# Interfering Expression of Chimeric TranscriptSEPT7P2-PSPH Promotes Cell Proliferation in Patients with Nasopharyngeal Carcinoma

Authors: Jing Wang; Guo-Feng Xie; Yuan He; Ling Deng; Ya-Kang Long; Xin-Hua Yang; Jiang-Jun Ma; Rui Gong; Wen-Jian Cen; Zu-Lu Ye; Yi-Xin Zeng; Hai-Yun Wang; Jian-Yong Shao
Journal: Journal of Oncology
 (2019)

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## Abstract

Introduction. Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer which is mostly prevalent in southern China. The development of NPC involves accumulation of multiple genetic changes. Chromosomal translocation is always thought to be accompanied with the fusion chimeric products. To data, the role of the fusion chimeric transcript remains obscure.Materials and Methods. We performed RNA sequencing to detect the fusion genes in ten NPC tissues. Sanger sequencing and quantitative RT-PCR were used to measure the level of the fusion chimeric transcript in NPC tissues and cell lines. The functional experiments such as CCK8 assay, colony formation, and migration/invasion were conducted to analyze the role of this transcript in NPCin vitro.Results. We demonstrated that the chimeric transcriptSEPT7P2-PSPH was formed by trans-splicing of adjacent genes in the absence of chromosomal rearrangement and observed in both NPC patients and cell lines in parallel. Low-expression of theSEPT7P2-PSPH chimeric transcript induced the protein expression ofPSPH and promoted cell proliferation, metastasis/invasion, and transforming abilityin vitro.Conclusions. Our findings indicate that the chimeric transcriptSEPT7P2-PSPH is a product of trans-splicing of two adjacent genes and might be a tumor suppressor gene, potentially having the role of anticancer activity.


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## Body

## 1. Introduction

Nasopharyngeal carcinoma (NPC) presents regional differences in incidence, such as high incidences in North Africa and Southern China [1]. More than 60% of NPC patients at diagnosis are in late stages or even present with metastasis although many biomarkers were reported to be useful for early detection such as Epstein-Barr virus (EBV) antibodies [2]. Therefore, there is still an urgent need to identify the biomarkers for cancer diagnosis and therapeutic guidance.Gene fusions derived generally from chromosomal rearrangements are considered as cancer-related genetic events, and their products including RNAs and proteins are identified as specific diagnostic and therapeutic targets for cancers such as hematologic malignancy and lung adenocarcinoma [3, 4]. However, certain chimeric transcripts can be formed without chromosomal rearrangements. Previous studies demonstrated that a fusion transcript namedSLC45A3-ELK4 was formed with exon 1 ofSLC45A3 and the last 4 exons ofELK4[5, 6], which acted as oncogenes in prostate tumor cells without chromosomal changes [7]. Additionally, many studies showed a plenty of fusion transcripts present in normal and/or tumor cells [8–12]. Therefore, fusion genes might be occurred in normal or tumors cells with/without chromosomal changes. Although previous studies have already reported some tumor specific fusion genes in NPC, all of them occur accompanied with the rearrangement of chromosomes [13, 14].Herein, we carried out RNA sequencing and discovered a novel type of fusion gene, trans-splicing chimeric transcriptSEPT7P2-PSPH. In addition, we performedin vitro studies to demonstrate that the ability of cell invasion, migration, proliferation, colony formation, and transforming were activated when the expression of the transcript was decreased, indicating thatSEPT7P2-PSPH act as the tumor suppressor gene role in NPC tumorigenesis.


## 2. Materials and Methods

### 2.1. Primary Tumors and Pair-End RNA Sequencing

Ten formalin fixed paraffin embedded (FFPE) NPC samples were retrieved from the Department of Pathology of Sun Yat-sen University Cancer Center (SYSUCC, Guangzhou, China). All tumor samples were reviewed by two experienced pathologists. Total RNA were extracted from ten FFPE tissues with RNeasy FFPE Kit (QIANGEN, Hilden, Germany) according to the manufacturer’s instructions for RNA sequencing. cDNA libraries were prepared according to the standard protocol provided by the mRNA-seq sample Pre Kit (Illumina Inc, California, USA) and sequenced (90 nt paired-end) on the Illumina Hi-seq 2000. To identify fusion genes from paired-end RNA sequencing, the data were analyzed by the computational pipeline called SOAPfusion, which uses clusters of discordant paired-end alignments to inform a split-read alignment analysis for finding fusion boundaries [15]. The UCSCH. sapiens reference genome (build hg19) was used for alignments. The written informed consent was obtained from each patient and this study was approved by Ethics Committee and Institutional Review Board of SYSUCC.


### 2.2. Cell Cultures, Reverse Transcription and Sanger Sequencing

CNE1 and 6-10B used in the study were cultivated in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco, California, USA) in a humidified 5% CO2 incubator at 37°C. All cultivations involved have been sustained within 6 months. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription was subsequently performed with PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Kusatsu, Shlga, Japan). The products were employed with specific primers for Sanger sequencing to confirm potential fusion transcripts. The primers were shown as follows:SEPT7P2-PSPH 1-sense: 5′-TTACGACTTCTCGGTCTTCGG-3′SEPT7P2-PSPH 1-anti-sense: 5′-CGTCCTCAACGCCACAGATT-3′


### 2.3. Quantitative RT-PCR

Quantitative RT-PCR was performed using the ABI 7500 Real-time PCR system (Applied Biosystems, California, U.S.A) and SYBR Premix EX Taq (TaKaRa, Kusatsu, Shlga, Japan). All reactions were carried out in triplicate. The housekeeping geneGAPDH was used as normalization control. The relative expression levels of target geneSEPT7P2-PSPH were calculated by 2-△△Ct method, in which △Ct = meanCt SEPT7P2-PSPH– meanCt control, whereCt values are the cycle threshold for each sample [16]. Specific primers were shown as follows:SEPT7P2-PSPH 2-sense: 5′-ACCTGAGCCTGGGAGGAAA-3′SEPT7P2-PSPH 2-anti-sense: 5′-GTCAACATCAAAACACACAGCATC-3′


