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Abstract

Splicing factor 3b subunit 4, a critical component of pre-message RNA splicing complex, has been reported to play an important part in the tumorigenesis. However, the expression pattern and biological role of splicing factor 3b subunit 4 in pancreatic cancer have never been investigated. In this study, we found that both the messenger RNA (p < 0.001) and protein level of splicing factor 3b subunit 4 were decreased significantly in pancreatic cancer specimens compared with their adjacent normal tissues. Overexpression of splicing factor 3b subunit 4 in pancreatic cancer cells inhibited cell growth and motility in vitro, while suppressing splicing factor 3b subunit 4 expression promoted the proliferation and migration of pancreatic cancer cells. In addition, splicing factor 3b subunit 4 was found to inhibit the activity of signal transducer and activator of transcription 3 signaling via downregulating the phosphorylation of signal transducer and activator of transcription 3 on a tyrosine residue at position 705. Taken together, these findings demonstrated that splicing factor 3b subunit 4 acted as a suppressive role in pancreatic cancer and indicated that restoring the function of splicing factor 3b subunit 4 might be a strategy for cancer therapy.

Keywords

Splicing factor 3b subunit 4 pancreatic cancer signal transducer and activator of transcription 3 signaling cell proliferation cell migration

Introduction

Pancreatic cancer is one of the common malignant digestive system tumors, with approximately 337,000 new cases annually worldwide.¹ It is the fourth leading cause of cancer-related death in United States and results in an estimated 40,000 deaths per year.² In China, the incidence of pancreatic cancer has increased gradually in last decade, and about 90,100 new cases and 79,400 deaths occurred in 2015.³ The early diagnosis of pancreatic cancer is pessimistic, and about 80% of patients already have local invasion or distant metastasis when initially diagnosed.⁴ Although 15%–25% of patients have opportunities to receive surgeries, the recurrence rate is up to 50%–86% even after radical resections.^5,6 Recently, the comprehensive treatments of pancreatic cancer including chemotherapy, radiation, molecular targeting therapy, and combinations have developed rapidly. However, the overall survival of pancreatic cancer patients does not improve significantly. Thus, it is vital to reveal the underlying mechanisms related to pancreatic tumorigenesis in order to seek biomarkers for early detection and provide novel therapeutic strategies.

Alternative splicing, the process of accurately excising introns of pre-message RNA and generating multiple transcripts from a common precursor, has been reported to have multiple effects on various types of cancer. The abnormal expression of splicing factors, for instance, ESRP1 and RBM5, has been shown to remarkably impact the biology of pancreatic cancer such as cell proliferation, migration, invasion, and even drug resistance.^7,8 Splicing factor 3b (SF3b) is an essential component of the U2 small nuclear ribonucleoprotein (snRNP) and the U11/U12 di-snRNP, which are combined into the major and the minor spliceosomes, respectively.⁹ SF3b recognizes the branch points within pre-message RNA to promote the assembly of precursor spliceosome during alternative splicing.^9,10 Splicing factor 3b subunit 4 (SF3B4) is one of the seven core proteins constituting SF3b complex, and it also participates in the regulation of cell cycle, cell differentiation, and immunodeficiency.^11–13 However, study of SF3B4 in cancer is rare. Recently, Xu et al.¹⁴ discovered that SF3B4, SF3B2, and HSPA1A were significantly upregulated in hepatocellular carcinoma (HCC), indicating that aberrant regulation of splicing pathway may participate in the initiation and progression of liver cancer. However, the expression and biological functions of SF3B4 in pancreatic cancer are largely unknown.

