Concise Review: LIN28/let-7 Signaling,a Critical Double-Negative Feedback Loop During Pluripotency,Reprogramming,and Tumorigenicity

Abstract

MicroRNAs (miRNAs) with 20–30 nucleotides have recently emerged as the multidimensional regulators of cell fate decisions. Recent improvement in high-throughput sequencing has highlighted the potential role of LIN28/let-7 regulatory network in several developmental events. It was proposed that this pathway might represent a functional signature in cell proliferation, transition between commitment and pluripotency, and regulation of cancer and tumorigenicity. LIN28/let-7 regulatory pathway is one of the excellent examples of the relationship between an miRNA and mRNAs. This review article highlights the potentials of LIN28/let-7 signaling in gene regulatory pathways during pluripotency, reprogramming, and tumorigenicity.

Introduction

The discovery of microRNAs (miRNAs) as short ribonucleic acid gene regulators is a critical step in cellular and molecular biology (Khoshnam et al., 2017b; Schmidt, 2014). miRNAs are endogenous small noncoding RNAs consisting of 20–30 nucleotides with diverse regulatory functions that target specific mRNAs and can lead to translational repression of mRNAs in animals (Bartel, 2004; Khoshnam et al., 2017a; Ran et al., 2017) and in plants (Zhang and Wang, 2015). However, it is now evident that a few miRNAs can activate the translation of mRNAs in unknown pathways (Mengardi et al., 2017; Vasudevan et al., 2007).

LIN28/let-7 regulatory pathway is an excellent example of the relationship between miRNAs and mRNAs. The lethal-7 (Let-7) miRNA belongs to the “Let-7 family” miRNAs and is one of the highly conserved miRNAs that is originally identified in genetic screening of heterochronic mutants in the nematode Caenorhabditis elegans (Büssing et al., 2008; Reinhart et al., 2000; Roush and Slack, 2008; Thornton and Gregory, 2012). This family is often present in multiple copies in the genome that share their seed sequence from nucleotides 2 to 8 at their 5′ ends and are considered as the important modulators of cell proliferation and differentiation during development (Büssing et al., 2008; Roush and Slack, 2008).

Let-7 miRNA is synthesized from primary miRNA (pri-miRNA Let-7) in two stages by the action of Drosha enzyme in the nucleus and Dicer enzyme in the cytoplasm (Wahid et al., 2010) (Fig. 1). Recent studies indicate that the post-transcriptional regulation of the Let-7 miRNA is controlled by the conserved RNA-binding protein Lin28, which binds to the terminal loop or the pre-element portion of the Let-7 family (Heo et al., 2008). In support of this notion, two mammalian homologues of Lin28 including Lin28a and Lin28b contain two RNA-binding domains named as cold shock domain and retroviral-type zinc finger domain that bind to the pre-Let-7 family and repress their biogenesis (Büssing et al., 2008; Heo et al., 2008; Nam et al., 2011). However, the inhibitory effect of Lin28a and Lin28b on the Let-7 biogenesis is achieved by different mechanisms (Piskounova et al., 2011).

FIG. 1.

LIN28/let-7 signaling, a critical double-negative feedback loop during pluripotency, reprogramming, and tumorigenicity. Let-7 miRNA is synthesized from primary miRNAs (pri-miRNAs Let-7) in two stages by the action of Drosha enzyme in the nucleus and Dicer enzyme in the cytoplasm. The Let-7 miRNA is controlled by the conserved RNA-binding protein Lin28 that binds to the terminal loop or pre-element portion of the Let-7 family. Let-7 expression is regulated through Lin28 after generation of ESCs. Also, LIN28/let-7 signaling pathway has a special role in developing miRNA-mediated reprogramming and can be an acute marker during tumorigenicity. ESCs, embryonic stem cells; miRNA, microRNAs.

