lncRNA MALAT1 Regulates Mouse Granulosa Cell Apoptosis and 17β-Estradiol Synthesis via Regulating miR-205/CREB1 Axis

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a known long noncoding RNA, was reported to play a crucial role in follicular growth and ovarian disease. However, the physiological function of MALAT1 in mouse granulosa cells (mGCs) remains largely unclear. The aims of this study were to determine the biological function and molecular mechanism of MALAT1 in mGCs. We knocked down MALAT1 in mGCs by using siRNA against MALAT1. We found that knockdown of MALAT1 promoted apoptosis and caspase-3/9 activities in mGCs. Enzyme-linked immunosorbent assay demonstrated that knockdown of MALAT1 significantly decreased the production of estradiol (E2) and progesterone (P4) in mGCs. Mechanistically, MALAT1 serves as a competing endogenous RNA (ceRNA) to sponge microRNA-205 (miR-205), thereby facilitating its downstream target of cyclic AMP response element- (CRE-) binding protein 1 (CREB1). Furthermore, CREB1 overexpression or miR-205 downregulation partially recovered the effect of MALAT1 depletion in mGCs. In summary, these findings suggested that MALAT1 regulated apoptosis and estradiol synthesis of mGCs through the miR-205/CREB1 axis.


Introduction
It is well known that most ovarian follicles (>99%) in the mammalian ovary undergo atresia and degeneration, and only <1% of the follicles suffer ovulation [1,2]. The high apoptosis ratio of GCs could impair folliculogenesis, leading to follicular atresia [3]. Especially, steroid hormones synthesized by GCs play an important role in ovarian follicular development, oocyte maturation, endometrial proliferation, and mammary gland development [4]. Therefore, exploring the molecular mechanism involved in GC progression is crucially important for decreasing follicular atresia and increasing ovulation rates and female fertility.
Long noncoding RNAs (lncRNAs) are a class of noncoding RNAs that are longer than 200 nucleotides in length and do not appear to have protein-coding potential [5]; they have been found to participate in various biological processes, such as early embryonic development [6], granulosa cell proliferation [7], and stem cell differentiation [8]. Moreover, some lncRNAs have been observed to be expressed in ovaries [9], mature and immature oocytes [10], and cumulus cells [11]. Growing evidence suggested that lncRNAs are involved in follicular growth and development through the regulation of GC proliferation, differentiation, apoptosis, and steroidogenesis [12][13][14][15]. Therefore, ovarian functional research related to lncRNAs was being performed extensively to clarify the molecular mechanism of follicular growth and development.
MALAT1, a key lncRNA, was reported to play pivotal roles in various malignancies, mainly by regulating the proliferation, migration, and invasion of cancer cells [16]. In the reproductive system, MALAT1 was shown to be associated with pregnancy loss [17], polycystic ovary syndrome (PCOS) [18], and endometriosis [19]. Although a study demonstrated that MALAT1 promoted human GC proliferation through the regulation of the ERK/MAPK pathway [20], the detailed function and regulatory mechanism of MALAT1 in mouse granulosa cells (mGCs) remain largely unclear. The present study is aimed at examining the potential involvement of MALAT1 in mGC proliferation, apoptosis, and estradiol synthesis. We also investigated the regulatory mechanism underlying the effect of MALAT1 on mGCs by conducting a series of molecular and cellular experiments in vitro.
2.3. RNA Extraction and qRT-PCR. Total RNA was isolated from mGCs using the TRIzol Reagent (Invitrogen) according to the standard protocol. One μg RNA was reverse transcribed to cDNA with the PrimeScript RT Reagent Kit (Takara Biotechnology Dalian Co. Ltd., Dalian, China). qRT-PCR was performed on an ABI Prism 7900 system (Applied Biosystems, CA, USA) using the SYBR Premix Ex Taq (Takara Biotechnology Dalian Co. Ltd., Dalian, China). The following thermocycling protocol was used: denaturation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 10 sec, annealing at 62°C for 20 sec, and extension at 72°C for 40 sec. The relative expression was calculated using the 2 −ΔΔCt method and normalized to GAPDH or U6. The sequences of primes are listed in Table 1 based on previous studies [22][23][24].
2.5. Caspase-3/9 Activity Assay. Caspase-3 Activity Assay Kit and Caspase-9 Activity Assay Kit (Beyotime, Beijing, China) were used to determine the activities of caspase-3/9 in mGCs 48 h after transfection according to the manufacturer's protocol. The OD value at 405 nm was measured using a microplate spectrophotometer (Thermo Labsystems, Vantaa, Finland).

