NPC tissues from 126 patients with NPC underwent primary surgery without any preoperative local or systemic antitumor treatment. All the enrolled cases were histopathologically confirmed. Studies on human NPC specimens were approved by the Ethical Committee of the Affiliated Hospital of Zhengzhou University (Henan, China).

Human NPC cell lines (5-8F, CNE-1, CNE-2) and nasopharyngeal epithelial cell line (NP69) were purchased from the National Collection of Authenticated Cell Cultures and authenticated by STR profiling. Cells were contained in RPMI1640 medium (C11875500BT, Gibco) with 10% FBS (C0232, Gibco) and 1% PS (V900929, Sigma-Aldrich). Cells were cultured at 37°C, 5% CO₂ incubator.

The interaction between lncRNAs and miRNAs was obtained from the Starbase database. The KEGG pathway enrichment analysis was performed for target mRNAs downstream of miR-490-3p by the “Clusterprofiler” package in R version 4.0.2. The target genes of miR-490-3p were acquired using TargetScan, miRDB, and miRTarBase.

Total RNA from cell lines and fresh NPC tissues (n = 12) was extracted using TRIzol reagent (15596026, Invitrogen). A QuantiTect Reverse Transcription Kit (205311, QIAGEN) was used for cDNA synthesis from the extracted RNA. SYBR Green Real-time PCR Master Mix (QPK-201, Solarbio) was used for qPCR. HCG11, miR-490-3p, miR-1297, miR-455-5p, MAP3K9, CLCC1, NUFIP2, PAPPA, RBPJ, and SMARCD4 levels were normalized ( Table S1 ).

NC siRNA, HCG11 siRNA, NC shRNA, HCG11 shRNA, miRNA mimics, and inhibitor were obtained from the RiboBio Co. Ltd. HCG11 siRNA sequence: GAATATCTGAGGTGACAAT. NPC cells were transferred using Lipofectamine (13778030, Thermo Fisher) for the indicated times. In addition, tumor cells were transduced with control pLKO.1 lentivirus or lentivirus encoding HCG11 shRNA, and stably expressing cells were screened using puromycin.

An RNA FISH kit (430601, Gene Pharma) was used for lncRNA expression analysis in NPC according to the instructions of the manufacturer. Briefly, tissue sections were dewaxed, treated with protease, preheated, and hybridized with HCG11 FISH probes (RiboBio) overnight. Thereafter, the tissue sections were washed and stained with DAPI. The images were captured using a fluorescence microscope. The HCG11 score was determined by multiplying the intensity of immunostaining and the percentage of HCG11-positive cells.

NPC cells were seeded in culture plates (500/well) for 1 week. Thereafter, tumor cells were washed for three times, fixed with paraformaldehyde (PFA, P0099, Beyotime) for 10 min, and stained for 10 min with 0.1% crystal violet (548-62-9, ChenSrc). Images were acquired and quantified by analyzing the colon numbers and size.

For the CCK8 assay, NPC cells were inoculated in 96-well culture plates (1000/well) and cultured for four days. The proliferation rate of NPC cells was determined by the CCK8 (CK04, Dojindo). CCK8 cells were incubated for 1 h and tested every 24 h using a microplate reader.

NPC cells were transfected with NC siRNA orHCG11 siRNA for 48 h. Thereafter, cells were incubated with or without cisplatin (HY-17394, MCE, 30 μM). Cell apoptosis was determined using an Annexin V-FITC/PI Kit (A211-01, Vazyme) according to the instructions of the manufacturer.

Briefly, cells were starved for 12 h in medium without serum, and 3 × 10³ cells were digested and then added to the upper compartment of Transwell (3422, Corning) with 200 μl medium containing 1% FBS. The lower compartment was filled with medium (with 10% FBS). After culturing for 48 h, NPC cells were removed from the inner membrane. The upper Transwell insert was fixed with PFA for 10 min, stained for 10 min with 0.1% crystal violet. The cells on the outer membrane were imaged under a microscope and analyzed.

First, 5-8F cells stably overexpressing the HCG11 shRNA-pLKO.1 vector were generated. For the in vivo assay, 1 × 10⁶ cells (WT, HCG11 shRNA) were subcutaneously injected into female BALB/c nude mice (7 weeks-old). The tumor volume was measured daily. Mice were euthanized 19 days after injection. Thereafter, tumors were obtained for further analysis. Animal experiments were approved by the Committee of Use of Animal Care at the Affiliated Hospital of Zhengzhou University.

