The Integrated Analysis of Differential Long NonCoding RNAs Expression Proles in Extracellular Vesicles Derived From Chemoradioresistant and Parental Nasopharyngeal Carcinoma Cells

The patients with nasopharyngeal carcinoma (NPC) suffer from poor outcomes after chemoradiotherapy. Extracellular vesicles (EVs) play crucial roles in regulating cancer progression and chemoradioresistance. To investigate the functional role of chemoradioresistant cell-derived EVs for parental cells, and long noncoding RNAs (lncRNAs) expression proles in EVs secreted by chemoradioresistant cells. The effect of chemoradioresistant NPC cell-derived EVs on migration and invasion abilities of parental cells were detected by Transwell assays. Human cancer lncRNA PCR array was performed on EVs released from chemoradioresistant NPC cells (CNE1R and CNE2R) and parental cells (CNE1 and CNE2). The concurrent chemoradioresistance of CNE1R and CNE2R was signicant higher than that of CNE1 and CNE2. Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western blotting results showed the CNE1, CNE2, CNE1R and CNE2R -derived EVs were successfully obtained. The chemoradioresistant cell-derived EVs remarkably promoted migration and invasion ability of parental cells with different differentiation. Notably, a total of 91 differentially expressed (DE) lncRNAs were regulated in EVs by chemoradiotherapy. Among them, the 4 upregulated (MALAT1, FAM212B-AS1, LINC-PINT and H19) and 7 down-regulated (SNHG16, CDKN2B-AS1, ZFAS1, CCAT1, SNHG6, GAPLINC and TUG1) DE lncRNAs associated with chemoradioresistance, were identied in EVs derived from chemoradioresistant cells with different differentiation. We found the EVs derived from chemoradioresistant NPC cells regulated an aggressive phenotype of NPC cells, and identied an altered lncRNAs expression pattern in chemoradioresistant NPC cell- derived EVs.


Introduction
Nasopharyngeal carcinoma (NPC) belongs to a squamous-cell carcinoma that located in the nasopharyngeal epithelium [1]. This carcinoma has a distinctive ethnic and geographic distribution, which has higher occurrence in southern China than in the rest of the world [2]. EBV infection and lymphoepithelial-like histologic are notable characteristics of NPC [2]. Non-keratinizing, undifferentiated NPC (WHO type III) is the is the most common subtype, and accounts for 63-95% of all NPC cases worldwide [3]. Prognosis is poor once the NPC cells migrate even if this carcinoma obtained advanced therapies [4]. The metastatic NPC is mainly caused by many factors such as Epstein-Barr virus infection, heredity and environmental factors [5,6]. These factors can result in disruption of cell junctions, epithelial-mesenchymal transition (EMT), invasion, and clonogenicity [7]. Although the application of radiotherapy or chemotherapy techniques have contributed to the improvement in the local control of NPC, chemoradioresistance is a major impediment to achieve longterm survival [8]. A majority of the NPC patients receiving radiotherapy treatment confront with local recurrence and distant metastasis within 1.5 years for radioresistance [9].
Long non-coding RNAs (lncRNAs) are a class of non-protein-coding transcripts above 200 nucleotides in length [10]. LncRNAs can function as competing endogenous RNAs and antisense RNAs, and are proved to act as either promoters or suppressors in various carcinomas. The lncRNAs mainly act in epigenetic level, transcriptional level and posttranscriptional level, among other processes [11]. The recent studies have showed the lncRNAs are abnormal various types of cancers treated with chemoradiotherapy. For instance, lncRNAs with aberrant expression values between the two groups of LARC patients have been identi ed in local advanced rectal cancer (LARC) with different neoadjuvant chemoradiotherapy downstaging depth score [12]. Wei Xiong et al has identi ed 2,662 differentially expressed lncRNAs in 5-FU-based colorectal cancer HCT116 cells relative to those in parental HCT116 [13] However, the abnormal lncRNA expression pro le in NPC receiving chemoradiotherapy, and in released EVs of that is still known few.
Extracellular vesicles (EVs) released from various types of cells, can be secreted to the extracellular space through fusion with plasma membrane, and contribute to intercellular communication [14]. Classically, EVs can be further classi ed on the basis of their biogenesis, size and biophysical properties, i.e., exosomes, microvesicles, and apoptotic bodies [15]. EVs carry amounts of bioactive proteins, mRNA, small RNA and lipids, and transfer their contents to recipient cells to involve in tumor cell progress [16], can play crucial role in phenotype alteration of sensitive cancer cells. For instance, NPC exosomes can regulate biological behaviour of NPC cells [17,18]. MiR-9 exists in human 5-8F cell-derived exosomes [19].
Decrease of miR-9 expression via promoter methylation results in increase of CXCR4 expression, stimulating biological behavior (e.g. proliferation, migration and invasion) of NPC cells via the activation of MAPK pathway [20]. NPC exosomes modulates epithelial-mesenchymal transition of cells via delivering mitotic cytokines (e.g., TGF-β), resulting in resistance to chemotherapy [21]. However, the lncRNA expression pro le from NPC-derived EVs is wanting. Whether the chemoradiotherapy-induced NPC EVs regulate aggressive phenotypes of NPC cells is on the way.
In our study, we explored the functional role of chemoradioresistant NPC cells-derived EVs on parental NPC cells with different differentiation, and for the rst time identi ed differentially expressed (DE) lncRNAs related with chemoradioresistance in chemoradioresistant NPC cell-derived EVs via PCR array. The effect of chemoradioresistant NPC cell-derived EVs on migration and invasion abilities of parental cells with different differentiation were evaluated. The shared lncRNAs associated with chemoradioresistance were further identi ed in EVs derived from chemoradioresistant cells with different differentiation.

