Expression profile of LncRNA ANRIL, miR-186, miR-181a, and MTMR-3 in patients with preeclampsia

Preeclampsia (PE) is a leading cause of maternal and neonatal morbidity and mortality worldwide. Several studies demonstrated the role of lncRNAs and miRNAs in the pathogenesis of preeclampsia; the aim was to detect the expression profiles of serum LncRNA ANRIL, miR-186, miR-181a, and MTMR-3 in patients with preeclampsia. The study included 160 subjects divided into 80 subjects considered as a control group, 80 patients with preeclampsia. We found that there was a significant difference between the preeclampsia and control groups with up-regulation of miR-186 median (IQR) = 4, 29 (1.35–7.73) (P < 0.0001), miR-181a median (IQR) = 2.45 (0.83–6.52) (P = 0.028), and downregulation of lncRNA ANRIL median (IQR) = 0.35(0.28–0.528) (P < 0.0001), MTMR median (IQR) = 0.32(0.155–1.11), (P < 0.0001). ROC curve of lncRNA ANRIL, miR-186, miR-181a, and MTMR-3 in preeclampsia patients showing the roles of these markers in the diagnosis of preeclampsia. In conclusion, serum LncRNA ANRIL, miR-186, miR-181a, and MTMR-3 could be promising biomarkers in the diagnosis of preeclampsia.


Introduction
Preeclampsia (PE) is a pregnancy-related disorder that affects 4.6% of all pregnancies, all over the world and is one of the most common causes of maternal and neonatal mortality [1]. In early-onset preeclampsia, the process starts with poor placentation and vascularization that occurs by an insufficient trophoblastic invasion of the uterine arteries then the condition is aggravated by activation of the immune system [2][3][4]. During normal pregnancies, placental autophagy (the degradation of damaged or dysfunctional cellular components and the recycling of cellular constituents) prevents aggregation of proteins in the trophoblasts; these aggregations could cause abnormal development of the placenta by activation of apoptosis and cellular senescence resulting in telomere shortening or dysfunction [5]. Many recent studies demonstrate the role of non-coding RNA (lncRNAs and miRNAs) in the regulation of autophagy in vitro and in vivo [6].
MiRNAs regulate autophagy by changing the levels of different proteins responsible for several steps of the autophagy process. MiR-181a was found to be a modulator of autophagy in gastric cancer cells by downregulation of Myotubularin-related protein 3 (MTMR3) [9]. MTMR3, a phosphatidylinositol 3-phosphate (PI3P) phosphatase, is one of the principal genes involved in autophagy pathway regulation [10,11]. MTMR3 has a multi-model function in the regulation of autophagy: inhibition of mTORC1 and reduction in local PI3P level [12].
MiR-186 has the potential to inhibit glioma-conditioned human cerebral microvascular endothelial cell autophagy by targeting Atg7 and Beclin1 [13].
Recent research reported the interaction between lncRNA ANRIL and microRNAs (181a and 186) in numerous diseases, in particular when it comes to cancer. ANRIL through inhibition of miR-181a promotes proliferation of laryngeal carcinoma [14]. ANRIL by sponging miR-186 could endorse cervical cancer development and enhance risk stratification and bad prognosis in multiple myeloma [15].
However, no previous study has investigated the relationship between ANRIL and miR-181a, and miR-186 in preeclampsia. Thus, we designed this case-control study to detect and correlate the expression profiles of lncRNA ANRIL, its target miRNAs (miR-186, miR-181a), and MTMR-3 (target for miR181a) as autophagy-regulated genes in pregnant women with and without preeclampsia and also relate their levels to relation to patients' clinical and biochemical investigations.

Subjects
The study was conducted on 160 subjects divided into 80 normally pregnant women as control subjects and another 80 women with preeclampsia. Preeclampsia was diagnosed by: systolic blood pressure >140 mmHg and/or diastolic blood pressure>90 mmHg, accompanied by a urinary protein level that was >0.3 g in a 24 h urine collection [16]. Patients were enrolled from the Department of Obstetrics and Gynecology, Fayoum University Hospital, Egypt. The study was approved by the Ethical Committee of the Faculty of Medicine (R256), Fayoum University.

Samples collection
8 ml of blood was taken and collected in 3 tubes one of them for complete blood count, the 2nd for estimation of prothrombin time, and the 3rd tube for serum separation which used for the measurement of molecular and serological analysis.

RNA extraction
By using (Qiagen, Valencia, CA, USA) extraction kits and as prescribed by the data supplied by the manufacturer, 200 μl of serum was added to 1000 μl QIAzol lysis reagent and then incubate for 5 min followed by 200 μl chloroform to each sample followed by centrifugation for 15 min at 12,000×g. RNA quality was estimated by NanoDrop2000.

