1D).Consistently, these primary transcripts were also not induced inb-catenin knockdown cells (Fig. 1E). These findings indicate that

the Wnt–b-catenin pathway utilises alternative mechanisms todownregulate multiple let-7 family members through a post-transcriptional pathway.

The Wnt–b-catenin pathway activates Lin28 expression inbreast cancer cells

Lin28 and Lin28B RNA binding proteins have beendemonstrated to bind to the stem loops of let-7 miRNAs and toinhibit their biogenesis by blocking Drosha- and Dicer-mediatedcleavage and accelerating the turnover of let-7 precursors (Heo

et al., 2008; Newman et al., 2008; Piskounova et al., 2008; Rybaket al., 2008). Therefore, we speculated that the Wnt–b-cateninpathway may inhibit let-7 family members through the

transactivation of Lin28 or Lin28B. To verify this hypothesis,we used semi-quantitative RT-PCR to examine Lin28 andLin28B expression in different breast cancer cell lines upon

LiCl stimulation. Lin28B expression was undetected in three offour cell lines tested, and no changes were found in the ZR-75-30cell line, but Lin28 expression was dramatically induced in all

cell lines treated with LiCl (Fig. 2A, left). Similar results wereachieved by qPCR analysis (Fig. 2A, right). Conversely,knockdown of b-catenin expression resulted in greatly reducedLin28 expression in ZR-75-30 cells (Fig. 2B). We previously

demonstrated that Pygo2, a newly found b-catenin interactionprotein, is overexpressed in breast cancer stem cells andaugments Wnt–b-catenin pathway activity (Chen et al., 2010).

We examined whether Pygo2 knockdown also affects Lin28expression. As shown in Fig. 2C, depletion of Pygo2 in ZR-75-30cells caused a remarkable decrease in Lin28 expression. Next, we

investigated whether the let-7 family members responded toLin28 in the breast cancer cells in our experiments. As expected,when Lin28 mRNA and protein expression were effectivelyknocked down (supplementary material Fig. S2A), the levels of

mature let-7a and let-7f transcripts were greatly increased(supplementary material Fig. S2B), whereas overexpression ofLin28 resulted in reduced mature let-7a and let-7f levels

(supplementary material Fig. S2C). In contrast, the primarytranscript levels of let-7a/let-7f/let-7d and let-7g were notchanged in either experiment (supplementary material Fig.

S2D,E). To further verify that both b-catenin and Lin28 couldinhibit the biological function of let-7s as microRNAs, weperformed a luciferase assay using a let-7 sensor containing a

constitutively expressed luciferase reporter bearing two copiesof sequences complementary to let-7a in the downstream 39

UTR. As shown in Fig. 2D, endogenous let-7 miRNAs in the

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MDA-MB-231 cells greatly inhibited let-7 sensor activity,

indicating that these miRNAs indeed target the 39 UTR of

luciferase and inhibit its expression. Enforced expression of b-

catenin and Lin28 both remarkably rescued the sensor activity

almost to the control vector levels, which suggests that the

biological function of let-7 is inhibited. Finally, we treated the

MDA-MB-231, ZR-75-30 and T-47D cells with recombinant Wnt1

to see weather the Wnt ligand also triggers the same activity. As

expected, b-catenin expression was upregulated in all cell lines

treated with recombinant Wnt1. Moreover, Lin28 was upregulated

whereas let-7a/let-7f was downregulated in these cells (Fig. 2E).

The regulatory consistency of let-7 suggests that the Wnt–b-catenin

pathway and Lin28 may share the same signalling cascade and

supports the hypothesis that the Wnt–b-catenin pathway inhibits let-

7 miRNAs by activating Lin28 expression in breast cancer cells.

Lin28 is a novel direct downstream target gene of the Wnt–

b-catenin pathway

To determine whether Lin28 is a direct Wnt–b-catenin pathway

target gene, we examined the genomic sequence within a 10-kb

window centred on the transcriptional start site (TSS) of Lin28.

The genomic sequence extending from 1.0 kb upstream to

114 bp (ATG site) downstream of the TSS contains seven

consensus LEF/TCF-binding sites of CTTTG or GAAAC, with

four sites showing conservation between human and mouse

(Fig. 3A, conserved sites indicated as black dots). This fragment

was cloned into a PGL3-basic promoter-less luciferase reporter

cassette. The fragment was sufficient to drive the luciferase

reporter activity in a dose-dependent manner when stimulated

with LiCl, b-catenin or Wnt-1 in 293T cells (Fig. 3B). Equivalent

Wnt-dependent reporter activity was conferred by a truncated

fragment without the last three sites, which indicates that the

conserved sites are responsible for the luciferase activity.

Although mutation of the site 2 or 3 binding sequence had no

effect, deletion or mutation of site 1 or 4 resulted in significantly

compromised luciferase activity induced with LiCl, albeit the

decrease for the site 1 mutation was minor (Fig. 3C). Similar

results were achieved in MDA-MB-231 breast cancer cells when

Wnt–b-catenin activity was stimulated with recombinant Wnt1

(Fig. 3D). In a chromatin immunoprecipitation (ChIP) assay,

both LEF1 and b-catenin were found to occupy the endogenous

Lin28 promoter at site 4 but not at site 1 in both MDA-MB-231

and T-47D cells (Fig. 3E), suggesting that only site 4 functions as

a true endogenous LEF1/b-catenin-binding site on the Lin28

promoter. Taken together, our findings establish that the Wnt–b-

catenin pathway activates Lin28 expression by direct binding to

Fig. 1. Post-transcriptional repression of let-7 family

miRNAs by the Wnt–b-catenin pathway. (A) Relative

mature microRNA (miRNA) levels of individual let-7 family

members in ZR-75-30 cells stably expressing lentiviral vector

(LV)-GFP (control) and LV-b-catenin (b-Cat; left), using

qPCR analysis. Western blotting results showing b-catenin

overexpression levels (right). (B) Relative levels of mature let-

7a, let-7f and let-7g miRNAs in control and LiCl- (36 h) or

LV-HA-Wnt1-treated ZR-75-30 cells using qPCR analysis

(top). Western blotting showing the indicated proteins

expression (bottom). (C) qPCR analysis of the mature let-7a,

let-7f miRNA levels in ZR-75-30 cells stably expressing lacZ

shRNA (control) and two different b-Cat shRNAs (top).