Subcellular Fractionation Assay.
The NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA) were applied with the isolation of the nuclear and cytoplasmic extracts from mGC cells. The distribution of MALAT1 was determined in the cytoplasm or nucleus using qRT-PCR. GAPDH and U6 were used as cytoplasmic and nuclear controls, respectively.
2.8. Luciferase Reporter Assay. The miRNAs that interact with MALAT1 were predicted with starBase2.0 (http:// starbase.sysu.edu.cn/). A dual-luciferase reporter gene assay was used to verify the target relationship between miR-205 and MALAT1. A wildtype (WT) or mutant-type (MT) MALAT1 fragment containing the miR-205 binding site was synthesized and inserted into the pGL3-basic vector (Promega, Madison, WI, USA). For luciferase reporter assay, 1 × 10 5 mGCs were cotransfected with the WT-MALAT1 reporter plasmid and the MT-MALAT1 reporter plasmid F-CTCGCTTCGGCAGCACA R-AACGCTTCACGAATTTGCGT Abbreviations: F = forward; mRNA = messenger RNA; PCR = polymerase chain reaction; R = reverse. 2 BioMed Research International and miR-205 mimics or miR-NC control. The luciferase reporter assay system (Promega) was applied to examine the luciferase activities at 48 h after cotransfection.
2.10. Western Blot Assay. The cultured mGCs were harvested and lysed using a radioimmunoprecipitation assay buffer (Sigma-Aldrich) according to the manufacturer's protocol.
2.11. Statistical Methods. All the statistical data are shown as mean ± standard deviation (SD) of at least three independent experiments and three parallel holes in each experiment (n = 9). All statistical analyses were performed with SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA). Student's t-test was used to compare differences between two groups, and multiple groups were compared with one-way ANOVA followed by Duncan's test as a post hoc test. P < 0:05 was taken into account as statistically significant.

Knockdown of MALAT1 Promotes Apoptosis of mGCs.
To investigate the expression of MALAT1 in follicles, we detected the expression levels of MALAT1 in HFs, EAFs, and PAFs by qRT-PCR. The results showed that the expres-sion of MALAT1 in EAFs and PAFs were significantly lower than those in HFs (both P < 0:01; Figure 1(a)), suggesting that MALAT1 might be involved in follicular atresia. To further clarify the biological roles of MALAT1 in mGCs, the endogenous MALAT1 in primary mGCs was knocked down by siRNAs and the cell apoptosis and caspase-3/9 activities were determined by flow cytometry and ELISA, respectively. As shown in Figure 1(b), MALAT1 was successfully knocked down by 84.2% by si-MALAT1. Meanwhile, we found that knocking down MALAT1 significantly promoted the apoptotic cell number (Figure 1(c)) and increased caspase-3/9 activities (Figures 1(d) and 1(e)).

Knockdown of MALAT1 Decreases the Concentrations of Estradiol and Progesterone in mGCs.
To assess whether MALAT1 affects steroid hormone levels in mGCs, we measured the concentrations of estradiol (E2) and progesterone (P4) in mGCs at 48 h postinfection by ELISA. The result revealed that the concentrations of E2 and P4 were significantly reduced in the si-MALAT1 group (both P < 0:01) compared with the si-NC group (Figures 2(a) and 2(b)). We further analyzed the expression of two genes encoding steroidogenic enzymes cytochrome P450 1B1 (CYP1B1) and CYP19A1 in mGCs transfected with si-MALAT1 or si-NC. The results revealed that MALAT1 depletion significantly reduced the CYP1B1 and CYP19A1 protein expression in mGCs (Figure 2(c)). These results suggest that MALAT1 might play a crucial role in the regulation of steroidogenesis in mGCs.

BioMed Research International
(ceRNA) by binding with miRNAs and regulating gene expression at the posttranscriptional level [25]. To determine the mechanism by which MALAT1 regulates mGC apoptosis, the miRNAs that directly interact with MALAT1 were predicted using the bioinformatics tool starBase2.0. Among the miRNAs, we select miR-205 as a study object based on its biological role in the reproduction field. MALAT1 harbors a recognition sequence of miR-205 (Figure 3(a)). This interaction was confirmed by dual-luciferase assay. As shown in Figure 3  BioMed Research International the WT-MALAT1 luciferase activity of mGCs, but not that of MT-MALAT1. To further investigate the potential direct binding between MALAT1 and miR-205, RNA pull-down assays were performed in mGC cells, and this revealed that biotin-labeled miR-205-wt could pull down MALAT1, while bio-miR-205-mut could not pull down MALAT1 (Figure 3(c)), indicating the direct interaction between MALAT1 and miR-205. In addition, we found that knockdown of MALAT1 obviously increased miR-205 expression in mGCs (Figure 3(d)). We also showed that transfection of miR-205 mimics significantly increased miR-205 expression in mGCs, whereas transfection of the miR-205 inhibitor obviously decreased miR-205 expression in mGCs (Figure 3(e)). However, miR-205 upregulation or downregulation failed to cause alteration of MALAT1 expression in mGCs (Figure 3(f)). These results demonstrate that MALAT1 could bind to miR-205 in mGCs.

