Long non‐coding RNA MALAT1 mediates hypoxia‐induced pro‐survival autophagy of endometrial stromal cells in endometriosis

Abstract Endometriosis is a common gynecological disease characterized by diminished apoptosis, sustained ectopic survival of dysfunctional endometrial cells. Hypoxia has been implicated as a crucial microenvironmental factor that contributes to endometriosis. It has been reported that long non‐coding RNA MALAT1 (lncRNA‐MALAT1) highly expressed in endometriosis and up‐regulated by hypoxia. Hypoxia may also induce autophagy, which might act as cell protective mechanism. However, the relationship between lncRNA‐MALAT1 and autophagy under hypoxia conditions in endometriosis remains unknown. In the present study, we found that both lncRNA‐MALAT1 and autophagy level were up‐regulated in ectopic endometrium from patients with endometriosis, and its expression level correlates positively with that of hypoxia‐inducible factor‐1α (HIF‐1α). In cultured human endometrial stromal cells, both lncRNA‐MALAT1 and autophagy were induced by hypoxia in a time‐dependent manner and lncRNA‐MALAT1 up‐regulation was dependent on HIF‐1α signalling. Our analyses also show that knockdown of lncRNA‐MALAT1 suppressed hypoxia induced autophagy. Furthermore, inhibiting autophagy with specific inhibitor 3‐Methyladenine (3‐MA) and Beclin1 siRNA enhanced apoptosis of human endometrial stromal cells under hypoxia condition. Collectively, our findings identify that lncRNA‐MALAT1 mediates hypoxia‐induced pro‐survival autophagy of endometrial stromal cells in endometriosis.

Hypoxia has a pathophysiological effect through the process of disease and regulation of gene expression. 4 Numerous studies have suggested that peritoneal hypoxia is intricately involved in the pathogenesis of ovarian endometriosis. 5,6 The key factor mediating cellular hypoxia response is the hypoxia-inducible factor-1 (HIF-1), which is a heterodimeric transcription factor composed of an O 2sensitive α-subunit and a constitutively expressed β-subunit. 7 In women susceptible to endometriosis, it is probable that retrograde menstruation of endometrial cells or debris that attach to the peritoneal wall may be exposed to hypoxic-ischaemic peritoneal microenvironment. 8 Previous studies reported that hypoxia may play a role in the survival and angiogenesis of retrograde endometrial cells in implanted ectopic endometriotic lesions. 9 Moreover, during this process HIF-1α regulate the expression of multiple target genes involved in different processes including angiogenesis, apoptosis, and autophagy. 10,11 Autophagy is an evolutionarily conserved cellular catabolic process by which cells capture bulk intracellular components like abnormal protein aggregates and damaged organelles and degraded them through delivery to lysosomes. 12 Autophagy is vital in a range of pathophysiological situations, and dysregulated autophagy is associated with various human diseases, including neurodegenerative diseases, infection, and malignancy. 13 It is generally believed that autophagy plays a crucial role in protecting cells to survival by preventing apoptosis under various metabolic stresses microenvironment, such as hypoxia or oxidative stress. [14][15][16] Interestingly, our previous studies as well as others revealed that autophagic process was up-regulated in ovarian endometriomas. 17 Long non-coding RNAs (lncRNAs) are a family of RNAs longer than 200 nucleotides with limited or no protein-coding potential. 19 An increasing number of lncRNAs have been reported to participate in various biological processes such as cell differentiation, apoptosis, and autophagy and are involved in the pathogenesis of endometriosis. 20,21 Among them, lncRNA metastasis-associated lung adenocarcinoma transcript 1 (lncRNA-MALAT1), which was first described to be associated with metastasis of lung tumours, is highly conserved in mammalian species. 22 Researchers found lncRNA-MALAT1 was robustly induced by hypoxia in several cancer cell types. [23][24][25] In addition, several studies reported that lncRNA-MALAT1 modulates the activity of autophagy. 26,27 More importantly, recent research indicated expression of lncRNA-MALAT1 was significantly increased in the ectopic endometrium of patients with ovarian endometriosis. 28 However, the correlation between lncRNA-MALAT1 and autophagy remains unclear in the context of endometriosis. Therefore, considering the fact that the elevated expression of lncRNA-MALAT1 in ovarian endometriosis and its connection with autophagy as well as the up-regulation of both lncRNA-MALAT1 and autophagy were upregulated by hypoxia, we have been suggested that up-regulation of lncRNA-MALAT1 by hypoxia may promote autophagy which acts as a cell pro-survival mechanism in endometriosis.  From the healthy control group an endometrial specimen (n = 30) was collected from healthy fertile women who were undergoing laparoscopic tubal ligation or reversal of tubal sterilization by hysteroscopy. The matched eutopic endometrium (n = 30) was collected from the same patient with ovarian endometriosis (n = 30) undergoing laparoscopic treatment for infertility and/or ovarian cysts.
Ovarian endometriotic tissues (cyst diameter measured by ultrasound ranged from 38 to 72 mm) were carefully stripped from the inner cyst wall avoiding contamination with ovarian tissues. In the endometriosis group, all patients showed stage III or IV endometriosis according to the American Society for Reproductive Medicine classification. 29 All specimens were immediately frozen and stored in liquid nitrogen for RNA and protein extraction or paraffin embedded for immunohistochemistry; they were all analyzed at the same time.
The clinical data of the patients are summarized in Table S1.

