Long non-coding RNA MEG3 inhibits cervical cancer cell growth by promoting degradation of P-STAT3 protein via ubiquitination

A total of 22 paired cervical cancer tissues and adjacent normal tissues were collected from patients who had undergone surgery between April 2016 and September 2016. Patients did not receive any preoperative cancer treatments, such as radiotherapy or chemotherapy. All tissues were evaluated by two pathologists and was frozen in liquid until use. Informed consent was obtained from all participating subjects and the study was approved by the Ethics Committee of Shenzhen People’s Hospital.

A lentiviral vector carrying MEG3 (MEG3 group) and its control lentiviral vector (vector group), as well as a lentiviral vector carrying MEG3-specific short-hairpin RNA (shRNA; MEG3 shRNA group) and its control lentiviral vector (NC shRNA group) were purchased from Shanghai GenePharma Co. Ltd. Cell transfection was performed according to the lentiviral protocol. Siha or HeLa cells in logarithmic growth phase were seeded into 12-well plates at a density of 0.5 × 10⁵ cells/well, and 40% confluent cells were transfected with different groups of lentiviral vectors. After screening with puromycin (1 μg/mL), cervical cancer cells stably expressing high or low level of MEG3 were obtained.

Twenty female NOD/SCID mice, 4–5 weeks old, were purchased from the animal center of Sun Yat-sen University and raised under specific pathogen-free (SPF) conditions. Cells from MEG3, vector, MEG3 shRNA, and NC shRNA groups were washed with phosphate-buffered saline (PBS) and resuspended at a density of 1 × 10⁶ cells/mL. Mice were randomly divided into four groups and subcutaneously injected with 100 μL of cell suspension at their lower right flanks. Tumor size was measured every 4 days. All mice were sacrificed on day 21. Tumor tissues were collected and fixed with 10% paraformaldehyde. Paraffin sections of 3-μm thickness were obtained and stained and observed under an optical microscope. All animal experimental procedures were evaluated and approved by the Ethics Committee of Shenzhen People’s Hospital.

Experimental procedures, reagents, and primers were the same as reported in our previous studies [5‐7]. P-STAT3, STAT3, cleaved caspase-3 and c-MYC antibodies were purchased from Cell Signaling Technology (1:1000; Massachusetts, USA). The pcDNA-STAT3 plasmid mediating overexpression and its blank control pcDNA-NC and siRNAs against STAT3 (si-STAT3) and the nonsense control (si-NC) were all synthesized by GenePharma (Shanghai, China).

P-STAT3 concentration in 22 samples of cervical cancer tissues and adjacent normal tissues was detected by P-STAT3 InstantOne ELISA™ kit (Thermo Scientific, Massachusetts, US), following the manufacturer’s instruction. Briefly, the protease inhibitor was added into the cervical cancer and adjacent normal tissues and then tissues were homogenized. After 30 min of 12,000 r/min centrifugation, total protein was extracted from the supernatant. The 50 mL of sample lysate, lysis mix and control lysate was added to microplate wells respectively. Then 100 μL of detection reagent was added to each assay well. After incubation for 20 min, the absorbance at 450 nm was measured using an ELISA microplate reader. The P-STAT3 level of cervical cancer tissue which relative to its pair adjacent normal tissue was calculated.

Siha and Hela cells were transfected with the P-STAT3-TA-luc plasmids (Beyotime Biotechnology, Shanghai, China) using the Lipofectamine 2000 (Invitrogen, California, US) following the manufacturer’s instruction. After transfection for 48 h, Firefly luciferase activities were assayed using the Luciferase Assay System (Promega, WI, USA) according to the manufacturer’s instructions.

Cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 20 min, treated with 0.5% Triton X-100 for 20 min, and washed again with PBS before treatment with goat serum for 30 min at room temperature. The cells were overnight incubated with a primary antibody against P-STAT3 (1:200, CST) at 4 °C. Following incubation, the cells were washed with PBS and treated with a secondary antibody for 2 h in the dark, followed by staining with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. Samples were rinsed with PBS and sealed with a mounting medium containing an anti-quenching agent.

The RNA pull-down assay was performed using the Pierce™ Magnetic RNA–protein pull-down kit (Thermo, MA, USA). MEG3 and its antisense transcript were synthesized and purified in vitro and labeled with biotin. The experimental procedures were carried out according to the manufacturer’s instructions. Briefly, 3 μg of biotin-labeled RNA was mixed with 1 mg of protein extract, and the mixture was incubated with Dynabeads Myone Streptavidin T1 beads overnight at 4 °C. The RNA–protein complex was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining, followed by western blot analysis.