The collaboration between Lin28 and Let-7 is highly conserved across evolution and is critical for regulating the developmental events (Pasquinelli et al., 2000). During mutagenesis screenings, Lin28 was first discovered in the C. elegans as an important regulator of developmental timing (Ambros and Horvitz, 1984; Jun-Hao et al., 2016; Moss et al., 1997; Thornton and Gregory, 2012; Tsialikas and Romer-Seibert, 2015). Several groups have reported that the LIN28/let-7 pathway regulates differentiation of embryonic stem cells (ESCs) (Shyh-Chang and Daley, 2013), cell growth and metabolism (Shyh-Chang and Daley, 2013), tumorigenic cell functions (Hikasa et al., 2016; Shi et al., 2008), and has also been implicated in various diseases (Kumar et al., 2016; Powers et al., 2016; Punga et al., 2016; Thornton and Gregory, 2012). Hence, these new insights might help to validate LIN28/let-7 as a therapeutic advance for cancer treatment and to illustrate a new dimension for regulating the developmental events and controlling animal cell behavior.

LIN28/let-7 Pathway During Pluripotency

ESCs are pluripotent cells with the ability to self-renew and to generate various differentiated cells (Gangaraju and Lin, 2009). Lin28 is involved in the regulation of OCT4, NANOG, and SOX2 that leads to stemness enhancement, proliferation and, suppression of ESC differentiation (Büssing et al., 2008; Shyh-Chang and Daley, 2013). Indeed, LIN28/let-7 signaling has emerged as a significant player in the translational regulation of ESC propagation and differentiation (Gangaraju and Lin, 2009). During ESC proliferation, Lin28a in association with Lin28b directly inhibits the Let-7 miRNA maturation. Lin28a specifically attaches to the tetranucleotide sequence motif (GGAG binding site) in the terminal loop of pre-Let-7 through a terminal uridylyl transferase enzyme or TUTase (Zcchc11/TUT4) and inhibits pre-Let-7 maturation (Heo et al., 2009; Wang et al., 2012).

In fact, TUTase adds oligouridine tail to the pre-Let-7, which suppresses Drosha/Dicer function (Heo et al., 2008, 2009; Shyh-Chang and Daley, 2013; Stefani et al., 2015). However, the level of Lin28 is decreased upon ESC differentiation (Melton et al., 2010). In this process, two heterochronic miRNAs including Lin-4 (mir-125) and Let-7 strongly suppress Lin28 transcription through direct binding to its 3′ UTR portion (which has four conserved binding sites for the Let-7 family) in an autoregulatory circuit that regulates miRNA processing during ESC differentiation (Roush and Slack, 2008; Rybak et al., 2008). In contrast, Lin28 can also function in an Let-7-independent manner by directly targeting the 3′ UTR of Hmga2 mRNA (a key chromatin-associated protein), which has important roles in physiological apoptosis of differentiating ESCs (Parisi et al., 2017).

Lin-4 was first identified in C. elegans and is known to control the timing of stem cell differentiation through appropriately regulating the transition time from symmetric to asymmetric cell divisions (Carrington and Ambros, 2003; Harandi and Ambros, 2015; Reinhart et al., 2000). Therefore, the mature Let-7 family members, Lin-4 and Lin28b, participate in regulating the ESC proliferation and differentiation (Rahkonen et al., 2016; Rybak et al., 2008).

LIN28/let-7 Pathway During Reprogramming

Transgenic expression of the ESC pluripotency factors, including Oct4, Sox2, Nanog, Klf4, and c-Myc, leads to reprogramming of normal or malignant somatic and cancer cells into induced pluripotent stem cell (Chien et al., 2015). It is well known that miRNAs have a crucial effect on regulating gene expression during reprogramming (Leonardo et al., 2012). For example, LIN28/let-7 signaling has been reported to play a special role in developing miRNA-mediated reprogramming in which through inhibiting Let-7 biogenesis, Lin28 influences mRNA translation (Rehfeld et al., 2015).