Signal transducer and activator of transcription 3 (STAT3) is a classic transcription factor that mediates intracellular signaling in many pathological and physiological processes such as inflammation, immunity, development, and metabolism.^15,16 Recent studies have demonstrated that aberrantly activated STAT3 signaling existed in a large number of clinical pancreatic cancer samples, and the expression level of phosphorylated STAT3 was an independent risk factor for the prognosis of pancreatic cancer patients who underwent surgical removal.^17,18 Although many researchers revealed that STAT3 signaling played a pivotal role in the development of pancreatic cancer via inhibiting apoptosis, inducing angiogenesis, promoting metastasis, and so on, little progress has been made in targeting this signaling pathway.^19–21 Nevertheless, results from recent experimental studies have implied that STAT3β, one of the two STAT3 isoforms generated by alternative splicing of exon 23, could exert tumor suppressor effects under specific conditions, which draw our attention to the value of RNA splicing in tumor therapies targeting STAT3.²²

In this study, we examined the expression of SF3B4 in pancreatic cancer tissues, investigated its biological functions, and revealed its influence on STAT3, which would facilitate STAT3-targeted therapy in pancreatic cancer.

Materials and methods

Patients and tissue samples

A total of 63 paired fresh pancreatic cancer samples were collected from patients who underwent radical pancreatic cancer resections in Zhongshan Hospital of Fudan University (Shanghai, China) during the period from 2010 to 2013. All the patients did not get any therapy prior to surgery and were confirmed by pathological diagnoses of the surgical specimens. The samples were placed into liquid nitrogen once collected and conserved in −80°C refrigerator for further study. Adjacent normal tissues were defined as the tissues collected from the area more than 3 cm away from the border of tumor tissues.

Pathological sections of pancreatic cancer were granted by the Department of Pathology, Zhongshan Hospital of Fudan University (Shanghai, China).

All the patients involved in this research signed the informed consents. This study was approved by the Institutional Review Board of Zhongshan hospital.

Cell lines and cell culture

HEK293T cells and five pancreatic cancer cells (PANC-1, MIA PaCa2, SW1990, CFPAC-1, and Capan-2) were obtained from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified eagle medium (DMEM, Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS, Invitrogen) and antibiotics (Sigma, St. Louis, MO, USA). Cells were kept in a humidified incubator containing 5% CO₂ at 37°C.

Real-time polymerase chain reaction

Total RNA from clinical samples was extracted using TRIzol reagent (Invitrogen) and then reverse transcribed to complementary DNA (cDNA). Real-time polymerase chain reaction (PCR) was performed in triplicate in an iCycler iQ System (Bio-Rad, Hercules, CA, USA) using LightCycler-DNA Master SYBR Green I mixture. Amplification reaction conditions were set as follows: 95°C for 3 min, 40 cycles of 95°C for 3 s, 62°C for 20 s, 72°C for 30 s, and 72°C for 10 min. All the primers were designed via the software Oligo 7: forward primer of SF3B4 5′-CTCCGAGCGGAATCAGGATG-3′ and reverse primer 5′-GGCATGTGGGTGTTGACTACT-3′, and forward primer of β-actin 5′-GATCATTGCTCCTCCTGAGC-3′ and reverse primer 5′-ACTCCTGCTTGCTGATCCAC-3′. The PCR products from each primer pair were analyzed through melting curves and 1.2% agarose gel electrophoresis to the amplification specificity.

Western blot

Cells were collected and lyzed in radioimmunoprecipitation assay (RIPA) buffer containing phosphatase inhibitors and protease inhibitors for 20 min. Then the cell lysates were centrifuged at 12,000 r/min for 20 min at 4°C. Protein quantitation was conducted using Bradford reagent (Sigma) according to the instructions. Same amounts of protein samples were mixed with loading buffer and heated at 100°C for 5 min. Proteins were then separated through 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) after electrophoresis. The membranes containing target proteins were cut out and blocked with 4% bovine serum albumin (BSA) dissolved in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 1 h at room temperature. And then, incubated with primary antibodies to SF3B4 (Abcam, Cambridge, UK), Flag, STAT3, Bcl2, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Tubulin (Santa Cruz, CA, USA), MUC1 (Absci, MD, USA), and p-STAT3 (Cell Signaling Technology, MA, USA) in appropriate dilution overnight at 4°C. After washed three times with TBST, the membranes were further incubated with corresponding secondary antibodies for 1 h at room temperature. The protein bands were detected by enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL, USA).

