CircKIAA0368 Promotes Proliferation,Migration,and Invasion by Upregulating HOXA10 in Nasopharyngeal Carcinoma

Abstract

Background

Nasopharyngeal carcinoma (NPC) represents a head and neck cancer caused by cancerization of nasal epithelial cells. HOXA10 has been identified to promote proliferation and invasion of NPC cells, but its regulatory mechanism has not been well discussed. Published research work has also pointed out that circular RNAs (circRNAs) could regulate mRNAs to affect NPC tumorigenesis and development.

Aim

To explore the roles of HOXA10 and its specific regulatory mechanism regarding circRNAs in NPC.

Methods

Reverse transcription polymerase chain reaction and western blot were applied to test gene expression. Functional assays were used to evaluate changes in NPC cell phenotypes. Mechanism assays were done to verify RNA-RNA or RNA-protein interaction.

Results

HOXA10 was highly expressed in NPC tissues and cell lines. Moreover, HOXA10 knockdown could restrict NPC cell proliferation, invasion, migration, and epithelial–mesenchymal transition. CircKIAA0368 was upregulated in NPC cells and could elevate HOXA10 expression by sponging miR-6838-5p. Furthermore, circKIAA0368 was unveiled to competitively bind to p300/CREB-binding protein-associated factor (PCAF) to repress acetylation and degradation of HOXA10 protein.

Conclusion

CircKIAA0368 upregulates HOXA10 expression via miR-6838-5p and PCAF, consequently promoting NPCcell and tumor growth.

Keywords

nasopharyngeal carcinoma HOXA10 circKIAA0368 miR-6838-5p PCAF

Introduction

Nasopharyngeal carcinoma (NPC) is a malignant tumor of the head and neck caused by cancerization of nasal epithelial cells, which is prevalent in Asia and North Africa.^1,2 Treatments for NPC patients include chemotherapy and radiotherapy. As the treatment protocols improve, overall survival rates of patients in nonmetastatic condition have climbed up to more than 80%.³ Although current treatment leads to a favorable survival rate, chemo-resistance emerges as a difficult problem in curing recurrent NPC patients.⁴ Messenger RNA (mRNA) has been proved to affect malignant behaviors of NPC.⁵ According to previous research work, HOXA10 promotes the proliferation and invasion of cancer cells in NPC,⁶ but the mechanism of HOXA10 in NPC remains largely unclear.

Lately, circular RNAs (circRNAs) have been identified to engage in the regulation of various physiological and pathological processes. Several studies have reported that circRNAs are involved in NPC tumor formation and development.^7,8 For example, hsa_circRNA_001387 has been reported to be a potential diagnostic and prognostic biomarker for NPC⁹; CircRNA ZNF609 has been confirmed to promote growth and metastasis of NPC by competing with microRNA-150-5p.¹⁰ Moreover, it has been found that circCTDP1 regulates HOXA10 in NPC cells,¹¹ but whether other circRNAs are involved in the regulation of HOXA10 expression in NPC cells remains indistinct. Therefore, it is of great significance to find out a new circRNA that could regulate HOXA10 expression and explore their molecular mechanisms in NPC.

It has been pointed out that circRNAs can act as competing endogenous RNAs (ceRNAs) to competitively bind with targeted microRNAs (miRNAs), thereby influencing the function of miRNAs and their downstream mRNAs.¹² The ceRNA model implicated with circRNA, miRNA, and mRNA is frequently discussed in various cancers, including NPC. For instance, circCRIM1 has been found to bind with miR-422a and repress the inhibitory impact of miR-422a on FOXQ1, thus resulting in NPC metastasis and docetaxel chemoresistance.¹³ It has also been revealed that circSETD3 promotes NPC cell invasive and migratory abilities via releasing MAPRE1 from the bondage of miR-615-5p and miR-1538.¹⁴ This study tried to figure out if ceRNA mode was involved in the regulation of our subject circRNA.