A recent report has confirmed that LIN28/let-7 pathway is the key regulator of Oct4 and Sox2 expression during reprogramming of oral squamous carcinoma cells (OSCCs) into a stem-like state (Chien et al., 2015). In this study, ectopic Lin28b expression increased the level of Oct4 and Sox2 expression in OSCCs, whereas overexpression of Let-7 reversed this event. In addition, during reprogramming, ARID3B (AT-rich interacting domain 3B) increased the transcription of Oct4 and HMGA2 domain enhanced the Sox2 expression, and both ARID3B and HMGA2 domains of Lin28 are direct targets of Let-7 miRNA (Liao et al., 2016). Therefore, LIN28/let-7 signaling induces OSCC reprogramming, tumor initiation, and regulates stemness (Chien et al., 2015).

LIN28/let-7 and Tumorigenicity

LIN28/let-7 signaling has been identified to play vital roles in several human cancers, so it can be an interesting regulatory pathway for drug discoveries (Ji and Wang, 2010; Jiang and Baltimore, 2016; Schmidt, 2014). A recent work on breast cancer stem cells (BCSCs) indeed confirmed that the LIN28/let-7 pathway in cooperation with the long noncoding RNA-H19 (H19) functions as heterochronic genes and has a critical task in the maintenance of BCSCs. After BCSCs propagation, H19 positively regulates the expression of Lin28. In this way, Lin28 with a double-negative feedback loop plays a central role in the suppression of mature Let-7 production and H19 expression in BCSCs. During this feedback loop, increased Lin28 expression in association with reduced let-7 activation is consistent with negative regulation of H19, a downstream repressor of Let-7.

Although Lin28 expression is shown to be reduced after Let-7 expression, so H19 seems to be an essential downstream target for BCSC maintenance (Peng et al., 2017). Recently, LIN28/let-7 pathway has been identified as a critical target for nonsmall-cell lung cancer (NSCLC) therapy, as well. Actually, it is shown that overexpression of Let-7 in lung adenocarcinoma cell line represses lung cancer cell proliferation in vitro (Takamizawa et al., 2004). In another interesting work, chemotherapeutic drugs that were employed on NSCLC cells led to disturbance of LIN28/let-7 double-negative feedback loop through the high level of Lin28 and low level of Let-7. Hence, LIN28/let-7 signaling can be an acute marker during radiotherapy and chemotherapy in NSCLC patients (Yin et al., 2017). Moreover, it has been shown that the percentage of the ALDH1 (a relatively rare aldehyde dehydrogenase 1) positive tumor cells has been correlated with the expression of Lin28 mRNA.

This pathway is fundamental in the maintenance of ALDH1-positive tumor cells with a double-negative feedback loop. Indeed, Let-7 miRNA appears to have an opposing function to Lin28 and negatively controls ALDH1-positive tumor cells. Also, Lin28 may control the ALDH1 cell population in an Let-7-independent pathway through activation of reprogramming factors, such as OCT4 (Vaz and Tanavde, 2012; Virant-Klun et al., 2015; Yang et al., 2010). Recent advances in the applied mechanisms of LIN28/let-7 pathway highlight that this complex modulates aerobic glycolysis and cancer progression through targeting pyruvate dehydrogenase kinase 1 in a hypoxia- or hypoxia-inducible factor-1-independent manner (Ma et al., 2014).

In addition, LIN28/let-7 pathway can regulate tumorigenesis in association with Merlin/NF2 (Moesin-ezrin-radixin-like protein, also known as schwannomin), a tumor suppressor protein encoded by the neurofibromatosis type 2 gene (NF2) (Petrilli and Fernández-Valle, 2016). At high cell density, this protein is dephosphorylated and directly binds to Lin28b and suppresses its activation. Therefore, pri-Let-7 permits to complete its maturation and can be activated as one of the downstream targets of Merlin/NF2 toward inhibiting cell growth. While, at low cell density, phosphorylated Merlin/NF2 does not bind to Lin28b and as a result pri-Let-7 maturation would be blocked in the nucleus which leads to increase of cell proliferation in a dose-dependent manner. Therefore, this mechanism is related to the cell density that triggers Merlin/NF2 dephosphorylation (Hikasa et al., 2016; Petrilli and Fernández-Valle, 2016).

