MiR‐192‐5p/RB1/NF‐κBp65 signaling axis promotes IL‐10 secretion during gastric cancer EMT to induce Treg cell differentiation in the tumour microenvironment

Abstract Background Regulatory T (Treg) cells are important components of the tumour microenvironment (TME) that play roles in gastric cancer (GC) metastasis. Although tumour cells that undergo epithelial‐mesenchymal transition (EMT) regulate Treg cell function, their regulatory mechanism in GC remains unclear. Methods The miR‐192‐5p was identified by examining three Gene Expression Omnibus GC miRNA expression datasets. RNA immunoprecipitation (RIP) and dual‐luciferase reporter assays were conducted to identify interactions between miR‐192‐5p and RB1. The role of miR‐192‐5p/RB1 in GC progression was evaluated based on EdU incorporation, wound healing and Transwell assays. An in vitro co‐culture assay was performed to measure the effect of miR‐192‐5p/RB1 on Treg cell differentiation. In vivo experiments were conducted to explore the role of miR‐192‐5p in GC progression and Treg cell differentiation. Results MiR‐192‐5p was overexpressed in tumour and was associated with poor prognosis in GC. MiR‐192‐5p bound to the RB1 3′‐untranslated region, resulting in GC EMT, proliferation, migration and invasion. MiR‐192‐5p/RB1 mediated interleukin‐10 (IL‐10) secretion by regulating nuclear factor‐kappaBp65 (NF‐κBp65), affecting Treg cell differentiation. NF‐κBp65, in turn, promoted miR‐192‐5p expression and formed a positive feedback loop. Furthermore, in vivo experiments confirmed that miR‐192‐5p/RB1 promotes GC growth and Treg cell differentiation. Conclusion Collectively, our studies indicate that miR‐192‐5p/RB1 promotes EMT of tumour cells, and the miR‐192‐5p/RB1/NF‐κBp65 signaling axis induces Treg cell differentiation by regulating IL‐10 secretion in GC. Our results suggest that targeting miR‐192‐5p/RB1/NF‐κBp65 /IL‐10 may pave the way for the development of new immune treatments for GC.


