miR-34

miR-34c

过表达可以抑制

Nanos2< /p>

在

转录后水平的表达。我们的实验证实，在老鼠精原干细胞中，< /p>

miR-34c

通过与

Nanos2

+

相互作用可能促

进减数分裂导致

Stra8

的上调。

Introduction

Spermatogenesis

is

a

highly

mediated

process

of

germ

cell

differentiation.

Taking

protamine

(Prm1)

as

an

example,

meiosis

is

known

for

their

high

transcriptional

and

low

translational

activities

during

spermatogenesis

in

male

germline

stem

cells

(mGSCs).

Therefore,

post-transcriptional

regulation

is

significant

for mammalian

spermatogonial stem

cells

(SSCs) [Wu et

al.,

2011]. MicroRNAs

(miRNAs) are

involved in nearly every biological process examined to date, but little is known of the identity or function of

miRNAs in their potential involvement in spermatogenesis [Curry et al., 2011; Hua et al., 2010]. There are

some

miRNAs,

which

play

critical

roles

in

the

process

of

spermatogenesis.

For

instance,

miR-383

is

associated with

male infertility

and promoted embryonal

testicular

carcinoma cell proliferation [Lize et

al.,

2010]. MicroRNA-184 down-regulates nuclear receptor co-repressor 2 in mouse spermatogenesis [Wu et al.,

2011].

Additionally,

miR-34

family

is

conserved

among

various

species,

functioning

in

the

processes

of

proliferation, apoptosis, and differentiation [Corney et al., 2007]. MiR-34c could play an essential role in late

spermatogenesis process [Bouhallier et

al.,

2010]. In 2011,

Brinster and

his

colleagues

found that

miR-34c

prohibited the most abundantly in SSC-enriched germ cell cultures by small RNA libraries construction and

analysis

[Niu

et

al.,

2011].

These

studies

highlighted

the

importance

of

miR-34c

expression

in

controlling

SSCs’

growth

and

differentiation.

SSCs

are

necessary

for

spermatogenesis,

although

they

constitute

one

in

thousand cells in testis [Niu et al., 2011]. NANOS2 belongs to Nanos family which contains evolutionarily

conserved zinc-finger motif encoding RNA-binding proteins,

required in mouse SSCs for maintaining their

self-renewal by preventing differentiation [Shen et al., 2010]. In mouse male germline stem cells (mGSCs),

Nanos2 suppresses meiosis and in turn is required in maintaining SSCs [Suzuki et al., 2008; Sada et al., 2012].

Stra8 is a proved signature of entering meiosis in both male and female germ cells; Nanos2 can suppress the

expression of Stra8 in mouse stem cells [Suzuki et al., 2008; Cannell et al., 2010]. miR-34c could up-regulate

Stra8

expression

in

dairy

goat

mGSCs

[Li

et

al.,

2013].

Nanos2

interacts

4

with

Cnot1,

a

component

of

CCR4-NOT

deadenylation

complex,

（

The

Ccr4-Not

complex

is

a

unique,

essential

and

conserved

multi-subunit

complex

that

acts

at

the

level

of

many

different

cellular

functions

to

regulate

gene

expression

）

which

could

be

co-localized with P bodies. NANOS2- interacting RNAs may be recruited to P-bodies and degraded by the

enzymes

contained

therein

through

NANOS2-mediated

deadenylation

[Cannell

et

al.,

2010;

Liang

et

al.,

2012].

In

P

bodies

(processing

bodies),

Dicer

enzymes

recognize

specific

double-stranded

RNA,

producing

small

fragment RNA whose 3’end has

two prominent bases. Double-stranded miRNAs nuclease combine to form

the RNA-induced silencing complex (RISC), miRNAs open double-stranded to activate RISC by base pairing

with

mRNA

combination,

then

make

mRNA

decay

or

translational

repression

[Olszewska

et

al.,

2012].