Immunohistochemistry

The tissue slides were roasted for 30 min at 60°C and then immersed in xylene quickly. After rehydration through graded ethanol, the slides were placed in a water bath with 0.01 mol/L citrate buffer (pH 6.0, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) at 98°C for 30 min for antigen retrieval. Endogenous peroxidase activity was eliminated by methanol containing 0.3% H₂O₂. The sections were then blocked with 2% BSA at room temperature for 60 min to eliminate nonspecific staining. After incubated with primary antibody to SF3B4 (1:200 dilution, Abcam, Cambridge, UK) in a moist chamber at 4°C overnight, the slides were washed with 0.01 mol/L phosphate-buffered saline (PBS; 8 mmol/L Na₂HPO₄, 2 mmol/L NaH₂PO₄, and 150 mmol/L NaCl) and covered with secondary antibody (Jie Hao Biotechnology, Shanghai, China) according to the manufacturer’s instructions for 1 h. The bound antibody was detected by 3,3′-diaminobenzidine tetrachloride (DAB; Sigma) chromogen solution. All sections were counterstained with hematoxylin and then dehydrated and mounted.

The immunohistochemical evaluation for the expression of SF3B4 in pancreatic cancer tissues and normal tissues was carried out by two independent experts according to the intensity and extent of staining. Staining intensity for SF3B4 was scored as 0 to 3 (0 = negative; 1 = weak; 2 = moderate; 3 = strong). Staining extent was also scored into five categories: 0 (0%), 1 (1%–24%), 2 (25%–49%), 3 (50%–74%), and 4 (75%–100%), depending on the percentage of positive-stained cells. The level of target protein was evaluated by immunoreactive score, which was the product of intensity score and percentage score.

Overexpression of SF3B4 in pancreatic cancer cells

To construct the SF3B4 expression vector, the coding sequence of SF3B4 gene was amplified by PCR. The primers were designed as follows: forward primer 5′-ATAGGATCCATGGCTGCCGGGCCGATCTC-3′ and reverse primer 5′-GCAAGCTTTTACTGAGGGAGAGGGCCTCGAAGTGG-3′. Then, the amplified sequence was inserted into the vector pCMV-Tag2B using BamHI and HindIII and combined with a flag tag. Again, the sequence of flag-SF3B4 fusion gene was amplified by PCR and the products were transferred to the lentiviral vector pCDH. The SF3B4 lentiviral vector and empty pCDH vector were transfected into HEK293T cells, and virus was collected from the culture medium. After infected by the virus for 24 h, the pancreatic cancer cells PANC-1 and MIA PaCa2 were selected with the culture medium containing puromycin. The resistant cells were pooled, and western bolt was carried out to check the infection efficiency.

Knocking down the expression of SF3B4 in pancreatic cancer cells

RNA interference (RNAi) lentiviral plasmids (si con and si SF3B4) were purchased from GeneChem (Shanghai, China). The target sequences of SF3B4 for RNAi were 5′-CCGTATCTTATGCCTTCAA-3′ (1#) and 5′-TCCTATCACCGTATCTTAT-3′ (2#), and the control sequence was 5′-TTCTCCGAACGTGTCACGT-3′. The RNAi and control plasmids were transfected into 293T cells, and culture medium containing virus was gathered after 24 and 48 h. Pancreatic cancer cells SW1990 and MIA PaCa2 were infected by the virus and then selected by puromycin.

Crystal violet assay

Equal amounts of pancreatic cancer cells (1 × 10³/well) were seeded in six-well plates and cultured in DMEM containing 10% FBS for 10 days. Culture medium was renewed every 2 days. The medium was removed after 10 days, and the cells were washed with PBS twice. Then, the cells were stained with 0.5% crystal violet solution in 20% methanol and washed with clean water after staining for 20 min and photographed. The stained cells were dissolved with 1% SDS solution, and the absorbance was measured using a microplate reader at 600 nm.