INTRODUCTION
Gastric cancer (GC) is one of the most prevalent tumours with a high mortality rate, particularly in China. 1,2 Metastasis is the mean cause of death in GC patients. The tumour microenvironment (TME), which consists of nonmalignant cells, immune cells and the inflammatory mediators they secrete, 3 plays a vital role in tumour metastasis. 4,5 Regulatory T cells (Tregs), as a subtype of CD4 + T cells, accumulate in the TME and play vital roles in tumour metastasis. 6 Large populations of FOXP3 + Tregs have been recognized in the TME, and their accumulation has been linked to poor prognosis in cancer. 7,8 Elevated FOXP3 + Tregs have been linked to poor overall survival and tumour metastasis in GC. 9,10 Therefore, investigation of the potential mechanisms underlying Treg cell and cancer cell interaction is essential for understanding the mechanisms of metastatic process in GC.
Epithelial-mesenchymal transition (EMT) is crucial for tumour metastasis. 11 Tumour cells undergoing EMT exhibit mesenchymal phenotypes that promote cell migration. EMT promotes tumour metastasis by modulating the TME, 12,13 it induces an immunosuppressive TME, which confers immune escape, and metastasis in tumour cells. During tumour EMT, tumour cells secret cytokines including TGF-β and interleukin-10 (IL-10) to induce T cells into Tregs, 14,15 thereby promoting tumour progression and metastasis. 16,17 Additionally, TGF-β-induced Tregs have been shown to promote tumour metastasis in B16-F10 mouse. 18 However, the specific mechanisms by which EMT tumour cells promote Treg cell differentiation in GC have not yet been evaluated.
MicroRNA (miRNA) is small non-coding RNA, 17-25nucleotide in lengths, that can bind to target mRNA resulting in mRNA translational inhibition or degradation. 19 MiRNAs are dysregulated in tumour cells undergoing EMT. 20,21 In GC, miRNAs promote tumour metastasis by modulating the tumour cell EMT. 22,23 MiRNAs in tumour cells promote Treg cell differentiation, thereby affecting tumour metastasis. 24,25 Whereas the potential mechanisms of miRNA dysregulation induce tumour cells to promote Treg cell differentiation remains largely unknown. Given the importance of miRNA in tumour EMT and Tregs, we speculate that miRNA induces EMT of tumour cells and secretes regulatory molecules to induce Treg cell differentiation.
RB1, as a tumour suppressor, plays important role in cell cycle and metastasis in many cancers. 26,27 The altered RB1 affected the production of cytokines and chemokines in tumour cells. 28 Through bioinformatics analysis, we found that RB1 may be a target of miR-192-5p. Since we unveiled that miR-192-5p promotes tumour progression and metastasis in GC. We investigated the possibility that miR-192-5p/RB1 could act as a mediator in the TME during GC EMT. Our findings might offer insights on how miRNA dysregulation induces tumour cells to promote Treg cell differentiation and facilitate the development of new immunotherapies for GC.
Target prediction tools (TargetScan, Microrna, Starbase, and miRWalk) were utilized to explore target gene of miR-192-5p; gene set enrichment analysis (GSEA) was performed on predicted genes ( Figure 1D-G). Interestingly, the predicted genes were enriched in cell cycle and EMT pathways such as nuclear factor-kappaB (NF-κB) pathway, JAT-STAT pathway, PI3K-Art signaling pathway and Th17 cell differentiation. Among the predicted genes, the predicted gene RB1, as a tumour suppressor that regulates the cell cycle, is known to participate in immune cell infiltration and multiple signaling pathways, such as the NF-κB signaling pathway. 30 Based on these findings, we further investigated the relationship between RB1 and miR-192-5p. We measured RB1 expression in 30 pairs of GC and ANT samples ( Figure 1H,I, Figure S1H). RB1 expression was substantially lower in GC ( Figure 1I, p < .001), and decreased RB1 expression was vastly associated with poor overall survival (OS) in GC ( Figure 1J, p < .001). In addition, miR-192-5p expression was inversely linked with RB1 expression ( Figure 1K, p = .0011).
To further prove that RB1 is the target of miR-192-5p, RNA immunoprecipitation (RIP) was performed using anti-Ago2 or control IgG, and the immunoprecipitates were analyzed by PCR ( Figure 2C). RIP results showed that RB1 was significantly enriched in Ago2-coated beads (p < .001). In addition, RB1 enrichment was up-regulated in MKN45 cells transfected with miR-192-5p compared to that in cells transfected with mimic negative control (NC) (p = .0157). Furthermore, dual-luciferase reporter assay was carried out by cloning RB1-3′-untranslated region (UTR) into the luciferase reporter plasmid. Sequence alignment showed that RB1 contains a miR-192-5p binding site ( Figure 2D). The luciferase activities were significantly decreased in GC cells transfected with the wide tyle (WT)-3′ UTR-RB1 and the miR-192-5p (p = .0220), while it was not significantly altered when GC cells were transfected with the mutant type (MUT)-3′ UTR-RB1 and the miR-192-5p ( Figure 2E).

2.4
MiR-192-5p/RB1 promotes Treg cell differentiation in the GC TME EMT in tumour induces immunosuppressive cells such as tumour-associated macrophage and Tregs by expressing cytokines, thereby shaping the TME to promote cancer metastasis. 31,32 We investigated whether miR-192-5p/RB1 induces Treg cell differentiation in the GC TME by performing bioinformatics analysis, and result revealed that The luciferase activities of BGC-823 cells or MKN45 cells co-transfected with miR-192-5p and luciferase vectors containing RB1 3′-UTR WT or RB1 3′-UTR MUT were determined by dual-luciferase reporter assay. Data are pooled from three independent experiments. Statistical analysis between two groups was conducted using two-tailed t-test. One-way analysis of variance (ANOVA) statistical tests were adopted for more than two groups. Error bars, standard deviation (SD). *p < .05, **p < .01, ***p < .001 RB1 was negatively correlated with the T helper cell 17 (Th17) differentiation pathway, which contains differentiation of TH17 and Tregs, respectively. We then investigated the relevance between miR-192-5p/RB1 and the genes representing Th17 cells and Tregs. The results showed FOXP3 was expressed at a significant level in GC ( Figure S1B,H), while IL17A expression was not different between GC and ANT samples ( Figure S1A,H). IF staining showed that tumour tissues exhibited almost no positive CD4IL17A expression. However, there was significantly positive CD4FOXP3 expression in tumour tissues ( Figure  S2A). Furthermore, miR-192-5p and RB1 expression levels were markedly correlated with FOXP3 but not with IL17A ( Figure S1C-F). The data suggest that miR-192-5p/RB1 is relevant to Tregs but not Th17 cells in GC. Therefore, we examined whether miR-192-5p promotes Treg (H) Total number of invasive cells in five fields was counted manually. Data are pooled from three independent experiments. One-way ANOVA statistical tests were adopted for more than two groups. Error bars, standard deviation (SD). *p < .05, **p < .01, ***p < .001 cell differentiation by inhibiting RB1. Since Programmed cell death protein 1 (PD-1) was a characteristic protein of Tregs, which could enhance the Tregs immunosuppressive function, 33,34 IF results demonstrated that tumour tissues expressed high expression of PD-1 ( Figure S3C), we detected PD-1 expression on Tregs. A GC cells and peripheral blood mononuclear cells (PBMCs) co-culture system was established ( Figure 4A). GC cells transfected with miR-192-5p or RB1 were co-cultured with activated PBMCs in a ratio of 1:1(2 × 105 cells/ml) ( Figure S7A,B). After

