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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2024 Sep 9;150(9):416. doi: 10.1007/s00432-024-05923-y

Paclitaxel hyperthermia suppresses gastric cancer migration through MiR-183-5p/PPP2CA/AKT/GSK3β/β-catenin axis

Xiansheng Yang 1,#, Chang Liu 3,#, Zheng Li 4,#, Juncai Wen 5, Jinfu He 4, Yunxin Lu 6, Quanxing Liao 7, Tian Wang 4, Hongsheng Tang 4,✉, Xianzi Yang 8,✉, Lisi Zeng 2,✉
PMCID: PMC11383839  PMID: 39249161

Abstract

Background

Gastric cancer (GC), a prevalent malignant tumor which is a leading cause of death from malignancy around the world. Peritoneal metastasis accounts for the major cause of mortality in patients with GC. Despite hyperthermia intraperitoneal chemotherapy (HIPEC) improves the therapeutic effect of GC, it’s equivocal about the mechanism under HIPEC.

Methods

MiR-183-5p expression was sifted from miRNA chip and detected in both GC patients and cell lines by qRT-PCR. Gene interference and rescue experiments were performed to identified biological function in vitro and vivo. Next, we affirmed PPP2CA as targeted of miR-183-5p by dual luciferase reporter assay. Finally, the potential relationship between HIPEC and miR-183-5p was explored.

Results

MiR-183-5p is up-regulated in GC and associated with advanced stage and poor prognosis. MiR-183-5p accelerate GC migration in vitro which is influenced by miR-183-5p/PPP2CA/AKT/GSK3β/β-catenin Axis. HIPEC exerts migration inhibition via attenuating miR-183-5p expression.

Conclusion

MiR-183-5p can be used as a potential HIPEC biomarker in patients with CC.

Keywords: Gastric cancer, MiR-183-5p, Migration, HIPEC

Introduction

Gastric cancer (GC) is the fifth common cancer and fourth leading cause of cancer deaths globally (Sung et al. 2021). Advanced GC often metastasizes to peritoneum, liver, lymph node. Peritoneal metastasis is the common form of distant metastasis with poor outcomes (Kang and Kim 2022). Hyperthermic intraperitoneal chemotherapy (HIPEC) is a treatment that perfuses heated chemotherapy drugs into the abdominal cavity to eliminate dissociative cancer cells. High temperature chemotherapeutic drugs can increase the permeability of drugs and inhibit the metastasis of cancer cells (Khan and Johnston 2022; Gronau et al. 2022). With many clinical studies proved that compared to cytoreductive surgery (CRS), CRS combined with HIPEC significantly improves the median survival and 3-years survival of GC patients with peritoneal metastasis (Yang et al. 2011; Chen et al. 2022a, b; Bonnot et al. 2019). Epithelial-mesenchymal transition (EMT) is a dynamic biologically process, which affects embryonic development, tissue repair and regeneration, and cancer metastasis (Nieto et al. 2016; Owusu-Akyaw et al. 2019). After stimulation of physiological and pathological factors, polar epithelial cells lose the adhesion of basement membrane, intercellular tight junction and transform into infiltrating and migratory mesenchymal cells (Gonzalez and Medici 2014). EMT is connected with tumor initiation, invasion, metastasis, recurrence and treatment resistance (Pastushenko and Blanpain 2019; Aiello and Kang 2019).