MTT assay

Equal amounts of pancreatic cancer cells (1 × 10³/well) were seeded in 96-well plates for various durations with three replicates each time; 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 20 µL/well) was added and incubated for 4 h. The treated cells were then dissolved with 200 µL dimethyl sulfoxide (DMSO; Sangon Biotech, Shanghai, China) and optical density (OD) was detected at 540 nm using a microplate reader.

Cell migration assay

Cell migration assay was conducted using a modified Boyden chamber. A total of 2 × 10⁵ cells (250 µL) suspended in DMEM supplemented with 1% FBS were placed in upper chamber, and 400 µL culture medium containing 10% FBS was added into the lower chamber. After incubated for 12 h, cells that remained on the upper surface of filter membrane were removed and invaded cells were fixed and stained by hematoxylin and eosin.

Luciferase reporter assay

Cells were seeded in 24-well plates at a suitable density and incubated for 24 h. A volume of 0.05 µg STAT3 reporter plasmid (granted by the lab of Chin YE, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China) and a volume of 0.02 µg Renilla luciferase pRL-TK as an internal control were co-transfected using lipofectamine 2000 and IL-6 (5 ng/mL) was added after transfected for 24 h. Cell lysates were prepared 8 h after treatment, and reporter activities were detected by the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

Statistical analysis

Data of three independent experiments were expressed as mean ± standard deviation (SD). All statistical analyses were performed with SPSS statistics v19.0 (IBM Corp., Armonk, NY, USA). The differences between experimental group and control group were analyzed by Student’s t-test. p < 0.05 was considered statistically significant.

Results

SF3B4 was downregulated in pancreatic cancer samples

To investigate the expression of SF3B4 in pancreatic cancer, we collected 63 paired tumor and adjacent normal tissues (Table 1). The messenger RNA (mRNA) level of SF3B4 was examined by real-time PCR. As the results shown in Figure 1(a), the expression of SF3B4 was decreased in 50 (79.4%) cancer tissues compared with matched adjacent noncancerous tissues, and the overall mRNA abundance was significantly reduced in tumor group (p < 0.001). Furthermore, western blot was performed to detect the protein level of SF3B4 in pancreatic cancer. Consistent with the results shown in real-time PCR, downregulation of SF3B4 was observed in all seven paired specimens in varying degrees (Figure 1(b)). In addition, immunohistochemical staining of pancreatic cancer sections further confirmed the downregulation of SF3B4 protein level in pancreatic cancer tissues (Figure 1(c)). Taken together, all the above observations indicated that the downregulation of SF3B4 might play an important role in pancreatic cancer.

Table 1.

Summary of clinicopathological variables.

Characteristics	Number of patients
Gender
Male	43
Female	20
Age (years)
≤60	25
>60	38
Tumor size
≤2 cm	8
>2 cm	55
Tumor location
Head	40
Body/tail	23
Differentiation
Well	15
Moderate to poor	48
Tumor-node-metastasis (TNM) stage
I	27
II	36
Lymph node metastasis
Yes	28
No	35
Smoking status
Yes	31
No	32

Figure 1.

SF3B4 was downregulated in pancreatic cancer. (a) The mRNA expression of SF3B4 was performed by real-time PCR in 63 pancreatic cancer specimens and paired normal tissues. The SF3B4 expression was normalized to that of β-Actin. Data were calculated from triplicates, (b) the protein expression of SF3B4 was conducted by western blot in seven paired pancreatic cancer samples, (c) the protein level of SF3B4 in pancreatic cancer samples was examined via IHC, and (d) the expression of SF3B4 was investigated in different pancreatic cancer cell lines (PANC-1, MIA PaCa2, SW1990, CFPAC-1, and Capan-2).

