Long non‐coding RNA MEG3 promotes autophagy and apoptosis of nasopharyngeal carcinoma cells via PTEN up‐regulation by binding to microRNA‐21

Long non‐coding RNAs (lncRNAs) have been highlighted as attractive markers for diagnosis and prognosis as well as new therapeutic targets in multiple cancers, including nasopharyngeal carcinoma (NPC). Here, we attempted to investigate the underlying regulatory role of the lncRNA maternally expressed gene 3 (MEG3) in NPC development. As determined by RT‐qPCR, MEG3 expression was down‐regulated in NPC cells. Online RNA crosstalk analysis predicted the binding of miR‐21 to MEG3 and PTEN, respectively. MEG3 was validated to bind to miR‐21 while PTEN was identified as a target of miR‐21 by dual‐luciferase reporter gene assay. Exogenous transfection was done to change the levels of MEG3, miR‐21 and PTEN in HK‐1 cells to investigate their effects on the autophagy and apoptosis of NPC cells. The results suggested that MEG3 overexpression in HK‐1 cells up‐regulated PTEN and down‐regulated miR‐21, by which MEG3 further inhibited autophagy and apoptosis ability of NPC cells. The tumour formation ability was tested after injecting the HK‐1 cells into nude, mice and tumour growth was monitored. Consistently, MEG3 overexpression inhibited the tumour formation in vivo. Collectively, MEG3 promotes the autophagy and apoptosis of NPC cells via enhancing PTEN expression by binding to miR‐21.

gene on DLK1-MEG3 locus in human chromosome 14q32.2. 6 MEG3 expresses poorly in various primary human tumours and tumour cell lines, and MEG3 inhibits proliferation of tumour cells. 7 Notably, ectopic expression of MEG3 leads to cell cycle arrest, and inhibition of colony formation, and cell proliferation in NPC. 8 Our prediction revealed that MEG3 could bind to a small non-coding RNA molecule microRNA-21 (miR-21). miRNAs are well known to inhibit their target gene expression in a sequence-dependent manner, therefore, functioning in diverse cellular processes of cancers. 9,10 Although, overexpressed miR-21 is associated with the progression of various cancers including NPCs, 11,12 scarce data on the downstream targets of miR-21 in NPCs have been obtained. Of note, our sequence analysis and mRNA-miRNA interaction prediction revealed that phosphatase and tensin homologue (PTEN) is a potential target gene of miR-21. Moreover, PTEN is one of the tumour suppressor genes with the most frequently inactivated expression in sporadic cancer. 13 Interestingly, the down-regulation of PTEN occurring in NPC cells, promotes the progress of tumour cell growth, migration and invasion. 1 Based on these aforementioned findings, we have been suggested an interaction between MEG3, miR-21 and PTEN, which may participate in the biology of NPCs.

| Ethics statement
The study protocol was approved by the Ethics Committee and Experimental Animal Ethics of Linyi People's Hospital. Informed written consent was obtained from each patient before the study.
All the experiments were conducted strictly in accordance with the Helsinki Declaration. The animal experiment strictly adhered to the principle to minimize the pain, suffering and discomfort to experimental animals.

| Study subjects
Fresh frozen NPC clinical tissue specimens and corresponding paracancerous tissue specimens were collected from 80 NPC patients who received operations in Linyi People's Hospital from December 2015 to November 2018. All these patients were followed up for 3 years. Four NPC cell lines (C666-1, HK-1, 5-8F and 6-10B) and nasopharyngeal epithelial cell line (NP69) from National Infrastructure of Cell Line Resource (http://www.cellr esour ce.cn/) were cultured in complete DMEM (Gibco) with 5% CO 2 and 95% saturated humidity at 37℃. Cells were passaged after reaching 90% confluence.

| Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
The total RNA was extracted and then reversely transcribed into cDNA. Then, the sample was loaded, followed by real-time qPCR in the ABI7500 qPCR instrument (ABI) using the SYBR Premix EX Taq kit (RR420A, Takara). Three replicate wells were set for each sample.
All primers were synthesized by Shanghai Sangon Biotechnology Co.
Ltd. (Table 1). The Ct value of each well was recorded with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 used as an internal reference. The relative expression of the RNA of interest was calculated using the 2-ΔΔCt method.

| Fluorescence in situ hybridization (FISH)
The subcellular localization of MEG3 was identified by FISH.