MicroRNAs (miRNA), a class of small non-coding RNA about 22-24nt in length, binds to the untranslated region of mRNA (3’-UTR) to inhibit translation of protein (Fabian and Sonenberg 2012). Deregulation of miRNA plays a vital role in various fundamental biological processes including tumorigenesis, cancer progression, tumor invasion and metastasis (Adams et al. 2014; Croce 2009). Continuous studies have explored the essential role of miRNA in GC(Wei et al. 2020; Hao et al. 2017; Wang et al. 2015). MiR-146b inhibits cell proliferation and promotes apoptosis by reducing the expression of PTP1B(Xu et al. 2020). MiR-3619-5p inhibits AMPK/PGC1α/CEBPB axis to affect stemness of cells and drug resistance (Wu et al. 2020). Mir-21-5p, miR-4646-5p and miR-27b are closely related to GC metastasis (Li et al. 2018; Yang et al. 2021; Feng et al. 2017). It has been reported that miR-183-5p can be used as a potential biomarker for the poor prognosis of patients with GC (Wang et al. 2021; Li et al. 2019). MiR-183 can reverse the function of ultraviolet radiation resistance associated gene (UVRAG), thereby inhibiting the levels of autophagy and apoptosis in GC cells (Yuan et al. 2018). In addition, miR-183 can inhibit cell proliferation, invasion and metastasis by inhibiting the expression of EZRIN (Cao et al. 2014) and ITGB1 (Cao et al. 2019). However, miR-183-5P targeted TPM1 to promote GC cell proliferation, invasion and metastasis (Lin et al. 2019). And miR-183 promote metastasis of GC cells by binding to PDCD4(Gu et al. 2014) or AKAP12 (Zhang et al. 2020). Although it has been found the effect of thermochemotherapy and important role of miR-183-5p in GC, the mechanism between miR-183-5p and hyperthermia has not been reported.

In this study, we aimed at further research the role of miR-183-5p in GC. We found that miR-183-5p regulated AKT/GSK3β/β-catenin axis to promote GC metastasis and targeted to protein phosphatase 2 catalytic subunit alpha (PPP2CA). What’s more, miR-183-5p attenuated the efficacy of HIPEC for paclitaxel, which provides more ideas for clinical treatment.

Materials and methods

Specimen collection

All GC tumor tissue specimens and non-tumor tissue samples were obtained from GC patients at Sun Yat-Sen University Cancer Center between March 2013 and May 2015. All tissues were frozen in -80℃ liquid nitrogen immediately after surgery. This study was supported and managed by the Ethics Committee of Sun Yat-Sen Memorial Hospital and Affiliated Cancer Hospital & Institute of Guangzhou Medical University.

Cell culture

Human normal gastric epithelial cell line (GES1), gastric cancer cell lines (AGS, BGC823, MGC803, SGC7901, MKN45, MKN74, NCI-N87, NUGC4 and HGC27) were purchased from Procell Life Science & Technology Co. Ltd (Wuhan, China). All cells were cultured in RPMI-1640 medium (NUGC4 in DMEM) supplemented with 10% or 20% (HCG27 only) fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C in 5% CO2.

RNA extraction and quantitative real-time PCR

Total RNA was extracted from GC cell lines and samples using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. The precipitated RNA was resuspended in 30 µl of RNase-free water. Then, 2 µg RNA was reverse-transcribed into cDNA using a reverse transcription kit (Promega, Madison, USA).

Expression levels of miR-183 were measured by qRT-PCR, which was carried out with SYBR® Green master mix kit (Promega Corporation). All experiments were performed in triplicate. Relative fold expression changes were calculated using the comparative CT method (2-ΔΔCT), and the endogenous control, U48, was used for normalization of miRNA expression and GAPDH was for mRNA expression. U48 (HmiRQP9001 and P01011A, GeneCopoeia), miR-183-5p (HmiRQP0244 and P01011A, GeneCopoeia).

Wound healing assay

Cells were plated on six-well plates and incubated at 37℃ overnight. A standardized scratch was made on the cell layer by a 200 µL micropipette tip when cells reached 90–100% confluence. Images were captured with an inverted microscope at 0, 24, 48, 72 and 96 h. The scratched wound distances were measured using Image J software.

Transwell assay

Migration assays were performed in 24-transwells. Briefly, cells were seeded in the upper chambers and starved overnight. The lower chamber was supplemented with 750µL RPMI-1640 medium containing 10% FBS. After 24-hour migration, filter was fixed with methanol and stained with crystal violet. The supernatant cells were scraped off gently with cotton swab and lower cells were imaged and counted.