Western blotting indicating the b-catenin knockdown

efficiency (bottom). (D) qPCR analysis of the let-7a/let-7f/let-

7d and let-7g pri-miRNA levels in ZR-75-30 cells stably

expressing LV-GFP and LV-b-Cat. (E) qPCR analysis of the

let-7a/let-7f/let-7d and let-7g pri-miRNA levels in ZR-75-30

cells stably expressing lacZ shRNA and two different b-Cat

shRNAs. All data are the means 6 s.d. of three independent

experiments.

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the Lin28 promoter, which provides compelling evidence that

Lin28 is a direct target gene of the Wnt–b-catenin pathway.

Activation of Lin28 is necessary for the Wnt–b-catenin

pathway-induced let-7 repression and cell proliferation

To examine whether Lin28 is necessary for the Wnt–b-catenin-

pathway-mediated repression of let-7 family members, we used

two different shRNAs to inhibit Lin28 expression in MDA-MB-

231 cells in the high Wnt state. Both of the shRNAs were

observed to knock down the Lin28 expression significantly to

levels comparable to those of negative control cells (Fig. 4A,

left). Knockdown of Lin28 completely reversed the b-catenin-

mediated repression of mature let-7a and let-7f, suggesting the

essential role of Lin28 in this process (Fig. 4A, right).

Conversely, overexpression of Lin28 almost completely

compromised the b-catenin knockdown-induced let-7a and let-7f

expression (Fig. 4B). As HRAS and HMGA2 are known let-7

targets, we examined the relationship between these proteins and the

Wnt–b-catenin pathway. The results from different experiments all

indicated that the Wnt–b-catenin pathway enhances HRAS and

HMGA2 protein expression and that this activity depends on Lin28

activation and let-7 repression (Fig. 4A–C).

The Wnt–b-catenin pathway is known to enhance the

proliferation of many cancer types including breast cancer

(MacDonald et al., 2009; Matsuda et al., 2009). As expected,

enforced expression of b-catenin resulted in a significantly

increased growth rate in MDA-MB-231 cells. The simultaneous

knockdown of Lin28 was found to almost completely

compromise b-catenin activity (Fig. 4D). When plated at a

clonal density, a significant decrease in the number and size of

Fig. 2. The Wnt–b-catenin pathway activates Lin28

expression in breast cancer cells. (A) Expression levels of

Lin28 and Lin28B mRNA upon Wnt–b-catenin pathway

stimulation in breast cancer cell lines. ZR-75-30, MDA-MB-

231, MCF7 and T-47D cell lines were treated with or without

LiCl for 36 hours. A no-cDNA template group was used as a

negative control (NC), and 18S rRNA was used as an internal

control. Semi-quantitative RT-PCR analysis was used to

measure the Lin28 and Lin28B levels (left). The expression

levels of Lin28 mRNA were confirmed using qPCR (right).

(B) Relative expression of Lin28 mRNA (left) and protein

(right) measured using qPCR and western blotting in ZR-75-

30 cells stably expressing lacZ shRNA (control) and two

different b-Cat shRNAs. (C) Relative expression of Lin28

mRNA measured by qPCR in ZR-75-30 cells stably

expressing lacZ shRNA and Pygo2 shRNA. (D) Luciferase

sensor assay. Schematic representation of the let-7 sensor,

which contained a constitutively expressed firefly luciferase

reporter bearing two copies of sequences complementary to

let-7a in the downstream 39UTR (left). Luciferase activity of

the let-7 sensor in b-catenin, Lin28 or GFP-transfected MDA-

MB-231 cells (right). The vehicle vector pMIR reporter was

used as a control reporter. (E) MDA-MB-231, ZR-75-30 and

T-47D cell lines were treated with or without Wnt1 protein

(100 ng/ml) for 48 hours. qPCR analysis was used to measure

the mature let-7a and let-7f levels (top); western blotting was

used to measure b-catenin and Lin28 levels (bottom). Data

are the means 6 s.d. of three independent experiments

(*P,0.001).

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colonies was observed for Lin28-depleted cells (Fig. 4E). These

results suggest that the transactivation of Lin28 is essential for

the Wnt–b-catenin-pathway-mediated let-7 inhibition and cell

proliferation. Moreover, overexpression of let-7a in Wnt-

activated MDA-MB-231 cells was also observed to completely

inhibit b-catenin-activated cell growth and colony formation

(Fig. 4C–E), which underscores the importance of let-7

miRNAs as Wnt–b-catenin pathway downstream targets in

regulating cell proliferation. Collectively, the above results

verify the hypothesis that the repression of let-7 through the

activation of Lin28 is necessary for Wnt–b-catenin-pathway-

induced cell proliferation.

Let-7 repression through activation of Lin28 is necessary

for Wnt–b-catenin-pathway-induced expansion of breast

cancer stem cell populations

Although previous studies have demonstrated that the Wnt–b-

catenin pathway, Lin28 and let-7 miRNAs all play central roles

in stem cell self-renewal and differentiation, their possible

involvement in the same signalling cascade that regulates cancer

stem or cancer-initiating cells has not been addressed. Such cells

in breast cancer can be enriched from established cancer cell lines

using either mammosphere cultures or FACS sorting for a CD44+

CD242 population (Al-Hajj et al., 2003; Ponti et al., 2005). Using

western blotting and qPCR, we detected higher levels of

Fig. 3. Lin28 is a novel direct

downstream target gene of the Wnt–b-

catenin pathway. (A) Schematic

representation of the genomic region near

the transcription start site of Lin28. The

dots represent the putative LEF/TCF-

binding sites CTTTG or GAAAC; those in

black are conserved between human and

mouse. (B) The luciferase activity of the

pGL3-Lin28 vector containing the

putative LEF/TCF-binding sites was

activated in a dose-dependent manner

upon LiCl (left), b-catenin (middle) or

Wnt-1(right) stimulation in 293T cells.

