The Long Noncoding RNA MEG3 Retains Epithelial-Mesenchymal Transition by Sponging miR-146b-5p to Regulate SLFN5 Expression in Breast Cancer Cells

More and more studies have shown that long noncoding RNAs (lncRNAs) play essential roles in malignant tumors. The lncRNA MEG3 serves as a crucial molecule in breast cancer development, but the specific molecular mechanism needs to be further explored. We previously reported that Schlafen family member 5 (SLFN5) inhibits breast cancer malignant development by regulating epithelial-mesenchymal transition (EMT), invasion, and proliferation/apoptosis. Herein, we demonstrated that MEG3 was downregulated in pan-cancers and correlated with SLFN5 expression positively in breast cancer by bioinformatics analysis of TCGA and UCSC Xena data. Intervention with MEG3 positively affected SLFN5 expression in breast cancer cells. MEG3 repressed EMT and migration/invasion, similar to our previously reported functions of SLFN5 in breast cancer. Through bioinformatics analysis of starBase and LncBase data, 12 miRNAs were found to regulate both SLFN5 and MEG3, in which miR-146b-5p was confirmed to be regulated by MEG3 using MEG3 siRNA and overexpression method. MiR-146b-5p could bind to both SLFN5 3′UTR and MEG3, and inhibit their expression in a competing endogenous RNA mechanism, assayed by luciferase reporter and RNA pull down methods. Therefore, we conclude that MEG3 positively modulates SLFN5 expression by sponging miR-146b-5p and inhibits breast cancer development.


Introduction
Although breast cancer (BRCA-)-related mortality has been declining over the past 20 years, BRCA has the highest morbidity and mortality in woman cancers worldwide to date [1,2].Treatment failure is mainly due to the extensive heterogeneity and distant metastases of BRCA [3].For cancer cell metastasis, epithelial-mesenchymal transition (EMT) is the initial step, through which cancer cells acquire mesenchymal morphologies, migratory and invasive capabilities, and can metastasize to other distal organs [4][5][6][7].In BRCA, EMT triggers primitive alterations in the tumor microenvironment, which increases the number of tumor cells and their migration, invasion potential, and chemoresistance [8,9].
Studies show that the expression or dysfunction of long noncoding RNAs (lncRNAs) is correlated to many serious diseases, such as degenerative neurological diseases, cardiovascular diseases, and cancer [10][11][12][13].Various lncRNAs play roles in regulating EMT and tumor progression in a variety of tumors, but the mechanisms are different [14].For example, the lncRNA SNHG7 promotes BRCA tumorigenesis and progression through the Notch-1 signaling pathway and EMT initiation [15].The lncRNA PANDAR promotes the EMT pathway by upregulating MMPs (2 and 9) levels in BRCA [16].Maternally expressed gene 3 (MEG3) affects cell growth and development in various tissues [17,18].MEG3 levels are downregulated in a variety of cancers [19][20][21].In BRCA, MEG3 inhibits BRCA growth by upregulating endoplasmic reticulum stress and activating p53 [22] and suppresses EMT of BRCA cells by targeting E-cadherin [23].However, the mechanism through which MEG3 regulates BRCA progression remains to be explored.
Schlafen-5 (SLFN5), a member of the Schlafen family, is abnormally expressed and involved in the progression of melanoma, renal cell carcinoma, gastric cancer, and glioblastoma [24][25][26][27].BRCA big data from the Cancer Genome Atlas (TCGA) shows that SLFN5 transcript level significantly decreases in BRCA.We previously found SLFN5 inhibited MT1-MMP expression associated invasion [28], and retained ZEB1 transcriptional expression associated EMT and progression in BRCA cells [29,30], so SLFN5 may play an inhibitory role in BRCA progression.However, the upstream regulatory mechanism of SLFN5 is rarely explored, and whether SLFN5 is regulated by noncoding RNA, such as lncRNAs and microRNA, in BRCA is unclear.
Herein, by means of bioinformatics analysis, we found that MEG3 and SLFN5 present the same expression pattern in BRCA and bind with the common mircoRNA, laying a foundation for the competitive endogenous RNA mechanism.Our hypothesis was proved by a series of biological experiments.MEG3 intervention can affect the SLFN5 level in BRCA cells and regulate the EMT process.Importantly, miR-146b-5p can bind both MEG3 and SLFN5 and regulate their expression via a competitive mechanism, suggesting a novel regulation mechanism for SLFN5 in BRCA.