CCK-8 assay

The cell counting kit-8 (CCK-8, Dojindo, Japan) was performed to measure the half-maximal (50%) inhibitory concentration (IC50) values of GC cells exposed to different drug concentrations. GC cells were seeded into 96-well plates. 24 h later, the cells were treated with different concentrations of paclitaxel (Taxol) ranging from 0 to 1000 ug/ml for 48 h. Then the CCK-8 solution (10µL) diluted by fresh RPMI-1640 medium was added to each well and incubated at 37 °C. Finally, the OD values were detected at the wavelength of 450 nm using a microplate reader.

Western blot

Cells were lysed in RIPA buffer and protein concentration of the supernatant was determined by Pierce™ BCA Protein Assay Kit (Thermo Scientific, 23227). 30 µg protein were separated by SDS-PAGE on 10% gel and transferred to PVDF membranes. PVDF membranes were overnight incubated with primary antibodies at 4 °C. Washed with Tris-Buffered Saline and Tween 20 (TBST) 3 times, the membranes were incubated with appropriate secondary antibodies at room temperature for 2 h. After washing, the chemiluminescence image system Tanon 2000 (Tanon, China) was utilized to visualize the immunoreactive bands on the membranes.

Tumor formation and thermochemotherapy treatment in vivo

All animal experiments were conducted in accordance with research ethics and were accepted by the Guangzhou Medical University Laboratory Animal Center Committee approvement. Female BALB/c nude mice (4–5 weeks old, weighing 12–14 g) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China), kept in SPF mice house. HGC27 miR-183-5p mimics cell suspension was injected subcutaneously into mice with 1 × 10^6. Intraperitoneal injection of PTX (15 mg/kg) or water bath hyperthermia starting from the 6th day, and the cycle was once every 6 days, a total of 3 times. The mice were killed at the end of 24 days experiment period and subcutaneous xenograft tumors were separated for qRT-RCR test.

Dual luciferase reporter assay

HEK293T cells were seeded in 96-well plates and transiently co-transfected with psiCHECK-PPP2CA plus hsa-miR-183-5p, psiCHECK-PPP2CA plus NC, psiCHECK-PPP2CA-mut plus hsa-miR-183-5p and psiCHECK-PPP2CA-mut plus NC. 48 h after co-transfection, luciferase reporter assay (Promega) was performed according to the manufacture’s specifications.

Statistical analysis

All experiments were independently repeated three times. The results are expressed as means ± standard deviation. All data analysis was performed by SPSS statistical software (SPSS 16.0). The differences in variables between groups were determined by Student’s t-test. Cox regression analysis was utilized for evaluated clinicopathological features and miR-183-5p expression. Survival curves were estimated by Kaplan Meier curves and log-rank tests. A value of p less than 0.05 was considered statistically significant.

Results

MiR-183-5p was down-regulated after HIPEC and positively associated with poor prognosis in GC patients

Serum samples were collected from 5 GC patients with peritoneal carcinomatosis before and after HIPEC. MicroRNA expression profiles were used to screen the abnormally expressed miRNAs. A total of 270 differentially expressed miRNAs were found in the group after HIPEC, including 101 downregulated and 169 upregulated miRNAs. Among all downregulated miRNAs, 25 of them were down-regulated by more than 1.5-fold and miR-183-5p had the highest fold of down-regulation (Fig. 1A).

Fig. 1.

Fig. 1

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MiR-183-5p was down-regulated after HIPEC and positively associated with poor prognosis in GC patients. (A) MicroRNA expression profiles predict the expression level of miRNAs in GC patients before and after HIPEC. (B) Expression of miR-183-5p in GC tissues and normal tissues. (C) Relative expression of miR-183-5p in GC tissues and normal tissues from ENCORI website. (D) Kaplan–Meier overall survival curves for 97 gastric cancer patients with low (n = 48) and high (n = 49) miR-183-5p expression. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Next, qRT-PCR analysis was conducted to examine the expression level of miR-183-5p in 97 tumor tissues and 38 normal tissues isolated from GC patients. Compared with GC normal tissues, miR-183-5p was significantly upregulated in GC specimens (Fig. 1B). In addition, ENCORI website (http://starbase.sysu.edu.cn/) was used to detect miR-183-5p levels in GC patients. The result showed that miR-183-5p levels were upregulated in 372 GC tissues comparing to 32 normal samples (Fig. 1C). Kaplan–Meier survival analysis suggested that GC patients with high expression of miR-183-5p had a lower overall survival rates than those with low miR-183-5p expression (Fig. 1D).