The empty pGL3-basic vector was used as

a negative control. (C) Luciferase activity

of various pGL3-Lin28 vectors (wild type,

deleted or mutated in LEF/TCF sites) in

untreated and LiCl-treated (24 hours)

293T cells. (D) Luciferase activity of

pGL3-Lin28 vectors (wild type or Del2)

in Wnt1 protein-treated (0, 10, 50, 100 ng/

ml for 48 hours) MDA-MB-231 cells.

(E) Chromatin immunoprecipitation

(ChIP) assay of LEF1 or b-catenin for the

Lin28 promoter in MDA-MB-231 (top)

and T-47D (bottom) cells using semi-

quantitative RT-PCR analyses.

(B–D) Data are the means 6 s.d. of three

independent experiments.

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b-catenin and Lin28 proteins but lower levels of let-7 members in

mammospheres of the MDA-MB-231, MCF7 and T-47D cell

lines in comparison to their corresponding adherent cultures

(Fig. 5A). Knockdown of b-catenin expression in MDA-MB-231

cells resulted in decreased Lin28 expression and increased let-7a

transcript levels in the mammospheres (Fig. 5B). Consequently,

b-catenin-depleted cells formed fewer, smaller mammospheres

(Fig. 5C). The expression trend of let-7 and Lin28 regulated by

b-catenin in mammospheres suggests a functional relevance of

these factors in breast cancer stem cells. Given our result that the

Wnt–b-catenin pathway/Lin28/let-7 cascade promotes breast

cancer cell proliferation, we reasoned that this phenomenon is

caused by stem cell expansion. To verify this hypothesis, we

infected MDA-MB-231 cells with b-catenin-expressing

lentiviruses and examined mammosphere formation. The enforced

expression of b-catenin in MDA-MB-231 cells resulted in

significantly more and larger mammospheres. Simultaneous

knockdown of Lin28 or overexpression of let-7a was found to

compromise the b-catenin-enhanced mammosphere formation almost

completely (Fig. 5D). We also examined whether the Wnt–b-catenin

pathway/Lin28/let-7 cascade affects the size of the CD44+ CD242

population in breast cancer cells. T-47D cells were used for this

analysis because ,90% of MDA-MB-231 cells are of this type,

making it difficult to score any potential increase. As shown in

Fig. 5E, the enforced overexpression of b-catenin in T-47D cells by

lentiviral infection yielded a greatly increased CD44+ CD242

Fig. 4. Activation of Lin28 is necessary for Wnt–

b-catenin pathway-induced Let-7 repression and

cell proliferation. (A) Knockdown of Lin28

completely reversed the b-catenin-mediated

repression of mature let-7a and let-7f. MDA-MB-

231 cells were infected with b-catenin lentiviruses

(LV-b-Cat) and treated with two different Lin28

shRNAs simultaneously. qPCR was used to

measure Lin28 levels (left, top); western blotting

was used to measure b-catenin, HRAS and HMGA2

levels (left, bottom); semi-quantitative RT-PCR

was used to measure let-7a and let-7f abundance

(right). (B) Overexpression of Lin28 completely

compromised b-catenin knockdown-induced let-7a

and let-7f expression. MDA-MB-231 cells were

infected with b-Cat shRNA and treated with Lin28

lentiviruses (LV-Lin28) simultaneously. Western

blotting was used to measure b-catenin, Lin28,

HRAS and HMGA2 protein levels (left); qPCR was

used to measure let-7a and let-7f abundance (right).

(C) Upregulation of HRAS and HMGA2 by b-

catenin is dependent on let-7 suppression. MDA-

MB-231 cells were infected with b-catenin

lentiviruses (LV-b-Cat) and treated with let-7a

lentiviruses (LV-let-7a) simultaneously. Semi-

quantitative RT-PCR was used to measure let-7a

abundance; western blotting was used to measure b-

catenin, HRAS and HMGA2 levels.

(D,E) Knockdown of Lin28 or enforced expression

of let-7a compromised b-catenin-induced cell

proliferation. MDA-MB-231 cells from A and C

were used. Cell growth rates (D) and colony

formations (E) were measured. (E) A representative

experiment of three independent experiments is

shown. Photographs of colonies were taken at day

10 (left), and the number of colonies was quantified

(right). Data are the means 6 s.d. of three

independent experiments (*P,0.001).

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population, but the overexpressed b-catenin was not able to induce an

increase in the CD44+ CD242 population in the Lin28-depleted or let-

7a-overexpressed cells. Collectively, these results demonstrate that

let-7 repression through the activation of Lin28 is necessary for Wnt–

b-catenin-pathway-induced expansion of the breast cancer stem cell

population.

In vivo analyses of the Wnt–b-catenin pathway/Lin28/let-7

cascade

To investigate whether the Wnt–b-catenin pathway is associated

with Lin28 expression in breast cancer patients, tissue

microarrays from 82 patients with breast cancer who had

undergone mammary gland resection were examined by

Fig. 5. Let-7 repression through activation of Lin28 is necessary for Wnt–b-catenin pathway-induced expansion of breast cancer stem cell populations.

(A) b-catenin and Lin28 expressions are enriched, whereas let-7 expression is reduced in breast cancer stem cells. Western blots for b-catenin and Lin28 protein

levels (left) and the qPCR analysis of let-7a transcripts (right) in mammospheres or corresponding adherent cells of different breast cancer cell lines.