| Western blot analysis
The total protein in tissues or cells was isolated using RIPA lysis buffer

| Immunofluorescence assay
Cells were fixed and blocked by 3% bovine serum albumin, which was followingly incubated overnight with rabbit anti-LC3II (ab48394, 1:1000, USA) at 4℃. Subsequently, cells were incubated with red fluorescent secondary antibody (Intertek) for 2 hours and sealed after staining for 5 minutes by DAPI (1 μg/mL), followed by observation and photographing under a fluorescence microscope (Olympus Optical Co., Ltd).

| TdT-mediated dUTP-biotin nick end-labelling (TUNEL) staining
The apoptosis of HK-1 cells was analysed by TUNEL staining. HK-1 cells were cultured as described previously, trypsinized and fixed in 1% paraformaldehyde in PBS at a concentration of 1 × 10 6 cells/mL.

| Flow cytometry
The

| Xenograft tumour in nude mice
A total of 24 BALA/c nude mice (aged 4 weeks, weighing 18-25 g) were purchased from Guangdong Experimental Animal Center (Guangdong, China) (http://www.gdmlac.com.cn/) and fed in a specific pathogenfree environment. These mice were subcutaneously injected with 1 × 10 6 HK-1 cells and stably transfected with oe-NC and oe-MEG3.
The weight and volume of xenograft tumours were recorded on the 7th, 14th, 21st and 28th days after inoculation using V = (A × B2)/ 2 (mm 3 , A, the long diameter, B, the short diameter). The graph of the average volume at each time point was plotted. The nude mice were killed by carbon dioxide asphyxiation to collect the tumour tissues.  And the survival difference of patients is shown by the log-rank analysis. * indicates P < .05 compared to paracancerous tissues or normal nasopharyngeal epithelial cells. The measurement data were described as mean ± standard deviation. A paired t test and one-way ANOVA were used in the comparison of two sets of data or data among multiple groups, respectively. In panel C, the overall survival of patients was described as the percentage through the Kaplan-Meier, and the log-rank analysis was used to calculate the p-value

| MEG3 is poorly expressed in NPC tissues
To verify the results in the previous study that MEG3 was downregulated in NPC, 8 RT-qPCR was performed to measure the expression of MEG3 in 80 pairs of NPC tissues and paracancerous tissues.
The expression of MEG3 in NPC tissues was lower than that in the paracancerous tissues (P < .05) ( Figure 1A). . The data were described as mean ± standard deviation. The difference was statistically significant when P < .05. Independent sample t test was used to compare data between two groups, while one-way ANOVA and Tukey's post hoc tests were applied for data comparison among multiple groups. Three independent experiments were performed with IgG. The data were described as mean ± standard deviation. An unpaired t test was used to test the comparison between two groups. Three independent experiments were performed expression of pro-apoptosis protein Bax and Cleaved-Caspase3 (P < .05) ( Figure 2D). Results from TUNEL staining indicated that the apoptosis of HK-1 cells was enhanced by MEG3 overexpression (P < .05) ( Figure 2E). Taken together, our data exhibited that MEG3 overexpression promoted the autophagy and apoptosis of NPC cells.

| MEG3 increases PTEN expression by binding to miR-21
To dissect the possible mechanism underlying MEG3 on NPC development, RNA-FISH was performed to detect the subcellular localization of MEG3 and identified that MEG3 mainly expressed in the cytoplasm of HK-1 cells ( Figure 3A). Binding sites between MEG3 and miR-21, miR-21 and PTEN were predicted on an available bioinformatics database ( Figure 3B). The potential binding between MEG3, miR21 and PTEN implies that MEG3 might regulate the expression of miR21 and/or PTEN. miR-21 was increased in NPC tumour tissues and NPC cells (P < .05) ( Figure 3C&D).

Consistent with the hypothesis, miR-21 expression was increased in cells after MEG3 knockdown (sh-MEG3). It was subsequently
verified that MEG3 overexpression in HK-1 cells markedly reduced the miR-21 expression while up-regulating the expression of PTEN ( Figure 3E).
Thereafter we set to determine the regulation of PTEN expression by MEG3 while the miR-21 level was further elucidated.