We also evaluated the correlation between miR-183-5p expression and clinical features. As showed in Table 1, miR-183-5p expression level was significantly associated with T stage and distant metastasis, and high miR-183-5p might promote gastric cancer progression. What’s more, univariate analysis revealed that N stage, M stage, TNM stage and miR-183-5p were significantly associated with OS in GC. Multivariate analysis indicated that TNM stage and miR-183-5p expression levels as predictors of prognosis for patients with gastric cancer (Table 2). Therefore, miR-183-5p may serve as a poor predictive factor in GC patients.

Table 1.

Correlations between mir-183-5p expression and the clinical features

Characteristics No. of patients has-miR-183-5p expression (%) p-value
Low High
Gender 0.337
Male 69 32(46.4%) 37(53.6%)
Female 28 16(57.1%) 12(42.9%)
Age (years) 0.114
< 60 59 33(55.9%) 26(44.1%)
>=60 38 15(39.5%) 23(60.5%)
Tumor Infiltration < 0.001
T1, T2 25 20(80.0%) 5(20.0%)
T3, T4 72 38(38.9%) 44(61.1%)
Lymph Node 0.481
N0, N1, N2 58 27(46.6%) 31(53.4%)
N3 39 21(53.8%) 18(46.2%)
Metastasis 0.006
M0 53 33(62.3%) 20(37.7%)
M1 44 15(34.1%) 29(65.9%)
TNM Stage 0.959
I, II 20 10(50.0%) 10(50.0%)
III, IV 77 38(49.4%) 39(59.6%)
Tumor Size (cm) 0.756
≤ 5 46 22(47.8%) 24(52.2%)
>5 51 26(51.0%) 25(49.0%)
Location 0.386
Upper 20 12(60.0%) 8(40.0%)
Middle 26 14(53.8%) 36(46.2%)
Down 51 48(49.5%) 49(50.5%)
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Table 2.

Univariate and multivariate Cox regression analysis of has-mir183-5p and survival in patients with gastric cancer (GC)

Variables Univariate analysis Multivariate analysis
HR 95%CI p-Value HR 95%CI p-Value
Overall Survival
Gender (male vs. Female) 1.147 0.671–1.959 0.617
Age ( > = 60 vs. <60) 1.169 0.719–1.901 0.528
Tumor Infiltration 1.29 0.735–2.263 0.375
Lymph Node 2.227 1.372–3.616 < 0.001
Metastasis 2.852 1.715–4.744 < 0.001
TNM Stage 5.959 2.376–14.947 < 0.001 6.155 2.448–15.474 < 0.001
Tumor Size (≥ 5 cm vs. <5 cm) 0.93 0.576–1.502 0.768
Location 1.196 0.874–1.636 0.263
has-miR-183-5p expression (high vs. low) 1.937 1.178–3.183 0.009 2.014 1.223–3.319 0.006
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MiR-183-5p promoted migration of GC cells

In order to investigate the function of miR-183-5p, we chose lower level of miR-183-5p cells AGS, HGC27 for miR-183-5p overexpression and higher cells MGC803, BGC823 for miR-183-5p knockdown (Fig. 2A). QRT-PCR assay showed that miR-183-5p was effectively upregulated in AGS and HGC27, while BGC823 and MGC803 were knocked down. (Fig. 2B). Wound healing assay and transwell assay revealed that high expression level of miR-183-5p in AGS and HGC27 promote cell migratory ability. On the contrary, ablation of miR-183-5p restrain cell migration in MGC803 and BGC823 (Fig. 2C, D). What’s more, western blot showed that E-Cadherin was opposite to the expression of miR-183-5p, but Vimentin has the synchronous change with miR-183-5p (Fig. 2E). In conclusion, these data confirmed that miR-183-5p enhanced GC cells migration in vitro.