| Immunohistochemistry
Immunohistochemical staining was performed as described in our previous study. Formalin-fixed paraffin-embedded specimens of normal, eutopic, and ectopic endometrium were cut into 5 μm sections.
Then sections were deparaffinized and antigen-retrieval was performed in sodium-citrate buffer (10 m mol L −1 , pH 6) for 10 minutes at 90°C. Endogenous non-specific peroxidase activity was quenched by treatment with 3% of hydrogen peroxide for 30 minutes at room temperature (RT). Then followed by 30 minutes incubation with 1% bovine serum albumin to block non-specific binding. Tissue sections were then incubated with the primary anti-HIF-1α (diluted 1:150; Affinity, USA) and microtubule-associated proteins 1A/1B light chain 3 (LC3) (diluted 1:100; Abcam, Cambridge, UK) overnight at 4°C.
Sections were subsequently washed with phosphate-buffered saline solution (PBS) and incubated with peroxidase-labelled goat anti-rabbit IgG (diluted 1:500; Servicebio Biotech, Wuhan, China) for 30 minutes at RT. 3,30-Diaminobenzidine tetrahydrochloride substrate (DAB) (Beyotime, Wuhan, China) was used as a substrate and sections were lightly counterstained with hematoxylin, dehydrated, and mounted. Negative control sections were incubated with an isotype-matched anti-rabbit antibody. Characteristics of antibody used for immunohistochemistry are listed in Table S3.
Scoring of protein expression was performed according to the intensity of staining and the percentage of positive cells. The intensity of staining was graded as 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The percentage of stained cells was graded as following; 0 = no staining, 1 ≤ 10%, 2 = 11%-50%, 3 = 51%-80%, and 4 ≥ 81%. The final score was calculated by multiplying the two scores. Scoring was done blindly and by two independent observers.

| Isolation and culture of primary human endometrial stromal cells
We purified primary human endometrial stromal cells as described previously with slight modification. 18

| Acridine Orange staining
We used AO staining for detection and quantification of acidic vesicular organelles (AVO) in autophagic cells. Acridine orange could be used as an indicator of autophagy, the volume of the cellular acidic compartment was visualized by Acridine orange staining. The cellular acidic compartment volume is increased in autophagy and therefore staining of the acidified autophagosome is used as a reliable autophagy marker. 30    FBS for another 24 hours, followed by further drug treatments.

| RNA interference
Sequences for siRNAs used in this study are shown in Table S2.

| Statistical analysis
Statistical analyses were performed using Graphpad Prism ® statistical analysis software (version 6.01; GraphPad Software Inc., CA, USA).
All data presented here are the mean ± standard deviations (SD).
The Student's t test was used to assess the significance of differences in protein levels of HIF-1α and LC3 in different type of endometrial tissues. Statistical comparison among multiple groups was performed using one-way analysis of variance (ANOVA). Differences with P values of <0.05 were considered statistically significant.
Each experiment was independently repeated at least three times.

| Morphology and identification of primary human endometrial stromal cells
Vimentin and E-cadherin are the specific markers of human endometrial stromal cells and human endometrial glandular epithelial cells, respectively. As shown in Figure 1A,B, human endometrial stromal cells displayed long-spindle and typical fibroblast-like shapes with positively expressing mesenchymal specific markers Vimentin and negatively expressing epithelial specific marker E-cadherin. The purity of primary human endometrial stromal cells in isolated cells was >95%.