2.5
MiR-192-5p/RB1 induces Treg cell differentiation by regulating IL-10 secretion in GC Based on the above results, we next sought out to illustrate the specific mechanism via which miR-192-5p/RB1 regulates Treg cell differentiation. First, we analyzed several cytokines expressions that were related to Treg cell differentiation. The results showed that IL-10 expression was notably increased in the miR-192-5p overexpressed cells (p = .0132), while profoundly decreased in the miR-192-5p depleted cells (p = .0128) ( Figure 5A). Then we detected secreted IL-10 levels in GC cell supernatant by ELISA. IL-10 secretion was markedly increased in the miR-192-5p overexpressed cells (p < .001), decreased in the miR-192-5pdeficient cells (p < .001) ( Figure 5B). MiR-192-5p reversed the decreased IL-10 level induced by RB1 in GC cells (p < .001), while miR-192-5p depletion has little impacts on the secretion of IL-10 in RB1 knockdown GC cells (p = .2317) ( Figure 5C,D). Collectively, the data demonstrated that miR-192-5p/RB1 could regulate IL-10 secretion in GC cells. In addition, ELISA and immunohistochemistry results showed that IL-10 levels were significantly elevated GC ( Figure S5A and B).

2.7
MiR192-5p/RB1 suppresses the transcriptional activity of NF-κBp65 and NF-κBp65 in turn promotes miR-192-5p expression RB1 played critical roles as transcriptional repressor and was reported to interact with the NF-κBp65. 35 Since we observed that miR-192-5p/RB1 could regulate IL-10 production through the NF-κBp65, we further investigated the underlying mechanism by which RB1 regulates NF-κBp65. Firstly, nuclear-cytoplasmic fractionation and coimmunoprecipitation (CoIP) assay showed an endogenous interaction between RB1 and NF-κBp65 ( Figure 7A,B). The RB1 expression was higher in the nucleus than that in the cytoplasm and the RB1-NF-κBp65 binding primarily occurred in the nucleus. The IF results also showed that RB1 was present in the nucleus ( Figure 7E). Thus, we speculated that RB1 binds to NF-κBp65 to regulate the transcription of the NF-κBp65 target gene in the nucleus. Luciferase activity was detected to confirm our observation ( Figure 7C,D). The results revealed NF-κBp65 activated the transcription activity that was driven by the IL-10 promoter (p < .001), whereas RB1 dramatically suppressed it (p < .001). RB1 depletion increased the firefly luciferase Statistical analysis between two groups was conducted using two-tailed t-test. One-way ANOVA statistical tests were adopted for more than two groups. Error bars, standard deviation (SD). *p < .05, **p < .01, ***p < .001 experiments. Statistical analysis between two groups was conducted using two-tailed t-test. One-way ANOVA statistical tests were adopted for more than two groups. Error bars, standard deviation (SD). **p < .01, ***p < .001 experiments. Statistical analysis between two groups was conducted using two-tailed t-test. One-way ANOVA statistical tests were adopted for more than two groups. Error bars, standard deviation (SD). *p < .05, **p < .01, ***p < .001  .001). Furthermore, after overexpression of NF-κBp65, RB1 abolished the transcription activity of luciferase (p < .001). However, after knockdown of NF-κBp65, RB1 depletion has little effect on the luciferase expression. In addition, miR-192-5p increased the firefly luciferase activity in GC cells with overexpressed RB1 (p < .001). After knockdown of RB1, miR-192-5p inhibitor has little impact on the luciferase activity (p = .061) ( Figure S9A,B). These results suggest that RB1 binds to NF-κBp65 in the nucleus to restrain the transcription of IL-10.
RB1 is a chromatin-associated tumour suppressor that can limit the transcription of cell cycle genes. 42 RB1 depletion has been shown to induce cell cycle defects, compromise G1/S cell cycle arrest and reduce senescence, which makes cells sensitive to oncogenic proliferation. 29 RB1 is significantly associated with metastasis of various cancer. 45 RB1 expression is reduced in GC, 46 which has also been confirmed in our research. Additionally, we revealed that the RB1 affected EMT, cell proliferation, migration and invasion. EMT is regulated by different pathways including the TGF-β, Wnt, PI3K/Akt and NF-κB signaling pathway, 47,48 which promote the expression of transcription factors to drive the occurrence of EMT. It was reported that RB1 depletion activates the NF-κBp65 and nuclear translocation of NF-κBp65. 35 RB1 specifically suppresses NF-κBp65 activity and inhibits the expression of NF-κB target genes, including PD-L1. 49 Additionally, it has been reported that RB1 affects the PI3K/Akt pathway to promote nasopharyngeal carcinoma EMT. 29 RB1 can interacts with E2F1 to regulate EMT related proteins in lung cancer. 50 Our study showed that knock down of NF-κBp65 lessened the miR-192-5p/RB1-mediated GC cell EMT, in addition, RB1 bound to NF-κBp65 and inhibited its transcriptional activity. Thus, miR-192-5p/RB1-mediated EMT may be attributed to NF-κBp65.