Materials and Methods
2.1.Bioinformatics Assay.All original data were downloaded from TCGA (https://cancergenome.nih.gov/) and UCSC Xena (http://xena.ucsc.edu/)websites and integrated using R 4.1.0to verify the results presented in the website for the database.The clinical characteristics associated with MEG3 expression in BRCA patients are listed in Table 1.We input MEG3 into the "Quick PanCAN Analysis" module of the UCSCXenaShiny and obtained the differences of MEG3 in tumors compared with adjacent normal tissues.Using the "Expression Analysis-Box Plot" module of GEPI A2 Web server (http://gepia2.cancer-pku.cn/#analysis),the box plot of BRCA tumor and normal tissue expression difference was obtained.The relationship between SLFN5 and MEG3 in BRCA was obtained by calculating the Pearson correlation coefficient.ESTIMATE, an algorithm designed for predicting TME tumor purity, provided both immune and stroma scores for this study.

2.3.
Real-Time PCR. 2 × 10 6 cells were lysed with 1 ml TRIzol reagent (Invitrogen) for total RNA isolation.RNA was reversely transcribed into cDNA using the PrimeScript RT reagent kit (TaKaRa, Japan), and expression difference of RNA was quantitatively analyzed by real-time PCR using a SYBR PremixEx Taq kit (Takara, Japan).Primer sequences used are shown in Table 3. Relative fold change of RNA was calculated using formula 2 −ΔΔCt with β-actin or U6 as loading control.
2.5.RNA Pull down Assay.MEG3 sequences were amplified and ligated into pcDNA3.1 vector, and recombinant vectors were transformed into JM109 competent cell.MEG3 plasmids and empty plasmids (NC) were purified, and plasmids were linearized with the restriction endonuclease SmaI.Linearized plasmids were used as templates for MEG3 RNA and NC transcriptions, respectively, using T7 RNA polymerase (Beyotime: D7069, China).Both MEG3 RNA and NC RNA were labeled with biotin at 3 ′ end using desthiobiotinylation kit (Pierce: 20163).The biotin-labeled RNAs were combined with streptavidin magnetic beads to pull down RNA isolated from MCF7 cells.Pulled down RNAs were isolated from magnetic beads with proteinase K treatment and reverse transcribed into cDNA used for the following realtime PCR to detect miR-146b-5p expression using SYBR Green (ABI: 4387406).

MEG3 Is Downregulated in Pan-Cancers and Positively
Correlated with SLFN5 Expression in BRCA.Firstly, we analyzed the difference of MEG3 expression between 33 tumor tissues and normal tissues, showing a decrease in 24 types of tumor tissues (such as BRCA, lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and ovarian cancer (OV)) and an increase in 4 types of tumor tissues (cholangio carcinoma (CHOL), large B-cell lymphoma (DLBC), testicular cancer (TGCT), and thymoma (THYM)), with no significant difference in 3 types of tumors (head and neck cancer (HNSC), pheochromocytoma and paraganglioma (PCPG) and sarcoma (SARC)) and no normal group information in the remaining 2 types of tumors (mesothelioma (MESO) and ocular melanomas (UVM)).These indicate that MEG3 may be negatively correlated with most tumor progression (Figure 1(a)).Subsequently, the expression level of MEG3 was further confirmed to be significantly lower in BRCA patients by bioinformatics (Figure 1(b)).It must also be mentioned that MEG3 level was negatively associated with TNM stage (Table 1 and Figure S1).Moreover, both MEG3 and SLFN5 present similar positive correlationships with immune infiltration in the TME of BRCA based on immune score and stromal score (Figure 1(c)).It should be pointed out that our previous study reported that SLFN5 level was negatively correlated with tumor stage in BRCA [29].The results of Pearson analysis revealed that the MEG3 transcript was correlated with SLFN5 mRNA level positively in BRCA (Figure 1(d)), indicating that MEG3 may be involved in progression and positively associated with SLFN5 regulation in BRCA.