Overexpression of SF3B4 inhibited the growth and migration of pancreatic cancer cells

Next, we assessed its biological functions in pancreatic cancer cells. Pancreatic cancer cells PANC-1 and MIA PaCa2 were infected with lentivirus containing either a pCDH vector inserted with full-length sequences of SF3B4 or an empty pCDH vector as a control. Puromycin-resistant cells were pooled, and the expression of SF3B4 was checked by western blot (Figure 2(a)). Crystal violet assay was performed to analyze the effect of SF3B4 on cell growth. The result showed that colony numbers of PANC-1 and MIA PaCa2 cells overexpressing SF3B4 were decreased significantly compared with control groups (Figure 2(b), p < 0.001 for both cell lines). A similar finding was obtained in MTT assay (Figure 2(c), p = 0.002 for PANC-1, p = 0.006 for MIA PaCa2). Moreover, migration assay was performed using a modified Boyden chamber. The upregulation of SF3B4 inhibited the mobility of cancer cells dramatically (Figure 2(d), p = 0.03 for PANC-1, p < 0.001 for MIA PaCa2). These results suggested that overexpression of SF3B4 showed suppressive effects on pancreatic cancer cells.

Figure 2.

Overexpression of SF3B4 suppressed the proliferation and migration abilities of pancreatic cancer cells. (a) Enhanced expression of SF3B4 in PANC-1 and MIA PaCa2 cells, (b) crystal violet assay to analyze the impacts of SF3B4 on the growth of PANC-1 and MIA PaCa2 cells, (c) MTT assay to evaluate the influences of SF3B4 on the proliferation of PANC-1 and MIA PaCa2 cells, and (d) enhanced expression of SF3B4 inhibited the migration of PANC-1 and MIA PaCa2 cells in the migration assay using a Boyden chamber. Each experiment was performed at least three times.

Knocking down the endogenous expression of SF3B4 promoted the growth and migration of pancreatic cancer cells

To further evaluate the effects of endogenous SF3B4 in pancreatic cancer, we knocked down the basal expression of SF3B4 in SW1990 and MIA PaCa2 cells with RNAi (Figure 3(a)). Similarly, crystal violet assay was carried out to measure the influence of downregulation SF3B4 on cell growth. It was found that knocking down the endogenous expression of SF3B4 promoted the growth of SW1990 and MIA PaCa2 cells (Figure 3(b), p < 0.001 for si1# and si2# in MIA PaCa2, p < 0.001 for si1# and si2# in SW1990). And, MTT assay showed a similar result (Figure 3(c), p < 0.001 for si1# and p = 0.001 for si2# in MIA PaCa2, p = 0.001 for si1# and p = 0.019 for si2# in SW1990). In addition, Boyden chamber assay revealed that the migration ability of pancreatic cancer cells was also enhanced (Figure 3(d), p < 0.001 for si1# and p = 0.001 for si2# in MIA PaCa2, p = 0.006 for si1# and p = 0.037 for si2# in SW1990). These observations demonstrated that downregulating the expression of SF3B4 might play an oncogenic role in pancreatic cancer.

Figure 3.

Knocking down the expression of SF3B4 promoted the proliferation and migration abilities of pancreatic cancer cells. (a) Knocking down the expression of SF3B4 in SW1990 and MIA PaCa2 cells by RNAi, (b) crystal violet assay to analyze the impacts of knocking down SF3B4 on the growth of SW1990 and MIA PaCa2 cells, (c) MTT assay to evaluate the influences of knocking down SF3B4 on the proliferation of SW1990 and MIA PaCa2 cells, and (d) knocking down the expression of SF3B4 promoted the migration of SW1990 and MIA PaCa2 cells in the migration assay using a Boyden chamber. Each experiment was performed at least three times.