The translation of mRNA into protein is essential for nearly every cellular process. Making sure that newly translated proteins function normally is a primary challenge for the cells.¹⁵ A large number of circRNAs have been uncovered to exert crucial biological functions via modulating protein function.¹⁶ For example, it has been demonstrated that circECE1 facilitates Warburg effect in osteosarcoma by preventing speckle-type POZ-caused c-Myc ubiquitination and degradation.¹⁷ In addition, it has been discovered that circRNA-SORE affects sorafenib resistance of hepatocellular carcinoma cells via blocking PRP19-mediated YBX1 degradation.¹⁸ Hence, the impact of a certain circRNA on HOXA10 protein is also worth investigating.

This study aimed to explore the role of HOXA10 in NPC as well as the specific molecular mechanisms for circRNAs to regulate HOXA10 expression, which might enrich the knowledge of NPC.

Materials and Methods

Cell Culture

Two NPC cell lines (C666-1 and 5-8F) were procured from the Chinese Academy of Sciences (Shanghai, China). The other 2 NPC cell lines (SUNE-1 and 6-10B) as well as normal nasal epithelial (NP-69) were provided by American Type Culture Collection (ATCC). Cells were cultivated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS; 10090, Gibco) and 100 U/mL penicillin/streptomycin. Incubators were stored in a humid environment with 5% CO₂ at 37 °C. Among these cell lines, only NP-69 cells were spontaneously immortalized.

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)

Total RNA was first extracted with the use of Trizol reagent (15596018, Invitrogen). PrimeScript™ II 1st Strand cDNA Synthesis Kit (6210A, Takara) was utilized for reverse transcription. Manufacturer's guidelines were strictly followed. After quantitative analysis, 2^−ΔΔCt method was further utilized for gene expression calculation. GAPDH or U6 was used as an internal control.

Cell Transfection

Short hairpin RNAs (shRNAs), provided by Gene Pharma, were used for the knockdown of HOXA10, circKIAA0368, hsa_circ_0093674, hsa_circ_0081534, and hsa_circ_0079557. PcDNA3.1 was utilized for overexpression of HOXA10. MiR-6838-5p mimics or miR-6838-5p inhibitor was bought for miR-6838-5p overexpression or inhibition. MiR-NC acted as a control vector. All plasmids were transfected into cells by Lipofectamine 3000 (L3000015, Invitrogen).

Cell Counting Kit-8 (CCK-8) Assay

CCK-8 kit (B34302, Bimake) was utilized following the manufacturer's protocol. The transfected cells were first seeded in 96-well plates. CCK-8 solution was added into indicated wells followed by 2-h incubation at 37 °C. Optical density (OD) value was detected at 450 nm every 24 h under a microplate reader (Synergy4, BioTek).

5-Ethynyl-2′-Deoxyuridine (EdU) Assay

The transfected cells were plated into 96-well plates and then treated with 5 μL EdU. Following cell fixation with 4% paraformaldehyde, DAPI (C1005, Beyotime) was added to stain cell nuclei. Ultimately, the cells were observed under the fluorescence microscope.

Transwell Assay

Transwell chambers (CLS3396-2EA) were used in transwell assays. Cell samples were seeded into the upper chamber which was covered with Matrigel (356234, BD Biosciences) in transwell invasion assays. Serum-free medium was added into the upper chamber. Lower chamber was supplemented with a culture medium containing 10% FBS. Twenty-four hours later, cells in the upper layer of the membrane were wiped off, while cells in the lower layer of the membrane were stained by 0.5% crystal violet. The images were captured under a microscope. Transwell migration assay was also conducted following the same procedures, but Matrigel was not used.

Western Blot

Cells were lysed by RIPA lysis buffer (89900, Thermo Fisher). Total protein was extracted from cells and transferred to PVDF membranes (3010040001, Sigma) after SDS-PAGE. Then membranes were blocked with 5% nonfat milk and incubated with primary antibodies against E-cadherin (ab40772, Abcam), N-cadherin (ab76011, Abcam), MMP-7 (ab207299, Abcam), HOXA10 (sc-271139), p300/CREB-binding protein-associated factor (PCAF; ab176316, Abcam), and GAPDH (ab181620, Abcam) overnight at 4 °C. Subsequently, membranes were washed and then incubated with secondary antibody for 2 h at room temperature. Protein quantification was achieved with the help of ECL Western Blotting Detection Kit (FP300, ABP Biosciences). GAPDH was regarded as the internal control.

