Interfering Expression of Chimeric Transcript SEPT7P2-PSPH Promotes Cell Proliferation in Patients with Nasopharyngeal Carcinoma

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 NPC in vitro. Results We demonstrated that the chimeric transcript SEPT7P2-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 the SEPT7P2-PSPH chimeric transcript induced the protein expression of PSPH and promoted cell proliferation, metastasis/invasion, and transforming ability in vitro. Conclusions Our findings indicate that the chimeric transcript SEPT7P2-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.


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 named SLC45A3-ELK4 was formed with exon 1 of SLC45A3 and the last 4 exons of ELK4 [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][9][10][11][12]. Therefore, fusion genes might be occurred in normal or 2 Journal of Oncology 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 transcript SEPT7P2-PSPH. In addition, we performed in 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 that SEPT7P2-PSPH act as the tumor suppressor gene role in NPC tumorigenesis.

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 UCSC H. 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.

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--actin antibody (Affinity, Ancaster, Canada) was acted as a protein control.

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.

Boyden Chamber Assays.
Invasion and migration abilities were examined after the siRNA of SEPT7P2-PSPH transfection. Boyden chambers (Corning, New York, USA) with 8 m inserts equipped with (invasion) or without (migration) Matrigel were placed in the 24-well plate. A total of 5 × 10 4 cells were suspended in 200 L 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.

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's t-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.

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 (Table 1). 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 named SEPT7P2-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)).
The transcript was formed with the exon 1 of SEPT7P2 and the exon 4 of PSPH. 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 of SEPT7P2 as well as the genome sequences between exon 3 and exon 4 of PSPH (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)).

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 (Figure 2(a)). Interestingly, we found that the relative expression of SEPT7P2-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][11][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-length PSPH was confirmed by western blot with the band at 25 kDa (Figure 2(c)).

Knocking Down SEPT7P2-PSPH Upregulated the Level of PSPH Protein and Promoted Cell Proliferation and Migration.
We carried out in vitro experiments to confirm the potential biologic function of the SEPT7P2-PSPH transcript. NPC cell lines CNE1 and 6-10B were selected to explore the effect of SEPT7P2-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 of SEPT7P2-PSPH at mRNA level was decreased sharply whereas the expression of PSPH 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 between SEPT7P2 and PSPH. 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 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   6 Journal of Oncology 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 assays in vitro have been conducted. As shown in Figure 3(d), knocking down SEPT7P2-PSPH with siSP1 and siSP2 markedly enhanced the migration and invasion of NPC cells (P < 0.05).

PSPH Expression and Prognosis of NPC Patients.
To investigate the expression status of PSPH in NPC, we performed IHC experiment for PSPH 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).

Discussion
The present study is the first to identify trans-splicing chimeric transcript SEPT7P2-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 gene PSPH. 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 cissplicing 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 the PSPH protein was translated from the downstream gene PSPH 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 of SEPT7P2 and another in exon 4 of PSPH. It seemed that the start codon in exon 1 of SEPT7P2 depressed the transcription of PSPH. 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 of PSPH is strongly relevant to the hepatocellular carcinoma patients' mortality as well as the critical importance on cMyc-induced cancer progression both in vitro and in vivo [23]. The level of PSPH has the relationship with breast cancer and lacrimal gland adenoid cystic carcinoma [24, 25]. In our study, high expression of PSPH was the tendency to NPC patients' poor survival though there was no statistically significant. Two previous studies indicated that PSPH was abundantly expressed in proliferating embryonic and hematopoietic stem cells and neural progenitors of the developing brain [26,27]. Furthermore, inhibition of PSPH 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 the PSPH overexpression induced when the pseudogene SEPT7P2 and PSPH 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 transcript SEPT7P2-PSPH in NPC warrants the further study.  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 of SEPT7P2-PSPH (n = 3; two-tailed Student's t-tests, * < 0.05, * * < 0.01 and * * * < 0.001). (b) and (c) Knocking down SEPT7P2-PSPH by siRNA can promote the colony-formation ability of CNE1 and 6-10B cells (n = 3; two-tailed Student's t-tests, * * < 0.01 and * * * < 0.001). (d) Cell migration and invasion assays revealed that the abilities of migration and invasion were enhanced after interfering with specific siRNA.

Conclusions
The trans-splicing chimeric transcript SEPT7P2-PSPH in our study might be a tumor suppressor gene in NPC tumorigenesis, potentially having the role of anticancer activity.

Data Availability
The raw data in this paper has been successfully uploaded and locked onto Research Data Deposit with RDD no. RDDB2019000527.