Long non-coding RNA CRNDE exacerbates NPC advancement mediated by the miR-545-5p/CCND2 axis

Background Previous studies indicated CRNDE to have a pivotal part within tumorigenesis. Notwithstanding, precise details on CRNDE activities within NPC are still uncertain. The investigation described in this article served to focus in greater depth on the mechanistics regarding CRNDE, together with all associated regulatory networks, on nasopharyngeal carcinoma (NPC) and its treatment possibilities. Methods Quantitative real-time polymerase chain reaction (RT-qPCR) analyzed CRNDE, miR-545-5p and CCND2 expression within NPCs and representative cell lineages. CCK-8 cell counting-, EdU-, wound-healing-/transwell-assays analyzed cellular proliferation, migrative, together with invasive properties. Apoptosis/cell cycle progression were scrutinized through flow cytometry. Dual-luciferase reporter assays validated CRNDE/miR-545-5p/CCND2 interplay. Proteomic expression of apoptosis-related protein, EMT-related protein and CCND2 protein were evaluated through Western blotting. In addition, Ki67 expression was evaluated through immunohistochemical staining. The effect of CRNDE in vivo was assessed by nude murine xenograft model studies. Results This study demonstrated up-regulated expression of CRNDE and CCND2 within NPC tissues/cell lines. Meanwhile, miR-545-5p was down-regulated. CRNDE knock-down or miR-545-5p over-expression drastically reduced NPC proliferative, migrative and invasive properties, promoted apoptosis/altered cell cycle, and inhibited CCND2 expression. However, miR-545-5p down-regulation had opposing effects. All inhibiting functions generated by CRNDE down-regulation upon NPC progression could be counterbalanced or synergistically exacerbated, depending on miR-545-5p down-regulation or up-regulation, respectively. Multiple-level investigations revealed CRNDE to serve as a sponge for miR-545-5p, and can target CCND2 within NPCs. Conclusions CRNDE increases CCND2 expression by competitive binding with miR-545-5p, thus accelerating the development of NPC. This provides potential therapeutic targets and prognostic markers against NPC.

emphasizing the importance of identifying and developing more effective NPC therapeutic measures. Therefore, more detailed pathogenesis of NPC and advanced treatment methods must be unraveled.
Long non-coding RNAs (lncRNAs) are > 200 bp and lack an open reading frame that cannot encode proteins [3]. Due to their vast spectrum of expression profiles and tissue-linked expression specificity, lncRNAs can be used as tumor markers and therapeutic targets [4]. Dysregulated expression/mutations in lncRNAs influences tumorigenesis and metastasis, with this trend occurring within multiple tumor models including colon [5], prostate [6] and oral cancer [7]. Colorectal neoplasia differential expression (CRNDE) is a long noncoding RNA with high-sensitivity/-specificity within plasma and tumor tissues. CRNDE was found to be implicated within multiple tumor processes [8]. Apart from colorectal cancer, CRNDE is also up-regulated in hepatic cancer [9], cervical cancer [10] and clear-cell-renal-cell carcinoma [11], promoting tumor expansion, invasiveness and metastases. In some head and neck tumors, CRNDE also plays a role in promoting cancer. For example, studies have found that CRNDE plays an oncogenic role in the development of TSCC by inhibiting the expression of miR-384 [12]. However, the mechanism/s employed by CRNDE to influence NPC malignancy is lacking.
MicroRNAs (miRNAs) entail small non-coding RNAs (20-22 nucleotides long), that possess pivotal regulatory functions upon physiological/developmental processes, within all cell-types [13]. MiRNA dysregulation is linked to a vast spectrum of human conditions, such as tumors. Previous literature demonstrated miR-545-5p to be down-regulated within colon adenocarcinoma and contributes to its proliferative, apoptotic, migrative and invasive properties [14]. However, previous literature reports regarding the influence of miR-545-5p over NPC tissue phenotypic characteristics are still scarce. In addition, cyclin D2 (CCND2), as a member of the highlyconserved cyclin family, has protein-independent periodicity throughout the cell cycle [15] and is closely linked to NPC manifestation and development [16]. However, no previous reports exist for confirming miR-545-5p to regulate CCND2.
LncRNAs have microRNA Responsible Elements (MRE), a sponge-binding site for miRNAs, in order to regulate specific miRNA-orchestrated target transcript down-regulation [17]. Chen and colleagues highlighted lncRNA CRNDE to promote angiogenesis within hepatoblastoma, through targeted activity on the miR-203/ VEGFA axis. Zhu et al. confirmed that CRNDE promotes proliferative/angiogenesis properties by pancreatic cancer, through regulation of miR-451a and CDKN2D [18]. Based on such studies, this investigation focused on the degree of expression and interactions of CRNDE, miR-545-5p and CCND2 within NPC, confirming that CRNDE can promote NPC pathogenesis and development through miR-545-5p/CCND2, consequently providing novel options for NPC therapies.