RNA Pull-Down Assay

RNA pull-down assay was done by use of Pierce Magnetic RNA-Protein Pull-Down Kit (20164, Thermo Fisher). The cell lysates were first incubated with biotinylated circKIAA0368 (bio-circKIAA0368) or bio-miR-6838-5p. Non-bio-circKIAA0368 or non-bio-miR-6838-5p worked as the control group. Afterward, M-280 streptavidin magnetic beads (S3762, Sigma) were added. After RNA-RNA or RNA-protein complex was obtained, the enriched RNAs or proteins were purified and analyzed through RT-qPCR or western blot/mass spectrometry.

RNA Binding Protein Immunoprecipitation (RIP)

RIP assay was conducted using Magna RIP™ Kit (17-700, Millipore). The cell lysates were incubated with anti-IgG, anti-Ago2, or anti-PCAF and magnetic beads. The RNAs were then isolated following immunoprecipitation. Ultimately, expression levels of the RNAs were subject to RT-qPCR analysis.

Luciferase Reporter Assay

Wild-type (WT) or mutant-type (Mut) sequence of circKIAA0368/HOXA10 3’UTR was cloned into pmirGLO vectors. Then, these vectors were cotransfected with miR-6838-5p mimics (labeled as miR-6838-5p) or negative control (NC) mimics (labeled as miR-NC) into NPC cells by Lipofectamine 3000 reagent. The sequence of HOXA10 promoter was cloned into pGL3 vectors. Dual-Luciferase Reporter Assay Kit was applied to detect the luciferase activity.

Subcellular Fractionation Assay

Nucleus and cytoplasm were first separated after centrifugation. RT-qPCR was involved to quantify the expression of circKIAA0368 in different parts of NPC cells. Western blot was conducted to examine the protein level of PCAF in cell nucleus and cytoplasm. U6 or GAPDH worked as the nuclear or cytoplasmic control for RNA. Histone or GAPDH acted as the nuclear or cytoplasmic control for protein.

Xenograft Assay

Male BALB/c mice (4 weeks old) were obtained from Beijing Cavins Animal Experimental Center. All animal experiment procedures were approved by the First Affiliated Hospital of Soochow University. SUNE-1 cells transfected with sh-NC, sh-circKIAA0368#1, sh-circKIAA0368#1 + pcDNA3.1, or sh-circKIAA0368#1 + pcDNA3.1/HOXA10 were subcutaneously inoculated into the nude mice. The volume of xenograft tumors was monitored every 3 days from the seventh day. After 28 days, tumors were excised from all sacrificed mice. Volume and weight of excised tumors were finally measured.

Immunohistochemistry (IHC)

IHC staining was operated following the previous description.¹⁹ In brief, paraffin-embedded xenograft tissues were collected. Next, these sections were deparaffinized and incubated with the primary antibody of Ki-67 and PCNA at 4 °C for a whole night. Afterward, tissues were incubated with indicated secondary antibodies conjugated with horseradish peroxidase. Color reaction was visualized following the treatment of diaminobenzidine solution. Counterstaining was achieved with hematoxylin.

Hematoxylin-Eosin (HE) Staining

HE staining was operated according to a previous study.²⁰ Xenograft tumors embedded in paraffin were cut into small fragments and then dehydrated with ethanol and xylol. Next, treated tissues were stained with HE solution. After being washed and dehydrated, the HE-stained sections were observed under a fluorescence microscope.

Statistical Analysis

Statistical analysis was done with the use of SPSS statistical software package and GraphPad Prism 7.0. Each experiment was carried out 3 times, and all data were demonstrated as mean ± standard deviation (SD). To analyze the statistical differences between 2 or more groups, Student t test, 1-way or 2-way analysis of variance (ANOVA) was involved. Data differences were statistically significant when P value was lower than .05.