However, there were little information on miR-34 effect on mouse SSCs and the real mechanism. To explore

the relationship between Nanos2, miR-34c and Stra8, we investigated the expression patterns of miR-34c and

found

that

miR-34c

could

play

critical

roles

in

regulation

of

mSSCs’

meiosis

differentiation

through

suppression its target-Nanos2, simultaneously, up-regulation of Stra8, Scp3.

Results

Characterization of SSCs derived from 6-12 d postnatal mouse testis

SSCs were derived

from

6 to 12 d postnatal

Kunming mouse. At first, the cultured SSCs were presented

paired or aligned, or 4 to 8 single cells aggregated colonies (Fig.1A). PCR indicated that the cultured SSCs

were positive for Oct4, CD90, Nanos2, while MEF cells were negative for

the SSC’s markers (Fig.1B).They

formed typical colonies at 2nd passage. From 3rd or 4th passages, SSCs were plated onto MEF layers. Most

SSCs

were

positive

for

GFR

α

1,

CD90

，

NANOS2,

PLZF

(spermatogonial

stem

cell

markers)

by

immunofluorescence (IF) assay.

V

ASA positive staining demonstrated that SSCs presented male germ cells

characters (Fig.1C).

MiR-34c was highly expressed in the adult mouse testis

To localize miR-34c expression in the developing testis, a miR-FISH probe in 14 dpp, 21 dpp, and 28 dpp and

adult mouse testis were used. A scrambled probe was used as a negative control. The results showed that the

miR-34c signal exhibited strongest, and the percentage of miR-34c positive spermatogonia reached the top in

adult

murine

testis

(Fig.2A,

B).

In

contrast,

the

signal

intensity

and

the

percentage

of

miR-34c

positive

in

2dpp,

7dpp

mouse

testis

were

significantly

weaker

compared

than

that

in

adult

testis

(data

not

shown).

According to the FISH results, in the adult mouse testis, the hybridization signal for miR-34c was detected in

pachytene spermatocytes and round spermatids (arrowheads in Fig.2A). These results were in consistent with

previous studies [Bouhallier et al., 2010; Liang et al., 2012; Zhang et al., 2012].

NANOS2 is a direct target of miR-34c

In

order

to

explore

how

miR-34c

regulates

mSSCs,

we

computationally

predicted

that

Nanos2

was

the

candidate of miR-34c targets from miRwalk database (/apps/zmf/mirwalk/).

Then miRDB (/miRDB/) provided the detailed interaction information between miR-34c and

Nanos2 (Fig.3A, 11 B). It was validated that they did have interaction analyzed by luciferase reporter assay.

The

predicted

binding

site,

3'UTR

of

Nanos2

was

then

inserted

downstream

from

the

Renilla

luciferase

coding region in the reporter vector (Fig.3C). Each reporter construct was separately co-transfected with the

miR-34c mimics into Hela cells. Compared to the mut-Nanos2- 3'UTR control, the luciferase activity declined

by

about

37.5 % after transfection with

miR-34c mimics

and Nanos2-3'UTR reporter vector

(Fig.3D). The

luciferase analysis showed that ectopic over-expression of miR-34c reduced Nanos2

protein

expression via

directly

binding

to

Nanos2

3'UTR,

indicating

that

Nanos2

is

one

target

of

miR-34c.

These

results

demonstrated

that

miR-34c

directly

regulates

Nanos2

protein

expression

through

its

binding

to

the

3'UTR

region of Nanos2.

MiR-34c over-expression inhibited Nanos2, and promoted meiosis in mSSCs

To

further

investigate

the

effects

of

miR-34c

on

mSSCs,

negative

control

small

RNAs,

miR-34c

mimic,

miR-34c inhibitor, and in combination with miR-34c mimic and its inhibitor were transfected into

mSSCs,

QRT- PCR

results

manifested

that

miR-34c

were

transfected

efficiently

into

SSCs

(Fig.4A),

and

over-expression

miR-34c

specifically

down-regulated

its

target-Nanos2.