### 2.4. Immunohistochemistry (IHC) Staining

4μm thick FFPE NPC slides were used to perform IHC staining. Firstly, the tumor slides were deparaffinized. Antigen retrieval procedure was then performed in a pressure cooker in 1 mM sodium citrate (Sangon Biotech, Shanghai, China) for 1.5 minutes.PSPH rabbit polyclonal (Proteintech, Wuhan, China) was diluted with SigalStain antibody diluent (Cell Signaling Technology, Danvers, MA, USA) at 1:400 for 1 h. Universal secondary antibody (Gene Tech, Shanghai, China) was applied for 15 minutes. Subsequently, diaminobenzidine was used as chromogens and slides were counterstained with haematoxylin before mounting.PSPH IHC was scored according to the scoring criteria including intensity and percentage as follows: 0, no staining; 1+, faint cytoplasmic reactivity without any background staining; 2+, moderate cytoplasmic reactivity; and 3+, granular cytoplasmic reactivity of strong intensity; 0, 0-25%; 1, 25%-50%; 2, 50%-75% [17].


### 2.5. siRNA Transfection

Small interfering RNAs which targeted the fusion gene (si-SP1 5′-GUUGUUTTTTCCTCCCUGG-3′; si-SP2 5′-UUGUUTTTTCCTCCCUGGC-3′) were bought from RIBOBIO Company (Guangzhou, China). CNE1 and 6-10B cells were transfected with oligonucleotides (20 nmol/L) using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.


### 2.6. Western Blotting

Cell lysis was carried out in RIPA buffer including protease inhibitor and phosphatase inhibitor cocktails (Biostool, Shanghai, China). The protein extracts were isolated from the same amounts using 12% SDS-PAGE gels. After that all the abstracts were transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). Subsequently, the membranes were incubated with rabbit polyclonal anti-PSPH antibody 1:1000 (Proteintech, Wuhan, China). And then, the incubation was performed with goat anti-rabbit IgG antibody 1:5000 (Abcam, Cambridge, UK). Anti-β-actin antibody (Affinity, Ancaster, Canada) was acted as a protein control.


### 2.7. Cell Proliferation Assay

Cell proliferation was evaluated by conducting CCK-8 assays. 1000 cells were seeded per well in 96-well plates in triplicate. After incubation with 10uL of CCK8 agent (Dojindo, Kumamoto, Japan) for 3 hours in the dark in an incubator at 37°C, the absorbance value was measured at 450 nm every 12 hours.


### 2.8. Boyden Chamber Assays

Invasion and migration abilities were examined after the siRNA ofSEPT7P2-PSPH transfection. Boyden chambers (Corning, New York, USA) with 8 μm inserts equipped with (invasion) or without (migration) Matrigel were placed in the 24-well plate. A total of 5 × 104 cells were suspended in 200 μL RPMI 1640 without serum in the upper chambers and cultured at 37°C for 24 hours. The cells which went through the inserts were stained with 0.5% crystal (Sangon, Shanghai, China). Then, the number of cells per field of view was counted under phase-contrast microscopy.


### 2.9. Colony Formation Assay

500 transfected cells were plated in six-well plates and cultured for 7 or 12 days. All the colonies were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet (Sangon, Shanghai, China) for quantification. All the experiments were performed in triplicate.


### 2.10. Anchorage-Independent Assay

In the 6-well plate, the bottom layers were the mixture of equivalent volumes of 1.2% agarose (Sangon, Shanghai, China) in PBS and medium containing 20% FBS. 5000 cells were suspended in the upper layer containing 1 ml 0.4% soft agarose.


### 2.11. Statistical Analysis

All statistical analyses were performed using SPSS 16.0 software (IBM, Armonk, NY, USA) and GraphPad Prism version 5.0 (GraphPad Software). The data are presented as the mean ± SEM from at least three independent experiments. Two-tailed Student’st-tests were conducted to compare the difference between groups. Progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan-Meier method, and the survival curves were compared with the log-rank test method. All tests were two-sided, and P < 0.05 was considered as statistically significant.


## 2.1. Primary Tumors and Pair-End RNA Sequencing

Ten formalin fixed paraffin embedded (FFPE) NPC samples were retrieved from the Department of Pathology of Sun Yat-sen University Cancer Center (SYSUCC, Guangzhou, China). All tumor samples were reviewed by two experienced pathologists. Total RNA were extracted from ten FFPE tissues with RNeasy FFPE Kit (QIANGEN, Hilden, Germany) according to the manufacturer’s instructions for RNA sequencing. cDNA libraries were prepared according to the standard protocol provided by the mRNA-seq sample Pre Kit (Illumina Inc, California, USA) and sequenced (90 nt paired-end) on the Illumina Hi-seq 2000. To identify fusion genes from paired-end RNA sequencing, the data were analyzed by the computational pipeline called SOAPfusion, which uses clusters of discordant paired-end alignments to inform a split-read alignment analysis for finding fusion boundaries [15]. The UCSCH. sapiens reference genome (build hg19) was used for alignments. The written informed consent was obtained from each patient and this study was approved by Ethics Committee and Institutional Review Board of SYSUCC.