EdU assay
Cells from each group of CNE-2Z and HNE-1, in logarithmic growth phase, were seeded into 6-well plates (1.5 × 10 4 cells/well). The cells in each group were labeled with EdU detection solution [Beyotime Biotechnology ™ , China] and consequently fix-treated using 4% paraformaldehyde [Beyotime Biotechnology ™ , China] at ambient temperature for 15 min. Following wash-steps using PBS, 200 µL/well reaction solution were introduced, and plates placed into incubation for 30 min in darkness at ambient temperature. Following another PBS wash-step, 1 mL Hoechst 33,342 [Beyotime Biotechnology ™ , China] were added to each well for DNA staining. Following a final PBS wash-step, wells were visualized by laser confocal microscopy.

Transwell assay
In the migration experiment, Materigel gel was not paved. In the invasion experiment, Materigel ® [Corning ™ , USA] and Opti-MEM ® I Reduced-Serum Medium [Thermo ™ , USA] were diluted at a ratio of 1: 8, with 50 μL of each chamber spread at the base of the upperchambers and incubated for 1 h to render it semi-solidified. CNE-2Z and HNE-1 cells were inoculated into the upper-chambers within serum-free RPMI-1640 medium, and 600 μL RPMI-1640 medium carrying 10% FBS were introduced into the lower wells. Regarding the migration experiment, cells were grown for 24 h. Regarding the invasion experiment, cells were grown for 48 h. The upper-chambers were consequently removed, fix-treated using 4% paraformaldehyde for 15 min, dyed using crystal violet for 10 min, subjected to PBS wash-step, followed by careful removal of excess dye using a cotton swab, and finally observed/counted. The cellular population density within five randomly selected fields-of-vision under light microscopy were determined, with all experimental runs performed on three separate occasions.

Wound-healing assay
Cells were labeled at the bottom of the six-well plate pre-inoculation. At 24 h post-transfection, once cultures obtained 80% confluence, cells were lined-perpendicular to the bottom-with a 100 μL sterile pipettor tip/gun. Linear changes at 0 h and 24 h were observed, and the cellular migrative ability was detected. The calculation used was:

Flow cytometric analyses
In order to assess apoptosis, cultures were collected and resuspended, centrifuged, supernatant discarded, and pre-cooled at 4 °C with D-Hanks [Phygene ™ , China] (pH = 7.2 ~ 7.4) washing cell precipitation. Consequently, 1 × binding buffer wash-step was performed on culture cells to precipitate once, followed by centrifugation and cell collection. Consequently, 200 μL 1 × binding buffer were used for resuspending cell precipitation. A total of 2 μL Annexin V-APC and PI staining were added, with cultures left at room temperature in darkness for 20-60 min. According to cell volume, 200-300 μL of 1 × binding buffer were added, followed by on-line detection.
In order to detect cell cycle, samples were harvested/ fixed overnight using 70% ethanol and pre-cooled at 4 °C. Fixative was then removed and cells washed using PBS at 4 °C once. Cell staining solution was prepared by adding 0.5 mL propidium iodide staining solution to each cell sample, slowly and fully resuspending cell precipitations, followed by 37 °C in darkness for 30 min and temporary storage at 4 °C, or in an ice bath. Following staining, flow cytometry was used to complete detection.

Western blotting
Protein

Xenograft tumor model
BALB/c nude murines (female) were segregated into two cohort-groups, sh-CRNDE NC (sh-NC) and sh-CRNDE. The tumor cells of each experimental group in the logarithmic growth phase were counted by blood cell counting plate, and finally suspended with the required volume of D-Hanks or PBS. Following preparation of tumor cells (5.00 E+06 cells/murine), a disposable sterile syringe was utilized to aspirate cells and consequently inject 200 µL into each nude murine. At 24-days following subcutaneous injection, all murines were euthanized using 2% pentobarbital sodium (0.5 mL), followed by cervical-dislocation sacrifice. Selected tumor samples were fix-treated with 4% paraformaldehyde for immunohistochemical analyses. All other samples were kept at − 80 °C for Western blot assays.

