Macrophage-derived exosomal miR-342-3p promotes the progression of renal cell carcinoma through the NEDD4L/CEP55 axis

Due to its difficulty in early diagnosis and lack of sensitivity to chemotherapy and radiotherapy, renal cell carcinoma (RCC) remains to be a frequent cause of cancer-related death. Here, we probed into new targets for its early diagnosis and treatment for RCC. microRNA (miRNA) data of M2-EVs and RCC were searched on the Gene Expression Omnibus database, followed by the prediction of the potential downstream target. Expression of target genes was measured via RT-qPCR and Western blot, respectively. M2 macrophage was obtained via flow cytometry with M2-EVs extracted. The binding ability of miR-342-3p to NEDD4L and to CEP55 ubiquitination was studied with their roles in the physical abilities of RCC cells assayed. Subcutaneous tumor-bearing mouse models and lung metastasis models were prepared to observe in vivo role of target genes. M2-EVs induced RCC growth and metastasis. miR-342-3p showed high expression in both M2-EVs and RCC cells. M2-EVs carrying miR-342-3p promoted RCC cell abilities to proliferate, invade and migrate. In RCC cells, M2-EV-derived miR-342-3p could specifically bind to NEDD4L and consequently elevate CEP55 protein expression via suppressing NEDD4L, thereby exerting tumor-promoting effects. CEP55 could be degraded by ubiquitination under the function of NEDD4L, and miR-342-3p delivered by M2-EVs facilitated the RCC occurrence and development by activating the PI3K/AKT/mTOR signaling pathway. In conclusion, M2-EVs promote RCC growth and metastasis by delivering miR-342-3p to suppress NEDD4L and subsequently inhibit CEP55 ubiquitination and degradation via activation of the PI3K/AKT/mTOR signaling pathway, strongly driving the proliferative, migratory and invasive of RCC cells.


Introduction
Renal cell carcinoma (RCC) represents the most frequent type of renal neoplasm, accounting for 90% of renal malignancies and 2% 3% of all adult malignant cancers [1,2]. It is the most lethal neoplasm of the urologic system, defined by an asymptomatic disease course, with late and uncharacteristic symptoms, leading to a poor survival prognosis [3]. Since the 1990s, the incidence of RCC has steadily increased year by year, with an annual increase of approximately 2% to 3% [2,4]. Patients suffering from localized RCC are frequently treated with nephrectomy [5]. Unfortunately, metastasis occurs in approximate onequarter of the patients, causing meager survival rates and severe social burden [6,7]. Therefore, identification of novel biomarkers is crucial for the treatment of RCC.
Macrophages are the most abundant immune cells in the tumor microenvironment (TME) and can be categorized into the classically activated (M1) and alternatively activated (M2) macrophages according to the polarization status [8,9]. M1 macrophages exhibit tumoricidal activities, whereas M2 macrophages facilitate tumor progression by enhancing tumor angiogenesis and metastasis [9,10]. Numerous types of cells, including macrophages, release extracellular vesicles (EVs), which are bioactive membrane-enclosed "packages" containing proteins, lipids and nucleic acids that are capable of modulating the TME and affecting the signaling pathway of recipient cells [11,12]. Increasing evidence suggests that cancers hijack EV-mediated communication to facilitate tumor progression by promoting cell proliferation, sustaining angiogenesis, reprogramming energy metabolism, evading immune response, and contributing to cancer cell invasion and metastasis [12][13][14]. In particular, M2 macrophage-derived EVs have been shown to stimulate the migration and invasion of various cancer types, including colorectal cancer, hepatocellular cancer and pancreatic cancer [15][16][17]. MicroRNAs (miRNAs) are one of the most studied classes of biomolecules carried by EVs. miRNAs, small non-coding RNAs, are capable of regulating gene expression post-transcriptionally, resulting in the attenuated translation of target mRNAs [18]. Several EV-derived miRNAs such as miR-19b-3p, miR-30c-5p and miR-210 have been studied and proposed as potential diagnostic and therapeutic targets in clear cell RCC (ccRCC, the most common subtype of RCC accounting for 75%-80% of all RCC cases) [19][20][21]. However, due to the novelty of this field, the role of M2 macrophage-derived EVs (M2-EVs) in regulating RCC progression and metastasis still needs to be further elucidated.