Results

HOXA10 Demonstrates the High Expression in NPC Cells and Promotes Malignant Phenotype of NPC Cells

At the very beginning, we searched on GEO dataset (GSE61218) to find differentially expressed genes in NPC tissues and normal tissues. Under the conditions of LogFC > 3 and adj. P < .01, the upregulated genes in NPC tissues were selected. GEPIA database (http://gepia.cancer-pku.cn/) was also utilized to identify dysregulated genes in Head and Neck Squamous Carcinoma (HNSC) tissues. With the conditions of Log2FC > 2 and adj. P < .01, the upregulated genes in HNSC tissues were screened out. After overlapping the above 2 searching outcomes, 7 genes were determined (Figure 1A). Subsequently, RT-qPCR measured the expression of these 7 genes in NPC cell lines (SUNE-1 and 5-8F) and normal nasal epithelial cell line (NP-69), and the heat map illustrated that though HOXA10 expression fluctuated in 3 SUNE-1 cell lines, it was generally higher in all involved NPC cell lines in comparison to NP-69 cells. The expression of CXCL10, CXCL11, CST1, and IFIT3 was basically lower in NPC cells than NP-69 cells. Additionally, the expression of the rest candidates was not differentially expressed in NPC cell lines, comparing with NP-69 cells (Figure 1B). Hence, HOXA10 was involved in the following assays, and other candidates were excluded. Subsequently, RT-qPCR was performed to quantify HOXA10 expression in 4 NPC cell lines (C666-1, 6-10B, 5-8F, and SUNE-1) and NP-69 cell lines. The collected data indicated that HOXA10 had a high expression level in NPC cell lines, especially in 5-8F and SUNE-1 cell lines (Figure 1C). Moreover, RT-qPCR results suggested that HOXA10 expression declined due to the transfection of sh-HOXA10 (Figure 1D). CCK-8 and EdU experiments revealed that the proliferation of NPC cells was restrained after HOXA10 knockdown (Figure 1E and F). Transwell assays verified that knockdown of HOXA10 resulted in the weakened invasive and migratory capabilities of NPC cells (Figure 1G and H). Western blot detected the protein levels of epithelial–mesenchymal transition (EMT) markers in NPC cells after HOXA10 knockdown. It was discovered that the protein level of E-cadherin increased after HOXA10 knockdown, while that of N-cadherin and MMP-7 decreased, which reflected that EMT process was hampered in these NPC cells due to HOXA10 knockdown (Figure 1I). In summary, HOXA10 manifests high expression in NPC cells, and HOXA10 knockdown inhibits proliferation, invasion, migration, and EMT of NPC cells.

CircKIAA0368 Is Upregulated and Positively Regulates HOXA10 Expression in NPC Cells

According to the previous study,²¹ the top 10 circRNAs significantly highly expressed in NPC tumor tissues were selected. Then, the levels of these 10 circRNAs in 2 NPC cell lines (SUNE-1 and 5-8F) and normal NP-69 cells were detected by RT-qPCR, and 4 circRNAs (hsa_circ_0093674, hsa_circ_0081534, hsa_circ_0088018, and hsa_circ_0079557) were found significantly upregulated in NPC cells (Figure 2A). Additionally, the data from RT-qPCR assays showed that the expression of 4 circRNAs decreased after their corresponding knockdown (Figure S1A). Next, we found knockdown of hsa_circ_0093674, hsa_circ_0081534 and hsa_circ_0079557 made almost no significant difference to HOXA10 protein level in NPC cells (Figure S1B). Only knockdown of hsa_circ_0088018 resulted in the significant downregulation of HOXA10 protein in NPC cells (Figure 2B). From circBase database (http://circrna.org/), we found that hsa_circ_0088018 (circKIAA0368) located at chr9: 114190321-114195652 and the length was 342nt (Figure 2C). As shown in Figure 2D, convergent primers could amplify cDNA and gDNA of circKIAA0368, while divergent primers could only amplify the cDNA in NPC cells, indicating the circular structure of circKIAA0368. Subsequently, NPC cells were treated with RNase R, and RT-qPCR experiments were performed. As the expression of circKIAA0368 was hardly changed, while the linear KIAA0368 was dramatically hydrolyzed by RNase R, which meant that circKIAA0368 was more stable than its linear host gene (Figure 2E). Next, the results of subcellular fractionation assay revealed that circKIAA0368 was in both cytoplasm and nucleus of NPC cells (Figure 2F). To conclude, circKIAA0368 is upregulated and positively regulates HOXA10 expression in NPC cells.