Conclusion and Perspective

LIN28/let-7 pathway is necessary for the regulatory network that governs pluripotency, reprogramming, and tumorigenicity. LIN28/let-7 signaling has emerged as a significant player in the translational regulation of ESC propagation and differentiation. Also, this pathway has a special role in developing miRNA-mediated reprogramming and can be an acute marker during tumorigenicity. The discovery of this pathway has highlighted its potential in modulating the proliferation and differentiation in normal and abnormal cells. Hence, targeted manipulation of this pathway may offer an appropriate opportunity as the therapeutic advances for cancer treatment.

Footnotes

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

References

Ambros

, and Horvitz

(1984). Heterochronic mutants of the nematode. Science, 226, 409–416.

Bartel

D.P.

(2004). MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell, 116, 281–297.

Büssing

, Slack

F.J.

, and Großhans

(2008). let-7 microRNAs in development, stem cells and cancer. Trends Mol. Med., 14, 400–409.

Carrington

J.C.

, and Ambros

(2003). Role of microRNAs in plant and animal development. Science, 301, 336–338.

Chien

C.-S.

, Wang

M.-L.

, Chu

P.-Y.

, Chang

Y.-L.

, Liu

W.-H.

, Yu

C.-C.

, Lan

Y.-T.

, Huang

P.-I.

, Lee

Y.-Y.

, Chen, and Y.-W. (2015). Lin28B/Let-7 regulates expression of Oct4 and Sox2 and reprograms oral squamous cell carcinoma cells to a stem-like state. Cancer Res. 75, 2553–2565.

Gangaraju

V.K.

, and Lin

(2009). MicroRNAs: Key regulators of stem cells. Nat. Rev. Mol. Cell Biol., 10, 116–125.

Harandi

O.F.

, and Ambros

V.R.

(2015). Control of stem cell self-renewal and differentiation by the heterochronic genes and the cellular asymmetry machinery in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U. S. A., 112, E287–E296.

Heo

, Joo

, Cho

, Ha

, Han

, and Kim

V.N.

(2008). Lin28 mediates the terminal uridylation of let-7 precursor MicroRNA. Mol. Cell, 32, 276–284.

Heo

, Joo

, Kim

Y.-K.

, Ha

, Yoon

M.-J.

, Cho

, Yeom

K.-H.

, Han

, and Kim

V.N.

(2009). TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell, 138, 696–708.

10.

Hikasa

, Sekido

, and Suzuki

(2016). Merlin/NF2-Lin28B-let-7 is a tumor-suppressive pathway that is cell-density dependent and hippo independent. Cell Rep. 14, 2950–2961.

11.

, and Wang

X.W.

(2010). A Yin-Yang balancing act of the lin28/let-7 link in tumorigenesis. J. Hepatol., 53, 974–975.

12.

Jiang

, and Baltimore

(2016). RNA-binding protein Lin28 in cancer and immunity. Cancer Lett. 375, 108–113.

13.

Jun-Hao

E.T.

, Gupta

R.R.

, and Shyh-Chang

(2016). Lin28 and let-7 in the metabolic physiology of aging. Trends Endocrinol. Metab., 27, 132–141.

14.

Khoshnam

S.E.

, Winlow

, Farbood

, Moghaddam

H.F.

, and Farzaneh

(2017a). Emerging roles of microRNAs in ischemic stroke: As possible therapeutic agents. J. Stroke, 19, 166.

15.

Khoshnam

S.E.

, Winlow

, and Farzaneh

(2017b). The interplay of MicroRNAs in the inflammatory mechanisms following ischemic stroke. J. Neuropathol. Exp. Neurol. nlx036, 76, 548–561.

16.

Kumar

, Haque

, and Nazir

(2016). Role of microRNA Let-7 in modulating multifactorial aspect of neurodegenerative diseases: An overview. Mol. Neurobiol., 53, 2787–2793.

17.

Leonardo

T.R.

, Schultheisz

H.L.

, Loring

J.F.

, and Laurent

L.C.

(2012). The functions of microRNAs in pluripotency and reprogramming. Nat. Cell Biol., 14, 1114–1121.

18.

Liao

T.-T.

, Hsu

W.-H.

, Ho

C.-H.