Herein, we first determined M2-EVs-related miRNAs by bioinformatics analysis in RCC and identified miR-342-3p as the significantly up-regulated miRNAs. The role of miR-342-3p in several cancers (such as hepatocellular and nasopharyngeal carcinoma) has already been investigated [22,23]. However, how EV communication involving miR-342-3p affects RCC cell is not fully understood. In this study, we explored the role of M2-EV-derived miR-342-3p in RCC and discussed the underlying mechanisms.
Cell culture RCC cell lines (ACHN and 769-P), human umbilical vein endothelial cells (HUVECs) and monocyte cell line THP-1 were purchased from the Cell Bank of the Typical Culture Preservation Committee of Chinese Academy of Sciences (Shanghai, China). These cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5%CO 2 humidified incubator.
Immunofluorescent staining M2 TAMs were digested and cultured in an immunofluorescence chamber (2 × 10 5 cells/per well). When the cell confluence reached about 90%, the cells were washed with cold PBS three times and fixed in 4% paraformaldehyde. After permeabilizing with 0.3% Triton X-100 and blocking with goat serum, the cells were incubated with the primary antibodies (diluted in PBS at a ratio of 1:100, Abcam, Cambridge, UK) CD68 (ab213363), CD206 (ab125028) and CD163 (ab156769) at 4°C overnight. Afterward, the cells were incubated with FITClabeled fluorescent secondary antibody (sheep anti-mouse IgG H&L) (Abcam, Cambridge, UK) at room temperature away from light for 1 h. The cells were then exposed to 4′,6diamidino-2-phenylindole (DAPI) (100 μL per well) for 15 min in dark conditions, sealed and observed under a fluorescence microscope (ECLIPSE E800, Nikon, Tokyo, Japan).
Isolation and identification of M2-EVs M2 TAMs were incubated overnight at 2 × 10 5 cells per well in 6-well plates. The culture medium was replaced with EV-free serum. After being cultured for another 48 h, CM was collected, and M2-EVs were isolated via differential centrifugation (500 g for 15 min; 2,000 g for 15 min; 10,000 g for 20 min) at 4°C. Following filtration by a 0.22 µM filter (on ice), the sample was centrifuged at 110,000 g for 70 min, resuspended using PBS (on ice) and ultracentrifuged. M2-EVs were finally obtained after resuspension in 100 μL sterile PBS.
A transmission electron microscope (TEM) was adopted to validate the successful extraction of M2-EVs. Briefly, 20 µL EVs were loaded onto the copper grid, followed by liquid suction with filter paper, counterstaining with the addition of 30 µL phosphotungstic acid solution (pH 6.8), baking with incandescent light and photographing. Particle size was determined by a Nanoparticle Tracking Analyzer (NS300, Malvern Instruments, Ltd., UK).
Fluorescent labeling and transfer of EVs M2-EV labeling was completed with the PKH67 Green Fluorescence Kit (UR52303, Umibio, Shanghai, Co., Ltd., China). Afterward, the harvested EVs were co-cultured with RCC cells, fixed in 4% paraformaldehyde, washed by PBS and exposed to DAPI (Sigma, MO, USA, D9542) for nuclear staining. The slides were photographed under the fluorescence microscope (ECLIPSE E800, Nikon, Tokyo, Japan).
Dual-luciferase reporter assay A dual luciferase reporter assay was done using a Promega dual-luciferase reporter kit (Madison, Wisconsin, USA). The sequences of NEDD4L containing predicted miR-342-3p binding sites and mutant binding sites were ligated to the pmirGLO Dual-Luciferase miRNA Target Expression Vectors (E1330, Promega, Madison, Wisconsin, USA) to build NEDD4L-WT and NEDD4L-MUT reporter vectors. HEK293 cells were co-transfected with the above-indicated plasmids and miR-342-3p mimic or miR-NC using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). After 48 h, the luciferase activities were detected.