(B) Knockdown of b-catenin expression resulted in decreased Lin28 expression and increased let-7a transcript levels in mammospheres. MDA-MB-231 cells

stably expressing b-Cat shRNA were cultured for mammosphere formation for 14 days, and qRT-PCR analysis was used to measure the expression levels of b-

catenin, Lin28 and let-7a. (C) b-catenin-depleted cells formed fewer, smaller mammospheres. Representative photographs of mammospheres were taken at day 14

(left); the number and size of spheres were quantified (right). (D) Knockdown of Lin28 or overexpression of let-7a compromised the b-catenin-enhanced

mammosphere formation. MDA-MB-231 cells were infected with LV-b-Cat and treated with Lin28 shRNA or LV-let-7a simultaneously, and the cells were

cultured for mammosphere formation for 12 days. Representative photographs of mammospheres were taken at day 12 (left); the number and size of spheres were

quantified (right). (E) Knockdown of Lin28 or overexpression of let-7a compromised the b-catenin-enhanced CD44+ CD242 population. MDA-MB-231 cells

were infected with the corresponding lentiviruses, and the percentage of CD44+CD242 cells was determined using FACS analysis. Representative FACS profiles

from a single pair are shown, and the diagrams show the mean values for three different pairs. For C and D data are the means 6 s.d. of four independent

experiments (*P,0.001). For A,B and E data are the means 6 s.d. of three independent experiments. Scale bars: 100 mm.

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immunostaining with b-catenin and Lin28 antibodies. The overallexpression levels of both the b-catenin and Lin28 proteins were

significantly higher in breast cancers than in adjacent tissues asindicated by the representative samples in the tissue microarrays(Fig. 6A, left) and the summary of all the samples (supplementarymaterial Table S2). Moreover, correlation analyses revealed strong

correlations between b-catenin expression and Lin28 levels(supplementary material Table S2; Fig. 6A, right). To analysefurther the relationship between the Wnt–b-catenin pathway,

Lin28 and let-7, 16 pairs of fresh tumour samples along with theiradjacent tissues were used to detect the expression of b-catenin,Lin28 and let-7a by western blotting and qPCR. As shown in

supplementary material Fig. S3, the b-catenin and Lin28expression levels in the cancers were higher than in the adjacenttissues, which is consistent with the tissue microarray results.Conversely, the let-7a expression levels in the cancers were

dramatically lower than those of the adjacent tissues. Moreover,Lin28 expression showed a positive relationship, whereas let-7aexpression was found to be inversely related to b-catenin. It has

been well documented for the mouse mammary tumor virus(MMTV)-wnt-1 mouse that the stem cell-enriched Lin2CD29hi

CD24+ population in premalignant mammary tissue is greatly

expanded (Shackleton et al., 2006), indicating the critical role forthe Wnt–b-catenin pathway in the expansion of breast cancer stemcells. We therefore measured the expression levels of b-catenin,

Lin28 and let-7a in this model using immunostaining, westernblotting and qPCR. As expected, b-catenin expression was greatlyincreased in premalignant mammary tissues compared with tissuesfrom wild-type littermates. Lin28 and let-7a showed coordinated

increased and decreased expression levels. Moreover, the let-7targets HRAS and HMGA2 also showed increased proteinexpression levels in premalignant mammary tissues of the

MMTV-wnt-1 mouse (Fig. 6B). To confirm the regulatingcascade in vivo in a second setting, we examined an additionalmodel with a high Wnt state. The model used, the Apcmin/+ mouse,

is a mouse intestine adenoma model bearing a truncated Apc allele.Results similar to those of the MMTV-wnt-1 mice were achievedand further confirm the relationship between the Wnt–b-cateninpathway, Lin28 and let-7a (Fig. 6C). Finally, the in vivo functional

relevance of the Wnt–b-catenin pathway/Lin28/let-7 cascade wasexamined. We intraductally injected cholesterol-, OMe-conjugatedLin28 siRNA into the premalignant mammary tissue of MMTV-

wnt-1 mice through and examined whether Lin28 knockdowncould rescue the let-7 repression and the stem cell phenotypedriven by the Wnt–b-catenin pathway. As illustrated in Fig. 6D,

premalignant mammary tissues exhibited decreased let-7a and let-7f levels and an expanded Lin2 CD29hi CD24+ population whencompared with tissues from wild-type littermates; however,

knockdown of Lin28 dramatically recovered the let-7 expressionand the size of the stem cell population when compared with thewild-type littermates. Similarly, delivery of a cholesterol-conjugated let-7a agomir into the premalignant mammary tissue

of MMTV-wnt-1 mice was also observed to dramatically rescuethe stem cell phenotype (Fig. 6E). Taken together, these resultsprovide solid evidence of the existence of a Wnt–b-catenin

pathway/Lin28/let-7 cascade in vivo.

DiscussionAberrant activation of the Wnt–b-catenin pathway has beenfound in a wide range of cancers, especially in cancers derivedfrom intestine, skin, mammary gland and haematopoietic cells.

Moreover, the Wnt–b-catenin pathway may preferentiallyinfluence stem/progenitor cell expansion in these cancers

(Wend et al., 2010). Although many downstream target genesfor both normal development and tumorigenesis have been

identified in different cellular contexts, the genes that mediate theWnt–b-catenin pathway activity in maintaining the stem cellproperties are still not very clear. c-Myc, a reprogramming factor

and a well-known Wnt target, was recently demonstrated to be animportant stem cell regulator in normal and cancerous cells (Kim

et al., 2010; Smith et al., 2011). However, no study hasestablished a convincing relationship between the Wnt–b-

catenin pathway and c-Myc in stem cells. In contrast, recentstudies on both iPS and ESCs suggest that the role of the Wnt–b-catenin pathway in reprogramming and maintaining stem cells

could be independent of c-Myc induction (Marson et al., 2008;Ying et al., 2008). In breast cancer, current studies indicate that

Hedgehog, Notch and the Wnt–b-catenin pathway, as well asBmi1 (Liu et al., 2006) and Lin28 (Yang et al., 2010)transcription factors are major regulators that affect cancer

stem cell properties. However, the downstream molecular eventsof the Wnt–b-catenin pathway responsible for this remain largely

unknown. In this study, we identified Lin28 as a noveldownstream target of the Wnt–b-catenin pathway and found

that Lin28 is necessary for Wnt–b-catenin-pathway-mediatedbreast cancer stem cell expansion, which highlights a molecularmechanism for the Wnt–b-catenin pathway in regulating the stem

cell properties of cancer cells. More recently, it was demonstratedthat the Wnt–b-catenin pathway acts as a new reprogramming

factor to promote somatic cells to pluripotency, although theunderlying molecular rationale is unclear (Ying et al., 2008).