CircKIAA0368 Depletion Accounts for the Suppression of NPC Cell Proliferation, Migration, Invasion, and EMT Process

In this section, we deeply explored the impact of circKIAA0368 downregulation in NPC cells. CCK-8 and EdU experiments revealed that the proliferation of NPC cells was repressed after circKIAA0368 knockdown (Figure 3A and B). Transwell assays verified that knockdown of circKIAA0368 resulted in inhibited invasion and migration of NPC cells (Figure 3C and D). Western blot assay measured the protein levels of EMT markers (E-cadherin, N-cadherin, and MMP-7). The obtained data suggested that EMT process was repressed in response to circKIAA0368 knockdown (Figure 3E). Taken together, the knockdown of circKIAA0368 inhibits malignant phenotype of NPC cells.

CircKIAA0368 Sponges miR-6838-5p to Upregulate HOXA10 in NPC Cells

After figuring out the effects of circKIAA0368 downregulation on NPC cell phenotype, we tried to dig into the regulatory mechanism of circKIAA0368 in NPC cells. RT-qPCR analysis indicated that circKIAA0368 knockdown resulted in a decrease of HOXA10 mRNA expression in SUNE-1 and 5-8F cells (Figure 4A). Then, we found that circKIAA0368 could not influence HOXA10 transcription because the luciferase activity of HOXA10 promoter had no significant variation after circKIAA0368 knockdown (Figure S1C). Moreover, these NPC cells were treated with MG132 to prevent protein degradation, and then a western blot assay was conducted. In the control group, circKIAA0368 knockdown resulted in a decrease in HOXA10 protein level. To contrast, no significant variation was observed in HOXA10 protein level after knockdown of circKIAA0368 in NPC cells treated with MG132 (Figure S1D). These results represented that circKIAA0368 could regulate the protein level of HOXA10 at the posttranslational level. The following RNA pull-down assay indicated that HOXA10 could be enriched by neither bio-circKIAA0368 nor non-bio-circKIAA0368 (Figure S1E). Considering nearly half of circKIAA0368 amassed in NPC cell cytoplasm, we inferred that circKIAA0368 might act as a ceRNA to affect HOXA10 mRNA level. As shown in Figure 4B, 52 candidate miRNAs with potential binding sites on both HOXA10 mRNA and circKIAA0368 were predicted through starBase (http://starbase.sysu.edu.cn/). The expression of candidate miRNAs in NPC cells and normal NP-69 cells was detected by RT-qPCR, and it was found that the top 3 downregulated miRNAs in NPC cells were miR-5590-3p, miR-6838-5p, and miR-4731-5p (Figure S2A). RNA pull-down assay demonstrated only miR-6838-5p was enriched by bio-circKIAA0368 (Figure 4C). With the use of bio-miR-6838-5p, we validated that HOXA10 mRNA was able to combine with miR-6838-5p (Figure 4D). As Ago2 works as one of RNA-induced silencing complex (RISC) components,²² an Ago2 protein within the RISC complex could bind to an miRNA, for which the binding affinities of miRNA-Ago2 complexes have been confirmed.^23,24 Hence, Ago2-RIP assays were performed, and we found that circKIAA0368, miR-6838-5p, and HOXA10 mRNA all existed in the RISC (Figure 4E). The binding sites of miR-6838-5p on circKIAA0368 and HOXA10 mRNA were predicted by starBase (Figure 4F). Subsequently, NPC cells were transfected with miR-NC or miR-6838-5p mimics, and the high overexpression efficiency of miR-6838-5p mimics was confirmed by RT-qPCR (Figure S3A). Next, luciferase reporter assay further confirmed that miR-6838-5p could interact with circKIAA0368 and HOXA10 (Figure 4G). Rescue experiments were then designed and implemented. The outcomes of RT-qPCR and western blot assays indicated that downregulating miR-6838-5p could counteract the influence of circKIAA0368 knockdown on HOXA10 expression (Figure 4H and I). Moreover, through RT-qPCR analysis, we found that circKIAA0368 and miR-6838-5p could not affect the expression of each other (Figure S3B). In short, circKIAA0368 regulates HOXA10 expression by sponging miR-6838-5p in NPC cells.

