Exosome-mediated long noncoding RNA (lncRNA) PART1 suppresses malignant progression of oral squamous cell carcinoma via miR-17-5p/SOCS6 axis

Background/aim Exosomes derived from oral squamous cell carcinoma (OSCC) could modulate OSCC development. This study aimed to explore effects of exosome-mediated lncRNA PART1 on OSCC cells. Materials and methods This study was performed in Tianjin Medical University Cancer Institute from February 2021 to March 2022. Bioinformatic analysis was performed on the public database GEPIA (http://gepia.cancer-pku.cn/). Exosomes isolated from cell lines squamous cell carcinoma 9 (SCC9) and Centre Antoine Lacassagne-27 (CAL27) were identified by transmission electron microscope and western blot. Exosome-mediated lncRNA PART1, microRNA-17-5p(miR-17-5p) and suppressor of cytokine signaling 6(SOCS6) RNA expressions were assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Cell counting kit-8(CCK-8), caspase-3 activity, and flow cytometry were applied to evaluate OSCC cell viabilities and apoptosis. Meanwhile, OSCC cell migratory ability and invasiveness were evaluated using transwell assay. Bindings between miR-17-5p and lncRNA PART1 or SOCS6 were validated using the luciferase reporter test. Western blot was used for detecting the protein levels of SOCS6, phosphorylated signal transducer and activator of transcription 3 (STAT3) and STAT3. Results According to GEPIA, lncRNA PART1 was downregulated in OSCC tissue samples and cells, and it had a positive correlation with the good prognosis of Head and neck squamous cell cancer (HNSCC) patients. After the exosomes from OSCC cells were isolated and verified, PART1 was then confirmed to be secreted by exosomes. Further, overexpression of exosome-mediated lncRNA PART1 inhibited OSCC cell viabilities, migration, and invasiveness but facilitated OSCC cell apoptosis. PART1 upregulated SOCS6 through sponging miR-17-5p. Moreover, exosome-mediated lncRNA PART1 suppressed the phosphorylation of STAT3. Conclusion Exosome-mediated lncRNA PART1 could mediate the OSCC progression via miR-17-5p/SOCS6 axis in vitro, suggesting that lncRNA PART1 might be a target for treating OSCC.


Cell culture
Human tongue squamous cell carcinoma cell lines (SCC9 and CAL27) and human normal oral epithelial cells (HOECs) were obtained from American type culture collection (ATCC, USA) and Procell (Wuhan, China), respectively. Human embryonic kidney 293(HEK-293T) cells were purchased from Procell, which were used in the luciferase reporter analysis. Thereafter, Roswell Park Memorial Institute 1640(RPMI-1640, Gibco, USA) supplemented with 10% FBS (Gibco) and 1% penicillin/ streptomycin was applied for incubating cells.

Exosomes isolation
Exosomes in SCC9 and CAL27 cells were isolated using the exoEasy Maxi Kit (Qiagen, Germany). First, cell culture was centrifuged for 20 min at 3000 × g to remove cells and cellular debris. Next, the supernatant was filtered to remove particles over 0.8 μm followed by mixing with Buffer XBP. Thereafter, the mixture was bound to exoEasy membrane affinity spin column. Extracted exosomes were rinsed by Buffer XWP and then washed by Buffer XE. Afterwards, exoRNeasy Midi and Maxi Kit(Qiagen) was applied for extracting RNAs.

Transmission electron microscopy
Exosomes were resuspended and fixed by 30 μL 2% paraformaldehyde. Afterwards, glow-discharged copper grid was used to absorb exosomes. Later, glow-discharged copper grids were fixed using 3% glutaraldehyde and then stained by uranyl acetate. Finally, a transmission electron microscope (JEM-1400, JEOL, Japan)was applied for observing exosomes at 100,000 x.

Cell viability detection
Transfected SCC9 and CAL27 cells (1 × 10 4 cells/well) were seeded in 96-well plates and 10 μL Cell counting kit 8(CCK-8, Beyotime, China) was added at 24 h, 48 h, and 72 h. Thereafter, cells were kept culturing for another 1 h. Optical density (OD) values at 450 nm were observed using a lab microplate reader.