Fig. 2.

Fig. 2

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MiR-183-5p accelerate GC migration in vitro. (A) MiR-183-5p expression levels in GC cell line. (B) Transfection efficacies of miR-183-5p mimics and inhibitor. Wound healing (C) and transwell assay (D) after HGC27 and AGS was transfected with miR-183-5p mimics and MGC803 and BGC823 with miR-183-5p inhibitor, the bar graph shows scratch-healing status. The number of cells that invade into the chamber below is shown as means ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) EMT-related proteins E-cadherin and Vimentin were detected by western blot analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

MiR-183-5p targeted PPP2CA and suppressed AKT/GSK3β/β-catenin signaling pathway

To explore the potential molecular mechanism of miR-183-5p, we identified PPP2CA as a potential target of miR-183-5p through online websites including PicTar, miRDB, DIANA MRMicroT and MiRGator (Fig. 3A). Dual-luciferase reporter assay indicated that co-transfection with the PPP2CA wild-type vectors and miR-183-5p mimics attenuated the luciferase activity in HEK 293T cells but not PPP2CA mutant-type (Fig. 3B). Besides, overexpression of miR-183-5p downregulated mRNA of PPP2CA, while inhibition of miR-183-5p upregulated mRNA levels of PPP2CA (Fig. 3C). Western blot assay also showed the opposite change between miR-183-5p and PPP2CA (Fig. 3D). What’s more, overexpression of PPP2CA reversed the migration ability caused by miR-183-5p overexpression in HGC27 and AGS cells (Fig. 3E, F).

Fig. 3.

Fig. 3

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MiR-183-5p negatively regulated PPP2CA and inhibited AKT/GSK3β/β-catenin pathway. (A) Venn diagram of miR-183-5p targeted genes and binding sites of miR-183-5p and PPP2CA. (B) Co-transfected with the PPP2CA wild-type or mutant-type and miR-183-5p in HEK 293T cells. (C) Expression of PPP2CA mRNA in miR-183-5p inhibitor or mimics GC cells. (D) Western blot analysis of the expression of PPP2CA and AKT/GSK3β/β-Catenin after miR-183-5p inhibition and overexpression. (E) Transwell and wound healing assay (F) in miR-183-5p mimics HGC27 and AGS transfected with PPP2CA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Subsequently we clarified the relationship between miR-183-5p and AKT/GSK3β/β-catenin signaling pathway. Western blot analysis showed that p-AKT(Thr308), p-GSK3β(Ser9) increased and p-β-Catenin (Ser33/37/Thr41) decreased in GC cells transfected with miR-183-5p mimics while these protein levels acted in opposite way in GC cells transfected with miR-183-5p inhibitor (Fig. 3G). Our study suggested that miR-183-5p targeted PPP2CA through binding to mRNA’s 3′-UTR and thus affected GC migration. And miR-183-5p also inhibited AKT/GSK3β/β-catenin signaling pathway.