CircKIAA0368 Competitively Binds to PCAF to Inhibit Acetylation and Degradation of HOXA10 Protein

Since the former experiments found that circKIAA0368 could regulate HOXA10 protein expression at the posttranslational level, the underlying mechanism was explored in this section. Firstly, through RNA pull-down assay, we found that PCAF was the binding protein of circKIAA0368 (Figure 5A). Previous research has reported that PCAF can bind to HOXA10 protein, leading to the acetylation and degradation of HOXA10 protein.²⁵ Therefore, we wondered if circKIAA0368 was able to regulate HOXA10 protein level through PCAF. RIP assay further confirmed the binding affinity of circKIAA0368 and PCAF protein in NPC cells (Figure 5B). Subcellular fractionation assay showed that PCAF was mainly distributed in the nuclei of NPC cells (Figure 5C). Furthermore, RT-qPCR and western blot assays suggested that circKIAA0368 knockdown led to no significant difference in PCAF expression (Figure S3C). According to the previous study, HAT domain of PCAF binds to HOXA10.²⁵ Therefore, we speculated that circKIAA0368 might be allowed to combine with HAT domain of PCAF, which prevented PCAF from binding to HOXA10. After RNA pull-down assay, only Myc-PCAF-WT was found to be enriched by circKIAA0368, rather than Myc-PCAF-HAT-Mut (Figure 5D). This indicated that circKIAA0368 is bound to PCAF at HAT domain. The binding affinity of HOXA10 and PCAF protein in NPC cells was further verified by co-immunoprecipitation (Co-IP) assay (Figure 5E). Subsequently, Co-IP assay was performed to examine the competitive relation between circKIAA0368 and PCAF. As shown in Figure 5F, the protein level of PCAF in SUNE-1 and 5-8F cells was raised after the knockdown of circKIAA0368. This finding indicated that circKIAA0368 could inhibit the binding relationship of PCAF and HOXA10. Considering the abovementioned report, this study tried to further understand the impact of circKIAA0368 on HOXA10 protein acetylation, for which Co-IP experiments were performed. The obtained data illustrated that circKIAA0368 knockdown resulted in the enhanced HOXA10 protein acetylation and reduced protein level of HOXA10 (Figure 5G). Furthermore, SUNE-1 and 5-8F cells were treated with cyclohexane (CHX) which can inhibit the protein synthesis.²⁶ Before and after circKIAA0368 knockdown, western blot assays detected the protein levels of HOXA10 at an indicated time interval. It was found that HOXA10 protein level dropped apparently after circKIAA0368 knockdown, representing that the stability of HOXA10 protein in NPC cells descended due to the downregulation of circKIAA0368 (Figure 5H). In conclusion, circKIAA0368 could bind to HAT domain of PCAF, which restrains the acetylation and degradation of HOXA10 protein.

CircKIAA0368 Facilitates Proliferation, Migration, Invasion, and EMT of NPC Cells via Regulating HOXA10 Expression

To investigate the regulatory relationship between circKIAA0368 and HOXA10 in NPC cell lines, a series of rescue experiments were operated. As shown in Figure 6A, the decreased HOXA10 expression caused by circKIAA0368 depletion was recovered by overexpressing HOXA10. Subsequently, CCK-8 and EdU assays evaluated the proliferation of SUNE-1 and 5-8F. It was found that the restricted proliferation of NPC cells caused by circKIAA0368 knockdown was allowed to be restored by upregulating HOXA10 (Figure 6B and C). The results of transwell assays reflected that invasion and migration of NPC cells were hampered owing to downregulation of circKIAA0368, while the inhibited phenotype was further restored after transfecting pcDNA3.1/HOXA10 into the NPC cells (Figure 6D and E). In addition, western blot data manifested circKIAA0368 knockdown impeded EMT process, and this result was reversed in the sh-circKIAA0368#1 + pcDNA3.1/HOXA10 group (Figure 6F). To summarize, circKIAA0368 promotes multiple malignant phenotype of NPC cells by upregulating HOXA10 expression.