## 2.2. Cell Cultures, Reverse Transcription and Sanger Sequencing

CNE1 and 6-10B used in the study were cultivated in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco, California, USA) in a humidified 5% CO2 incubator at 37°C. All cultivations involved have been sustained within 6 months. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription was subsequently performed with PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Kusatsu, Shlga, Japan). The products were employed with specific primers for Sanger sequencing to confirm potential fusion transcripts. The primers were shown as follows:SEPT7P2-PSPH 1-sense: 5′-TTACGACTTCTCGGTCTTCGG-3′SEPT7P2-PSPH 1-anti-sense: 5′-CGTCCTCAACGCCACAGATT-3′


## 2.3. Quantitative RT-PCR

Quantitative RT-PCR was performed using the ABI 7500 Real-time PCR system (Applied Biosystems, California, U.S.A) and SYBR Premix EX Taq (TaKaRa, Kusatsu, Shlga, Japan). All reactions were carried out in triplicate. The housekeeping geneGAPDH was used as normalization control. The relative expression levels of target geneSEPT7P2-PSPH were calculated by 2-△△Ct method, in which △Ct = meanCt SEPT7P2-PSPH– meanCt control, whereCt values are the cycle threshold for each sample [16]. Specific primers were shown as follows:SEPT7P2-PSPH 2-sense: 5′-ACCTGAGCCTGGGAGGAAA-3′SEPT7P2-PSPH 2-anti-sense: 5′-GTCAACATCAAAACACACAGCATC-3′


## 2.4. Immunohistochemistry (IHC) Staining

4μm thick FFPE NPC slides were used to perform IHC staining. Firstly, the tumor slides were deparaffinized. Antigen retrieval procedure was then performed in a pressure cooker in 1 mM sodium citrate (Sangon Biotech, Shanghai, China) for 1.5 minutes.PSPH rabbit polyclonal (Proteintech, Wuhan, China) was diluted with SigalStain antibody diluent (Cell Signaling Technology, Danvers, MA, USA) at 1:400 for 1 h. Universal secondary antibody (Gene Tech, Shanghai, China) was applied for 15 minutes. Subsequently, diaminobenzidine was used as chromogens and slides were counterstained with haematoxylin before mounting.PSPH IHC was scored according to the scoring criteria including intensity and percentage as follows: 0, no staining; 1+, faint cytoplasmic reactivity without any background staining; 2+, moderate cytoplasmic reactivity; and 3+, granular cytoplasmic reactivity of strong intensity; 0, 0-25%; 1, 25%-50%; 2, 50%-75% [17].


## 2.5. siRNA Transfection

Small interfering RNAs which targeted the fusion gene (si-SP1 5′-GUUGUUTTTTCCTCCCUGG-3′; si-SP2 5′-UUGUUTTTTCCTCCCUGGC-3′) were bought from RIBOBIO Company (Guangzhou, China). CNE1 and 6-10B cells were transfected with oligonucleotides (20 nmol/L) using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.


## 2.6. Western Blotting

Cell lysis was carried out in RIPA buffer including protease inhibitor and phosphatase inhibitor cocktails (Biostool, Shanghai, China). The protein extracts were isolated from the same amounts using 12% SDS-PAGE gels. After that all the abstracts were transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). Subsequently, the membranes were incubated with rabbit polyclonal anti-PSPH antibody 1:1000 (Proteintech, Wuhan, China). And then, the incubation was performed with goat anti-rabbit IgG antibody 1:5000 (Abcam, Cambridge, UK). Anti-β-actin antibody (Affinity, Ancaster, Canada) was acted as a protein control.


## 2.7. Cell Proliferation Assay

Cell proliferation was evaluated by conducting CCK-8 assays. 1000 cells were seeded per well in 96-well plates in triplicate. After incubation with 10uL of CCK8 agent (Dojindo, Kumamoto, Japan) for 3 hours in the dark in an incubator at 37°C, the absorbance value was measured at 450 nm every 12 hours.


## 2.8. Boyden Chamber Assays

Invasion and migration abilities were examined after the siRNA ofSEPT7P2-PSPH transfection. Boyden chambers (Corning, New York, USA) with 8 μm inserts equipped with (invasion) or without (migration) Matrigel were placed in the 24-well plate. A total of 5 × 104 cells were suspended in 200 μL RPMI 1640 without serum in the upper chambers and cultured at 37°C for 24 hours. The cells which went through the inserts were stained with 0.5% crystal (Sangon, Shanghai, China). Then, the number of cells per field of view was counted under phase-contrast microscopy.


## 2.9. Colony Formation Assay

500 transfected cells were plated in six-well plates and cultured for 7 or 12 days. All the colonies were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet (Sangon, Shanghai, China) for quantification. All the experiments were performed in triplicate.


## 2.10. Anchorage-Independent Assay

In the 6-well plate, the bottom layers were the mixture of equivalent volumes of 1.2% agarose (Sangon, Shanghai, China) in PBS and medium containing 20% FBS. 5000 cells were suspended in the upper layer containing 1 ml 0.4% soft agarose.


## 2.11. Statistical Analysis

All statistical analyses were performed using SPSS 16.0 software (IBM, Armonk, NY, USA) and GraphPad Prism version 5.0 (GraphPad Software). The data are presented as the mean ± SEM from at least three independent experiments. Two-tailed Student’st-tests were conducted to compare the difference between groups. Progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan-Meier method, and the survival curves were compared with the log-rank test method. All tests were two-sided, and P < 0.05 was considered as statistically significant.