MALAT1 Exerts a Biological Role in mGCs by
Regulating the miR-205/CREB1 Axis. A previous study demonstrated that miR-205 promoted mGC apoptosis by targeting CREB1 [23]. To investigate whether MALAT1 exerts a biological role in mGCs by modulating the miR-205/CREB1 axis, we first investigated the association with MALAT1, miR-205, and CREB1 in mGCs. We found that knockdown of MALAT1 significantly decreased CREB1 expression in mGCs, while transfection with the miR-205 inhibitor partially reversed this trend (Figures 4(a) and 4(b)). Finally, we found that downregulation of miR-205 or upregulation of CREB1 partially reversed the effect mediated by MALAT1 knockdown on proliferation, apoptosis, caspase-3/9 activities, and E2 and P4 production in mGCs (Figures 4(c)-4(g)). These results determine convincingly that MALAT1 exerts its biological function via the miR-205/CREB1 axis.

Discussion
Granulosa cells (GCs), a basic functional unit of the ovary, were reported to be involved in the growth and maturation of follicles by regulating proliferation, cell cycle, apoptosis, and synthesis of steroid hormones in female mammals [26]. Growing evidence indicates that the initiation of follicular atresia is mainly due to GC apoptosis [3,22,27]. Several lncRNAs have been identified as regulators of GC apoptosis in mammals. For example, the lncRNA NORFA inhibited granulosa cell apoptosis through regulating miR-126/transforming growth factor-β (TGF-β) axis [12]. Knockdown of PVT1 promoted cell proliferation, as well as inhibition of apoptosis of ovarian granulosa cells by regulating miR-17-5p and PTEN [28]. The lncRNA HOTAIR aggravated the endocrine disorders and granulosa cell apoptosis through competitive binding to miR-130a to upregulate the expression of IGF1 [14]. In this study, we prove that MALAT1 could affect apoptosis and estradiol synthesis of mGCs. We also demonstrate that MALAT1 plays an important role in regulating apoptosis and estradiol synthesis of mGCs through regulating the miR-205/CREB1 axis. MALAT1, as a conserved lncRNA, was shown to be involved in multiple biological processes, such as tumorigenesis, pre-mRNA splicing, and DNA repair [16,29,30]. Although a study has revealed that MALAT1 might affect human GC proliferation through P21/P53-dependent control of the cell cycle and the ERK/MAPK pathway [20], the biological function of MALAT1 in follicular atresia remains largely unclear. In the present study, we observed MALAT1 reduction in EAFs and PAFs compared with HFs. Downregulation of MALAT1 decreased the centration of E2 and P4 in mGCs. We also showed that knockdown of MALAT1 significantly induced apoptosis and promoted caspase-3/9 activities in mGCs. These results suggested that MALAT1 might play a key role in follicular atresia. Accumulating evidence suggested that dysregulated lncRNAs exert crucial regulatory functions in multiple processes through acting as ceRNAs to sponge microRNAs (miRNAs) [31]. MALAT1 was identified to sponge multiple miRNAs, such as miR-124-3p [32], miR-150-5p [33], miR-503 [34], miR-384 [35], and miR-22 [36]. To investigate the regulatory mechanism of MALAT1 in mGCs, we selected miRNA interactions with MALAT1 using starBase2.0 software. Among the miRNAs, we select miR-205 as a study object based on its biological function. Recently, a study demonstrated that miR-205 overexpression promoted apoptosis and decreased estradiol synthesis in mGCs [23]. Here, we confirmed that miR-205 could directly bind with MALAT1 in the luciferase reporter activity assay and RNA pull-down assays. We also found that knockdown of MALAT1 significantly increased miR-205 expression in mGCs, while overexpression or downregulation of MALAT1 did not alter the expression of MALAT1 in mGCs. In addition, miR-205 inhibition partially reversed the effect of MALAT1 depletion in mGCs. These results suggest that MALAT1 functions as a ceRNA by sponging miR-205 in mGCs.
Accumulating evidence confirmed that lncRNAs functioned as ceRNAs of miRNAs that cause the modulation of derepression of miRNA's target gene expression [25,31]. CREB1 was confirmed to be a direct target of miR-205 in mGCs [23]. Importantly, knockdown of CREB1 could induce apoptosis and decrease the production of E2 and P4 in mGCs [37]. Thus, we investigate the association with CREB1, miR-205, and MALAT1 in mGCs. We found that knockdown of MALAT1 in mGCs resulted in a reduction in the expression of CREB1, while this trend was reversed by CREB1 overexpression. Moreover, MALAT1-mediated effects on apoptosis, caspase-3/9 activities, and E2 and P4 production were effectively rescued by CREB1 overexpression. These findings suggest that MALAT1 affects mGC apoptosis and estradiol synthesis via the miR-205/CREB1 axis.
In conclusion, the present study revealed that MALAT1 could regulate mGC apoptosis and steroid synthesis, at least to a certain extent by regulating miR-205/CREB1 axis. These findings contribute to an understanding of the molecular mechanisms underlying the regulation of ovarian follicular atresia by MALAT1.

Data Availability
The data from this study are available in this published article.

Conflicts of Interest
The authors declare that they have no competing interests.