Intervention with MEG3 Positively Affects SLFN5
Expression in BRCA In Vitro.The above data demonstrated that MEG3 is positively correlated with SLFN5 expression in BRCA, and to investigate the potential mechanism, MEG3 levels were detected in BRCA cell lines with different invasive capabilities.The results showed that MEG3 RNA levels were evidently lower in high-invasive BRCA cells (BT549 and MDA-MB-231) than that in low-invasive BRCA cells (T-47D and MCF7) (Figure 2(a)).Then, to research the function of MEG3 in BRCA cells, low-invasive BRCA cells were transfected with si-MEG3 plasmid, si-MEG3-1, and si-MEG3-2; meanwhile, high-invasive cells were transfected with MEG3 plasmid.Interestingly, in the lower invasive cells, the mRNA and protein levels of SLFN5 were both significantly downregulated after si-MEG3 transfection (Figures 2(b)-2(c)), si-MEG3-2 showing a higher transfection efficiency so chosen to be used in the following experiments.In contrast, MEG3 transfection led to SLFN5 mRNA and protein expression evidently upregulated in highinvasive BRCA cells (Figures Our previous study revealed that high or low expression levels of SLFN5 lead to epithelial or interstitial morphology in some cancer cells, which indicates that SLFN5 may exert an important function on cellular EMT and invasion [28,29].Since the above results show that MEG3 can influence SLFN5 expression both at mRNA and protein levels and influence BRCA cell EMT and invasion, so we need to confirm SLFN5 function on EMT to explore their relationship further.Herein, T-47D and MCF7cells were transiently transfected with SLFN5-specific siRNA (si-SLFN5) or negative control siRNA (si-NC).Si-SLFN5-treated cells displayed       Subsequently, a sequence-based comparison between MEG3 and miR-146b-5p using RNAhybrid revealed that the MEG3 contains a target site for miR-146b-5p (Figure 4(e)).MEG3 overexpression obviously decreased miR-146b-5p level in MCF7 cells (Figure 4(f)).Similarly, MEG3 levels were greatly suppressed by miR-146b-5p mimic (Mi), while evidently elevated by miR-146b-5p inhibitor (AMO) (Figure 4(g)).Further, luciferase activity was detected, and the results showed that miR-146b-5p Mi suppressed the luciferase activity of MEG3-WT in MCF7 cells, but not the mutant one (Figure 4(h)).Then, RNA pull down assay revealed that a higher level of miR-146b-5p was pulled down by biotinlabeled MEG3 (MEG3-Bio) than by NC-Bio (Figure 4(i)), providing evidence for MEG3 binding with miR-146b-5p.
This study analyzed the MEG3 expression in pancancers by bioinformatics analysis, and the results demonstrated that MEG3 was decreased in most of cancers.And the downregulation of MEG3 in BRCA is consistent with the previous Dong's research [21].It brought to our attention that the expression of MEG3 was positively related to SLFN5 by bioinformatics analysis in BRCA patients.Our previous studies revealed that SLFN5 is a tumor suppressor in BRCA [28][29][30].SLFN5 can suppress BRCA cell invasion through the downregulation of MT1-MMP [28] and inhibit BRCA cell EMT through the downregulation of ZEB1 [29].In this study, knockdown of MEG3 can decrease SLFN5 expression, while elevated MEG3 can increase SLFN5   13 Journal of Immunology Research expression in BRCA cells; moreover, loss-and gain-offunction studies showed that MEG3 can inhibit the EMT of BRCA cells similar to SLFN5's function.Furthermore, the overexpression of MEG3 partially reversed morphological/functional changes induced by knockdown of SLFN5.Subsequently, EMT markers (E-cadherin, vimentin, and ZEB1) were analyzed to validate MEG3's roles in keeping epithelial morphology in BRCA cells, which indicates that MEG3 exerts inhibitory functions on BRCA progression by regulating SLFN5.Some studies validated our results; for example, MEG3 may be involved in regulating EMT process to inhibit breast cancer and pituitary development [23,36].We also found previously that SLFN5 can inhibit BRCA cell proliferation and promote their apoptosis [30] and that presumably MEG3 may involve in these regulation.
To determine the mechanisms underlying the relationship of MEG3, SLFN5, and EMT, we speculated that the ceRNA network might be a potential regulatory mechanism of MEG3-miRNA-SLFN5 axis.LncRNAs can serve as ceR-NAs to sponge miRNAs to regulate the gene expression targeted by miRNAs in diverse cancers [23,[38][39][40].Here, our study obtained 12 miRNAs related to both MEG3 and SLFN5 through bioinformatics screening, and the binding sites between miR-146b-5p and MEG3/SLFN5 were found.Subsequently, it is predicted that miR-146b-5p regulates both SLFN5 and MEG3 by RNA hybridization and confirmed that MEG3 can directly bind to miR-146b-5p by RNA pull down assay.However, miR-146b-5p showed the dual functions of promoting or inhibiting tumorigenesis in cancer [41][42][43][44][45].For instance, Qu L et al. reported that miR-146b-5p can promote Ewing's sarcoma malignancy by inhibiting B-cell translocation gene 2 expression [45].Notably, the role of miR-146b-5p regulating EMT is controversial in BRCA.Li S et al. reported that lncRNA NEAT1 promotes the BRCA progression by binding miR-146b-5p [46], completely contrary to the results of this study.Meanwhile, Akkiprik M et al. reported that miR-146b-5p is upregulated in the peripheral blood of patients with locally advanced BRCA [47], consistent with the results of this study, but the function of miR-146b-5p in cancers needs be more explored in the near future.Thus, this study confirmed that MEG3 could serve as a ceRNA involved in the potential mechanism to positively modulate SLFN5 expression through miR-146b-5p, thereby affecting EMT and invasion capability in BRCA cells (Figure 5).