## 3. Results

### 3.1. Novel Fusion Genes Discovered in NPC

We found 60 transcripts of fusion genes using RNA sequencing including 28 inter- and 32 intrachromosomal fusions from ten FFPE NPC tissues (Table1). Paired-end reads featured by two uncorrelated genes or spanned exon-exon junctions were nominated as putative chimeric genes (Figures 1(a) and 1(b)). Fusion genes recurrently occurred in more than one sample were selected for subsequent investigation. A novel fusion gene namedSEPT7P2-PSPH was discovered in 5 out of 10 samples and located on chromosome 7 (Figure 1(b)). To validate this newly found transcript, we designed specific primers matching the region that contained the fusion point at the mRNA level. The mRNA level of this fusion gene was validated using Sanger sequencing in the 5 samples previously and consistent with that identified by RNA sequencing (Figure 1(c)).Table 1
60 transcripts of fusion genes discovered by NGS including 28 inter- and 32 intrachromosomal fusions from ten FFPE NPC tissues.
up_gene up_chr down_gene down_chr Fusion_Type IGKC chr2 CALR chr19 INTERCHR-DS IGKJ5 chr2 CALR chr19 INTERCHR-DS MALAT1 chr11 RFWD2 chr1 INTERCHR-DS MRPS18A chr6 C5orf25 chr5 INTERCHR-DS RN7SL1 chr14 IGKC chr2 INTERCHR-DS RN7SL1 chr14 IGKJ5 chr2 INTERCHR-DS RNU6-1 chr15 SNORD3A chr17 INTERCHR-DS RNU6-33 chr4 SNORD3A chr17 INTERCHR-DS RNU6-42 chr3 RPS3 chr11 INTERCHR-DS RNU6-42 chr3 SNORD15A chr11 INTERCHR-DS RNU6-42 chr3 SNORD3A chr17 INTERCHR-DS SAMD12 chr8 ATP10D chr4 INTERCHR-DS ATP1B2 chr17 HSD17B12 chr11 INTERCHR-SS E2F4 chr16 RPL14 chr3 INTERCHR-SS EFCAB4A chr11 RN7SK chr6 INTERCHR-SS IGHG1 chr14 NINJ1 chr9 INTERCHR-SS IGKC chr2 CSNK2A2 chr16 INTERCHR-SS IGKJ5 chr2 CSNK2A2 chr16 INTERCHR-SS MIDN chr19 ALMS1 chr2 INTERCHR-SS PDE4B chr1 PPP6R3 chr11 INTERCHR-SS POU2AF1 chr11 PARP11 chr12 INTERCHR-SS RNU6-2 chr10 SNORD3A chr17 INTERCHR-SS RNU6-36 chr12 SNORD3A chr17 INTERCHR-SS RNU6-42 chr3 SNHG12 chr1 INTERCHR-SS RPL14 chr3 GLS chr2 INTERCHR-SS SLC7A5P2 chr16 PHC3 chr3 INTERCHR-SS SMARCA2 chr9 RPL14 chr3 INTERCHR-SS ZNF827 chr4 ZNF318 chr6 INTERCHR-SS ADCK4 chr19 NUMBL chr19 INTRACHR-SS-OGO-0GAP AP5S1 chr20 MAVS chr20 INTRACHR-SS-OGO-0GAP C12orf74 chr12 PLEKHG7 chr12 INTRACHR-SS-OGO-0GAP COL7A1 chr3 UCN2 chr3 INTRACHR-SS-OGO-0GAP CTBS chr1 GNG5 chr1 INTRACHR-SS-OGO-0GAP CTSD chr11 IFITM10 chr11 INTRACHR-SS-OGO-0GAP EEF1D chr8 NAPRT1 chr8 INTRACHR-SS-OGO-0GAP JAK3 chr19 INSL3 chr19 INTRACHR-SS-OGO-0GAP KIAA0101 chr15 CSNK1G1 chr15 INTRACHR-SS-OGO-0GAP LSM10 chr1 STK40 chr1 INTRACHR-SS-OGO-0GAP MAPK7 chr17 RNF112 chr17 INTRACHR-SS-OGO-0GAP MRPS31P2 chr13 TPTE2 chr13 INTRACHR-SS-OGO-0GAP NPL chr1 DHX9 chr1 INTRACHR-SS-OGO-0GAP PIK3R2 chr19 IFI30 chr19 INTRACHR-SS-OGO-0GAP POLA2 chr11 CDC42EP2 chr11 INTRACHR-SS-OGO-0GAP PPCS chr1 CCDC30 chr1 INTRACHR-SS-OGO-0GAP PROM2 chr2 KCNIP3 chr2 INTRACHR-SS-OGO-0GAP PSTPIP2 chr18 EPG5 chr18 INTRACHR-SS-OGO-0GAP RRM2 chr2 C2orf48 chr2 INTRACHR-SS-OGO-0GAP STYXL1 chr7 TMEM120A chr7 INTRACHR-SS-OGO-0GAP TTTY15 chrY USP9Y chrY INTRACHR-SS-OGO-0GAP VMAC chr19 CAPS chr19 INTRACHR-SS-OGO-0GAP WDFY1 chr2 AP1S3 chr2 INTRACHR-SS-OGO-0GAP WNT10B chr12 ARF3 chr12 INTRACHR-SS-OGO-0GAP EEF1DP3 chr13 FRY chr13 INTRACHR-SS-OGO-1GAP HACL1 chr3 COLQ chr3 INTRACHR-SS-OGO-1GAP IRF6 chr1 C1orf74 chr1 INTRACHR-SS-OGO-1GAP TMSB4Y chrY KALP chrY INTRACHR-SS-OGO-1GAP RMPP chr9 SMU1 chr9 INTRACHR-SS-OGO-53GAP FGFR3 chr4 TACC3 chr4 INTRACHR-SS-RGO SEPT7P2 chr7 PSPH chr7 INTRACHR-SS-RGO SRGAP2B chr1 SRGAP2C chr1 INTRACHR-SS-RGO chr, chromosome.Figure 1
RNA sequencing was used to discover SEPT7P2-PSPH fusion gene in NPC. (a) Detection ofSEPT7P2-PSPH fusion genevia RNA sequencing. The reads are aligned across the junction of the predicted fusion transcripts. (b) Circos plot ofSEPT7P2-PSPH fusion gene revealed the fusion existing in chromosome 7. (c) The fusion breakpoint was verified using Sanger sequencing. (d) A schematic illustration of the amplified regions of fusion gene with exons 1-2 ofSEPT7P2 and exons 3-4 ofPSPH. (e) The sequences between exons 1 and 2 ofSEPT7P2 and the sequences between exons 3 and 4 ofPSPH were all amplified by RT–PCR.
(a) (b) (c) (d) (e)The transcript was formed with the exon 1 ofSEPT7P2 and the exon 4 ofPSPH. The practical distance between the two exons are 10.2 Mb on chromosome 7. To estimate if there existed a genomic DNA rearrangement with the chimeric RNA, we designed 30 primer pairs to amplify the entire genome sequences between exon 1 and exon 2 ofSEPT7P2 as well as the genome sequences between exon 3 and exon 4 ofPSPH(Supplementary Table 1 and Figure 1(d)). However, all the segments were amplified by PCR indirectly indicating the fact that chromosomal arrangement was absent (Figure 1(e)).