Tumor tissue slices were prepared to prepare paraffin sections. The slices were subsequently subjected to H&E staining and immunostaining for Ki67 (9129, Cell Signaling Technology) by Servicebio Co., Ltd.

The binding sites of three miRNAs (miR-490-3p, miR-1297, and miR-455-5p) in HCG11 were cloned into the PGL3-CMV-LUC-plasmid (Promega) and constructed as PGL3-CMV-LUC-lncRNA HCG11 WT (HCG11-WT) or PGL3-CMV-LUC-lncRNA HCG11 mutant (HCG-Mut) plasmids. The MAP3K9 promoter containing miR-490-3p binding sites was cloned into the PGL3-CMV-LUC-plasmid (Promega) and constructed as PGL3-CMV-LUC-MAP3K9. Thereafter, 5-8F cells were transfected with PGL3-CMV-LUC and pRLTK (Promega) with miRNA mimics, miRNA inhibitors, or miR-NC using Lipofectamine 2000. A dual-luciferase reporter assay kit (DL101-01, Vazyme) was used to detect the luciferase activity.

Total protein was extracted from RIPA-lysed cells. Proteins were subjected to SDS-PAGE (P0012A; Beyotime), conducted transmembrane with polyvinylidene fluoride (IPVH00010; Millipore), blocked with milk, and incubated with primary antibodies at 4°C overnight. The primary antibodies used were anti-MAP3K9 (ab154506, Abcam) and anti-alpha tubulin (ab18251, Abcam). After primary antibody incubation, secondary antibody incubation was conducted for 1 h. The protein bands were analyzed using a chemiluminescence system (GE Healthcare).

All data are presented as mean ± SEM. The Student’s t-test was used to analyze the differences between the two groups. One-way analysis of variance was used to analyze the differences among multiple groups. p <0.05 was considered statistically significant.

First, this study used FISH to test HCG11levels in NPC pathological tissues (n = 126) and normal adjacent tissues (n = 12). FISH analysis revealed that HCG11 was highly expressed in NPC tissues ( Figures 1A, B ), which was further verified by qPCR in fresh NPC samples (n = 12) and normal adjacent tissues (n = 12) ( Figure 1C ). HCG11 levels were consistently elevated in NPC cell lines (CNE-1, CNE-2, and 5-8F) compared to those in normal NP69 cells ( Figure 1D ).

To investigate the potential clinical significance of HCG11, we measured the level of HCG11 in clinical NPC tissues (n = 126) and analyzed the correlations between HCG11 and clinicopathological parameters of NPC patients ( Table 1 and Figure 2 ). We found that HCG11 was dramatically higher in advanced-stage NPC tumors ( Figures 2A, B ) and lymphatic metastasis ( Figures 2C, D ). Correlation analysis showed that HCG11 expression was positively associated with tumor size ( Figure 2E ). NPC patients with higher HCG11 levels showed a poor prognosis and shorter survival time ( Figure 2F ). The results indicate that HCG11 may be an important prognostic marker for patients with NPC.

The biological functions of HCG11 in NPC were further explored. HCG11 siRNA was transfected into 5-8F and CNE-2 cells and found that HCG11 expression was significantly silenced ( Figure 3A ). Colony formation and CCK8 assays showed that cell proliferation capacity was inhibited by silencing HCG11 in 5-8F and CNE-2 cells ( Figures 3B–D ). Moreover, flow cytometry analysis indicated that HCG11 knockdown significantly increased the apoptotic rate of 5-8F and CNE-2 cells ( Figure 3E ). We found a positive correlation between HCG11 expression and lymph node metastasis ( Figures 2C, D ) and further investigated the role of HCG11 in cell migration. Transwell assays showed that 5-8F and CNE-2 cells with HCG11 silencing exhibited lower migration ability ( Figures 3F, G ).