Simultaneously,

the

mRNA

expression levels of Nanos3, Stra8 (the pre-meiotic markers) and Scp3 (meiotic marker) were up-regulated by

miR-34c over-expression at 48 h after transfection into mSSCs (Fig.4B). Furthermore, we found that mSSCs

transfected miR-34c mimics become irregular edged compared with that transfected NC. Immunofluorescence

analysis

revealed

the

expression

level

of

miR-

34c’s

target

-NANOS2

was

significantly

down-regulated

by

miR-34c,

and

the

pre-meiotic

marker

STRA8

and

meiotic,

germ

cell

marker

SCP3

were

significantly

up-regulated in

over- expression of miR-34c compared with

NC. Additionally,

germ cell marker-V

ASA was

little up- regulated, however, PLZF (self-renewal marker of SSCs) was down-regulated by miR-34c (Fig.5A,

B).

Nanos2

siRNA

analysis

showed

that

the

meiosis

markers:

Stra8

and

Scp3

expression

levels

were

specifically

upregulated

in

Nanos2

siRNA

group

compared

with

control

analysed

by

QRT- PCR

and

immunofluorescence (Supplemented Fig.1,2).

12

MiR-34c over-expression effect in seminiferous tubules

To assess how miR-34c function

in vivo, lentiviral particles of pLL3.7-CMV-34c were cultured with mouse

seminiferous

tubules

[Chu

et

al.,

2012].

Through

immunofluorescence

microscope,

the

transduced

GFP

positive cells were observed in seminiferous tubules (Fig.6A). PCR analysis and western blot showed that the

expression of Scp3 and Stra8 in mRNA and protein levels were

significantly increased in over-expression of

miR-34c.

Additionally,

expression

of

Nanos2

was

significantly

down-regulated

by

miR-34c

(Fig.6B).

Moreover,

whole

mount

staining

demonstrated

the

meiotic

related

proteins:

NANOS3,

DAZL,

SCP3

and

STRA8

were

significantly

increased

by

miR-34c,

however,

NANOS2

and

OCT4

were

significantly

down- regulated, respectively (Fig.6C, D).

Discussion

Some miRNAs play critical roles in life process by targeting 3'UTR of

their specific mRNAs. Studies have

showed that miRNAs, as a kind of newly found small RNAs, might play an important role in spermatogenesis

in mammals [McIver et al., 2012a; Bjork et al., 2010; Luo et al., 2010; Ro et al., 2007; McIver et al., 2012b;

Tong

et

al.,

2012].

miR-146

modulates

the

effects

of

RA

on

spermatogonial

differentiation

[Huszar

et

al.,

2013].

miR-122

expression

is

associated

with

abnormal

sperm

development.

miR-122

may

influence

spermatozoa-like cells by suppressing TNP2 expression and inhibiting the expression of proteins associated

with sperm development [Liu et al., 2013]. Expressions of Hsa-miR-34c were regarded differences between

immature and mature testes and they

regulated

a series

of

gene expression, which is

essential for different

types

of

cells

(mainly

spermatocytes

and

spermatids)

formation

and

differentiation

during

primates’

spermatogenesis

[Yan

et

al.,

2009].

MicroRNA-34c

expressed

highly

in

adult

testis,

and

by

transfection

of

miR-34c into vasa-overexpressed Hela cells, spermatogenesis-related genes (even containing some late-stage

expressed genes) were detected in these cells and miR-34c might be involved in the control of the late steps of

spermatogenesis [Bouhallier et al., 2010]. 13

Sperm-borne miR-34c is important for the first cell division via modulation of Bcl-2 expression [Liu et al.,

2012]. In our study, miR-34c FISH and QRT-PCR analysis demonstrated that miR-34c was testis-specific and

most highly expressed in testis of sexually matured mice, exactly in spermatogenic cells. miR-34c might play

an important role in mammalian spermatogenesis [Bouhallier et al., 2010;Liu et al., 2012]. These results were

almost

in

consistent

with

previous

studies

[Bouhallier

et

al.,

2010;

Niu

et

al.,

2011;

Zhang

et

al.,

2012].