### 3.2. The Expression of the Fusion Genes in Cell Lines and Tissues

In addition, we have investigated several tumor cell lines including Hela, Hep3B, NP69, CNE1, 6-10B, SUNE2, CNE2, HONE1, and 5-8F. All the cell lines were confirmed to harbor the fusion gene (Figure2(a)). Interestingly, we found that the relative expression ofSEPT7P2-PSPH was higher in the cohort of nasopharyngitis than that in NPC patients (Figure 2(b), P < 0.05). Collectively, these results indicated that the newly found transcript was not peculiar to tumor cells. Analogous studies have been published to demonstrate the noncanonical chimeric transcript [10–12]. Simultaneously, we performed western blotting assay to discover if there would be a new type of fusion protein accompanied by the transcript. Anti-PSPH polyclonal antibody was applied in the nine tumor cell lines. It was shown that the putative fusion protein did not exist and only the protein of full-lengthPSPH was confirmed by western blot with the band at 25 kDa (Figure 2(c)).Figure 2
SEPT7P2-PSPH expression. (a) By RT–PCR, theSEPT7P2-PSPH fusion transcripts were verified in nine cell lines. (b) The relative expression ofSEPT7P2-PSPH was higher in nasopharyngitis compared with those in NPC patients using quantitative RT-PCR (P < 0.05). (c) Full-lengthPSPH protein was expressed in nine cell lines by western blotting and no relevant chimeric fusion protein was detected. Expression ofSEPT7P2-PSPH was knocked down by the specific siRNA. The suppression effect was verified by quantitative RT-PCR and western blotting, respectively (d, e, n = 3; two-tailed Student’s t-tests, P∗∗ < 0.01 and P∗∗∗ < 0.001).
(a) (b) (c) (d) (e)


### 3.3. Knocking DownSEPT7P2–PSPH Upregulated the Level ofPSPH Protein and Promoted Cell Proliferation and Migration

We carried outin vitro experiments to confirm the potential biologic function of theSEPT7P2-PSPH transcript. NPC cell lines CNE1 and 6-10B were selected to explore the effect ofSEPT7P2-PSPH on NPC tumorigenesis. When compared with the negative control group, the expression of the chimeric transcript substantially decreased after two specifically designed siRNAs, siSP1 and siSP2, were transiently transfected into the cell lines. Also, there was no novel fusion protein synthesized. After transfection for 48 hours, the expression ofSEPT7P2-PSPH at mRNA level was decreased sharply whereas the expression ofPSPH protein was upregulated in both cell lines (Figures 2(d)-2(e) and Supplementary Figure 1). This may indirectly show the phenomenon that the chimeric RNA fusion did not produce a new fusion protein.However, the expression level of down-stream gene’s protein was reversely influenced by the occurrence of the fusion betweenSEPT7P2andPSPH. We subsequently performed cell proliferation assays to explore the effects of the fusion gene on the growth of CNE-1 and 6-10B. The cells transfected with si-SEPT7P2–PSPH-1 (siSP1) and si-SEPT7P2–PSPH-2 (siSP1) grew faster than those with negative control (Figure 3(a), P < 0.05). Low expression ofSEPT7P2-PSPH may contribute to tumor cell proliferation. For investigating its influence on the transforming ability of NPC cells, we conducted both colony formation assay and anchorage-independent growth assay. Strikingly, CNE1 and 6-10B cells with siSP1 and siSP2 formed larger colonies than the negative control groups, which was reversely correlated with the expression of the chimeric RNA (Figures 3(b) and 3(c), P < 0.05). To evaluate its biological function with migration and invasion, Boyden chamber assaysin vitro have been conducted. As shown in Figure 3(d), knocking downSEPT7P2-PSPH with siSP1 and siSP2 markedly enhanced the migration and invasion of NPC cells (P < 0.05).Figure 3
Knocking down SEPT7P2-PSPH can promote cell proliferation and colony formation of NPC cells. (a) CCK8 assays showed that cell proliferation was significantly enhanced after the specific siRNA interfered with the expression ofSEPT7P2-PSPH (n = 3; two-tailed Student’s t-tests, P∗ < 0.05, P∗∗ < 0.01 and P∗∗∗ < 0.001). (b) and (c) Knocking downSEPT7P2-PSPHby siRNA can promote the colony-formation ability of CNE1 and 6-10B cells (n = 3; two-tailed Student’s t-tests, P∗∗ < 0.01 and P∗∗∗ < 0.001). (d) Cell migration and invasion assays revealed that the abilities of migration and invasion were enhanced after interfering with specific siRNA.
(a) (b) (c) (d)