, Hwang

W.-L.

, Lan

H.-Y.

, Lo

, Chang

C.-C.

, Tai

S.-K.

, and Yang

M.-H.

(2016). let-7 modulates chromatin configuration and target gene repression through regulation of the ARID3B complex. Cell Rep. 14, 520–533.

19.

, Li

, Sun

, Huang

, Li

, He

, Wu

, Yang

, Zhong

, and Song

(2014). Lin28/let-7 axis regulates aerobic glycolysis and cancer progression via PDK1. Nat. Commun., 5, 1–13.

20.

Melton

, Judson

R.L.

, and Blelloch

(2010). Opposing microRNA families regulate self-renewal in mouse embryonic stem cells. Nature, 463, 621–626.

21.

Mengardi

, Limousin

, Ricci

E.P.

, Soto-Rifo

, Decimo

, and Ohlmann

(2017). microRNAs stimulate translation initiation mediated by HCV-like IRESes. Nucleic Acids Res. 45, 4810–4824.

22.

Moss

E.G.

, Lee

R.C.

, and Ambros

(1997). The cold shock domain protein LIN-28 controls developmental timing in C. elegans and is regulated by the lin-4 RNA. Cell, 88, 637–646.

23.

Nam

, Chen

, Gregory

R.I.

, Chou

J.J.

, and Sliz

(2011). Molecular basis for interaction of let-7 microRNAs with Lin28. Cell, 147, 1080–1091.

24.

Parisi

, Passaro

, Russo

, Musto

, Navarra

, Romano

, Petrosino

, and Russo

(2017). Lin28 is induced in primed embryonic stem cells and regulates let-7-independent events. FASEB J. 31, 1046–1058.

25.

Pasquinelli

A.E.

, Reinhart

B.J.

, Slack

, Martindale

M.Q.

, Kuroda

M.I.

, Maller

, Hayward

D.C.

, Ball

E.E.

, Degnan

, and Müller

(2000). Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature, 408, 86–89.

26.

Peng

, Li

T.-T.

, Wang

K.-L.

, Xiao

G.-Q.

, Wang

J.-H.

, Zhao

H.-D.

, Kang

Z.-J.

, Fan

W.-J.

, Zhu

L.-L.

, and Li

(2017). H19/let-7/LIN28 reciprocal negative regulatory circuit promotes breast cancer stem cell maintenance. Cell Death Dis., 8, e2569.

27.

Petrilli

A.M.

, and Fernández-Valle

(2016). Role of Merlin/NF2 inactivation in tumor biology. Oncogene, 35, 537–548.

28.

Piskounova

, Polytarchou

, Thornton

J.E.

, LaPierre

R.J.

, Pothoulakis

, Hagan

J.P.

, Iliopoulos

, and Gregory

R.I.

(2011). Lin28A and Lin28B inhibit let-7 microRNA biogenesis by distinct mechanisms. Cell, 147, 1066–1079.

29.

Powers

J.T.

, Tsanov

K.M.

, Pearson

D.S.

, Roels

, Spina

C.S.

, Ebright

, Seligson

, de Soysa

, Cahan

, and Theißen

(2016). Multiple mechanisms disrupt the let-7 microRNA family in neuroblastoma. Nature, 535, 246–251.

30.

Punga

, Bartoccioni

, Lewandowska

, Damato

, Evoli

, and Punga

A.R.

(2016). Disease specific enrichment of circulating let-7 family microRNA in MuSK+ myasthenia gravis. J. Neuroimmunol., 292, 21–26.

31.

Rahkonen

, Stubb

, Malonzo

, Edelman

, Emani

M.R.

, Närvä

, Lähdesmäki

, Ruohola-Baker

, Lahesmaa

, and Lund

(2016). Mature Let-7 miRNAs fine tune expression of LIN28B in pluripotent human embryonic stem cells. Stem Cell Res. 17, 498–503.

32.

Ran

, Xiao

C.-H.

, Xiang

G.-M.

, and Ran

X.-Z.

(2017). Regulation of embryonic stem cell self-renewal and differentiation by MicroRNAs. Cell. Reprogram., 19, 150–158.