Reverse transcription and RT-PCR reactions
Regarding miR-181a and miR-186, the total volume was 25 μL per reaction using the miScript miRNA PCR primers assay (Qiagen, Valencia, CA). SNORD 68 was used as an internal control for miR-181a and miR-186. Regards to LncRNA ANRIL 20 μL was used.GADPH was used as an internal control for LncRNA ANRIL. The qRT-PCR was programmed as follows: Heating at 95 • C for 15 min, followed by 50 cycles of 94.0 • for 15 s, 60.0 • for 30 s, and 72 • C for 30 s for denaturation, annealing, and extension of DNA. The relative expression of RNAs was calculated by the 2 -ΔΔCt method [17] Regarding MTMR3 mRNA, 100 ng of total RNA was used for reverse transcription using a High-Capacity cDNA Reverse Transcriptase kit (Applied Biosystems, USA) with a total volume of 20 μl, GAPDH was used as internal control, and a Maxima SYBR Green PCR kit (Thermo, USA), The primer sequences were as follows: MTMR3-forward 5′ AGCA-GAGTGGGCTCAGTGTT-3′, MTMR3-reverse 5′-ACTGTCCACGTTTGGT -CCTC-3′, Real-time PCR was performed in 20 μl reaction mixtures using Rotor-Gene Q System (Qiagen).

Statistical analysis
SPSS software version 22 (SPSS Inc, USA) was used. The mean, median, standard deviation, and range were calculated for quantitative data, the Mann-Whitney-U test was used if the variables were not normally distributed, meanwhile, the Independent-t-test was used if the variables were normally distributed in the comparison between different groups. Qualitative data were presented as numbers and percentages, and chi-square (χ2) was used as a test of significance. Spearman correlation was done to detect the relation of miR-186, miR-181a, LncRNA ANRIL, and MTMR3 with study parameters. ROC curve was used to determine the cut-off points, significance was adopted at P ≤ 0.05.