### 3.4.PSPH Expression and Prognosis of NPC Patients

To investigate the expression status ofPSPH in NPC, we performed IHC experiment forPSPH in 72 FFPE NPC specimens (Figure 4). The result revealed that the patients with high PSPH expression showed a tendency with shorter PFS and OS in NPC patients though there was no statistically significant (Figures 5(a) and 5(b), P = 0.205, P = 0.106).Figure 4
Representative IHC staining with negative (a), low (b), moderate (c), high, and (d)PSPH expression. Scale bar, left panel 500 μm; right panel 100 μm.
(a) (b) (c) (d)Figure 5
Survival analysis. (a) and (b) Kaplan-Meier analysis of progression-free survival and overall survival for NPC patients with low (n=27) versus high (n=45) expression ofPSPH through immunohistochemistry (IHC) staining.P value was determined by the log-rank test (P > 0.05).
(a) (b)


## 3.1. Novel Fusion Genes Discovered in NPC

We found 60 transcripts of fusion genes using RNA sequencing including 28 inter- and 32 intrachromosomal fusions from ten FFPE NPC tissues (Table1). Paired-end reads featured by two uncorrelated genes or spanned exon-exon junctions were nominated as putative chimeric genes (Figures 1(a) and 1(b)). Fusion genes recurrently occurred in more than one sample were selected for subsequent investigation. A novel fusion gene namedSEPT7P2-PSPH was discovered in 5 out of 10 samples and located on chromosome 7 (Figure 1(b)). To validate this newly found transcript, we designed specific primers matching the region that contained the fusion point at the mRNA level. The mRNA level of this fusion gene was validated using Sanger sequencing in the 5 samples previously and consistent with that identified by RNA sequencing (Figure 1(c)).Table 1
60 transcripts of fusion genes discovered by NGS including 28 inter- and 32 intrachromosomal fusions from ten FFPE NPC tissues.
up_gene up_chr down_gene down_chr Fusion_Type IGKC chr2 CALR chr19 INTERCHR-DS IGKJ5 chr2 CALR chr19 INTERCHR-DS MALAT1 chr11 RFWD2 chr1 INTERCHR-DS MRPS18A chr6 C5orf25 chr5 INTERCHR-DS RN7SL1 chr14 IGKC chr2 INTERCHR-DS RN7SL1 chr14 IGKJ5 chr2 INTERCHR-DS RNU6-1 chr15 SNORD3A chr17 INTERCHR-DS RNU6-33 chr4 SNORD3A chr17 INTERCHR-DS RNU6-42 chr3 RPS3 chr11 INTERCHR-DS RNU6-42 chr3 SNORD15A chr11 INTERCHR-DS RNU6-42 chr3 SNORD3A chr17 INTERCHR-DS SAMD12 chr8 ATP10D chr4 INTERCHR-DS ATP1B2 chr17 HSD17B12 chr11 INTERCHR-SS E2F4 chr16 RPL14 chr3 INTERCHR-SS EFCAB4A chr11 RN7SK chr6 INTERCHR-SS IGHG1 chr14 NINJ1 chr9 INTERCHR-SS IGKC chr2 CSNK2A2 chr16 INTERCHR-SS IGKJ5 chr2 CSNK2A2 chr16 INTERCHR-SS MIDN chr19 ALMS1 chr2 INTERCHR-SS PDE4B chr1 PPP6R3 chr11 INTERCHR-SS POU2AF1 chr11 PARP11 chr12 INTERCHR-SS RNU6-2 chr10 SNORD3A chr17 INTERCHR-SS RNU6-36 chr12 SNORD3A chr17 INTERCHR-SS RNU6-42 chr3 SNHG12 chr1 INTERCHR-SS RPL14 chr3 GLS chr2 INTERCHR-SS SLC7A5P2 chr16 PHC3 chr3 INTERCHR-SS SMARCA2 chr9 RPL14 chr3 INTERCHR-SS ZNF827 chr4 ZNF318 chr6 INTERCHR-SS ADCK4 chr19 NUMBL chr19 INTRACHR-SS-OGO-0GAP AP5S1 chr20 MAVS chr20 INTRACHR-SS-OGO-0GAP C12orf74 chr12 PLEKHG7 chr12 INTRACHR-SS-OGO-0GAP COL7A1 chr3 UCN2 chr3 INTRACHR-SS-OGO-0GAP CTBS chr1 GNG5 chr1 INTRACHR-SS-OGO-0GAP CTSD chr11 IFITM10 chr11 INTRACHR-SS-OGO-0GAP EEF1D chr8 NAPRT1 chr8 INTRACHR-SS-OGO-0GAP JAK3 chr19 INSL3 chr19 INTRACHR-SS-OGO-0GAP KIAA0101 chr15 CSNK1G1 chr15 INTRACHR-SS-OGO-0GAP LSM10 chr1 STK40 chr1 INTRACHR-SS-OGO-0GAP MAPK7 chr17 RNF112 chr17 INTRACHR-SS-OGO-0GAP MRPS31P2 chr13 TPTE2 chr13 INTRACHR-SS-OGO-0GAP NPL chr1 DHX9 chr1 INTRACHR-SS-OGO-0GAP PIK3R2 chr19 IFI30 chr19 INTRACHR-SS-OGO-0GAP POLA2 chr11 CDC42EP2 chr11 INTRACHR-SS-OGO-0GAP PPCS chr1 CCDC30 chr1 INTRACHR-SS-OGO-0GAP PROM2 chr2 KCNIP3 chr2 INTRACHR-SS-OGO-0GAP PSTPIP2 chr18 EPG5 chr18 INTRACHR-SS-OGO-0GAP RRM2 chr2 C2orf48 chr2 INTRACHR-SS-OGO-0GAP STYXL1 chr7 TMEM120A chr7 INTRACHR-SS-OGO-0GAP TTTY15 chrY USP9Y chrY INTRACHR-SS-OGO-0GAP VMAC chr19 CAPS chr19 INTRACHR-SS-OGO-0GAP WDFY1 chr2 AP1S3 chr2 INTRACHR-SS-OGO-0GAP WNT10B chr12 ARF3 chr12 INTRACHR-SS-OGO-0GAP EEF1DP3 chr13 FRY chr13 INTRACHR-SS-OGO-1GAP HACL1 chr3 COLQ chr3 INTRACHR-SS-OGO-1GAP IRF6 chr1 C1orf74 chr1 INTRACHR-SS-OGO-1GAP TMSB4Y chrY KALP chrY INTRACHR-SS-OGO-1GAP RMPP chr9 SMU1 chr9 INTRACHR-SS-OGO-53GAP FGFR3 chr4 TACC3 chr4 INTRACHR-SS-RGO SEPT7P2 chr7 PSPH chr7 INTRACHR-SS-RGO SRGAP2B chr1 SRGAP2C chr1 INTRACHR-SS-RGO chr, chromosome.Figure 1
RNA sequencing was used to discover SEPT7P2-PSPH fusion gene in NPC. (a) Detection ofSEPT7P2-PSPH fusion genevia RNA sequencing. The reads are aligned across the junction of the predicted fusion transcripts. (b) Circos plot ofSEPT7P2-PSPH fusion gene revealed the fusion existing in chromosome 7. (c) The fusion breakpoint was verified using Sanger sequencing. (d) A schematic illustration of the amplified regions of fusion gene with exons 1-2 ofSEPT7P2 and exons 3-4 ofPSPH. (e) The sequences between exons 1 and 2 ofSEPT7P2 and the sequences between exons 3 and 4 ofPSPH were all amplified by RT–PCR.
(a) (b) (c) (d) (e)The transcript was formed with the exon 1 ofSEPT7P2 and the exon 4 ofPSPH. The practical distance between the two exons are 10.2 Mb on chromosome 7. To estimate if there existed a genomic DNA rearrangement with the chimeric RNA, we designed 30 primer pairs to amplify the entire genome sequences between exon 1 and exon 2 ofSEPT7P2 as well as the genome sequences between exon 3 and exon 4 ofPSPH(Supplementary Table 1 and Figure 1(d)). However, all the segments were amplified by PCR indirectly indicating the fact that chromosomal arrangement was absent (Figure 1(e)).


