Musashi2 promotes the progression of pancreatic cancer through a novel ISYNA1‐p21/ZEB‐1 pathway

Abstract Our previous studies found overexpression of Musashi2 (MSI2) conduced to the progression and chemoresistance of pancreatic cancer (PC) by negative regulation of Numb and wild type p53 (wtp53). Now, we further investigated the novel signalling involved with MSI2 in PC. We identified inositol‐3‐phosphate synthase 1 (ISYNA1) as a novel tumour suppressor regulated by MSI2. High MSI2 and low ISYNA1 expression were prevalently observed in 91 PC tissues. ISYNA1 expression was negatively correlated with MSI2 expression, T stage, vascular permeation and poor prognosis in PC patients. What's more, patients expressed high MSI2 and low ISYNA1 level had a significant worse prognosis. And in wtp53 Capan‐2 and SW1990 cells, ISYNA1 was downregulated by p53 silencing. ISYNA1 silencing promoted cell proliferation and cell cycle by inhibiting p21 and enhanced cell migration and invasion by upregulating ZEB‐1. However, MSI2 silencing upregulated ISYNA1 and p21 but downregulated ZEB‐1, which can be rescued by ISYNA1 silencing. Moreover, reduction of cell migration and invasion resulting from MSI2 silencing was significantly reversed by ISYNA1 silencing. In summary, MSI2 facilitates the development of PC through a novel ISYNA1‐p21/ZEB‐1 pathway, which provides new gene target therapy for PC.

and chemoresistance of PC through negative regulation of Numb and wild type p53 (wtp53). 8,9 Myoinositol, a kind of water-soluble vitamin synthesized in cells, exhibits various functions, such as seawater acclimation, glucose-lipid metabolism and tumour suppression. [10][11][12][13] Inositol-3-phosphate synthase 1 (ISYNA1) encodes a necessary limit-enzyme in myoinositol biosynthesis and is a direct target of wtp53 in HCT116 and HepG2 cells. 15 Meanwhile, ISYNA1 is closely related with the chemotherapeutic resistance in malignant melanoma. 16 However, the coordinate role of ISYNA1 and MSI2 in the initiation of PC, to our knowledge, has not been reported, which would be investigated in current study.

| Western blot
The whole protein lysates extracted by BCA method were boiling with loading buffer, added into 10% SDS-polyacrylamide gels for electrophoresis, next proteins were transferred into polyvinylidene difluoride membranes(PVDM), then membranes were and β-actin (Proteintech) antibodies at 4°C at least 6 hours or overnight. Membranes were immersed with secondary antibodies (Santa Cruz) at room temperature for 2 hours and finally detected by the ECL kit. The grey values of each band were analysed by Quantity One software. The experiments were repeated three times.

| qRT-PCR
1ml TRIzol reagent was added into each cell and tissue sample for extraction of total RNA under the manufacturer. Using nucleotide determination, RNA level of each sample was firstly maintained at the same level. Then total RNA was used to synthesize cDNA by the Expand Reverse Transcriptase Kit. Finally, ISYNA1 and GAPDH expression were detected by a Light Cycler 2.0 with the Light Cycler kit. The reaction conditions were denaturation at 95°C for 30 seconds, 45 cycles of amplification at 95°C for 5 seconds and dissolution at 60°C for 45 seconds. The primer sequences were seen in Table S1. The relative ISYNA1 expression was counted by ΔCT: ΔCT(ISYNA1)-ΔCT(GAPDH). The experiments were repeated three times.

| Cell proliferation assays
After transfection with siRNA control and si-ISYNA1-1 for 48-72 hours, the Capan-2, SW1990 and Miapaca-2 cells were inoculated to 96-well plates at a suitable density (5000-6000 cells per well) and cultured for 1-4 days. Next, 20 μL MTT solution (5 mg/ mL) was added into each well for 4 hours. Then removing cell supernatants and cells were treated with 100 μL dimethyl sulphoxide. The optical density (OD) value at 560 nm was finally calculated by the microplate reader. The experiments were repeated three times.  calculated under five random fields at 200× magnification. 5 × 10 6 cells/mL and 2.5 × 10 6 cells/mL of SW1990 cells and 2 × 10 6 cells/ mL and 1 × 10 6 cells/mL of Capan-2 cells were used to perform cell migration and invasion experiments, respectively. The experiments were repeated three times.