| Expression of MALAT1, HIF-1α, and LC3 in ectopic endometrium of endometriosis
To investigate whether lncRNA-MALAT1, HIF-1α, and autophagyassociated marker LC3 play a role in endometriosis, we measured their expression in ovarian endometriosis specimens. Firstly, using qRT-PCR, we found that lncRNA-MALAT1 expression was higher in ovarian endometriosis tissues than that in normal and eutopic endometrium with endometriosis ( Figure 2A). Then, Western blot assay was performed to detect the expression levels of HIF-1α and autophagy marker LC3. As shown in Figure 2B,C, the data revealed that increased protein expression of HIF-1α and total accumulation of LC3-II were present in ovarian endometriosis samples compared with normal and eutopic endometrium from women with endometriosis.
Afterwards we further investigated the location and protein levels of HIF-1α and LC3 by immunohistochemical staining. HIF-1α was predominantly localized in the nuclei of epithelial and stromal cells; however, LC3 was expressed within the cytoplasm of both cell types. The expression levels of HIF-1α and LC3 in the ectopic endometrium were significantly greater than those in normal endometrium and eutopic endometrium from women with endometriosis ( Figure 2D). Taken together, the data provided evidence that the autophagy process occurred during ovarian endometriosis, and elevated expression of HIF-1α and lncRNA-MALAT1 may be involved in the pathological process.

| Hypoxia induced activation of autophagy in human endometrial stromal cells
To explore whether autophagy activation was triggered by hypoxia stress in human endometrial stromal cells, we firstly examined the protein expression levels of HIF-1α, Beclin1, and LC3-II, which are considered reliable indicators of autophagy. As shown in Figure 3A

| LncRNA-MALAT1 is induced by hypoxia and regulated by HIF-1α in human endometrial stromal cells
In order to reveal the mechanism underlying the up-regulation of lncRNA-MALAT1 in human ovarian endometriosis tissues, human endometrial stromal cells were treated with hypoxia for various time periods. As shown in Figure 4A, qPCR analysis revealed a time-dependent increase of lncRNA-MALAT1 in human endometrial stromal cells.
As HIF-1 is the master regulator of the cellular response to hypoxia, it  Endometriosis is considered to be a polygenically inherited disorder with a complex, multi-factorial aetiology. 33 Many different theories relating to the pathogenesis of endometriosis have been put forward and to date they all remain to be conclusively confirmed.
Sampson's transplantation and implantation theory, which is the most widely accepted assumption, indicates that endometriosis occurs due to the reflux of endometrial debris by retrograde menstruation. 34 However, because retrograde menstruation occurs in most women but only 5%-10% develop endometriosis, other factors such as peritoneal hypoxia microenvironment must contribute to the development of this disease. 8 and to lesion maintenance. 17 In this study, we first proved that the levels of LC3 was elevated in ectopic endometrium tissues of endometriosis. Our data further demonstrated that autophagy activity was induced by hypoxia at a time-dependent manner in human endometrial stromal cells. Taking these data together, we demonstrate that hypoxia can up-regulate endometrial cell autophagy level. F I G U R E 7 Schematic diagram of the functions of lncRNA-MALAT1 during hypoxia induced pro-survival autophagy in human endometrial stromal cells. In normoxia condition, HIF-1α protein is rapidly degraded by the ubiquitin-proteasome system. In hypoxia condition, HIF-1α is stabilized and translocated into the nucleus by importin α/β and dimerizes with HIF-1β. The HIF heterodimer affects transcription of downstream target lncRNA-MALAT1, which promotes the induction of pro-survival autophagy However, there are several limitations of our study that must be taken into account. First of all, endometrium samples were only collected during the proliferative phase and the sample size was relatively small, which may limit the interpretable power of the current results. Thus, in future studies, the larger sample sizes collected from the entire menstrual cycle should be used. Second, the exact molecular mechanisms underlying lncRNA-MALAT1 mediated autophagy in human endometrial stromal cells remains to be established. Thus, future research is needed to gain deeper insight into these questions.
In summary, to the best of our knowledge, this is the first report showing that lncRNA-MALAT1 contributes to hypoxia triggered protective autophagy, which is crucial for cell survival in endometriosis ( Figure 7). Altogether, these findings illustrate a new function of lncRNA-MALAT1 and the "hypoxia/lncRNA-MALAT1/protective autophagy" pathway may serve as a novel target for developing potential therapeutic targets for the treatment of endometriosis.