Material And Methods
Cell culture Human NPC cell lines CNE-1 (well differentiation) and CNE-2 (poor differentiation) used in this study were obtained from China Center for Type Culture Collection (Wuhan, China), cultured in RPMI-1640 medium (GIBCO), added with 10% fetal bovine serum (FBS),100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) in 5% CO2 at 37°C.

Establishment of concurrent chemoradioresistant NPC cell lines
When CNE-1 and CNE-2 cells were up to the con uence of 60%, they were cultured in the complete medium containing 0.05ug/ mL cisplatin, and were exposed to 6Gy irradiation using a 6-MV X-ray linear accelerator (ELEKTA, Beijing, China) at 200 cGy/min dose rate. After 24h, the cells were cultured in the fresh complete medium containing 0.05ug/ mL cisplatin (Sigma-Aldrich, Saint Louis, MO, USA). When the cell density reached 80-90%, they were further passaged and cultured. The next chemoradiotherapy was performed when the cell growth was in stable state for a total of 10 times. The CNE-1 and CNE-2 cell lines treated with chemoradiotherapy were named as CNE1R and CNE2R, respectively.

Colony formation assay
Each cell (CNE-1, CNE-2, CNE1R and CNE2R) were respectively inoculated in 6-well plates at a density of 300, 400, 800, 1000, 2000, 4000 cells/well and cultured in 200 μL cell culture medium for 24h. Subsequently, the cells were removed the original medium and cultured in the fresh complete medium containing 0.05ug/ mL cisplatin. At the same time, these cells were respectively treated with gradient doses of irradiation (0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy and 10 Gy) according to the gradient cell numbers under the above radiation condition. After 14 days, the cells were washed, xed for 30 min using methanol, and stained with 0.1% crystal violet (Sigma, MO, USA) for 10 min. The number of clones >50 was counted under a microscope (DMIRB, Leica, Germany) to calculate the plating e ciency (PE) (%) that is equal to (number of colonies/inoculated cell number) ×100%. Surviving fraction (SF) that is equal to number of colonies/ (number of cells seeded × PE) ×100%. We plotted the cell survival curve using the multi-target single-hit model: SF=1-(1-e -D/D0 ) N , and the radiobiological parameters D0 (the dose that gave an average of one hit per target), Dq (quasi-threshold dose) and SF2 (the SF following exposure to 2 Gy radiation) were determined using the survival curve. The experiments were repeated in triplicate.