Western blot analysis indicated an increased level of PTEN protein
in HK-1 cells expressing MEG3 (P < .05) whereas the PTEN level was reduced in HK-1 cells with MEG3 knocked down ( Figure 3F). These results suggested that MEG3 negatively regulated PTEN expression. By conducting similar gain-and loss-of-function assays, we found that miR-21 could positively regulate the PTEN expression ( Figure 3G&H). and mut-PTEN showed no difference (P > .05) ( Figure 3I&J).
Interestingly, the binding of miR-21 to MEG3 and PTEN was detected by RIP assay, which implied that the combination of miR-21 with MEG3 and PTEN in cells treated with anti-Ago2 was significantly increased as compared to the cells treated with anti-IgG (P < .05) ( Figure 3K).  Figure 4D). Hence, these findings suggested that highly expressed MEG3 can reverse the inhibitory effect of miR-21 on the apoptosis of HK-1 cells, which were further verified by flow cytometric data ( Figure 4E). The measurement data were described as mean ± standard deviation. Independent sample t test was used to compare data between two groups, while one-way ANOVA and Tukey's post hoc tests were applied for data among multiple groups. Three independent experiments were performed. The difference was defined as statistically significant when P < .05

| MEG3 promoted the autophagy and apoptosis of NPC cells by increasing the expression of PTEN
Immunohistochemical staining exhibited a higher PTEN expression in NPC tissues than paracancerous tissues (P < .05) ( Figure 5A&B).
The expression of PTEN in normal nasopharyngeal epithelial cells NP69 and NPC cell line HK-1 was monitored by RT-qPCR, which showed that PTEN was poorly expressed in HK-1 cells relative to NP69 cells (P < .05) ( Figure 5C).
The pro-apoptotic effect of MEG3 was reversed by PTEN knockdown.

| MEG3 overexpression inhibits oncogenicity of NPC cells in vivo
In vivo experiments were conducted to verify the function of MEG3, which showed that a smaller volume of xenograft tumours and slower tumour growth for nude mice injected with the NPC cells stably transfected with oe-MEG3 (P < .05) ( Figure 6A&B). RT-qPCR identified a higher MEG3 expression and lower miR-21 expression in xenograft tumours from nude mice injected with the oe-MEG3-transfected NPC cells, as compared to nude mice with the oe-NC-transfected NPC cells (P < .05) ( Figure 6C). Additionally, PTEN, Bax and Beclin1 protein levels were up-regulated whereas Bcl-2 and p62 protein levels were downregulated in xenograft tumours from nude mice when MEG3 was overexpressed (P < .05) ( Figure 6D). Hence, these above-reported results indicated that the oncogenicity of NPC cells in nude mice was inhibited by MEG3 overexpression.

| D ISCUSS I ON
Based on the status quo, targeted therapy provides a promising opportunity for NPC treatment. 3 Highly expressed MEG3 has been  Collectively, these above-reported findings support the inference that interaction of MEG3, miR-21 and PTEN could be the key mechanism in the regulation of cellular processes in NPC. Although the anti-autophagic function of miR-21 has been elaborated in the hypoxia/reoxygenation-exposed cardiomyocytes, 28 and renal tubular epithelial cells during renal ischaemia-reperfusion, 29 its involvement in the human cancer cells remains largely unknown. miR-21 was recently proposed as an inhibitor of autophagy in gastric cancer and therefore to enhance drug resistance. 30 The present study emphasized the tumour-promotive role of miR-21 in NPC by suppressing the cell autophagy. Notably, PTEN has been reported to be frequently mutated in human cancers F I G U R E 7 MEG3 overexpression can promote autophagy and apoptosis of NPC cells by increasing PTEN expression through interacting with miR-21 and is a well-known anti-oncogene that encodes a dual-specificity phosphatase antagonizing the phosphatidylinositol 3-kinase (PI3K) class I/AKT/mTOR pathway. 31 mTOR is a key checkpoint that negatively regulates the autophagy and inhibition of the PI3K/AKT/mTOR pathway potentially stimulates autophagy for prevention of cancer progression. 32 Considering the close relationship between PTEN and autophagy, our study further substantiated that PTEN knockdown reversed the promotive effect of MEG3 on NPC cell autophagy and apoptosis.
Those data suggested the importance significance of a MEG3/miR-21/ PTEN axis in the regulation of autophagy and apoptosis in NPC cells.

| CON CLUS ION
In summary, MEG3 acts as a tumour suppressor in NPC that can

ACK N OWLED G EM ENTS
The authors would like to acknowledge the helpful comments on this paper received from the reviewers.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
Baotao Lv designed the study. Liqiang Lin and Xiaoli Liu: Data collation, data analyses and initial draft of the manuscript. Baotao Lv: Drafting of the manuscript. All authors have read and approved the final submitted manuscript.

E TH I C A L A PPROVA L
The study protocol was approved by the Ethics Committee and Experimental Animal Ethics of Linyi People's Hospital. Informed written consent was obtained from each patient before the study. All the experiments were conducted strictly in accordance with the Helsinki Declaration. The animal experiment strictly adhered to the principle to minimize the pain, suffering and discomfort to experimental animals.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated/analysed during the current study are available.