Piezo type mechanosensitive ion channel component 1 facilitates gastric cancer omentum metastasis

Abstract The peritoneum, especially the omentum, is a common site for gastric cancer (GC) metastasis. Our aim was to expound the role and mechanisms of Piezo1 on GC omentum metastasis. A series of functional assays were performed to examine cell proliferation, clone formation, apoptosis, Ca2+ influx, mitochondrial membrane potential (MMP) and migration after overexpression or knockdown of Piezo1. A GC peritoneal implantation and metastasis model was conducted. After infection by si‐Piezo1, the number and growth of tumours were observed in abdominal cavity. Fibre and angiogenesis were tested in metastatic tumour tissues. Piezo1 had higher expression in GC tissues with omentum metastasis and metastatic lymph node tissues than in GC tissues among 110 patients. High Piezo1 expression was associated with lymph metastasis, TNM and distant metastasis. Overexpressed Piezo1 facilitated cell proliferation and suppressed cell apoptosis in GC cells. Moreover, Ca2+ influx was elevated after up‐regulation of Piezo1. Piezo1 promoted cell migration and Calpain1/2 expression via up‐regulation of HIF‐1α in GC cells. In vivo, Piezo1 knockdown significantly inhibited peritoneal metastasis of GC cells and blocked EMT process and angiogenesis. Our findings suggested that Piezo1 is a key component during GC omentum metastasis, which could be related to up‐regulation of HIF‐1α.


| INTRODUC TI ON
Gastric cancer (GC) is one of the most common gastrointestinal malignancies globally. 1,2 Tumour metastasis is a pivotal biological feature of GC and is also the main cause of deaths for GC patients.
About 53%-66% of distant metastatic GC occurs in the peritoneum, especially the omentum. 3 When the GC cells spread to the abdominal cavity, they may grow in the abdominal viscera and omentum. 3 Surgical treatment often fails to eradicate these cells. Therefore, patients' prognosis is still poor. Postoperative chemotherapy can destroy metastasis and residual tumours, which has been considered as an effective treatment for postoperative GC metastasis. 4 However, because of the presence of the peritoneal serosal barrier, most intravenous chemotherapy drugs cannot reach the abdominal cavity.
Also, chemotherapy drugs have a small molecular weight and are easily absorbed in the abdominal cavity. Thus, the efficacy of postoperative chemotherapy is relatively low. However, the mechanism by which GC cells transform from abscission to omental metastasis is unclear.
Piezo type mechanosensitive ion channel component 1 (Piezo1) protein is a mechanically sensitive cation channel. 5,6 As morphological changes occur in epithelial cells, cell homeostasis becomes imbalanced, and Piezo1 is activated, thereby accelerating Ca 2+ influx and cell cycle arrest. 7,8 In the early stages of tissue development, Piezo1 is activated by shearing force and Calpain is then activated in endothelial cells (ECs), leading to the division of focal adhesions and angiogenesis. 9 Piezo1 can promote vascular EC migration by activation of Ca 2+ channels, thereby driving embryonic angiogenesis. 10 Cell mobility is a key characteristic to ensure that tumour cells invade surrounding tissues and occur distant metastasis. Hypoxia may enhance the migrant ability of tumour cells that is closely related to tumour metastasis. In the hypoxic tumour microenvironment, hypoxia inducible factor 1α (HIF-1α), as a transcription factor, is notably elevated in tumour cells. 11 More importantly, its up-regulation can accelerate epithelial-mesenchymal transition (EMT) and angiogenesis via vascular endothelial growth factor (VEGF), thereby promoting tumour growth and metastasis. Thus, HIF-1α has been considered as an upstream regulatory factor of VEGF. Moreover, mitochondrial calcium ion influx could promote VEGF expression via up-regulation of HIF-1α, inducing angiogenesis. 12 It has been found that Piezo1 could mediate HIF-1α through Ca 2+ influx, which contributes to cyclical hydrostatic pressure. 13 In this study, we described the role and underlying molecular mechanisms of Piezo1 on GC omental implantation and metastasis.
High Piezo1 expression was identified in GC tissues with omentum metastasis and metastatic lymph node tissues. Consistent with previous studies, Piezo1 could accelerate cell proliferation and migration in GC cells. 14,15 Furthermore, Piezo1 could promote cell migration and Calpain1/2 expression via up-regulation of HIF-1α in GC cells. In peritoneal metastatic GC mouse model, Piezo1 knockdown could notably inhibit peritoneal metastatic tumour growth, block EMT process and angiogenesis. Therefore, Piezo1 could become a potential therapeutic target for GC patients with omentum metastasis.