Thermochemotherapy inhibit GC cell migration by inhibiting miR-183-5p

MiR-183-5p exerted migration ability by PPP2CA targeted degradation. Moreover, the expression of miR-183-5p decreased after HIPEC. Therefore, we further explored the role of miR-183-5p in thermochemotherapy for GC. QRT-PCR revealed that miR-183-5p was downregulated and PPP2CA was upregulated in MGC803, BGC823 after thermochemotherapy (Fig. 4A). Wound healing and transwell assays were performed to elucidate that thermochemotherapy significantly attenuated GC cells migration ability compared with hyperthermia or PTX alone (Fig. 4B, C). Nude mice experiment also verified thermochemotherapy inhibited tumor growth distinctly (Fig. 4D, E). What’s more, compared to mice treated with hyperthermia or PTX, expression of miR-183-5p was lower in mice tumor tissues with thermochemotherapy therapy (Fig. 4F). Interestingly, when miR-183-5p was increased in GC cells after thermochemotherapy, cell migration ability was reversed (Fig. 5A, B). In addition, inhibition of miR-183-5p in MGC803 and BGC823 after thermochemotherapy could further inhibited cell migration (Fig. 5C, D). Elevation of miR-183-5p also exerted higher LT50 (median lethal temperature) and IC50 (half maximal inhibitory concentration, Fig. 5E). Western blot suggested that PPP2CA and p-β-Catenin (Ser33/37/Thr41) were markedly up-regulated while p-AKT(Thr308)、p-GSK3β(Ser9) levels were prevalently downregulated after thermochemotherapy treatment (Fig. 5F).

Fig. 4.

Fig. 4

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Thermochemotherapy suppressed miR-183-5p and attenuated GC cell migration. (A) MGC803 and BGC823 cells were treated with 37℃, 37℃+PTX, 43℃, 43℃+PTX. QRT-PCR were used to detect relative expression of miR-183-5p and PPP2CA. (B) Wound healing and transwell assay (C) in MGC803 and BGC823 cells after treated with 37℃, 37℃+PTX, 43℃, 43℃+PTX. (D) Subcutaneous tumor implantation volume in mice injected with HGC27 miR-183-5p mimics cells. (E) Tumor volume was measured after HGC27 miR-183-5p mimics injection and mice were treated at the day of 9, 12, 15, 18 and 21. (F) Expression of miR-183-5p in different groups of mice tissue. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5.

Fig. 5

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Downregulation of miR-183-5p by thermochemotherapy inhibited cell migration. (A, B) MiR-183-5p reversed MGC803 and BGC823 migration ability inhibited by thermochemotherapy. (C, D) Thermochemotherapy combined with miR-183-5p inhibition further restricted migration of MGC803 and BGC823. (E) MiR-183-5p promoted gastric cancer cells resistance to hyperthermia and paclitaxel chemotherapy. (F) Chemotherapy elevated expression of PPP2CA, p-β-Catenin (Ser33/37/Thr41) and reduced p-AKT (Thr308) and p-GSK3β (Ser9). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Discussion

Despite previous studies has showed miRNAs, even miR-183 or miR-183-5p play a prominent role in GC, it’s still ambiguous about the relationship between miR-183-5p and thermochemotherapy. In this study we found miR-183-5p was significantly downregulated in GC serum chip after HIPEC. Both of fresh tissues and online database ENCORI revealed miR-183-5p’s high expression in tumor group and positive correlation with poor progress, further tumor infiltration and distant metastasis. MiR-183-5p also served as an independent risk factor for GC patients OS.

We further constructed miR-183-5p mimics and inhibitor cells to explore its function in vitro. Wound healing and transwell assays indicated miR-183-5p accelerated cells migration. Besides, cells with higher miR-183-5p had synchronous Vimentin and lower E-cadherin which act as cell migration and intracellular tight junction respectively (Nieto et al. 2016; Satelli and Li 2011). It is well known that miRNAs target and degrade 3’-UTR of mRNA to participate in tumorigenesis process (Michlewski and Cáceres 2019). Online databases PicTar, MRMicroT, miRDB and miRGator were utilized to screen possible combination sequences of miR-183-5p. Fortunately PPP2CA was found in four prediction databases and there are two binding sites with miR-183-5p. Dual-luciferase reporter assay confirmed miR-183-5p weakened fluorescence value of PPP2CA wide-type. Protein and mRNA of PPP2CA were also identified in stabilized cells that overexpressed or inhibited miR-183-5p. These results suggested miR-183-5p degrades PPP2CA by targeting its mRNA. Protein phosphatase 2 A (PP2A) is a conserved serine, threonine phosphatase in eukaryotes consisting of catalytic subunit C and scaffold subunit A (Janssens and Goris 2001; Longin et al. 2007). Catalytic subunit C including α (PPP2CA) and β, α subtype affects PP2A activity through regulating subunit B (Longin et al. 2007; Tolstykh et al. 2000). Xiang Tan etc. uncovered that PPP2CA probably related to lung cancer metastasis (Tan and Chen 2014). PPP2CA also suppressed prostatic cancer (PC) migration and invasion in vitro and PC progress in mice (Bhardwaj et al. 2014). Therefore, we further investigated whether miR-183-5p regulates GC migration ability through PPP2CA. While PPP2CA was recovered in AGS and HGC27 miR-183-5p mimics cells, the numbers of cells that migrated were reversed. Consequently, miR-183-5p degrades PPP2CA to affect the migration of GC.