33.

Rehfeld

, Rohde

A.M.

, Nguyen

D.T.T.

, and Wulczyn

F.G.

(2015). Lin28 and let-7: Ancient milestones on the road from pluripotency to neurogenesis. Cell Tissue Res. 359, 145–160.

34.

Reinhart

B.J.

, Slack

F.J.

, Basson

, Pasquinelli

A.E.

, Bettinger

J.C.

, Rougvie

A.E.

, Horvitz

H.R.

, and Ruvkun

(2000). The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature, 403, 901–906.

35.

Roush

, and Slack

F.J.

(2008). The let-7 family of microRNAs. Trends Cell Biol. 18, 505–516.

36.

Rybak

, Fuchs

, Smirnova

, Brandt

, Pohl

E.E.

, Nitsch

, and Wulczyn

F.G.

(2008). A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment. Nat. Cell Biol., 10, 987–993.

37.

Schmidt

M.F.

(2014). Drug target miRNAs: Chances and challenges. Trends Biotechnol. 32, 578–585.

38.

Shi

X.-B.

, Tepper

C.G.

, and deVere White

R.W.

(2008). Cancerous miRNAs and their regulation. Cell Cycle, 7, 1529–1538.

39.

Shyh-Chang

, and Daley

G.Q.

(2013). Lin28: Primal regulator of growth and metabolism in stem cells. Cell Stem Cell, 12, 395–406.

40.

Stefani

, Chen

, Zhao

, and Slack

F.J.

(2015). A novel mechanism of LIN-28 regulation of let-7 microRNA expression revealed by in vivo HITS-CLIP in C. elegans. RNA, 21, 985–996.

41.

Takamizawa

, Konishi

, Yanagisawa

, Tomida

, Osada

, Endoh

, Harano

, Yatabe

, Nagino

, and Nimura

(2004). Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res. 64, 3753–3756.

42.

Thornton

J.E.

, and Gregory

R.I.

(2012). How does Lin28 let-7 control development and disease?. Trends Cell Biol. 22, 474–482.

43.

Tsialikas

, and Romer-Seibert

(2015). LIN28: Roles and regulation in development and beyond. Development, 142, 2397–2404.

44.

Vasudevan

, Tong

, and Steitz

J.A.

(2007). Switching from repression to activation: MicroRNAs can up-regulate translation. Science, 318, 1931–1934.

45.

Vaz

, and Tanavde

(2012). Role of let-7 networks in stem cell maintenance and differentiation. In MicroRNA Let-7. Dahiya

, ed. (Nova Science Publishers, Hauppauge, NY), pp. 43–56.

46.

Virant-Klun

, Ståhlberg

, Kubista

, and Skutella

(2016). MicroRNAs: From female fertility, germ cells, and stem cells to cancer in humans. Stem Cells Int. 2016, 3984937.

47.

Wahid

, Shehzad

, Khan

, and Kim

Y.Y.

(2010). MicroRNAs: Synthesis, mechanism, function, and recent clinical trials. Biochim. Biophys. Acta, 1803, 1231–1243.

48.

Wang

, Cao

, Wang

, Liu

, and You

(2012). Regulation of let-7 and its target oncogenes. Oncol. Lett., 3, 955–960.

49.

Yang

, Lin

, Zhong

, Kaur

, Li

, Liang

, Lassus

, Wang

, Katsaros

, and Montone

(2010). Double-negative feedback loop between reprogramming factor LIN28 and microRNA let-7 regulates aldehyde dehydrogenase 1-positive cancer stem cells. Cancer Res. 70, 9463–9472.

50.

Yin

, Zhao

, Hu

, Yang

, Yu

, Wang

, Zhang

, Fu

, and Dai

(2017). Disturbance of the let-7/LIN28 double-negative feedback loop is associated with radio-and chemo-resistance in non-small cell lung cancer. PLoS One, 12, e0172787.

51.

Zhang

, and Wang

(2015). MicroRNA‐based biotechnology for plant improvement. J. Cell Physiol., 230, 1–15.