NANOS2

is

a

Nanos

family

protein

that

mediates

a

pivotal

role

in

SSC’s

self

-renewal

and

differentiation

[Sada et

al.,

2012;

Sada et

al.,

2009]. Bioinformatics

analysis and

Luciferase reporter assay

evidenced that

Nanos2 3'UTR has a specific miR-34c binding sequence. Further, the morphology of mouse spermatogonial

stem

cells

ectopic

over-expressed

miR-34c

could

not

maintain

the

typical

colony

formation,

but

promoted

SSCs differentiation trend. The expression levels of meiotic prophase marker and germ cell markers in mRNA

level

were

up-regulated,

accompanied

with

the

down- regulation

of

Nanos2.

In

contrast,

these

markers

exhibited downward expression patterns compared with NC group in treated with miR-34c inhibitor. These

results

further

indicated

that

Nanos2

is

one

target

of

miR-34c,

and

over-expression

miR-34c

influenced

spermatogonial stem cells’ differentiation by suppressing Nanos2 expression, and promoting the expression

genes associated with meiosis including Nanos3, Scp3 and Stra8.

Stra8 (stimulated by retinoic acid gene 8),

which is required for meiotic initiation in both sexes [Koubova et al., 2006; Anderson et al., 2008; Zhou et al.,

2008]. miR-34c mimics were synthesized and transfected into mSSCs. Moreover, miR-34c lentiviral vector

was

constructed,

virus

particles

were

collected,

and

cultured

with

seminiferous

tubules

in

vitro.

RT-PCR,

western

blot,

and

whole

mounting

of

seminiferous

tubules

demonstrated

that

miR-34c

over-expression

promoted

the

expression

of

meiosis

associated

markers

including

Nanos3,

Scp3

and

Stra8,

through

suppressing

its

target-Nanos2

expression.

NANOS3

have

been

directly

shown

to

function

in

germ

cell

development

across

diverse

species

from

flies,

worms,

frogs

and

mice

to

humans

[Julaton

et

al.,

2011].

NANOS3 was expressed in germ cells throughout

。

spermatogenesis and oogenesis [Kee et al., 2009]. DAZL and SCP3 are meiosis regulated genes [Koubova et

al.,

2006;

Anderson

et

al.,

2008;

J?

rgensen

et

al.,

2013].

In

our

study,

the

evidences

in

vivo

and

in

vitro

demonstrated that miR-34c plays a critical

role in regulation SSC’s differentiation, through NANOS2. Thus,

we first summarized the function model for the role of miR-34c in regulating mouse spermatogenesis (Fig.7).

In undifferentiated spermatogonia, NANOS2 play a role in inhibiting NANOS3, SCP3 and STRA8 expression

to

make

SSC

or

spermatogonia

maintain

an

undifferentiated

state.

When

mouse

testis

is

mature,

miR-34c

abundance removes the suppression of NANOS3, SCP3 and STRA8 by targeting NANOS2,

and promotes

SSC

or

spermatogonia

transition

to

a

differentiating

state.

This

study

further

extends

the

mechanism

of

meiosis in mammalian spermatogenesis. Taken together, our study first shows miR-34c functions by targeting

the

Nanos2

in

spermatogonial

stem

cells

meiosis

differentiation,

providing

a

novel

mechanism

with

involvement of miRNAs in the regulation of male germ cell differentiation.

研究表明

miRNAs< /p>

，作为一种新类型的

RNA

在哺乳动物精 子生成过程中起到很重要的作用。

miR-146

在精子生成过 程中调节

RA

的效应。

miR-122

表达与异常精子的发育有关。

miR-122

< br>通过抑制

TNP2

表

达和抑制与 精子发育有关蛋白的表达影响精子细胞。

Hsa-miR-34c

在成熟和不成熟的睾丸中表达是不同

的。它们调节一系列的基因表达，对于精子形成之 前不同类型细胞的分化和形成是很重要的，主要是

精母细胞和精子。

miR-34c

在成年睾丸中高表达，

miR-34c

转染进

V

ASA

过 表达的

hela

细胞中。与精

子的生成 有关的基因在这些细胞中检测到

。

miR-34c

可能参与控制晚期精子生成的过程。