| Statistical analysis
Statistical analyses were conducted by SPSS software 20.0 (SPSS).
The differences between expression of MSI2 and ISYNA1 in pancreatic cancer and adjacent normal pancreatic tissues were analysed by t test. The clinicopathological significance of ISYNA1 and MSI2, and their relationship were estimated by chi-squared and correlation analysis, respectively. Survival of patients was analysed by the Kaplan-Meier curve, while Log-rank test was used to evaluate the differences. The differences of cell proliferation and cycle, cell migration and invasion were analysed by t test. P < .05 was considered to be statistically significant.
Similarly, in 57 paired specimens, ISYNA1 expression in PC tissues cells compared with other cell lines ( Figure 1E). Immunofluorescence staining showed ISYNA1 was expressed in both nucleus and cytoplasm in pancreatic cancer cell lines ( Figure 1F), which was consistent with the results in IHC assays.

| Association between MSI2 and ISYNA1 expression with patients' parameters and survival time
In our previous study, we proved that MSI2 overexpression was positively associated with tumour size, UICC stage and poor prognosis of pancreatic cancer patients. 8 In current study, ISYNA1 expression was negatively related to T stage (P = .035) and vascular permeation (P = .030) in 91 PC patients (Table 1). Moreover, high expression of MSI2 with low ISYNA1 expression was found in serial pancreatic cancer sections and vice versa (Figure 2A). Correlation analysis identified a negative correlation between their expression in pancreatic cancer tissues (P = .032) ( Table 2). Kaplan-Meier indicated that patients expressed high MSI2 or low ISYNA1 had a worse survival time (P = .005 and P = .015, respectively) ( Figure 2B,C).
Moreover, patients expressed high MSI2 and low ISYNA1 underwent a much worse prognosis (P = .001) ( Figure 2D). In addition, univariate analysis showed lymph nodes metastasis and UICC stage were also related to the prognosis of pancreatic cancer patients. And in multivariate analysis, the expression of MSI2 and ISYNA1 were independent unfavourable prognostic indicators for pancreatic cancer (Table 3).

| ISYNA1 was downregulated by wtp53 in Capan-2 and SW1990 cells
Capan-2 and SW1990 cells expressed wtp53 while Miapaca-2 cell expressed mutant p53. The relationship between p53 and ISYNA1 in PC cells remained unclear. WB showed p53 silence decreased ISYNA1 expression in wtp53 Capan-2 and SW1990 cells ( Figure 3A,B), but not in mtp53 Miapaca-2 cell ( Figure 3C). And similar outcomes were also obtained in PCR assays ( Figure 3D). It indicated that wtp53 but not mtp53 had a positive regulation targets ISYNA1.

| ISYNA1 silencing promoted cell migration and invasion by upregulating ZEB-1 in Capan-2 and SW1990 cells
Transwell assay demonstrated that cell migration and invasion was prominently increased in si-ISYNA1 group in comparison to siRNA control group in Capan-2 and SW1990 cells ( Figure 5A,B).
Myoinositol, as a kind of water-soluble vitamin synthesized, acts as a tumour suppressive role in various cancers. However, its function remains unclear in PC. We found that myoinositol significantly suppressed cell migration and invasion in vitro ( Figure 5C,D). WB showed an evident upregulation of ZEB-1 in si-ISYNA1 groups compared with control, but other proteins (MSI2, p53, E-cadherin, β-Catenin, N-cadherin and snail-1) were unchanged ( Figure 5E,F).