SF3B4 inhibited STAT3 signaling in pancreatic cancer cells

In order to study the underlying molecular mechanisms, we conducted the screening via luciferase reporter assay. And the outcome indicated that SF3B4 overexpression inhibited the reporter gene of STAT3 signaling, and more obvious suppression was observed by the treatment of IL-6 (Figure 4(a), p = 0.013, p = 0.002). As many studies reported, IL-6 is an important pro-inflammatory cytokine strongly linked to pancreatic cancer and IL-6-JAK signaling is crucial for persistent STAT3 activation in cancer.²³ Thus, we treated tumor cells with IL-6 to further activate the phosphorylation of STAT3, under which background we could detect more obvious inhibition of SF3B4 on STAT3. To confirm the above results, we collected the protein samples in luciferase reporter assay treated with IL-6 and western blot showed the significant suppression of phosphorylated STAT3 (tyrosine residue at position 705 (Tyr705)) by SF3B4 (Figure 4(b)). Moreover, we transfected SF3B4 plasmid into 293T cells and found that forced expression of SF3B4 decreased the phosphorylation level of STAT3 (Figure 4(c)). Similar observations were found in PANC-1 and MIA PaCa2 cells. In addition, the expressions of the STAT3 target genes, Bcl2 and MUC1, were reduced as well (Figure 4(d)). Moreover, knocking down of endogenous SF3B4 elevated both the phosphorylated level of STAT3 and downstream genes of STAT3 signaling (Figure 4(e)). Taken together, all these data suggested that SF3B4 inhibited STAT3 signaling.

Figure 4.

SF3B4 inhibited the activity of STAT3 signaling pathway. (a) Overexpression of SF3B4 decreased the activity of STAT3 reporter in MIA PaCa2 cells and inhibited more obviously stimulated by IL-6, (b) overexpression of SF3B4 suppressed the phosphorylation of STAT3 in MIA PaCa2 under the condition of IL-6 stimulation, (c) overexpression of SF3B4 inhibited the phosphorylation of STAT3 in HEK293T cells, (d) enhanced expression of SF3B4 in MIA PaCa2 and PANC-1 cells inhibited the phosphorylation of STAT3 and decreased the expression of STAT3 target genes, MUC1 and Bcl2, and (e) knocking down the expression of SF3B4 in MIA PaCa2 promoted the phosphorylation of STAT3 and increased the expression of STAT3 target genes.

Discussion

Alternative splicing is a crucial process bringing about the diversity of protein, which undertakes the vast majority of biological functions in organism. Emerging evidence has manifested that splicing factors involve in various malignant tumors.^24–26 In the present study, we investigated the expression and functions of SF3B4 in pancreatic cancer. Our results showed that the expression of SF3B4 was downregulated in pancreatic cancer tissues compared with paired adjacent normal tissues both on the mRNA and protein level. Enhancing expression of SF3B4 in pancreatic cancer cells inhibited cell growth and mobility, while silencing the endogenous SF3B4 expression promoted the growth and migration of pancreatic cancer cells. Altogether, these findings suggested that SF3B4 might act as a repressor in pancreatic cancer. However, recent researches revealed that SF3B4 expression was significantly increased in HCC and associated with poor outcome in patients with HCC, which seemed to be contradictory with what we found in pancreatic cancer.^14,27 We surmised that the gene expression spectrums might be different in various tumors. Previous studies of lung cancers discovered that splicing factors SFRS5 and SFRS6 were dramatically upregulated in tumors compared with normal tissues, whereas the expression level of the splicing factors was decreased in pancreatic cancer.^28,29 This also suggested that the regulation of alternative splicing might differ according to tumor types. Carrigan et al.²⁹ detected the expression patterns of 92 genes encoding splicing factors in pancreatic cancer and found 28 tested genes got downregulated expressions in tumor samples. And interestingly, none of the splicing factors were increased in cancer tissues. These findings implied that splicing factors might serve as a tumor suppressor “family” in pancreatic cancer.