## 3.2. The Expression of the Fusion Genes in Cell Lines and Tissues

In addition, we have investigated several tumor cell lines including Hela, Hep3B, NP69, CNE1, 6-10B, SUNE2, CNE2, HONE1, and 5-8F. All the cell lines were confirmed to harbor the fusion gene (Figure2(a)). Interestingly, we found that the relative expression ofSEPT7P2-PSPH was higher in the cohort of nasopharyngitis than that in NPC patients (Figure 2(b), P < 0.05). Collectively, these results indicated that the newly found transcript was not peculiar to tumor cells. Analogous studies have been published to demonstrate the noncanonical chimeric transcript [10–12]. Simultaneously, we performed western blotting assay to discover if there would be a new type of fusion protein accompanied by the transcript. Anti-PSPH polyclonal antibody was applied in the nine tumor cell lines. It was shown that the putative fusion protein did not exist and only the protein of full-lengthPSPH was confirmed by western blot with the band at 25 kDa (Figure 2(c)).Figure 2
SEPT7P2-PSPH expression. (a) By RT–PCR, theSEPT7P2-PSPH fusion transcripts were verified in nine cell lines. (b) The relative expression ofSEPT7P2-PSPH was higher in nasopharyngitis compared with those in NPC patients using quantitative RT-PCR (P < 0.05). (c) Full-lengthPSPH protein was expressed in nine cell lines by western blotting and no relevant chimeric fusion protein was detected. Expression ofSEPT7P2-PSPH was knocked down by the specific siRNA. The suppression effect was verified by quantitative RT-PCR and western blotting, respectively (d, e, n = 3; two-tailed Student’s t-tests, P∗∗ < 0.01 and P∗∗∗ < 0.001).
(a) (b) (c) (d) (e)


## 3.3. Knocking DownSEPT7P2–PSPH Upregulated the Level ofPSPH Protein and Promoted Cell Proliferation and Migration

We carried outin vitro experiments to confirm the potential biologic function of theSEPT7P2-PSPH transcript. NPC cell lines CNE1 and 6-10B were selected to explore the effect ofSEPT7P2-PSPH on NPC tumorigenesis. When compared with the negative control group, the expression of the chimeric transcript substantially decreased after two specifically designed siRNAs, siSP1 and siSP2, were transiently transfected into the cell lines. Also, there was no novel fusion protein synthesized. After transfection for 48 hours, the expression ofSEPT7P2-PSPH at mRNA level was decreased sharply whereas the expression ofPSPH protein was upregulated in both cell lines (Figures 2(d)-2(e) and Supplementary Figure 1). This may indirectly show the phenomenon that the chimeric RNA fusion did not produce a new fusion protein.However, the expression level of down-stream gene’s protein was reversely influenced by the occurrence of the fusion betweenSEPT7P2andPSPH. We subsequently performed cell proliferation assays to explore the effects of the fusion gene on the growth of CNE-1 and 6-10B. The cells transfected with si-SEPT7P2–PSPH-1 (siSP1) and si-SEPT7P2–PSPH-2 (siSP1) grew faster than those with negative control (Figure 3(a), P < 0.05). Low expression ofSEPT7P2-PSPH may contribute to tumor cell proliferation. For investigating its influence on the transforming ability of NPC cells, we conducted both colony formation assay and anchorage-independent growth assay. Strikingly, CNE1 and 6-10B cells with siSP1 and siSP2 formed larger colonies than the negative control groups, which was reversely correlated with the expression of the chimeric RNA (Figures 3(b) and 3(c), P < 0.05). To evaluate its biological function with migration and invasion, Boyden chamber assaysin vitro have been conducted. As shown in Figure 3(d), knocking downSEPT7P2-PSPH with siSP1 and siSP2 markedly enhanced the migration and invasion of NPC cells (P < 0.05).Figure 3
Knocking down SEPT7P2-PSPH can promote cell proliferation and colony formation of NPC cells. (a) CCK8 assays showed that cell proliferation was significantly enhanced after the specific siRNA interfered with the expression ofSEPT7P2-PSPH (n = 3; two-tailed Student’s t-tests, P∗ < 0.05, P∗∗ < 0.01 and P∗∗∗ < 0.001). (b) and (c) Knocking downSEPT7P2-PSPHby siRNA can promote the colony-formation ability of CNE1 and 6-10B cells (n = 3; two-tailed Student’s t-tests, P∗∗ < 0.01 and P∗∗∗ < 0.001). (d) Cell migration and invasion assays revealed that the abilities of migration and invasion were enhanced after interfering with specific siRNA.
(a) (b) (c) (d)