| ISYNA1 silencing reversed reduction of cell migration and invasion caused by MSI2 silence in Capan-2 and SW1990 cells
Crispr-cas9 mediated MSI2 silencing Capan-2 and SW1990 stable cells were successfully constructed ( Figure 7A,B), which were cotransfected with ISYNA1siRNA. Transwell assays indicated cell migration was prominently decreased in MSI2 silencing Capan-2 and SW1990 cells compared with control group. However, ISYNA1 silence obviously reversed reduction of cell migration caused by MSI2 silence (Figure 6A). Similarly, ISYNA1 silence reversed the reduction of cell invasion resulted from MSI2 silence in Capan-2 and SW1990 cells ( Figure 6B). Taken together, MSI2 promoted cell migration and invasion by downregulating ISYNA1 protein in Capan-2 and SW1990 cells.

| MSI2 and ISYNA1 coordinately regulated p21 and ZEB-1 in vitro
We next researched the potential relationship of MSI2, p53,

| D ISCUSS I ON
Our previous studies indicated that MSI2 overexpression made for the progression of pancreatic cancer by negative regulation of Numb and wtp53 with the stimulation of gemcitabine or cisplatin. 8,9 In current study, we further investigated the novel signalling in terms of MSI2 in PC tissue and cell level. We identified ISYNA1 as a novel candidate tumour suppressor regulated by MSI2 and wtp53, which has to been reported, to our knowledge.
ISYNA1 encodes an inositol-3-phosphate synthase enzyme and plays a vital role in cellular myoinositol biosynthesis, which is closely related to the tumorigenesis. 14 In current study, ISYNA1 was downregulated in pancreatic cancer tissues and was negatively related to MSI2 expression, T stage, vascular permeation and poor prognosis of PC patients. Meanwhile, patients expressed high MSI2 and low ISYNA1 underwent a worse prognosis all which is not reported previously to our knowledge. Previous studies focus on ISYNA1 function in gene differential methylation and isoforms identification. 19,20 Recently, a high concentration of myoinositol in tumours is found related to high levels of ISYNA1. 21 However, studies involved the role of ISYNA1 in malignant tumours remain scarce and controvertial. The expression of ISYNA1 and myoinositol in gliomas was significantly higher than that in primary central nervous system lymphomas. 22 Overexpression of ISYNA1 suppressed cell proliferation in HCT116 and HepG2 cells. 15 The inconsistent outcomes might be due to the difference in cancer types and environment.
Our previous study reported that MSI2 silencing upregulated wtp53 only under chemotherapy stimulus. 9 Here, we first found that MSI2 silencing upregulated ISYNA1 and identified ISYNA1 as a direct target of wtp53 in Capan-2 and SW1990 cells. However, in mtp53 Miapaca-2 cell, p53 silence had no effects on ISYNA1. Also, MSI2 silence had no effect in both wtp53 and mtp53 in normal PC cells in our current and previous studies. 9 Thus, ISYNA is a down- ZEB-1, a member of ZHF family, was closely associated with epithelial-mesenchymal transition in many tumours, such as colorectal cancer and PC. [27][28][29] Its abnormal expression contributed to the invasion and metastasis of carcinoma by inducing EMT. 30 Here, we firstly reported that ISYNA1 silence promoted cell migration and invasion via upregulating ZEB-1 in PC. Furthermore, ISYNA1 silence reversed the reduction of cell migration, cell invasion and ZEB1 protein resulted from MSI2 silence in vitro. Thus, the overexpression of MSI2 facilitates cell migration and cell invasion in pancreatic cancer by regulating ISYNA1-ZEB-1 pathway.
In summary, MSI2 accelerates the development and progression of pancreatic cancer through a novel ISYNA1-p21/ZEB-1 pathway, which supplies a novel gene targets in the PC treatment.

ACK N OWLED G EM ENTS
The study was sustained by the Chinese National Science

DATA AVA I L A B I L I T Y S TAT E M E N T
All data will be available upon reasonable request.