| Tissue specimens
In total, 110 formalin-fixed and paraffin-embedded gastric cancer specimens were gathered from North China University of Science

| qRT-PCR
Extracted RNA from tissues or cells was reverse transcribed into cDNA by a reverse transcriptase kit (Invitrogen, #K1622, USA). qRT-PCR was presented via TB Green® Premix Ex Taq™ II kit (TAKARA, RR820A, Japan). The relative expression levels were quantified with the 2 −ΔΔCt method. Primer sequence information is listed in Table 1.

| Western blot and co-immunoprecipitation
Cells or tissues were extracted using RIPA lysis plus protease inhibitor on the ice. For co-immunoprecipitation, cell lysates were extracted and incubated with 2 μg specific antibodies for 18 hours at 4°C. Then, samples were incubated with 50 μL protein G agarose beads for 3 hours. After washing the beads for 5 times by K E Y W O R D S Ca 2+ influx, gastric cancer, HIF-1α, migration, omentum metastasis, Piezo1 lysis buffer, the precipitated proteins were resuspended in 30 μL SDS sample buffer and boiled at 95°C for 10 minutes. Proteins were separated by SDS-PAGE and transferred onto PVDF membrane (Merck Millipore, Germany). The membrane was blocked by 0.5% skimmed milk for 2 hours at room temperature. Then, the membrane was incubated with primary antibodies at 4 ℃ overnight, followed by incubation with secondary antibodies at room temperature for 2 hours. Primary antibodies were as follows: anti-Piezo1 (1:500; ProteinTech, #15939-1-AP, China), anti-Calpain1 anti-β-actin (1:1000; Abcam, #ab179467). Protein blots were visualized using ECL luminescence kit.

| Immunohistochemistry and immunofluorescence staining
For immunohistochemistry staining, formalin-fixed and paraffinembedded specimens were incubated with primary antibodies at

| Clone formation assay
Cells were inoculated into 6-well plates (1000 cells/well), followed by 10 days' culture. Cells were then fixed with paraformaldehyde for 15 minutes and stained with 1% crystal violet for 20 minutes.
The number of cells was counted under a microscope (Olympus, Japan).

| Wound healing assay
Cells were seeded onto 6-well plates. When the cell confluence reached 80%, the cells were scratched with a 200 μm pipette tip.
Then, the cells were incubated in a serum-free medium for 48 hours and the results were observed under a microscope.

| Construction of a nude mouse model of GC peritoneal metastasis
All nude mice were randomly assigned to two groups, including control group (n = 5) and si-Piezo1 group (n = 5). The nude mice were anaesthetized by 0.5% pentobarbital sodium, which were intraperitoneally injected with 300 μL serum-free medium resus-

| Statistical analysis
The data were analysed using SPSS 23.0 software (IBM SPSS, Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad, San Diego, CA). Each assay was repeated in triplicate. Data were expressed as F I G U R E 1 High Piezo1 expression in GC tissues along with omentum metastasis. A, Piezo1 was highly expressed in GC tissues than in normal tissues. Red represents gastric cancer samples, and grey represents normal samples. B, Overall survival analysis suggested that high Piezo1 expression predicted poorer overall survival time for patients with GC. C-E, Western blot and qRT-PCR assays were used to examine the expression of Piezo1 in normal tissues, GC tissues and GC tissues with omentum metastasis. F-I, Immunohistochemistry and immunofluorescence results showed the expression and distribution of Piezo1 normal tissues, GC tissues, GC tissues with omentum metastasis or metastatic lymph node tissues. *P < .05; **P < .01; ***P < .001; ****P < .0001 mean ± standard deviation (SD). The comparison of two groups was carried out by student's t test, whereas multiple comparisons were analysed using ANOVA. The correlation between Piezo1 expression and clinical parameters of GC was analysed using Chi test. The Pearson's correlation analysis of the two factors was presented.