Thus, our studies have shed light on the connection between theWnt–b-catenin pathway and the reprogramming process.

In regulating stem cell functions, miRNAs are proposed to be

important factors because expression levels of certain miRNAs inembryonic stem cells are different from those of differentiated

embryoid bodies (Suh et al., 2004). Consistently, tumoursanalysed by miRNA profiling have shown significantlydifferent miRNA profiles of stem cells compared with

differentiated cells from the same sample (Papagiannakopoulosand Kosik, 2008; Sun et al., 2010). In breast cancer, the inhibition

of let-7, miR-30 and miR-200 and the induction of miR-181 andmiR-495 are required for the maintenance of stem cell properties(Hwang-Verslues et al., 2011; Iliopoulos et al., 2010; Shimono

et al., 2009; Wang et al., 2011; Yu et al., 2010; Yu et al., 2007),suggesting miRNAs are important in regulating breast cancer

stem cells. As the Wnt–b-catenin pathway is a dominantregulator of breast cancer stem cells, we speculate that this

pathway may affect the stem cell properties through modulationsin miRNA expression. The relationship between the Wnt–b-catenin pathway and miRNAs has been verified in different

cellular contexts, although it remains obscure in stem cells,especially cancer stem cells. In contrast to the rare downstream

target miRNAs, many upstream modulated miRNAs of the Wnt–b-catenin pathway have been identified, of which miR-135a/b

(Nagel et al., 2008) and miR-315 (Silver et al., 2007) upregulate,whereas miR-200a (Korpal et al., 2008; Saydam et al., 2009),miR-21 (Hashimi et al., 2009), miR-203 (Thatcher et al., 2008)

and miR-8 (Kennell et al., 2008) downregulate b-catenintranscriptional activity through targeting the Wnt–b-catenin

pathway components. Among the few Wnt-regulated miRNAs,the Wnt–b-catenin pathway regulates miR-15/16 maturation,

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rather than its transcription. However, the underlying mechanism

is unknown (Martello et al., 2007). Another study showed

that miR-122a expression is downregulated in APC-driven

gastrointestinal cancers. The mechanisms by which the Wnt–b-

catenin pathway regulates miR-122a and 122a inhibition are

unknown (Wang et al., 2009). Additionally, miR-375 is

downregulated by b-catenin. However, the function of miR-375

and the transcriptional mechanism through which miR-375 is

regulated by the Wnt–b-catenin pathway are not clear and require

further investigation (Ladeiro et al., 2008). In an attempt to

identify novel downstream target miRNAs of the Wnt–b-catenin

pathway, we performed screening experiments in breast cancer

cells. In our screening, 15 miRNAs were upregulated, whereas 48

miRNAs were downregulated upon stimulation of the Wnt–b-

catenin pathway, which is consistent with the notion that an overall

downregulation of miRNAs occurs in many cancers, compared

with their normal tissue counterparts (Hammond, 2006). It is noted

that two known downregulated miRNAs, miR-15 and miR-375,

Fig. 6. See next page for legend.

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are present in our list, suggestive of a successful screening strategy

for our experiment. The obvious downregulation of let-7 members

is remarkable, because let-7 miRNAs have been demonstrated to

play an important role in breast cancer stem cells. Both qPCR and

reporter assays of several cell lines verified that let-7 miRNAs are

novel downstream target miRNAs of the Wnt–b-catenin pathway.

Furthermore, we found that the Wnt–b-catenin pathway regulates

let-7 miRNAs through a post-transcriptional pathway by directly

transactivating Lin28 expression. To the best of our knowledge,

this report is the first to find that the Wnt–b-catenin pathway

inhibits let-7 miRNAs through Lin28 transactivation.

Recent advances in the characterisation of stem cells in

mammary epithelium and breast cancer cells have opened the

door to understanding the signalling and transcriptional

programming underlying both the development of normal

mammary stem cells and the proliferation/differentiation of

their malignant counterparts (Al-Hajj et al., 2003; Ponti et al.,

2005; Shackleton et al., 2006; Stingl et al., 2006). The Wnt–b-

catenin pathway, Lin28 and let-7 are defined as crucial

mammary/breast cancer stem cell regulators. However, the

relationship between them is unknown. In this study, we have

functionally established a connection between the Wnt–b-catenin

pathway and Lin28/let-7 that is essential for Wnt–b-catenin-

pathway-mediated breast cancer stem cell expansion. This model

is strongly supported by: (1) the converging effects of the Wnt–b-

catenin pathway, Lin28 and let-7 on breast cancer stem cell

properties; (2) the dependence of the Wnt–b-catenin pathway in

let-7 inhibition and breast cancer stem cell expansion on Lin28

activation; (3) the dependence of the Wnt–b-catenin pathway in

breast cancer stem cell expansion on let-7 inhibition; and (4) the

expression pattern and functional relevance of b-catenin, Lin28

and let-7a in cancer samples and tumour models. As such, our

study presents the first mechanistic characterisation of the Wnt–

b-catenin pathway function in breast cancer stem cells and points

to Lin28 and/or let-7 as important downstream targets in these

cells in vitro and in vivo. As the Wnt–b-catenin pathway is

aberrantly activated in many cancer types, future work should

address whether the Wnt–b-catenin pathway/Lin28/let-7 cascade

is also involved in the regulation of other cancers. Moreover, re-

expression of let-7 miRNAs might be a new therapeutic option in

treating cancers with an aberrantly activated Wnt–b-catenin

pathway.