Discussion
Preeclampsia (PE) is a pregnancy-related disorder that can seriously threaten the safety of the mother and infant throughout the perinatal period [1], Autophagy is an important intrinsic process responsible for the turnover of old cellular proteins and organelles. and it is the principal process for adequate trophoblast invasion and normal placentation [6]. Recently, non-coding RNAs, such as long non-coding RNAs (lncRNAs) and miRNAs were demonstrated to regulate cell autophagy in vitro and in vivo [6]. The initial objective of our study is to evaluate for the first time the expression profiles of lncRNA ANRIL, its target miRNAs (miR-186, miR-181a), and MTMR-3 (target gene for miR-181a) in preeclampsia patients. Up and down-regulation of specific lncRNAs and microRNA could have a role in the PE pathogenesis by interrupting normal trophoblast proliferation, invasion, and apoptosis [18][19][20][21].
As for the answer to the principle question of the study, we found that the relative expression of lncRNA ANRIL was significantly decreased by 0.35 (0.28-0.528), As regards miR-181a and MTMR-3, The relative expression level of miR-181a was up regulated 2.45 (0.83-6.52) (P = 0.028) and downregulation of the level of MTMR 0.32 (0.155-1.11). Meanwhile, the relative expression level of miR-186 was upregulated in preeclampsia patients 4, 29 (1.35-7.73).
The role of LncRNA ANRIL in autophagy was illustrated by Kang et al who reported that the increased expression level of antisense noncoding RNA in the INK4 locus (ANRIL) directly inhibited PLD (phospholipase D) and led to the induction of autophagy [8].
Recent research reported a negative interaction between LncRNA ANRIL and microRNAs (181a and 186) in numerous diseases, in particular when it comes to cancer [14,15]. These microRNAs are directly related to autophagy [13,22]. So ANRIL could modulate autophagy either in a direct way or indirectly by regulating autophagy-related microRNAs (181a and 186), a negative significant correlation between the expression level of lncRNA ANRIL with miR-181a (r = − 0.355, P = 0.023) or with miR-186 (r = − 0.335, P = 0.032) was found enforced this link.
MiR-181a was found to target many genes in the autophagy pathway; one of them is Atg5 which is a target of miR-181a in suppressing the autophagy of tumor cells. Atg5 is a gene that encodes autophagy protein 5, which is necessary for autophagosome elongation in autophagy [23]. Another study found that miR-181a targeting high-mobility group box 1 protein gene (HMGB1); Wang et al reported  Fold change values show target genes expressions compared to controls, as determined using 2 -ΔΔCt . Levels of control fold change are equal to 1.
that ANRIL exacerbates the resistance of the chemotherapeutic drug gemcitabine in pancreatic cancer through inhibition of miR-181a thus activates HMGB1-induced autophagy [24]. The most recent gene is MTMR3; Lin et al found that miR-181a can inhibit autophagy in AGS gastric cancer cells by downregulating MTMR3 [9]. Also, Senousy et al found a link between MTMR3 rs12537 at miR-181a binding site with rheumatoid arthritis and systemic lupus erythematosus [25]. This study focused on MTMR3 as a principal gene in autophagy that linked miR-181a to PE.
Myotubularin-related protein 3 (MTMR3). MTMR3 has been reported to have opposite functions in the regulation of autophagy; a) MTMR3 inhibits mTORC1. MTORC1 controls every stage of the autophagy procedure; it prevents initiation and autophagosome formation by inactivating the autophagy regulatory complex (ULK1,Atg13and FIP200). mTORC1 also interferes with autophagy nucleation by targeting components of the class III PI3K complex I (PI3KC3-CI). In recent years, mTORC1 has been found regulate the autophagosome elongation. Furthermore, mTORC1 regulates autophagosome-lysosome fusion and autophagic flux termination via lysosomal tubulation. Thus, inhibition of mTORC1 by MTMR3 induces autophagy [26,27].MTMR3 has inositol lipid 3 phosphatase activity that dephosphorylates PI3P forming phosphatidyl inositol. MTMR3 reduces local phosphatidylinositol 3 phosphate (PI3P). PI3P is a lipid mediator of membrane trafficking and signaling that also has a role in autophagy initiation and formation of autophagosome. Thus, MTMR3 reduction was shown to promote autophagosome formation, however upregulation of MTMR3 resulted in smaller autophagosomes, which disabled autophagy [28].
These functions have opposing effects on autophagic flux. As a result, we concluded that MTMR3 regulates autophagy in multiple ways and that more research is needed to determine the precise significance of MTMR3in autophagy.
MTMR3 was found to be a direct target of miR-181a hence linking this miRNA to autophagy [9]. However, we did not find a correlation between serum miR-181a and MTMR3 expression in the PE group. Senousy et al reported a significant negative correlation between miR-181a and MTMR3 in SLE patients but not in rheumatoid arthritis patients [25]. The possible explanation for this unexpected finding is that MTMR3 is regulated by variables other than miR-181a that make the regulation of MTMR3 more complex mechanisms. The results showed a borderline significant positive correlation between MTMR3 and miR-186 for further review.
The relation detected between miR-186 and autophagy was controversial, previous two studies showed that miR-186 inhibits autophagy through decreased expression of Atg7 and Beclin1 [13] or by targeting inhibition of ATG14 [29] which are autophagy-induced genes. However, the most recent study demonstrated that miR-186 could induce autophagy by inhibiting the TLR4/MAPKs/NF-κB pathway [30].
The current study findings demonstrated a negative significant correlation between the expression level of lncRNA ANRIL and miR-186 (r = − 0.335, P = 0.032). This could be explained by that lncRNAs and miRNAs share a complementary pairing sequence at 3′-UTR, allowing molecular-level binding and counteraction which modulates various physiological and pathological activities [31]. By using the   [32] demonstrated the targeting of miR-186 with seven complementary binding sites for ANRIL. Besides they showed that miR-186 could reverse the function of ANRIL in cervical cancer cells. Also, the results showed that there was a negative significant correlation between the expression level of miR-181a with LncRNA ANRIL (r = -0.355, P = 0.023). A study done by Wang et al examined the role of the LncRNA-ANRIL/miR-181//HMGB1 axis in regulating the autophagy of pancreatic cancer cells [24]. Also, Ying et al investigated the mechanism of lncRNA-ANRIL/miR-181b in autophagy of the cardiac cells in mice with uremia by targeting ATG5 [33] Lin et al found a link between miR-181a and autophagy was demonstrated through its direct target gene, myotubularin-related protein 3 (MTMR3) [34]; overexpression of miR181a depresses MTMR3 and vice versa.
From the abovementioned findings, we suppose that the four target genes are all linked to a cascade of events that end in inhibition of autophagy; for more details, we suggested that decreased LncRNA ANRIL stimulate upregulating miR-186 and miR-181a, thus, in turn, increased miR181a decreased MTMR3 resulting in suppressing of autophagy which is a beneficial physiological process responsible for the degradation of damaged or dysfunctional cellular components and the recycling of cellular constituents thus preventing protein aggregation in trophoblasts and placental malfunction anomalies.
The results presented in this featured article sought to identify ANRIL, miR-186, miR181a, and MTMR3 as new diagnostic biomarkers of preeclampsia with therapeutic potential. Further research is needed to confirm our findings and to overcome the current study's limitations, which involve (1) a relatively small sample size and (2) a deficient scientific functional publication demonstrating the key roles or molecular basis of target genes in the pathogenesis of preeclampsia. Larger-scale investigations that investigate the precise roles of these genes in the disease pathogenesis are necessary.

Conclusion
Serum lncRNA ANRIL, miR-186, miR-181a, and MTMR-3 could be used as potential biomarkers for the diagnosis of preeclampsia that may be used as therapeutic targets.

Institutional review board statement
The study was approved by the Ethical Committee of the Faculty of Medicine (R256), Fayoum University. This study is consistent with the Declaration of Helsinki.

Informed consent statement
Informed consent was obtained from all subjects involved in the study.

Data availability statement
All relevant data are included in the article.

Funding information
No financial support is relevant to this study.

Declaration of competing interest
No conflict of interest.