Protein half-life assay RCC cells were treated with 10 μM cycloheximide (CHX, MedChemExpress, Shanghai, China) for various periods (0, 0.5, 1 and 2 h), or treated with MG132 to block protein synthesis at the same time. Cell extracts were prepared for Western blot assay.
The cell viability curve was drawn with time as abscissa and optical density value as ordinate.
Transwell assay Cells were added to the upper Transwell chambers (24-well insert; pore size, 8 μm; Corning, Tewksbury, MA, USA) precoated without (migration assay) or with (invasion assay) 50 μL of diluted Matrigel (1:8 dilution; BD Biosciences). Medium containing 10% FBS was added to the lower Transwell chambers. After 48 h, the transmigrated cells were fixed with 4% paraformaldehyde, treated with 0.2% Triton X-100 (Sigma, Saint Louis, MO, USA) solution and stained with 0.05% gentian purple. The number of stained cells was counted under an inverted microscope (XDS-800D, Shanghai Caikang Optical Instrument Co., Ltd., Shanghai, China) to evaluate the cell migration and invasion capabilities. Five visual fields were randomly selected to count, and the number of cells was expressed by mean.

RT-qPCR
Total RNA extracted from tissues or cells using Trizol (Thermo Fisher Scientific, Waltham, MA, USA) was taken to perform reverse transcription to obtain cDNA or miRNA cDNA with a reverse transcription kit (Fermentas Inc., Ontario, CA, USA) or miRcute Plus miRNA First-Strand cDNA Synthesis Kit (TIANGEN, China). Synthetic exogenous internal reference cel-miR-39 (1 pmol per sample; TIANGEN, Beijing, China) was supplemented to the culture medium (350 μL) or EVs (100 μg) before the experiment. Subsequently, miRNA was isolated from these samples using the mirVana PARIS kit (Ambion, Austin, Texas, USA). RT-qPCR for mRNA was completed with SYBR Ò Premix Ex Taq (Takara, Tokyo, Japan) on the ABI StepOne real-time PCR system (Applied Biosystems, Dublin, CA, USA). Meanwhile, miRcute Plus miRNA qPCR detection kit (TIANGEN, Beijing, China) was adopted for RT-qPCR of miRNA. GAPDH was used as the internal control for measuring mRNA expression in cells and tissues, while U6 for measuring miRNA expression. In addition, the miRNA level of culture medium and EVs was normalized to exogenous cel-miR-39. The universal reverse primer of miRNA was obtained from miRcute Plus miRNA qPCR detection kit (TIANGEN, Beijing, China), and other primers were provided by Shanghai Sangon (Shanghai, China). All primer sequences were displayed in Table 1. All experiments were processed in triplicates, and the results were analyzed following the 2 (−ΔΔCT) method.

Xenograft tumor and lung metastasis in nude mice
Sixty-four 4-5-week immunodeficient nude mice (Bal B/c, nu/nu) were housed under non-pathogenic conditions at 20°C-26°C and humidity 50%-65%. ACHN cells or ACHN cell lines stalely transfected with pcDNA-3.1-NEDD4L were collected, counted, and resuspended in PBS with the final concentration of 2 × 10 7 cells/mL. In addition, trypan blue exclusion determined that 95% of the cells were viable prior to injection. The prepared ACHN cells were subcutaneously injected into the back of nude mice. Eight days after injection, DiI-labeled EVs or PBS (blank control) were injected through the tail vein. After that, the tumor volume was measured at six-day intervals with a vernier caliper and calculated as W = 1/2 * a * b 2 (a, long diameter; b, short diameter). Four weeks later, CO 2 was used to execute nude mice, and tumor tissues was isolated and weighed. The nude mice were randomly assigned to: i. Control (tail vein injection of saline); ii. M2-EVs (tail vein injection of 10 μg M2-EVs); iii. M2-EVs-anti miR-342-3p (tail vein injection of 10 μg EVs isolated from M2 macrophages infected with 10 μg anti-miR-342-3p lentivirus); iv. Overexpressed (oe)-NEDD4L (injection of stable pcDNA-3.1-NEDD4L transduced ACHN cell line and tail vein injection of saline); v. oe-NEDD4L+M2-EVs (injection of stable pcDNA-3.1-NEDD4L transduced ACHN cell line and tail vein injection of M2-EVs) (n = 8 for each treatment). To establish lung metastasis model, ACHN cells or ACHN cells stably transfected with pcDNA-3.1-NEDD4L were injected into female Bal B/C nude mice through the tail vein (1 × 10 6 cells/100 μL). After 14 days, 10 μg DiIlabeled EVs or an equal amount of PBS was repeatedly injected into nude mice via a caudal vein twice a week for one month. Subsequently, all nude mice were euthanized. Then, metastatic pulmonary nodes were counted, and the lung weight was measured. Lung tissues were harvested to evaluate the expressions of miR-342-3p, NEDD4L and CEP55 via RT-qPCR and Western blot. The lung was split for further H&E staining.