Immunohistochemistry
The transplanted tumor was fixed with formalin for 48 h, soaked in 4% paraffin and sliced. Three xylene washing cycles were performed every 15 min, followed by three washing cycles using a differing ethanol concentration in each wash cycle (100%/95%/-80%) every 5 min. Consequently, high-pressure antigen repair was performed, using a high-pressure cooker containing 2 L of doubledistilled water treated with 40 mL of pH 8.0 EDTA repair solution. This solution was consequently heated to boiling point. Following from this step, tumor slices (together with the dyeing frame) were placed into the repair solution for two minutes in order to allow boiling repair. The procedure was then halted, and the solution was allowed to cool by adding a moderate volume of distilled water. Tumor slices were consequently extracted individually. Tissue-regions from each tumor slice were marked using an immunohistochemical pen and placed in an incubator.
EMT-linked proteins Snail, E-cadherin, N-cadherin and Vimentin were identified via Western blotting. In comparison to the control group and NC, E-cadherin levels within the si-CRNDE group were elevated, with down-regulated Snail, N-cadherin and Vimentin levels (P < 0.01) (Fig. 2c). Such findings suggest CRNDE to exacerbate NPC progression.

Down-regulation of CRNDE inhibits apoptosis of NPC cells and affects cell cycle
CRNDE influences on NPC pathology were studied by detecting apoptotic activities and NPC cell cycle status. Flow cytometry demonstrated that apoptotic rates in the si-CRNDE group were elevated, in comparison to control/NC groups, and the si-CRNDE groups of CNE-2Z/ HNE-1 cell lines exhibited G0/G1 phase arrest (P < 0.01) (Fig. 3a, b).

Knockdown of CRNDE inhibits xenograft growth associated with miR-545-5p/CCND2 axis
In order to study the effect of CRNDE knockdown in vivo, a murine xenograft model was developed via injection of sh-CRNDE or its related negative control (sh-NC) (Fig. 9a). In comparison to the sh-NC group, tumor volume/weight in sh-CRNDE group was decreased (P < 0.01) (Fig. 9b). Immunohistochemistry demonstrated that the brown color of sh-CRNDE group was significantly reduced in comparison to NC group, post-Ki67 staining and DAB coloring (Fig. 9c). Western blotting demonstrated downregulation of CCND2 within sh-CRNDE group, together with up-regulation of Bax/Cleaved Caspase 3. Bcl-2 was also down-regulated. In addition, E-cadherin was up-regulated while Snail, N-cadherin and Vimentin were downregulated (P < 0.01) (Fig. 9d). It was further confirmed that