In the further study of molecular mechanism, we found that SF3B4 could affect the activity of STAT3 signaling pathway via inhibiting the phosphorylation of STAT3 (Tyr705). As far as we know, this is the first report that SF3B4 can suppress STAT3 signaling. STAT3, as a critical mediator in the signaling pathway, is mainly located in the cytoplasm in inactive state.³⁰ STAT3 forms dimers and then translocates into the cell nucleus to regulate the expressions of target genes, once activated by phosphorylation of Tyr705, which catalyzed by various tyrosine kinases such as VEGFR, EGFR, PDGFR and nonreceptor tyrosine kinases such as JAK, Src.^31,32 Numerous studies have demonstrated that persistently activated STAT3 signaling took a pivotal part in tumorigenesis by regulating cellular proliferation, migration, apoptosis, and angiogenesis.^33,34 And many attempts have been done to block this signaling for the treatment of cancer, such as the development of tyrosine kinase inhibitors AG490, cucurbitacin, and small molecule modulators FILL31 and FILL32.^35–37 However, litter therapeutic effect has been achieved, which is ascribed to the complicated regulation of STAT3 signaling.

Alternative splicing has been demonstrated to play multiple roles in STAT signaling by generating various protein isoforms of the pathway, such as the C-terminally truncated isoforms of STAT1, 3, 4, 5A, and 5B that lack the transactivation domain.³⁸ Shchelkunova et al.³⁹ developed a new approach to inhibit the expression of full-length STAT5B (a proto-oncogene) while enhancing STAT5ΔB (a tumor suppressor) through alternative splicing, which brought a novel strategy for anti-tumor treatment. In addition, interleukins (ILs), the major stimulators of inflammatory signaling mediated by STATs, can be regulated by alternative mRNA exon splicing in a tissue-specific manner. Lee et al.⁴⁰ found IL-15ΔE7, an alternatively spliced IL-15 isoform, could inactivate the JAK/STAT5 signaling pathway and decrease the expression of downstream target Bcl2 by suppressing the phosphorylation of STAT5, which indicated the competitive inhibitor role of this isoform. Other studies indicated that the isoforms of IL-15 receptor, IL-15Rα2 and α4, could inhibit the activity of STAT3 significantly even upon IL-15 stimulation.⁴¹ As we all know, inflammatory pathways take an important part in the initiation and progression of pancreatic cancer. It is possible that the isoforms of ILs or their receptors, which are alternatively spliced by SF3B4, inhibit the phosphorylation of STAT3 in pancreatic cancer. And we will verify the assumption in our further research.

Some limitations still exist in this research, such as the biological function experiments of SF3B4 have not been conducted in vivo. In addition, the exact mechanism through which SF3B4 regulates STAT3 need to be further investigated. In conclusion, our study revealed the expression and functions of SF3B4 in pancreatic cancer and presented a promising strategy for tumor treatment.

Footnotes

W.Z.,N.M.,and H.J. contributed equally to this work.

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

This research was supported by grants from the National Natural Science Foundation of China (No. 81401923).

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SF3B4 is decreased in pancreatic cancer and inhibits the growth and migration of cancer cells

Abstract

Keywords

Introduction

Materials and methods

Patients and tissue samples

Cell lines and cell culture

Real-time polymerase chain reaction

Western blot

Immunohistochemistry

Overexpression of SF3B4 in pancreatic cancer cells

Knocking down the expression of SF3B4 in pancreatic cancer cells

Crystal violet assay

MTT assay

Cell migration assay

Luciferase reporter assay

Statistical analysis

Results

SF3B4 was downregulated in pancreatic cancer samples

Overexpression of SF3B4 inhibited the growth and migration of pancreatic cancer cells

Knocking down the endogenous expression of SF3B4 promoted the growth and migration of pancreatic cancer cells

SF3B4 inhibited STAT3 signaling in pancreatic cancer cells

Discussion

Footnotes

Declaration of conflicting interests

Funding

References