CircKIAA0368 Promotes NPC Tumor Growth via Regulating HOXA10

In this section, in vivo experiments were implemented via subcutaneously injecting transfected SUNE-1 cells into mice to explore the effect of circKIAA0368 and HOXA10 on NPC tumor growth. As shown in Figure 7A, the tumor growth rate was retarded by circKIAA0368 knockout but was recovered on account of HOXA10 upregulation. Moreover, the volume and weight of xenograft tumors were decreased by circKIAA0368 knockdown, while being restored by HOXA10 augment (Figure 7B and C). The expression of circKIAA0368, miR-6838-5p, and HOXA10 mRNA in each group was detected by RT-qPCR. The results indicated that the expression of circKIAA0368 and HOXA10 remarkably decreased upon circKIAA0368 depletion, while only HOXA10 expression was recovered by HOXA10 upregulation. Meanwhile, no significant change was seen in the expression of miR-6838-5p under different conditions (Figure 7D). Western blot assays measured the expression of HOXA10 and EMT markers in the xenograft tumors excised from different groups. We found that the protein levels of HOXA10, N-cadherin, and MMP-7 decreased after the knockdown of circKIAA0368, while that of E-cadherin increased. The above trends were reversed by overexpressing HOXA10 (Figure 7E). Data from IHC assay showed that in response to the knockdown of circKIAA0368, Ki-67 and PCNA expression in xenograft tumors declined, while the expression of these proliferation makers was recovered due to HOXA10 overexpression (Figure 7F). In addition, HE assay results reflected the suppressive influence of circKIAA0368 knockdown on the number of metastatic nodules was abrogated by HOXA10 overexpression (Figure 7G). To sum up, circKIAA0368 promotes NPC tumor growth by regulating HOXA10.

Discussion

NPC, as one of the malignant head and neck tumors, is reported to result from genetic variation, environmental factors, and Epstein-Barr virus (EBV) infection, etc.²⁷ Despite that NPC is sensitive to radiotherapy and complementary therapy, recurrence and metastasis are still challenges in treating NPC. The elevation of oncogenes expression and the decline of tumor suppressor genes expression are regarded as the key factors of tumorigenesis.²⁸ Reviewing previous literature, HOXA10 has been identified to promote proliferation and invasion of NPC cells, but its regulatory mechanism has not been well discussed.⁶ In this study, we revealed HOXA10 displayed a high expression in NPC through GEO, GEPIA databases, and RT-qPCR assay. We also unveiled that downregulating HOXA10 reduced the viability, proliferation, invasion, and migration of NPC cells, which were consistent with the abovementioned literature.

Increasing evidence has suggested that circRNAs play pivotal parts in the initiation and progression of malignant tumors, including NPC. For instance, Shuai et al have pointed out upregulation of circRNA_0000285 serves as a prognostic biomarker for NPC.²⁹ Fan et al have manifested circARHGAP12 could facilitate the malignant biological phenotypes of NPC, which might be conducive to developing targeted therapy for NPC.³⁰ Herein, hsa_circ_0088018 (circKIAA0368) was uncovered to be significantly upregulated in NPC tissues,²¹ and it could positively regulate HOXA10 expression. Subsequently, we verified that circKIAA0368 knockdown restricted cell proliferative, invasive, and migratory abilities of NPC cells. Moreover, data from in vivo and in vitro rescue assays confirmed circKIAA0368 facilitated NPC cell and tumor growth via upregulating HOXA10, which signified that HOXA10 and circKIAA0368 might be used as potential biomarkers in NPC. Reviewing previous research, transcription factors could upregulate multiple oncogenes important to NPC carcinogenesis.^31,32 Hence, we speculated a certain transcription factor might account for the upregulation of circKIAA0368 in NPC, which will be further explored in our future study.