EV isolation, quantitation and characterization
EVs isolation and identi cation referred to the MISEV2018 guidelines [22]. NPC cells were cultured until 80%-90% con uence was reached, and then changed with serum starvation. After incubation for 48 h, the supernatants were centrifuged at 300 g for 10 min to remove dead cells and cell debris. Subsequently, the supernatants were ltered using a 0.22 μm membrane and concentrated to 1 ml using Amicon Ultra 15 Centrifugal Filter Unit (Millipore, USA). The lipids left was transferred to a fresh microcentrifuge tube. EVs were isolated using the ExoQuick TC kit (System Biosciences, USA) referring to manufacturer's protocol. The isolated EVs were resuspended in PBS. The EVs were xed using 4% paraformaldehyde and loaded onto a TEM copper grid (Agar Scienti c Ltd., Stansted, UK), and then washed with 1% glutaraldehyde, PBS, and distilled water., Next, EVs were incubated with 4% uranyl acetate and observed under TEM (Hitachi H7500 TEM, Tokyo, Japan).
Western Blotting EV protein quanti cation was obtained using Pierce BCA Protein Assay Kit (Life Technologies, USA) following the manufacturer's instructions..The proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies for mouse anti-CD63 (Cat# ab108950, Abcam, 1:1000) and GAPDH (Proteintech, Chicago, UK; 1:1000) at 4°C overnight, and then washed and incubated with secondary antibody (Santa Cruz, Dallas, USA) for 2 hours. Protein bands were visualized using enhanced chemiluminescence reagent (ECL AdvanceTM; GE Healthcare) and images were obtained via a ChemiDoc MP system (Bio-Rad, Hercules, CA, USA).

Migration and invasion assays
For the migration assays, 1×10 5 cells were seeded onto the upper chamber (Cat#353097, FALCON,) added with 500 μl of serum-free DMEM and incubated with or without EVs. Subsequently, the 700 μl DMEM with 10% FBS was supplemented into the lower chambers. After 24 h of incubation, the cells underneath the membrane were xed using 4% paraformaldehyde and stained with 800μl crystal violet (Cat# C0121; Beyotime, Shanghai, China) for 30 min at room temperature. The number of migrated cells was counted under a microscope. For the invasion assays, the basic steps were the same as above, except that chambers precoated with matrigel (Cat #354480, BioCoat,).
The RNA extraction, cDNA Synthesis Using an RT2 First Strand Kit and human cancer lncRNA PCR array Total RNA derived from NPC cells was obtained using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and its concentration was measured by Nanodrop 2000 (Tiangen, Beijing, China). RNA were performed with agarose electrophoresis for RNA quality control. The cDNA was synthesized using RT 2 rst strand kit by reference to the manufacturer's manual (Cat:330401, Qiagen, Hilden, Germany). The 10 μl reverse transcription mixture was prepared and thoroughly mixed with the genomic elimination mixture (10μl) and performed an incubated at 42℃ for 15 min. After the reaction termination at 95℃ for 3 min, a 91 μl of RNase-free water was added into each reaction mixture and mixed for following assays.
The RT 2 SYBR Green Master mix was used for human cancer lncRNA PCR array (Yingbiotech, Shanghai, China). Brie y, a real-time PCR mixture was prepared by adding the 650 μl of 2X PCR master mix, 102µl diluted cDNA, and 548µl RNase free water. The membrane on the PCR Array was carefully, and 10 µl of the PCR mixture was added to each of the wells. Finally, PCR Array was performed in triplicates. GAPDH was used as endogenous control. The 2 -∆∆Ct method was applied to calculate the relative lncRNAs expression.

Statistical analysis
All data were expressed as mean ± SD in three duplicate experiments. GraphPad Prism 8 (GraphPad Software, Inc.) was applied for statistical analysis. Student's T-test was utilized to calculate the differences. The statistically signi cant differences were considered at p < 0.05.

Results
Identi cation of the chemoradioresistant CNE1R and CNE2R cells To identify whether CNE1R and CNE2R cells were resistant to concurrent chemoradiotherapy compared to parental cells, we performed colony formation assay. The results showed that the morphology of CNE1R and CNE2R cells presented the cell body elongated, the extended pseudopod and large intercellular space compared with the CNE1 and CNE2 cells (Fig. 1A). With the increase of radiation dose, the number of cell colonies signi cantly decreased. At the same radiation dose, the number of CNE1 and CNE2 cell colonies were lower than that of CNE1R and CNE2R cell colonies, respectively (Fig. 1B). The SF value of CNE1R and CNE2R was higher than that of CNE1 and CNE2, respectively (Fig. 1C). Additionally, there was an increase in D0, Dq and SF2 in the CNE1R and CNE2R relative to the CNE1 and CNE2, respectively (Table I), indicating an obvious chemoradioresistance were found in CNE1R and CNE2R as compared to CNE1 and CNE2. These results showed that the chemoradioresistanct NPC cells with different differentiation were successfully obtained.