The TME plays a vital role in immune regulation. Through GSEA analysis, we found that miR-192-5p/RB1 was linked to the Th17 cell differentiation pathway. Despite playing opposing roles in immune regulation, Treg and Th17 cells share a common differentiation pathway. We found that miR-192-5p/RB1 is correlated with Tregs but not Th17 cells in tumour tissues. Tregs can be classified into two types: natural/thymic-derived (nTreg) and peripherally induced cell (pTreg). 51 FOXP3 is a key regulatory gene for Treg development; CD4 + CD25 + FOXP3 + Tregs, either derived from nTreg or induced from pTreg, can suppress immune responses in TME. 52 A wealth of evidence suggests that FOXP3 + Tregs infiltrate GC. Some studies showed that tumour-infiltrating Treg cells can promote GC development and metastasis. [54][55][56][57] Tumourinfiltrating Tregs inhibit anti-tumour immune response via several pathways, including the production of immunosuppressive cytokines, PD-1 checkpoint inhibition and up-regulation membrane protein PD-1 and CTLA-4, 58,59 ultimately facilitating tumour metastasis. 60 Tregs promote cancer cell invasion by affecting the EMT status. 61,62 In addition, Tregs promote tumour angiogenesis in the TME through the secretion of VEGF-A and IL-10, thereby supporting tumour metastasis. 63 In vivo experiments, the tumour volume was significantly smaller after eliminating Tregs with anti-IL-10 or anti-CD25, which confirmed the effect of Tregs on tumour progression. Several studies have reported compelling evidence that tumour cells produce various cytokines and chemokines to promote the proliferation of Tregs and induce FOXP3 -T cells into FOXP3 + Tregs. 64,65 It was well established that the Treg cell differentiation is induced by the TGF-β and IL-10 cytokines. 66 Therefore, the expression levels of these cytokines in the supernatants of GC cells co-cultured with Tregs were detected. IL-10 was significantly increased in the supernatants of the co-culture system. NF-κBp65 binds to upstream of the IL-10 and has a role in enhancing IL-10 expression. 68 We found that there is an RB1/NF-κBp65/IL-10 axis in GC cells that induces Treg cell differentiation. EMT tumour cells have a close interaction with Tregs, resulting in further tumour growth and metastatic spread. 16 Our study provides evidence for the vital role of IL-10 induced by EMT tumour cells in the creation of a favourable TME for GC progression through regulating Treg cell differentiation.
Our data showed that miR-192-5p/RB1 can activate the PD-1/PDL1 pathway. Zhang et al. revealed that PD-1 is indispensable for Tregs suppressive functions, and loss of PD-1 expression impaired the function of Tregs. 69 Nair et al. found that PD-1 enhances the stability of Tregs by increasing FOXP3 expression. 70 Cancer cells can evade immune surveillance by expressing co-inhibitory molecule PDL1; Tregs express inhibitory molecule such as PD-1, which interacted with co-inhibitory molecule PDL1, resulting in attenuated CD8 + T cell responses. 71 We found that miR-192-5p/RB1 promoted the PDL1 expression on tumour cells and increased the percentage of PD-1 + FOXP3 + Tregs, which may enhance the immunosuppressive capacity of Tregs and promote immune evasion in GC.
Although some studies have confirmed that Tregs promoted GC metastasis, there is a primary limitation of our study that an absence of corroboration that Tregs deficiency inhibits tumour progression by affecting immune cells and tumour cells. Therefore, additional work is needed to understand its underlying mechanism. Additionally, since we found that miR-192-5p/RB1 could activate the PD-1/PDL1 pathway, further studies are needed to explore the mechanism by which miR-192-5p/RB1 regulates PD-1/PDL1 pathway and might uncover additional therapeutic target in GC.