Many functions of circRNAs have been reported, including their roles as miRNA sponges to indirectly modulate the expression of miRNA's downstream target genes.^33,34 For instance, Zhu et al have proved circRNA ZNF609 promotes growth and metastasis of NPC by competing with microRNA-150-5p and upregulating Sp1 expression.¹⁰ In addition, Wei et al have discovered circ_0008450 enhances CXCL9 expression via competitively binding to miR-577, which finally regulates NPC cell proliferation and invasion.³⁵ In this study, it was confirmed that circKIAA0368 acted as a miR-6838-5p sponge to regulate HOXA10 expression.

CircRNAs have also been revealed to modulate protein-protein interactions to exert their functions in cancers. For instance, Li et al have uncovered that circNDUFB2 facilitates the interaction between TRIM25 and IGF2BPs to induce ubiquitination and degradation of IGF2BPs, thus promoting non-small cell lung cancer progression.³⁶ Herein, it was found circKIAA0368 competed with HOXA10 for the binding of PCAF. Moreover, due to the knockdown of circKIAA0368, more PCAF bound to HOXA10, which increased the acetylation of HOXA10. Previous research has reported that PCAF could bind to HOXA10 protein, leading to the acetylation and degradation of HOXA10 protein.²⁵ In line with the former study, we also found PCAF could acetylate and degrade HOXA10 protein. According to a published report, HOXA10 could activate the transcription of downstream genes, eventually promoting carcinogenesis.³⁷ We will try to uncover the downstream mechanism of HOXA10 in our future study.

In conclusion, this study shows that circKIAA0368 regulates HOXA10 through competitively binding with miR-6838-5p and PCAF, consequently promoting NPC cell and tumor growth. Reviewing previous literature, the NPC field suffers from a limited number of authenticated cell lines, many of which are contaminated by HPV or EBV virus.^38,39 Considering the main research targets and limited resources in the current study, this study did not explore whether HPV E6/E7 infection and EBV infection are associated with the expression of our target genes in NPC cells, which is a limitation of the current study. We will try to investigate the link between the expression of these target genes in NPC cells and HPV E6/E7 infection/EBV infection in our future study. We hope that our experimental results can provide new ideas for the future targeted therapy of NPC.

Supplemental Material

sj-tif-1-ajr-10.1177_19458924221100960 - Supplemental material for CircKIAA0368 Promotes Proliferation, Migration, and Invasion by Upregulating HOXA10 in Nasopharyngeal Carcinoma

Supplemental material, sj-tif-1-ajr-10.1177_19458924221100960 for CircKIAA0368 Promotes Proliferation, Migration, and Invasion by Upregulating HOXA10 in Nasopharyngeal Carcinoma by Zhiping Chen, Qiaoying Gong, Daojing Li and Juying Zhou in American Journal of Rhinology & Allergy

Supplemental Material

sj-tif-2-ajr-10.1177_19458924221100960 - Supplemental material for CircKIAA0368 Promotes Proliferation, Migration, and Invasion by Upregulating HOXA10 in Nasopharyngeal Carcinoma

Supplemental material, sj-tif-2-ajr-10.1177_19458924221100960 for CircKIAA0368 Promotes Proliferation, Migration, and Invasion by Upregulating HOXA10 in Nasopharyngeal Carcinoma by Zhiping Chen, Qiaoying Gong, Daojing Li and Juying Zhou in American Journal of Rhinology & Allergy

Supplemental Material

sj-tif-3-ajr-10.1177_19458924221100960 - Supplemental material for CircKIAA0368 Promotes Proliferation, Migration, and Invasion by Upregulating HOXA10 in Nasopharyngeal Carcinoma

Supplemental material, sj-tif-3-ajr-10.1177_19458924221100960 for CircKIAA0368 Promotes Proliferation, Migration, and Invasion by Upregulating HOXA10 in Nasopharyngeal Carcinoma by Zhiping Chen, Qiaoying Gong, Daojing Li and Juying Zhou in American Journal of Rhinology & Allergy

Footnotes

Author Contributions

Zhiping Chen carried out the experiment of molecular biology and drafted the manuscript. Qiaoying Gong, Daojing Li carried out the animal experiment. Juying Zhou participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript. All animal experiment procedures were approved by the First Affiliated Hospital of Soochow University.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Juying Zhou

Supplemental Material

Supplemental material for this article is available online.

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