We performed RIP assay using the Magna RNA-binding protein immunoprecipitation kit (Thermo, MA, USA). The experimental procedures were in accordance with the manufacturer’s instructions. Briefly, 3 μg of P-STAT3 and IgG control antibodies were overnight incubated with cell lysates at 4 °C. A total of 25 μL of protein A/G beads were incubated with the mixture for another 2 h. The co-precipitated RNAs were extracted for RT-qPCR and 2% agarose gel electrophoresis.

Cells were transfected with ubiquitin (Ubbiotech, Changchun, China) and P-STAT3 plasmids using jetPRIME (Polyplus, Strasbourg, France). After 36 h of transfection, 20 μM of MG132 (Selleck Chemicals, Houston, TX, USA) was added to the medium for 4 h, followed by protein extraction for western blot analysis. Cell lysates were immunoprecipitated (IP) with the labeled antibodies and overnight incubated at 4 °C. The eluted proteins were determined by western blotting.

CHX-chase assay was performed using CHX (Selleck Chemicals), an inhibitor of protein synthesis. The cells in each group were mixed with 12.5 μg/mL of CHX and the expression of P-STAT3 protein was determined by western blot analysis at 0, 3, 6, 12, and 24 h.

Statistical analysis of the data was performed using the SPSS 19.0 software. Measurement data were expressed as mean ± standard error of mean (mean ± SEM), and the difference was considered statistically significant at P < 0.05.

Mice were divided into four groups and subcutaneously injected with cervical cancer cells from the MEG3, vector, MEG3 shRNA, or NC shRNA group. The results showed that the tumor size was smaller and the tumor formation rate was slower in MEG3 group than in vector group. MEG3 shRNA group had larger subcutaneous tumors and faster rate of tumor formation than NC shRNA group. The difference was statistically significant (Fig. 1a, b, P < 0.05).

The tumor tissues were collected for RT-qPCR and MEG3 expression was confirmed to be high in MEG3 group and low in MEG3 shRNA group (Fig. 1b, P < 0.05). The results of immunohistochemistry showed that the expression of P-STAT3 and Ki-67 proteins significantly decreased and the rate of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells significantly increased in MEG3 group than in the control group. On the contrary, the expression of P-STAT3 and Ki-67 proteins significantly increased and the number of TUNEL-positive cells significantly decreased in MEG3 shRNA group (Fig. 1c, P < 0.05). The difference was statistically significant. However, there was no significant difference in STAT3 expression between MEG3 group and the control group, or between MEG3 shRNA group and the control group (Fig. 1c, P > 0.05).

We first used ELISA and RT-qPCR to detect the relative expression of P-STAT3 and MEG3 in cervical cancer tissues. Then Pearson correlation analysis indicated that the expression of MEG3 was negatively correlated with that of P-STAT3 in cervical cancer tissues (Fig. 2a, P < 0.01, R = − 0.807). Next, we validated it in cervical cancer cell lines. The high-level expression of MEG3 in Siha cells from MEG3 group was confirmed by RT-qPCR. The expression of MEG3 in HeLa cells from MEG3 shRNA group was low, and the difference was statistically significant, indicative of the successful establishment of the stable cell lines following transfection (Fig. 2b, P < 0.05). The expression levels of STAT3, P-STAT3, caspase-3, cleaved caspase-3, and c-MYC were determined by western blot analysis. As a result, the expression of P-STAT3 and c-MYC was significantly lower in Siha cells from MEG3 group than Siha cells from the control group. On the other hand, the expression of caspase-3 and cleaved caspase-3 significantly increased in the cells from MEG3 group. In comparison with the control group of HeLa cells, MEG3 shRNA group of HeLa cells showed a significant decrease in the expression of P-STAT3 and c-MYC and a significant increase in the expression of caspase-3 and cleaved caspase-3 (Fig. 2c). However, the difference of STAT3 expression was not significant (Fig. 2c). The expression level of P-STAT3 protein was evaluated with immunofluorescence. The results showed that the fluorescence intensity was significantly lower in cells from MEG3 group than in cells from the control group. The fluorescence intensity from HeLa cells in MEG3 shRNA group was significantly increased as compared with that from the cells in the control group (Fig. 2d).