Materials and MethodsCell culture

HEK 293T (human embryonic kidney 293T) and human breast cancer cell lines

ZR-75-30 (human breast ductal carcinoma), MDA-MB-231 (human breast

adenocarcinoma), MCF7 (human breast adenocarcinoma) and T-47D (humanbreast ductal carcinoma) were obtained from the American Type Culture

Collection and grown in Dulbecco’s modified Eagle’s medium (DMEM)

supplemented with 10% foetal calf serum (Gibco), penicillin and streptomycin.

To induce the Wnt–b-catenin pathway, cells were grown in the presence of 20 mMLiCl (Sigma) for 24–36 hours or 100 ng/ml Wnt1 protein (Abcam, cat. no.

ab84080) for 48 hours. All cell lines were grown at 37 C with 5% carbon dioxide.

Reagents

The following antibodies were used: anti-Lin28 (Abcam, cat. no. ab46020), anti-b-

catenin (Cell Signaling Technology, cat. no. 9562), anti-b-actin (Sigma-Aldrich,

cat. no. A1978), anti-HRAS (Proteintech Group, cat. no. 18295-1-AP), anti-HMGA2 (R&D Systems, cat. no. AF3184), anti-LEF-1 (Santa Cruz

Biotechnology, cat. no. sc-8591), anti-HA (Sigma-Aldrich, cat. no. H9658). The

human cell surface markers utilised were CD24-PE (BD Biosciences) and CD44-PE-Cy5 (eBioscience). Cholesterol-conjugated mmu-let-7a agomir and negative

control agomir as well as cholesterol-, 29-O-methyl (OMe)-conjugated mouse

lin28 siRNA and negative control siRNA for in vivo delivery were obtained from

Ribobio Co. (Guangzhou, China).

Quantitative real-time PCR

Total RNAs were isolated using TRIzol reagent (Invitrogen). cDNAs wereprepared from these RNAs using a ReverTra Ace qPCR RT kit from Toyobo.

Quantitative PCR was performed using a Rotor gene 6000 Sequence Detection

System with the Thunderbird SYBR qPCR Mix (Toyobo). Mature let-7a, let-7f andlet-7g miRNAs were quantified using a predesigned miRCURY LNATM Universal

RT microRNA PCR Assay (Exiqon). A eukaryotic 18S rRNA or U6 snRNA

endogenous control was used as an internal standard, and the results were

calculated using the DDCT (where CT is threshold cycle) method. Primersequences are provided in supplementary material Table S3.

Cell proliferation

Stable transfected cells were plated in 96-well dishes at 1000 cells/well. Cellgrowth rates were monitored using a Cell Counting Kit-8 (CCK-8, Dojindo

Molecular Technologies) and an MTT Assay Kit (Promega).

Colony formation assays

Stable transfected cells were plated in six-well dishes at 500 cells/well. After 10

days, the cultures were washed twice with PBS, incubated with methanol for20 minutes, stained with Crystal Violet for 30 minutes, and washed with tap water.

The colonies were counted under a low magnification microscope and a group of

more than 10 cells was defined as a colony.

Fig. 6. In vivo analyses of the Wnt–b-catenin pathway/Lin28/let-7

cascade. (A) Breast cancer samples derived from 82 patients, in a tissue

microarray along with their adjacent normal tissues were immunostained with

b-catenin and Lin28 antibodies. Left: representative immunostaining for b-

catenin and Lin28 is shown for three patient samples. Right: correlations

between b-catenin and Lin28 protein levels in tumour and normal tissues were

measured using the Spearman rank correlation test (r50.7640,

P51.8461028). The three representative samples are marked in the

correlation map. Scale bar: 50 mm. (B) Expression levels of b-catenin, Lin28

and let-7a in MMTV-Wnt-1 transgenic mouse tissue (Wnt-1 TG).

Immunohistochemical analysis of b-catenin and Lin28 expression in

premalignant mammary tissues derived from a Wnt-1 TG mouse and

mammary tissues from its wild-type (WT) littermate, at 14 weeks (left). Scale

bar: 50 mm. Western blotting and semi-quantitative RT-PCR analysis of b-

catenin, Lin28, HRAS, HMGA2 and let-7a levels in the same mammary

tissues (right). (C) Immunohistochemical analysis of b-catenin and Lin28

expression in small intestine tissues derived from an Apcmin/+ mouse at the

age of 16 weeks. Black arrows indicate adenoma tissues; red arrows indicate

villus tissues (left). Scale bar: 200 mm. Western blotting and RT-PCR analysis

of b-catenin/Lin28, HRAS, HMGA2 and let-7a levels in small intestine

mucosa (free of muscle) derived from an Apcmin/+ mouse and its wild-type

(WT) littermate, at an age of 16 weeks (right). (D) Knockdown of Lin28

compromised the let-7 repression and the stem cell expansion of premalignant

mammary tissues from the Wnt-1 transgenic (TG) mice. WT and Wnt-1 TG

mice (n53) at 14 weeks of age were administered cholesterol-, OMe-

conjugated negative control (NC) or Lin28 siRNA through intraductal

injection. Let-7 expression was detected by qPCR (top, left) and the

percentage of Lin2 CD29hi CD24+ cells in cell suspensions of mammary

tissues was determined by FACS analysis (top, right). Shown are

representative FACS profiles from a single pair, and the diagram indicates the

mean values of three different pairs (bottom, left). Western blots show a

successive knockdown of Lin28 in Wnt-1 TG mammary cells (bottom, right).

(E) Enforced expression of let-7a rescued the stem cell population of

premalignant mammary tissues from the Wnt-1 TG mice. WT and Wnt-1 TG

mice (n53) at 14 weeks of age were administered cholesterol-conjugated

negative control (NC) or let-7a agomir by intraductal injection, and the

percentage of Lin2 CD29hi CD24+ cells in cell suspensions of mammary

tissues was determined by FACS analysis. Shown are representative FACS

profiles from a single pair (top), and the graph shows the mean values of three

different pairs (bottom, left). The qPCR shows a successive intake of let-7a in

Wnt-1 TG mammary cells (bottom, right). (B–E) A representative experiment

of three independent experiments are shown; data in D and E are the means 6

s.d. of three independent experiments.