| Piezo1 is highly expressed in GC tissues with omentum metastasis
In accordance with the gene expression profiling interactive analysis (GEPIA) database (http://gepia.cance r-pku.cn), the expression of Piezo1 in GC tissues was distinctly higher than that in normal tissues ( Figure 1A). Totally, 110 GC patients were recruited in our study, divided into high-and low-expression groups based on the median value of Piezo1 expression. Overall survival (OS) analysis results suggested that Piezo1 was negatively correlated with shorten OS time of gastric cancer patients ( Figure 1B). Also, Piezo1 expression was in association with lymphatic metastasis and with TNM staging (Table 2). At the mRNA and protein levels, Piezo1 expression was significantly higher in GC tissues than that in normal tissues ( Figure 1C-E). A higher expression level of Piezo1 was found in GC tissues with omentum metastasis in comparison with GC tissues (Figure 1C-E).

| Piezo1 facilitates cell proliferation and suppresses cell apoptosis in GC cells
Two siRNAs targeting Piezo1 were designed and synthesized. The transfection efficiency of si-Piezo1 was assessed in two GC cells (SNU-1 and HGC-27) using qRT-PCR and Western blot. We found that Piezo1 mRNA and protein expression was prominently suppressed in two GC cells after transfection with the two siRNAs (Figure 2A-C  with Yoda1 with a concentration-dependent manner ( Figure 2H, I).
The p53/p21 complex may regulate invasion and apoptosis for tumour cells. 16 Thus, we detected the expression of p53 and p21 proteins in two GC cells transfected with si-Piezo1 by Western blot. As shown in Figure 2J-L, the expression of P53 and P21 proteins was distinctly decreased in GC cells after transfection with si-Piezo1. Other cell cycle regulators including CDK4, CDK6 and CyclinD1 were also detected using Western blot. Our data suggested that CDK4, CDK6 and CyclinD1 expression was all significantly lowered following transfection with si-Piezo1 ( Figure 2J-L). Furthermore, our immunohistochemistry results confirmed that Ki-67-positive cells were remarkedly decreased after treatment with si-Piezo1 ( Figure 2M, N). These results suggested that Piezo1 could facilitate cell proliferation and suppress cell apoptosis in GC cells.

| Piezo1 induces the expression of HIF-1α, VEGF and EMT-related proteins in GC cells
In Consistent with qRT-PCR results, E-cadherin protein expression was distinctly decreased after treatment with Yoda1 in GC cells, which was elevated by si-Piezo1 (Supplementary Figure S1).

| Piezo1 could promote cell migration via upregulation of HIF-1α in GC cells
We focused on the effect of dysregulated Piezo1 on HIF-1α and VEGF expression. As expected, immunohistochemistry results suggested that Piezo1 overexpression remarkedly activated the expression of HIF-1α and VEGF proteins in SNU-1 and HGC-27 two GC cells ( Figure 4A-D). In converse, their expression was suppressed following transfection with si-Piezo1. Above results were verified by immunofluorescence ( Figure 4E-H). To explore the relationship between Piezo1 and HIF-1α, we constructed two GC cells stably transfected with HIF-1α and Piezo1 knockdown ( Figure 5A, B). Western blot confirmed that HIF-1α was efficiently silenced in GC cells ( Figure 5C, D).
Transwell results showed that the migrant ability of GC cells was distinctly suppressed by si-Piezo1 and promoted by Yoda1 ( Figure 5E-H).
Furthermore, we found that HIF-1α also inhibited GC cell migration.

| Piezo1 physically interacts with HIF-1α in GC cells
We further evaluated whether Piezo1 could physically interact with HIF-1α. Our co-immunoprecipitation results showed that Piezo1 and HIF-1α existed in the same protein complex in SNU-1 and HGC- Figure S2). Piezo1 knockdown decreased, whereas Piezo1 overexpression increased the interaction between Piezo1 and HIF-1α. Hence, our data indicated that Piezo1 could physically interact with HIF-1α.