Discussion
NPC is a highly prevalent head and neck malignant tumor with significant regionality [19]. Investigation of the pathogenesis and development mechanism/s of NPC can open a broader idea for the treatment of NPC. Following the above-described experiments, our investigation found that in NPC cells, miR-545-5p was down-regulated, together with CRNDE and CCND2 up-regulation. CRNDE can upregulate CCND2 through sponge-binding of miR-545-5p, affecting NPC proliferation, migration, invasion and apoptotic activities.
LncRNAs represent a non-protein-coding RNA molecular class which have pivotal parts within tumor Up-regulation/down-regulation influences by miR-545-5p on NPC proliferative/migrative properties. a miR-545-5p expression within multiple NPC cellular lineages (CNE-2Z, HNE-1, 5-8F). b miR-545-5p expression within NPC (CNE-2Z, HNE-1) following transfection of miR-545-5p mimics/inhibitor. c CCK-8 assays analyzed NPC proliferative property (CNE-2Z, HNE-1). d The relationship between the expression of miR-545-5p and the migration of NPC cells (CNE-2Z, HNE-1) was studied by wound healing assay (×40). e The proliferation of NPC cells was analyzed by the EdU assay (CNE2Z, HNE-1) (×200) *p < 0.05; **p < 0.01 development, treatment and prognosis. In recent years, CRNDE has been confirmed as up-regulated within colon cancer and has promoted the development of other tumors. Notwithstanding, CRNDE roles within the process of NPC is rarely reported. This study demonstrated that CRNDE was up-regulated within carcinoma tissues of NPC patients. In comparison to normal nasopharyngeal epithelial cells, CRNDE was up-regulated within NPC lineages and promotes NPC proliferative, migrative and invasive properties, together with inhibiting apoptosis. This ability to promote cancer was suppressed when CRNDE was knocked down. In mitochondrial apoptosis-related pathways, antiapoptotic gene Bcl-2 and pro-apoptotic Bax are two key Fig. 6 Effects of up-regulation/down-regulation of miR-545-5p upon invasion and migration of NPC cell lines. a The effect of miR-545-5p on migration and invasion of NPC cells (CNE-2Z, HNE-1) was detected by transwell assay (×200). b Western blot was used to detect the effect of miR-545-5p mimics or miR-545-5p inhibitor transfection on the expression of EMT-related proteins in NPC cells (CNE-2Z, HNE-1). c Western blot was used to detect the expression of EMT-related proteins when si-CRNDE was down-regulated and miR-545-5p mimics were co-transfected. *p < 0.05; **p < 0.01 influencing genomic factors [20]. Caspases are essential components of the apoptotic mechanism, especially caspase-3 [21]. Following knock-down of CRNDE, Bcl-2 was drastically down-regulated, with concomitant up-regulation of Bax and caspase-3. Epithelialmesenchymal transition (EMT) describes the gradual event of epithelial cells obtaining mesenchymal profiles, with consequently serious contributions to cancer progression, metastasis and drug resistance [22,23]. Snail can endow tumors with stem cell-type characteristics and exacerbate chemoresistance, patient relapses and EMT [24]. The reduction of E-cadherin, cytokeratin cytoskeleton replacement by Vimentin, together with mesenchymal cell morphological manifestations, are The effect of miR-545-5p expression on the cell cycle progression of NPC cells (CNE-2Z, HNE-1) was detected by flow cytometry. c Western blot was used to detect the expression of apoptosis-related proteins when miR-545-5p was overexpressed or knocked down. d The expression of apoptosis-related proteins was detected by Western blot following co-transfection of si-CRNDE and miR-545-5p inhibitor. *p < 0.05; **p < 0.01 all considered to be EMT characteristics. E-cadherin increase, coupled with Vimentin reductions, can partially regulate NPC metastasis and invasiveness. Therefore, we further studied the influence of CRNDE on EMT in NPC cell lines. This investigation demonstrated that post-CRNDE knock-down, E-cadherin was drastically down-regulated, with concomitant up-regulation of N-cadherin and Vimentin. In essence, such outcomes indicate that CRNDE accelerates NPC progression.
The competitive endogenous RNA (ceRNA) hypothesis of lncRNAs shows that when miRNA response elements (MRE) bind to miRNA, miRNA-regulated genes can be silenced [25]. Based on this, we launched a series of studies on CRNDE and ceRNA regulatory networks. Bioinformatics-based approaches were used for predicting CRNDE and miR-545-5p downstream targeting interplays, and ultimately postulating that CRNDE can affect NPC development through miR-545-5p modulation-as confirmed by dual-luciferase reporter assay. Consequently, this study describes the first report on miR-545-5p overexpression leading to thwarting of the proliferative, migrative and invasive properties of NPC, together with enhanced apoptotic activity. When miR-545-5p was inhibited, the opposite results were obtained. This study thus revealed that miR-545-5p is intimately linked to NPC tumorigenesis and development.
CCND2 is a cyclin D family protein, and its kinase activity promotes tumorigenesis by enhancing signal transduction, mediated by cyclin-dependent kinase (Cdk) [26]. Bioinformatics predicted CCND2 was a downstream target for miR-545-5p, and we further verified whether CRNDE regulated CCND2 as a ceRNA. This study demonstrated that CCND2 down-regulation occurred following CRNDE downregulation, with this influence rescued by anti-miR-545-5p. Once miR-545-5p is overexpressed, it will form a synergistic effect with CRNDE knockdown. Therefore, CRNDE can regulate tumorigenesis and development of NPC by serving as ceRNA of CCND2. Nevertheless, additional in vivo studies are required to further validate such findings. In addition, more detailed mechanisms for CRNDE reporter assays demonstrated that CCND2 was a target of miR-545-5p. b CCND2 was expressed in a human normal nasopharyngeal epithelial cell line (NP69) and NPC cell lines (CNE-2Z, HNE-1, 5-8F). c Downregulating CRNDE or upregulating miR-545-5p can affect expression of CCND2 in NPC cells (CNE-2Z, HNE-1). d Western blot was used to detect the changes of CCND2 expression in NPC cells (CNE-2Z, HNE-1) when miR-545-5p was up-regulated or down-regulated. e Western blot was used to detect the expression of CCND2 when CRNDE was down-regulated and miR-545-5p was up-regulated or down-regulated. *p < 0.05; **p < 0.01 regarding NPC tumorigenesis and development must be explored further.

Conclusion
This study identified that CRNDE, as an oncogene for NPC, was highly expressed within NPC biopsies and cellular lineages. In addition, CRNDE and CCND2 have binding sites with miR-545-5p. CRNDE knockdown can inhibit CCND2 within NPC, and this phenomenon is reversed when miR-545-5p is down-regulated. Therefore, CRNDE acts as a ceRNA for sponging miR-545-5p, resulting in up-regulation of CCND2 expression. In summary, CRNDE/miR-545-5p both have potential application value in the treatment of NPC, which can be employed as novel tumor biomarkers for NPC and provide new strategies for early NPC diagnosis and its targeted therapy.

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