Conclusion
Our present research discovered that MEG3 is weakly expressed in BRCA and regulates SLFN5 expression positively in vitro.Importantly, MEG3 modulates SLFN5 expression via sponging miR-146b-5p and inhibits EMT of BRCA cells, which indicates that MEG3/miR-146b-5p/SLFN5 axis may be a potential therapeutic target in BRCA treatment.

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Journal of Immunology Research a mesenchymal phenotype compared to si-NC-treated cells (Figure3(k) in Figure3 continued).Si-SLFN5 cells exhibited a decrease in vimentin and ZEB1 and an increase in Ecadherin at both mRNA and protein expression (Figures3(l) and 3(m) in Figure3continued), confirming that SLFN5 knockdown can inhibit EMT in BRCA.These results showed that MEG3 and SLFN5 have similar func-tions on EMT in BRCA and, together with MEG3, can positively regulate SLFN5 expression, providing a full possibility for the regulatory mechanism of competitive endogenous RNAs (ceRNA).

Figure 2 :Figure 3
Figure 2: Intervention with MEG3 affects SLFN5 expression in BRCA cells.(a) Real-time PCR analysis of MEG3 RNA level in BRCA cell lines with different invasive capabilities, high-invasive capability cell lines MDA-MB-231 and BT549, and low-invasive capability cell lines MCF7 and T-47D.(b) Expression change of MEG3 RNA and SLFN5 mRNA in T-47D cells and MCF7 cells interfered with si-MEG3 or negative control siRNA (si-NC) analyzed by real-time PCR.(c) SLFN5 protein change in T-47D cells and MCF7 cells interfered with si-MEG3 or si-NC analyzed by Western blotting.(d) Expression change of MEG3 RNA and SLFN5 mRNA in BT-549 cells and MDA-MB-231 cells treated with MEG3 plasmids or control plasmids (NC) analyzed by real-time PCR.(e) SLFN5 protein change in BT-549 cells and MDA-MB-231 cells treated with MEG3 or NC plasmids analyzed by Western blotting.* * P < 0:01.