| Piezo1 knockdown could significantly inhibit peritoneal metastasis of GC
The peritoneal metastasis of GC model was established in nude mice.
No significant differences in bodyweight or diet were observed after nude mouse models injected with si-Piezo1 or control during 14 days ( Figure 6A, B). si-Piezo1 remarkedly reduced the number and volume of intraperitoneal implanted GC tumours in nude mice ( Figure 6C-E).
Haematoxylin & eosin (H&E) staining results showed that the tumour area in the si-Piezo1 group was significantly decreased than that in the control group ( Figure 6F). Furthermore, the peritoneal tumour tissues in the control group had tight structures with dark nuclear staining and small clusters of tumour cell aggregation. In the si-Piezo1 group, the tumour cells had a loose structure, light nuclear staining, nuclear pyknosis in some cells and were scattered ( Figure 6G, H). Our data results demonstrated that Piezo1 knockdown significantly inhibited peritoneal implantation and metastasis of GC cells.

| Piezo1 knockdown could block EMT process and angiogenesis in peritoneal metastatic GC
qRT-PCR and Western blot assays were used to examine the expression levels of Piezo1, HIF-1α, VEGF, vimentin and E-cadherin in F I G U R E 4 Piezo1 promotes the expression of HIF-1α and VEGF proteins in GC cells. A, B, Representative images of immunohistochemistry for SNU-1 and HGC-27 two GC cells treated with Yoda1 and si-Piezo1. C, D, The expression of Piezo1, HIF-1α and VEGF was quantified in GC cells according to immunohistochemistry. E-H, Immunofluorescence assay results showed the expression of Piezo1, HIF-1α and VEGF in GC transfected with Yoda1 and si-Piezo1. *P < .05; **P < .01; ***P < .001; ****P < .0001 F I G U R E 5 Piezo1 could promote cell migration via up-regulation of HIF-1α in GC cell. A, B, Representative images of SNU-1 and HGC-27 two GC cells stably transfected with HIF-1α and Piezo1 knockdown. C, D, Western blot was used to assess the transfection efficiency of HIF-1α knockdown in GC cells. E-H, Transwell assay was performed to detect the migrant ability of GC cells after treatment with si-Piezo1, Yoda1 or si-Piezo1 + Yoda1. I-L, Wound healing assay was used to measure the wound distance for GC cells transfected with si-Piezo1, Yoda1 or si-Piezo1 + Yoda1. *P < .05; **P < .01; ****P < .0001 peritoneal metastatic GC tumour tissues. As shown in Figure 7A

| D ISCUSS I ON
In this study, highly expressed Piezo1 was found both in GC tissues with or without omentum metastasis. Studies have shown that Piezo1 promotes the proliferation and migration of GC cells. 15 Thus, we studied the role and mechanism of Piezo1 in the omentum implantation and metastasis of GC cells in depth.
We  24 Our co-immunoprecipitation confirmed the interactions between HIF-1a and Piezo1 in GC cells.
Silenced Piezo1 significantly suppressed the migration ability of GC cells. However, the migration of GC cells was notably promoted by Piezo1 activation induced by Yoda1, which was reversed by HIF-1α knockdown. Therefore, Piezo1 may induce the cells to adapt to the hypoxic environment and accelerate the migration and metastasis of tumour cells by up-regulation of HIF-1α. [25][26][27] This discovery requires more in-depth experimental and clinical verification.
Numerous studies have shown that EMT is an important early process of invasion and metastasis for GC cells.

| CON CLUS ION
Piezo1 was highly expressed in GC tissues with omentum metastasis. The up-regulation could accelerate cell migration and Calpain1/2 expression by up-regulation of HIF-1α in GC cells. In peritoneal metastatic GC mouse model, we found that silencing Piezo1 could notably suppress peritoneal metastatic tumour growth, block EMT process and angiogenesis. Thus, Piezo1 could become a therapeutic target for GC patients with omentum metastasis, which requires to be validated by clinical and basic experiments.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

E TH I C S A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
The study was approved by the Ethics Committee of North China University of Science and Technology Affiliated Hospital (2014025).

CO N S E NT FO R PU B LI C ATI O N
All patients were informed consent.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets analysed during the current study are available from the corresponding author on reasonable request.