Caspase-3 activity detection
Based on the manufacturer's protocols, a caspase-3 activity assay kit (Beyotime) was applied for evaluating caspase-3 activities in SCC9 and CAL27 cells. Cells were incubated in 96-well plates. Thereafter, detection buffer and Ac-DEVD-pNA (2 mM) were added to the cell culture and the mixture was incubated for 2 h at 37 o C. The absorbance of caspase-3 was measured at 405 nm using a lab microplate reader.

Flow cytometry
Apoptosis of SCC9 and CAL27 cells was evaluated using an Annexin V-FITC assay kit (Beyotime). First, cells after exosome treatment and transfection were collected and resuspended by Annexin V-FITC binding buffer. Then, cells were mixed with 5 μL Annexin V-FITC and 10 μL PI followed by incubation in darkness for 20 min at 23 o C. Finally, Accuri C6 Plus flow cytometer (BD Biosciences, USA) was used for detecting apoptosis of SCC9 and CAL27 cells.

Bioinformatics tools
Based on GEPIA(http://gepia.cancer-pku.cn/), the boxplot about lncRNA PART1 expressions in HNSCC was generated. Moreover, the survival analysis correlated with lncRNA PART1 expression in HNSCC patients was performed using the Kaplan-Meier method on GEPIA database. All the clinical data were from the public database GEPIA.

Statistical analysis
GraphPad Prism 9 (GraphPad, USA) and SPSS 19.0 (USA) were utilized to analyze statistical data. If the data distribution fit a normal distribution with equal variance, differences between the two groups were examined using the student's t-test. One-way ANOVA was applied for analyzing differences between two groups while two-way ANOVA was carried out to analyze data in the CCK-8 assay. Dunnett's T3 test was applied for adjusting p values in multiple tests. P < 0.032 was defined to be statistically meaningful.

LncRNA PART1 was downregulated in OSCC tissues and cells
To explore the effects of lncRNA PART1 on OSCC, we first analyzed its expressions in HNSCC tissues. Based on data of GEPIA (http://gepia.cancer-pku.cn/), lncRNA PART1 expressions were lower in HNSCC tissues ( Figure 1A). Kaplan-Meier analysis based on GEPIA database indicated that OSCC patients with higher lncRNA PART1 expression had better prognosis ( Figure 1B). We also examined lncRNA PART1 expressions in OSCC cells, which was downregulated in SCC9 and CAL27 cells compared with human oral epithelial cells ( Figure 1C). These results suggested that lncRNA PART1 might play a critical role in OSCC.

LncRNA PART1 was transmitted extracellularly via exosomes
Exosomes can be secreted by cells actively, and RNAs in exosomes can be transmitted. To investigate whether lncRNA PART1 was packaged and transferred by exosomes, we extracted exosomes from mediums and observed them using TEM, which showed round and oval membranes and the diameter was 100-150 nm ( Figure  2A). Then, exosomes indicators, CD63, CD9 and TSG101, and Calnexin, a negative indicator for exosomes were examined and results showed that their protein expressions of CD63, CD9, and TSG101 were higher in exosomes than cell extracts while Calnexin was low in exosomes, confirming the presence of exosomes ( Figure 2B).
Furthermore, no significant difference in lncRNA PART1 levels in exosomes was observed after RNase treatment, while RNase and Triton X-10 treatment downregulated lncRNA PART1 expressions, suggesting that lncRNA PART1 was encapsulated by exosomes ( Figure 2C).

Overexpression of exosome-mediated lncRNA PART1 inhibited OSCC cell progression
Cells transfected with oe-PART1, or its negative control, and respective exosomes extracted from the cell medium were examined for lncRNA PART1 expression. RT-qPCR results indicated that lncRNA PART1 expression was not only upregulated in cells transfected with oe-PART1, but also increased in exosomes extracted from the oe-PART1 cell group (oePART1-Exo) ( Figure 3A). This confirmed that lncRNA PART1 is mainly presented in exosomes. Furthermore, CCK8 results revealed that exosomes from SCC9 and CAL27 cells with PART1 upregulation suppressed viabilities (Figures 3B-3C). In contrast, caspase-3 activity and apoptosis rates in SCC9 and CAL27 cells were increased in cells cultured with respective oePART1-Exo ( Figures 3D-3E). Additionally, migratory and invasive capacities of OSCC were inhibited by oePART1-Exo ( Figures 3F-3G).

Disclaimer
No funding is involved in this study. No conflict of interest is involved.