Identi cation of the chemoradioresistant and parental NPC cell -derived EVs
To identify the collected EVs derived from the well and poorly differentiated NPC cells receiving with and without chemoradiotherapy, TEM and NTA were performed. As presented in Fig 2A and 2B, the obtained particles were consistent with EVs in size (100-150 nm) and morphology (round-shaped).

The chemoradioresistant NPC cells released-EVs potentiated migration and invasion ability of parental cells
To clarify the functional roles of chemoradioresistant cell-released EVs in parental cells, we rst identify wthether chemoradioresistant NPC cell -secreted EVs were e ciently taken up by parental cells. As expected, CNE1 and CNE2 taken up DiI-labelled EVs derived from CNE1R and CNE2R (Fig. 3). The further results showed that CNE1R-EVs and CNE2R-EVs signi cantly promoted the migration and invasion ability of CNE1 and CNE2 cells, respectively ( Fig. 4 and Fig. 5 (Fig. 6A). Moreover, a total of 38 downregulated DE lncRNAs were identi ed in the CNE2R-EVs, and 31 downregulated DE lncRNAs were found in the CNE1R-EVs, among which 7 downregulated DE lncRNAs were shared (Fig. 6B). Hierarchical clustering was utilized to analyze the expressed pattern of the 11 shared lncRNAs (Fig. 6C). Chemoradioresistant NPC cells and parental cells had a high degree of heterogeneity. The similar spectral clustering was observed in NPC cells with or without receiving chemoradiotherapy.

Discussion
NPC is considered to be a distinct endemic disease in Southern China [1]. Chemoradiotherapy is a paramount approach as the main treatment strategy for NPC, but the effectiveness of chemoradiotherapy is limited for radiation resistance and drug resistance [23]. Chemotherapy can advance EV secretions in cancer cells, leading to the transfer of chemoresistance-related molecules to the adjacent cancer cells to induce susceptibility to chemotherapy [24,25]. To elucidate the functional effect of EVs released from chemoradioresistanct NPC cells, the chemoradioresistant CNE1R and CNE2R cells were established from their parental cell line. The chemoradioresistant NPC cell-derived EVs were internalized in parental cells, and promoted cell migration and invasion. Subsequently, to delineate the lncRNAs associated with chemoradioresistance, the lncRNA expression pro le in EVs derived from chemoradioresistant NPC cells was revealed.
EVs may contribute to chemoradioresistance via transferring genetic information into sensitive cancer cells, and further affect the biological function (e.g. growth, migration, invasion and distant metastasis) of sensitive cancer cells [26,27]. For instance, recent literature elucidates that exosomal miR-20a-5p derived from radio-resistant NPC cells promoted the adjacent NPC cells radio-resistance through repression of Rab27B by targeting Rab27B 3′-UTR [28]. EBV infection-derived exosomes may activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway to promote the stemness and chemoradioresistance of NPC through transferring LMP1 [29,30]. NPC-exosomes modulates epithelial-tomesenchymal transition(EMT) of cells through delivering growth factor beta, resulting in resistance to chemotherapy [31,32]. Here, chemoradioresistant NPC cells-derived EVs facilitated the migration and invasion ability of parental cells, suggesting these EVs promoted an aggressive phenotype of parental NPC cells, and might further affect chemoradioresistance and EMT of parental cells.
Several studies have shown that NPC-derived EVs carry various contents and play crucial roles in progression of NPC [33,34]. However, the effect of chemoradioresistant NPC-derived EVs on progression of NPC remains unknown via carrying lncRNAs. Linc-ROR facilitates NPC invasion and metastasis via enhancing EMT [35]. H19 has been veri ed to be involved in NPC metastasis [36]. Knockdown of XIST leads to a remarkable decrease of cell proliferation, a marked increase of radiosensitivity in NPC cells [37]. These ndings suggest that chemoradioresistant NPC cells-derived EVs, containing active lncRNAs (i.e. MALAT1, FAM212B-AS1, LINC-PINT and H19), might exert functional role in NPC progression. This will be our future research direction.

Conclusion
In the present study, the chemoradioresistant NPC cells-derived EVs facilitated migration and invasion ability of cells with different differentiation. The 4 upregulated and 7 down-regulated lncRNAs were shared in EVs derived from well-differentiated and poor-differentiated NPC cells receiveing chemoradiotherapy. We propose this study provides the clue that secreted EVs of chemoradioresistant NPC cells may carry lncRNAs to affect aggressive phenotype of parental cells. Ethics approval Not applicable.

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