MATERIALS AND METHODS
Detailed materials and methods are provided in Supplemental Materials.

Patient samples
Thirty paired GC samples were obtained from patients diagnosed with GC based on pathological examinations. GC patients had received tumour radical surgical treatment at the Zhongnan Hospital, which is affiliated with Wuhan University. No patient had received radiotherapy or chemotherapy before surgery. Peripheral Blood (PB) samples (3 ml) were collected in tube containing EDTA from 30 GC patients and 40 healthy volunteers without any malignancy (Table S2). The study was approved by the Ethics Committee of Zhongnan Hospital, Wuhan University. All patients gave informed consent.

4.4
Luciferase gene reporter assay

CoIP assay
IP/CoIP Kit (#abs955, Shanghai, China) was used to perform CoIP assay. GC cells were lysed on ice and centrifuged at 4 • C for 10 min. Supernatants were collected and incubated with anti-RB1 (CST, #9309, USA) or normal rabbit IgG (CST, #3900, USA) with rotating at 4 • C overnight. On the following day, Protein A/G was added to the supernatants with rotation at 4 • C for 3 h. Afterward, supernatants were subjected to WB analysis.

Subcutaneous tumourigenesis experiment
Animal experiment was conducted in line with the Guide for the Care and Use of Laboratory Animals of Wuhan University. BALB/c nude mice (female, 4-week-old) and C57BL/6 mice (female, 4-week-old) were purchased from Hubei Research Center of Laboratory Animals (Wuhan, China,). Note that 5 × 10 6 BGC-823 cells were injected subcutaneously on the right flank region of the nude mice. After 12 days, miR-192-5p antagomir/NC (RiboBio, #miR30000222-4-5, China) was injected into the tumour at a dose of 5 nmol/50 μl PBS every 3 d for four times. Then PBMCs obtained from healthy volunteers were injected into tumour at the dose of 2.5 × 10 7 cells/50 μl after a week. Ten days after BGC cell injection, tumour volumes were measured with a caliper every 5 days and calculated by the formula (width 2 × length)/2. The mice were euthanized after 5 weeks of BGC cell injection. The tumour tissues were collected for tumour weight and other analyses.

Bioinformatics assay and statistics analysis
MiRNA expression was obtained from the GEO website and was analyzed by GEO2R. Heatmap was performed by Funrich v3.1.3. GSEA was conducted by Sangerbox (http://www.sangerbox.com/). The survival curve of RB1 was obtained from the Kaplan-Meier Plotter website (http://kmplot.com/analysis/). Eight hundred seventyfive patients were included, and six patients were excluded in the Kaplan-Meier Plotter website. 72 The association between miR-192-5p and clinical pathology parameters were analyzed by χ 2 test or Fisher exact test. Univariable and multivariable Cox regression analyses were applied to detect potential risk factor. Spearman correlation was performed to study the correlation of two continuous variables. Kaplan--Meier analysis was used for evaluating survival curves. Data were shown as mean ± standard deviation. Statistical analyses were performed by SPSS 17.0 (SPSS Inc., USA). Statistical significance was determined by Student's t-test. p < .05 was determined statistically significant.

A C K N O W L E D G E M E N T S
This work was supported bygrants from the Health Commission of Hubei Province Scientific Research Project (WJ2019H012), Improvement Project for Theranostic ability on Difficulty miscellaneous disease (Tumor)(ZLYNXM202018), National Natural Science Fund Youth Fund of China (81702411).

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in Gene Expression Omnibus (GEO), reference number GSE78775, GSE86226, GSE164174. Other data that support our findings are available at the Clinical and Translational Medicin's website.