## 3.4.PSPH Expression and Prognosis of NPC Patients

To investigate the expression status ofPSPH in NPC, we performed IHC experiment forPSPH in 72 FFPE NPC specimens (Figure 4). The result revealed that the patients with high PSPH expression showed a tendency with shorter PFS and OS in NPC patients though there was no statistically significant (Figures 5(a) and 5(b), P = 0.205, P = 0.106).Figure 4
Representative IHC staining with negative (a), low (b), moderate (c), high, and (d)PSPH expression. Scale bar, left panel 500 μm; right panel 100 μm.
(a) (b) (c) (d)Figure 5
Survival analysis. (a) and (b) Kaplan-Meier analysis of progression-free survival and overall survival for NPC patients with low (n=27) versus high (n=45) expression ofPSPH through immunohistochemistry (IHC) staining.P value was determined by the log-rank test (P > 0.05).
(a) (b)


## 4. Discussion

The present study is the first to identify trans-splicing chimeric transcriptSEPT7P2-PSPH without chromosomal rearrangement in NPC patients. Knocking down of this transcript may promote cell proliferation and metastasis/invasion possibly due to the upregulation expression of the downstream genePSPH.Traditionally, chromosome rearrangements can generate fusion genes which are in an intimate relationship with tumor carcinogenesis. To date it makes possible to discover more and more valuable fusion genes acting as a crucial regulator in cancer formation, maintenance, and evolution. Previous studies have shown that chimeric RNAs in normal and cancer cells can be generated by intergenic splicing in the absence of chromosome rearrangements [7–9, 18, 19]. This kind of fusion gene can be used as “noncanonical chimeras”. It has been reported that there are two methods for “noncanonical chimeras” formation involving cis-splicing and trans-splicing of adjacent genes [10]. Cis-splicing fusions tend to occur between two genes that are located within 30kb in the same chromosome. Trans-splicing can be classified into two types: intragenic trans-splicing taking place between two pre-mRNAs transcribed from the same genome loci and intergenic trans-splicing that is from two different genome loci.SEPT7P2-PSPH can be classified as intergenic trans-splicing located on chromosome 7 aligned with the fact that the up- and downstream genes are 10.2Mb distance.Furthermore, we found that metastasis/invasion and proliferation of the tumor cells were inversely enhanced after the transcript had been knocked down. This was different from previously analogous studies [14, 20]. The western blot result showed that there was no newly synthetic fusion protein accompanied with the chimeric RNA. Only thePSPH protein was translated from the downstream genePSPH and was overexpressed after the chimeric RNA had been knocked down. The sequence of the chimeric RNA indicated that both of the two genes’ start codons were retained after the occurrence of the fusion, one in exon 1 ofSEPT7P2 and another in exon 4 ofPSPH. It seemed that the start codon in exon 1 ofSEPT7P2 depressed the transcription ofPSPH. The reason why fusion genes have the ability of oncogenesis can be illuminated that the new synthesized fusion products possess the tumorigenic function. Another reason is that the preoncogene could be upregulated after connected to a stronger promoter or the antioncogene could be downregulated after fused with a weaker promoter [21].SEPT7P2 is a pseudogene which has been proved to be related to SEPT7 and expressed in all tissue types [22], playing a role in multiple biological processes including vesicle trafficking, apoptosis, remodeling of the cytoskeleton, neurodegeneration, and neoplasia.Some researchers have reported that abnormal expression ofPSPH is strongly relevant to the hepatocellular carcinoma patients’ mortality as well as the critical importance oncMyc-induced cancer progression bothin vitroandin vivo[23]. The level ofPSPH has the relationship with breast cancer and lacrimal gland adenoid cystic carcinoma [24, 25]. In our study, high expression ofPSPH was the tendency to NPC patients’ poor survival though there was no statistically significant. Two previous studies indicated thatPSPH was abundantly expressed in proliferating embryonic and hematopoietic stem cells and neural progenitors of the developing brain [26, 27]. Furthermore, inhibition ofPSPH induced programmed cell death in tonsillar cell cultures [28]. In another words,PSPH plays an important oncogenic role in regulating the ability of cell proliferation and apoptosis. Hence, we put forward the hypothesis that it could be thePSPH overexpression induced when the pseudogeneSEPT7P2 andPSPH fuse with each other. Recently, some studies demonstrated that trans-splicing can become one of the tumor-targeting methods in cancer related treatment, in which trans-splicing ribozyme may function as a potential anticancer agent via stimulating anticancer gene activity [29]. However, such role of the transcriptSEPT7P2-PSPH in NPC warrants the further study.


## 5. Conclusions

The trans-splicing chimeric transcriptSEPT7P2-PSPH in our study might be a tumor suppressor gene in NPC tumorigenesis, potentially having the role of anticancer activity.



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