Furthermore, the expression level of P-STAT3 mRNA was evaluated with RT-qPCR. The results showed that the difference in STAT3 mRNA between MEG3 group and the control group, or between MEG3 shRNA group and the control group, was no significant (Fig. 2e, P > 0.05). Additionally, we evaluated the effect of MEG3 on STAT3 gene expression by luciferase assay. But we did not observed a significant difference of the luciferase signal in MEG3 group or in MEG3 shRNA group, compared to their control respectively (Fig. 2e, P > 0.05).

Moreover, the results of western blot analysis showed that STAT3 expression was high in HeLa cells transfected with pcDNA-STAT3 but low in Siha cells transfected with siRNA-STAT, indicative of the successful cell transfection (Fig. 2f). Then RT-qPCR was performed to determine the expression level of MEG3. The results revealed the absence of any significant difference in the expression of MEG3 between HeLa cells transfected with pcDNA-STAT3 and Siha cells transfected with siRNA-STAT3 (Fig. 2g, P > 0.05).

The bands specific to MEG3 were obtained by RNA pull-down assay and subjected to western blot analysis for protein identification. P-STAT3 protein level was significantly higher in MEG3 group than in the antisense group (Fig. 3a). Anti-P-STAT3 (IgG for control) was used for RIP assay. Both electrophoresis and RT-qPCR results of RIP products showed that anti-P-STAT3 could significantly enrich MEG3 (Fig. 3b, P < 0.05).

The cells from MEG3, vector, MEG3 shRNA, and NC shRNA groups were treated with CHX, and the expression level of P-STAT3 protein was determined at 0, 3, 6, 12, and 24 h. The results showed that the degradation rate of P-STAT3 protein was significantly higher in MEG3 group than in the control group. The degradation rate of P-STAT3 protein significantly decreased in MEG3 shRNA group as compared with the control group. The difference was statistically significant (Fig. 3c, d, P < 0.05).

The cells from MEG3, vector, MEG3 shRNA, and NC shRNA groups were treated with MG132 or 3-MA. The difference in P-STAT3 protein expression was determined by western blot analysis. The results revealed the absence of any significant change in the expression of P-STAT3 protein between MEG3 + MG132 (+) group and vector + MG132 (+) group, suggesting that MG132 affected the regulatory effect of MEG3 on P-STAT3 protein. In comparison with vector + MG132 (+) group, MEG3 + 3-MA (+) group showed a significant increase in the expression of STAT3 protein, suggesting that 3-MA had no significant effect on the regulation of P-STAT3 protein by MEG3 (Fig. 3e).

Ubiquitination assay results suggested that P-STAT protein ubiquitination significantly increased in MEG3-overexpressing cells as compared with the control cells, whereas P-STAT protein ubiquitination significantly reduced in the cells exhibiting low expression of MEG3 (Fig. 3f).

To investigate whether the effects of MEG3 on the proliferation and apoptosis of cervical cancer cells are exerted through the regulation of P-STAT3 protein expression, cells were treated with niclosamide, an inhibitor of STAT3 protein phosphorylation. Cervical cancer cells were divided into MEG3 shRNA + dimethyl sulfoxide (DMSO) group, MEG3 shRNA + niclosamide group, NC shRNA + DMSO group, and NC shRNA + niclosamide group. The expression level of P-STAT3 protein was determined by western blot analysis. As a result, we found that the level of P-STAT3 protein significantly decreased in NC shRNA + niclosamide group as compared with NC shRNA + DMSO group, indicating that niclosamide effectively inhibited STAT3 phosphorylation (Fig. 4a). The level of P-STAT3 protein significantly decreased in MEG3 shRNA + niclosamide group as compared with MEG3 shRNA + DMSO group, suggesting that niclosamide could antagonize the stabilization of P-STAT3 protein by MEG3 shRNA (Fig. 4a).

The changes in the cellular functions were evaluated. Clone formation assay suggested that the number of clones formed by cells from MEG3 shRNA + niclosamide group was significantly lower than that formed by cells from MEG3 shRNA + DMSO group (Fig. 3b, d, P < 0.05). Flow cytometry results showed that the ratio of early apoptotic cells in MEG3 shRNA + niclosamide group was significantly higher than the ratio of apoptotic cells in MEG3 shRNA + DMSO group (Fig. 3c, e, P < 0.05). The difference was statistically significant.