Because of the down-regulation of miR-183-5p in GC serum specimens after hyperthermia chemotherapy, we concentrated on clarifying the association between paclitaxel thermochemotherapy and miR-183-5p. Thermochemotherapy decreased miR-183-5p expression and rose PPP2CA regardless mRNA or protein in vitro. Moreover, miR-183-5p promoted hyperthermia and PTX resistance in gastric cancer. Thermotherapy combined with PTX could further inhibited GC migration and healing. Nude mice transplanted tumor model validated that compared with hyperthermia or PTX chemotherapy group, thermochemotherapy can better inhibit tumor growth. What’s more, the lowest expression of miR-183-5p was detected in thermochemotherapy group tumors. These results hinted PTX thermochemotherapy may play a tumor inhibition role in GC through miR-183-5p descent. Hence, we restored miR-183-5p in BGC823 and MGC803 after thermochemotherapy, both migrations were more pronounced. In contrast, it’s more restricted while miR-183-5p inhibition in AGS and HGC27 after PTX hyperthermia.

Although miR-183-5p targeted PPP2CA to make impact on GC migration, we sought the possible downstream mechanisms of PPP2CA. Arun Bhardwaj etc. indicated inactivation of PP2A could activate AKT, ERK (Bhardwaj et al. 2011) and EMT evolution with tumor migration (Grille et al. 2003; Yoo et al. 2011). As a downstream molecule of AKT, GSK3β can induce proteasome hydrolysis of β-Catenin by mediating β-Catenin phosphorylation (Moon et al. 2004). Activation of β-catenin causes epithelial marker E-cadherin deletion, which means missing contact of tumor cells and more likely to metastasize (Yang et al. 2022; Chen et al. 2022a, b; Zhang et al. 2018). Not only can GSK3β phosphorylate β-catenin, but also p-AKT phosphorylate β-catenin, promoting it translocate to nucleus and initiating EMT gene expression (Moon et al. 2004). Therefor, we detected AKT/GSK3β/β-Catenin signal axis and fortunately found PTX thermochemotherapy rose PPP2CA, p-β-catenin (Ser33/37/Thr41) and p-AKT (Thr308), p-GSK3β (Ser9) were down regulation. Thus, we identified PTX thermochemotherapy inhibit GC metastasis through miR-183-5p/PPP2CA/AKT/GSK3β/β-catenin axis, which may provide a novel biomarker for HIPEC therapy and observation.

Acknowledgements

The authors would like to thank Affiliated Cancer Hospital & Institute of Guangzhou Medical University for its support.

Author contributions

XS Y, C L and Z L performed all the experiments and contributed equally to this research. J W, JF H, YX L, QX L and T W were responsible for collecting samples and clinical information. LS Z, XZ Y and HS T designed this study and draft the manuscript.

Data availability

The data that support the findings of this study are available on request from the corresponding author or first author, upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xiansheng Yang, Chang Liu and Zheng Li contributed equally to this work.

Contributor Information

Hongsheng Tang, Email: 15913139343@163.com.

Xianzi Yang, Email: yangxz@gzhmu.edu.cn.

Lisi Zeng, Email: zenglisi2017@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author or first author, upon reasonable request.

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