We also investigated the role of HCG11 in NPC progression in vivo. First, we stably transfected 5-8F cells with the pLKO.1-empty vector and pLKO.1-HCG11-shRNA vector ( Figure 4A ). Control 5-8F or HCG11-shRNA 5-8F cells (1 × 10⁶ cells) were inoculated subcutaneously into BALB/c nude mice. During the establishment of the 19 days, 5-8F cells with HCG11 knockdown showed a decreased growth rate ( Figure 4B ), which was further confirmed by the smaller average volume and lighter tumor weight in the HCG11shRNA group ( Figures 4C, D ). HCG11 expression in resected tumor tissues was significantly downregulated in the HCG11 shRNA group, as detected by FISH. The percentage of Ki67-postive cells was reduced in HCG11 knockdown tumor tissues, as determined by immunohistochemistry ( Figure 4E ). Moreover, we found a positive correlation between HCG11 levels and tumor weight ( Figure 4F ), which is consistent with previous results ( Figure 2E ). In summary, these results indicated that HCG11 plays an oncogenic role in NPC.

It has been established that lncRNAs can sponge miRNAs to regulate specific genes and affect cancer malignant phenotypes (7), whereas the correlation between miRNAs and HCG11 in NPC remains to be explored. In this section, we first obtained the interaction between HCG11 and miRNA from the StarBase database and database analysis. The results showed that miR-490-3p, miR-1297, and miR-455-5p are potential targets of HCG11 ( Figure 5A ). qPCR analysis revealed higher levels of miR-490-3p, but not miR-455-5p or miR-1297, were observed in 5-8F and CNE-2 cells transfected with HCG11 siRNA ( Figure 5B ). To further confirm whether miR-490-3p is a target of HCG11, we co-transfected miR-490-3p mimics with PGL3-CMV-LUC.miR-490-3p expression was significantly upregulated, and the luciferase activity of HCG11 WT was significantly inhibited, but not the HCG11 mutant ( Figures 5C, D ). The luciferase reporter assay further suggested that HCG11 does not contain binding sites for miR-455-5p or miR-1297 ( Figure S1 ). Moreover, we confirmed the correlation between HCG11 and miR-490-3p in NPC tissues and found that miR-490-3p was downregulated in NPC tumors ( Figure 5E ), which was negatively associated with HCG11 ( Figure 5F ). In summary, these results indicated that miR-490-3p is negatively regulated by HCG11.

We then investigated whether HCG11 regulated NPC progression by inhibiting miR-490-3p. In colony formation and CCK8 assays, cell proliferation capacity was significantly inhibited by downregulating HCG11 in 5-8F cells, which could be partially rescued by miR-490-3p inhibitor ( Figures 6A–D ). We also investigated the role of HCG11/miR-490-3p in cell migration. 5-8F cell lines transfected with HCG11 siRNA showed lower migration ability compared to the control siRNA groups, while the miR-490-3p inhibitor significantly reversed the impaired migration ability of 5-8F cells with HCG11 silencing ( Figures 6E, F ). Therefore, HCG11 can regulate NPC cell proliferation and migration via the negative regulation of miR-490-3p.

It is known that ncRNA–mRNA interactions are involved in various tumor processes (7). miRNAs bind to the 3-UTR of mRNAs at partially complementary sequences to regulate the blocking/degradation of mRNA translation. To confirm the oncogenic genes, the KEGG pathway enrichment analysis was performed for target mRNAs downstream of miR-490-3p by the “Clusterprofiler” package in RStudio. We also identified the downstream targets of miR-490-3p using TargetScan, miRDB, and miRTarBase. A bioinformatics analysis showed that MAP3K9 may be a potential target of miR-490-3p ( Figures 7A, B ). As a serine/threonine protein kinase, MAP3K9 functions as an upstream activator of MAPK signaling, which is involved in tumor progression. A qPCR analysis revealed that the miR-490-3p mimic significantly downregulated MAP3K9 ( Figure 7C ). To confirm the relationship of miR-490-3p and MAP3K9, we co-transfected miR-490-3p mimic or miR-490-3p inhibitor with PGL3-CMV-LUC-MAP3K9, the luciferase activity of MAP3K9 was significantly inhibited by miR-490-3p mimic and enhanced by amiR-490-3p inhibitor ( Figure 7D ).

To further investigate whether HCG11 regulates miR-490-3p/MAP3K9 signaling in NPC cells, we co-transferred HCG11 siRNA and miR-490-3p inhibitor in 5-8F cells. MiR-490-3p was significantly inhibited by the miR-490-3p inhibitor ( Figure 7E ) and thus upregulated MAP3K9 levels ( Figures 7F–H ). HCG11 knockdown upregulated miR-490-3p expression ( Figure 7E ) and significantly downregulated MAP3K9 expression ( Figures 7F–H ). In summary, HCG11 regulates NPC cells through miR-490-3p/MAP3K9 signaling.