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FACS analysis

Confluent cells were trypsinised into single-cell suspensions. These culture cellsuspensions and the tissue digested cell suspensions were washed withfluorescence-activated cell sorting (FACS) buffer (2% foetal bovine serum inPBS), counted, and stained with fluorophore-conjugated antibodies. A total of 106

cells in 100 ml of FACS buffer were incubated with antibodies for 30 minutes at4 C. Unbound antibodies were washed off, and the cells were sorted using aBeckman EPICS XL instrument.

Mammosphere culture

Cells (104 cells/ml) were cultured in ultra-low attachment plates in serum-freeDMEM/F12 (Invitrogen) supplemented with B-27 (1:50; Invitrogen), 20 ng/mlepidermal growth factor (EGF; BD Biosciences), 20 ng/ml basic fibroblast growthfactor (bFGF; BD Biosciences) and 4 mg/ml insulin (Sigma), and fed every 3 days.

miRNA microarrays

Total RNAs were harvested using TRIzol (Invitrogen) and an RNeasy mini kit(Qiagen) according to the manufacturer’s instructions. The samples were labelledusing a miRCURYTM Hy3TM/Hy5TM Power labelling kit and hybridised on amiRCURYTM LNA Array (v.14.0). Scanning was performed on an Axon GenePix4000B microarray scanner. GenePix pro V6.0 was used to read the raw intensity ofthe images. Signals that were more than twice the background were removed, anddatasets were median-centred before calculating fold change values.

Chromatin immunoprecipitation assay

A chromatin immunoprecipitation (ChIP) assay was performed following theUpstate Biotechnology protocol. Briefly, T-47D cells were fixed with 1%paraformaldehyde at room temperature for 10 minutes, washed, and lysed withSDS lysis buffer (50 mM Tris-HCl, 1% SDS, 10 mM EDTA, and proteaseinhibitors). The lysates were sonicated to reduce DNA lengths to between 300 and600 bp. The soluble fraction was diluted, precleared with salmon sperm DNA–protein-A–agarose, divided into two tubes and incubated with specific antibodiesor control IgG. The immune complexes were then precipitated with protein A/Gbeads and eluted with elution buffer (0.1 M NaHCO3 and 1% SDS). The elutedsamples were reverse cross-linked and treated with proteinase K. DNA waspurified by phenol–chloroform extraction and dissolved in distilled water. Real-time PCR quantification of the ChIP samples was performed in triplicate using theThunderbird SYBR qPCR Mix (Toyobo). Primer sequences for the Lin28 promoterare provided in supplementary material Table S3.

Generation of miRNA-, shRNA- or cDNA-expressing lentiviruses

Oligonucleotides encoding let-7a1 pre-miRNA or shRNA targeting Lin28, b-catenin, Pygo2 or lacZ (control) were synthesised by Invitrogen and cloned underthe control of the U6 promoter in the lentiviral vector lentilox pLL3.7. Foroverexpression of Lin28, b-catenin S33Y, HA-Wnt1 or GFP (control), the cDNAswere cloned under the control of the EF1a promoter in the lentiviral vector pLV-CS2.0. The generation of lentivirus vectors was performed by co-transfectingpLL3.7 or pLV-CS2.0 carrying the expression cassette with helper plasmidspVSV-G and pHR into 293T cells using Lipofectamine 2000 (Invitrogen). Theviral supernatant was collected 48 hours after transfection, and viral titres weredetermined by transducing HeLa cells at serial dilutions and analysing the GFPexpression using flow cytometry. Cells at 50–70% confluency were infected withviral supernatants containing 10 mg/ml Polybrene for 24 hours, after which freshmedium was added to the infected cells, which were later selected with puromycinor G418. The oligonucleotide sequences are provided in supplementary materialTable S4.

Let-7 luciferase assay

To evaluate the miRNA function of let-7, a pMIR-REPORTTM luciferase reportervector (Ambion) with two copies of sequences complementary to let-7a clonedinto its 39 UTR (let-7 sensor) was used. The reporter vector plasmid wastransfected into MDA-MB-231 using Lipofectamine 2000 according to themanufacturer’s instructions. To correct for the transfection efficiency, a luciferasereporter vector without the let-7 target was transfected in parallel. The luciferaseactivity was assayed using a luciferase assay kit (Promega). let-7 miRNA functionwas expressed as a percentage reduction in the luciferase activity of cellstransfected with the reporter vector containing the let-7 target sequences comparedwith cells transfected with the vector without the let-7 target.

Lin28 promoter luciferase assay

To generate the luciferase reporter vectors, the Lin28 promoter fragment wasamplified from human genomic DNA and cloned into the KpnI–HindIII site of thefirefly luciferase plasmid pGL3-basic-IRES. For reporter assays, HEK 293T cellsin 24-well plates were transfected at 50–60% confluency using the calciumphosphate method. Both Lin28 promoter constructs (50 ng) and cytomegalovirus(CMV)–b-galactosidase (25 ng) reporter plasmids were co-transfected with the b-catenin S33Y or Wnt-1 expression plasmids or in the presence of LiCl (10 to

20 mM) for 24 hours. The total amount of plasmid DNA transfected was madeequivalent by adding empty vectors. Cells were harvested after 24 hours andprocessed for luciferase and b-galactosidase assays, and the data were normalisedto the b-galactosidase levels.

In vivo stem cell assay

Fourteen-week-old virgin female MMTV-wnt-1 (FVB) and wild-type (FVB) micewere anesthetized. The keratin plugs were removed from the surface of the nipple,revealing the duct orifice. The mammary ducts were cannulated using a 1.0-cm,34-gauge, blunt-ended needle attached to a 1-ml tuberculin syringe. Next, 5 nmolof cholesterol-conjugated microRNA agomir or cholesterol-, OMe-conjugatedsiRNA in 25 ml PBS was infused into the mammary gland once every 3 days for atotal of 12 days (four times). Twenty days after the first injection, the mammaryglands of the injected site were dissected from the female mice. The animalexperiments were reviewed and approved by the Institutional Animal Care and UseCommittee of Xiamen University College of Medicine.

After mechanical dissociation with surgical scissors, the tissue was placed inculture medium 1640 supplemented with 5% bovine calf serum and containing300 U/ml collagenase (Sigma) and 100 U/ml hyaluronidase (Sigma) and digestedfor 3 hours at 37 C. The resultant organoid suspension was vortexed and red bloodcells lysed in NH4Cl. A single-cell suspension was obtained by sequentialdissociation of the fragments by gentle pipetting for 1–2 minutes in 0.25% trypsinand then for 2 minutes in 5 mg/ml dispase II plus 0.1 mg/ml DNase I (Sigma-Aldrich) followed by filtration through a 40-mm mesh. Cells were resuspended to106 cells in 100 ml of cold FACS buffer. Staining was performed using thefollowing antibodies and reagents on ice for 30 minutes as follows: biotin-conjugated Lin (CD45, CD31, TER119, 50 ml/ml; EasySep, cat. no. 19757), PE-conjugated CD24 (10 ml/100 ml; StemCell Technologies, cat. no. 10814), FITC-conjugated CD29 (10 ml/100 ml; BioLegend, cat. no. 102205), PE-conjugated IgGisotype control (10 ml/100 ml), FITC-conjugated IgG isotype control (10 ml/100 ml). Unbound antibodies were washed off, propidium iodide (2 mg/ml finalconcentration) was added and the mixture incubated on ice for 20 minutes. Thecells were sorted using a Beckman EPICS XL instrument. Forward scatter (FS) andside scatter (SS) were used to select most of the single cell populations. The PI-positive, non-viable cells were excluded from the final analysis. Lin+ cells weregated out of the final analysis. Single stained samples were used as compensationcontrols. Cells incubated in non-specific IgG isotype control (conjugated to PE orFITC) were used to set the gates that define Lin2, CD24+ or CD29hi cells,respectively.

Histological analysis

Tissue microarrays from 82 patients with breast cancer were purchased fromShanghai Outdo Biotech Co., Ltd. The human tissue preparation and analysis wereapproved by the Institutional Review Board of The First Affiliated Hospital ofXiamen University. The tissues were fixed in 10% formalin, embedded in paraffin,and sectioned. After dewaxing and rehydration, the sections were pretreated withperoxidase blocking buffer (Maxim, Fuzhou, China) for 20 minutes at roomtemperature. Antigen retrieval was performed by boiling in Tris-EDTA (pH 9.0)for 20 minutes. After treatment with a blocking buffer (5% normal goat serum inPBS) for 1 hour at room temperature, sections were incubated with anti-Lin28antibody (1:300, overnight at 4 C, Abcam) and anti-b-catenin antibody (1:200,overnight at 4 C; Cell Signaling Technology) in the blocking buffer. Secondaryantibody reagents were obtained from the SABC kit (Boster BiologicalTechnology, Wuhan, China) or DAB kit (Maxim Biological Technology,Fuzhou, China).

The pictures were taken using a computerized imaging system (LeicaMicrosystems, Imaging Solutions Ltd, Cambridge, UK). Under high-powermagnification (2006), photographs of three representative fields were capturedusing Leica QWin Plus v3 software; identical settings were used for eachphotograph. For the reading of each antibody staining, a uniform setting for all ofthe slides was applied. The mean integrated optical density (IOD) was used torepresent the relative expression of indicated protein. IOD is a computer-assistedmethod which is commonly used to quantify both the area and the intensity of thepositive staining in immunohistochemistry. We calculated the IOD of eachphotograph acquired from the tissue microarray sections by using Image-Pro Plusv6.2 software (Media Cybernetics Inc., Bethesda, MD). The typical positivestaining area was located, with the help of a pathologist, in the segmentation panel,standard optical density was chosen in the intense calibration panel, andbackground was subtracted in the optical density calibration panel, which waschosen to exclude all the areas without epithelial or tumour tissues. We alsosubtracted the background in serial sections by using a non-specific IgG instead ofprimary antibody. The ‘mean IOD’ (IOD/tissue area) represents the expressionlevel of indicated protein.

Statistical analysis

For the tissue microarrays, Fisher’s exact test was used to compare qualitativevariables; quantitative variables were analysed using t-tests and Spearman’s rankcorrelation tests. The data are presented as the means 6 s.d. All statistical tests

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were two-sided Student’s t-tests, and a P,0.05 was considered to be statisticallysignificant.

Author contributionsW.-Y.C., T.-Z.W. and Q.-C.L. performed the experiments andanalyzed the data. Q.-W.W. provided the tumour samples andperformed the experiments. Q.-F.L., M.Y., G.-D.Y., J.-F.W., Y.-Y.C., G.-B.S. and Y.-J.L. performed the experiments. W.-X.Z.provided the tumour samples. Z.-M.Z. designed the experiments.B.-A.L. designed the experiments and wrote the manuscript.

FundingThis work was supported by the ‘973’ Project of the Ministry ofScience and Technology [grant numbers 2009CB52220,2013CB530600 to B.-A.L.]; the National Natural ScienceFoundation of China [grant numbers U1205023, 81272384,90919037, 81201617, 81201616 to B.-A.L.]; the 2013 Major Projectof Science and Technology from the Department of Education [grantnumber 313051 to B.-A.L.]; the Natural Science Foundation of FujianProvince [grant numbers 13111125 to Z.-M.Z.]; the Key Projects ofFujian Province [grant number 2011Y01010460 to Z.-M.Z.]; theproject of the Fujian Health Department [grant number 2011-CXB-34to Z.-M.Z.]; the project of the Xiamen Technique Bureau [grantnumber 3502Z20114003 to Z.-M.Z.]; and ‘Project 111’ sponsored bythe State Bureau of Foreign Experts and Ministry of Education [grantnumber B06016 to B.-A.L.].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.123810/-/DC1

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