Statistics
All data were processed by SPSS 21.0 statistical software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 8.0.2 software (GraphPad Software, Inc., La Jolla, CA, USA). Measurement data were expressed in the form of mean ± standard deviation. Data of the two groups were compared using the independent samples t-test. One-way ANOVA (followed by Tukey posthoc test) was adopted for data comparison between multiple groups. Data were compared between groups at different time points using repeated measures ANOVA and Tukey posthoc test. Values of p < 0.05 were considered statistically significant.

M2-EVs promote malignant biological behaviors of RCC cells
M2-EVs bear great responsibility for cell migration and metastasis in several cancers [24]. To investigate the pivotal biological effects of M2-EVs on RCC, M2 TAMs were firstly extracted (Fig. S1A) and presented with positive expressions of markers CD68, CD163 and CD206 in immunofluorescence staining (Fig. S1B). M2-EVs were subsequently extracted. TEM manifested that M2-EVs showed round or oval shapes, which was uneven in size with a complete membrane structure, and contained low-density substances (Fig. S1C). The particle diameter of the EVs was 30-100 nm (Fig. S1D). Western blot assay obtained positive Alix, CD63 and LAMP2 while negative calnexin proteins in EVs (Fig. S1E). PKH67 labeled M2-EVS was co-cultured with RCC cells. PKH67 labeled green fluorescence could be observed in RCC cells (Fig. 1A), indicative of the internalization of M2-EVs by RCC cells. The biological effect of M2-EVs was further analyzed. Compared to the control group, M2-CM and M2-EVs groups showed a significant increase in cell proliferation (Fig. 1B), invasion (Fig. 1C, Fig. S2A) and migration (Fig. 1D,  Fig. S2B), accompanied by distinctly down-regulated E-cadherin level and up-regulated Vimentin level (Fig. 1E,  Fig. S3A). These results identify that M2-EVs can promote RCC cell abilities to proliferate, migrate and invade.
To further investigate whether CEP55 was involved in M2-EVs-miR-342-3p dependent regulation in the tumorigenesis and development of RCC, potential downstream target genes for miR-342-3p were obtained on ENCORI and were then intersected with the differentially down-regulated genes in GSE36895 dataset and the E3 ubiquitin ligase of CEP55 predicted by the UbiBrowser database (Figs. 4B-4C). In the meantime, ENCORI data revealed a negative correlation between NEDD4L and CEP55 in RCC (Fig. 4D). Therefore, NEDD4L was eventually selected as the target gene.
In vivo animal experiment was then conducted. Empty RCC cells or cells overexpressing NEDD4L were respectively subcutaneously injected into the nude mice, along with the tail injection of M2-EVs. It was found that NEDD4L overexpression resulted in reduced tumor growth speed and weight, while the presence of M2-EVs exerted a tumorpromoting effect. Additionally, the tumor growth speed and weight tended to be significantly reduced in oe-NEDD4L +M2-EVs group and M2-EVs-anti miR-342-3p group vs. M2-EVs group (all p < 0.001) (Figs. 6A-6C).
Co-IP and GST pull-down assays were performed in the presence of MG132 to test the relationship between NEDD4L and CEP55 at the protein level. In the meantime, endogenous CEP55 was detected after expression of Myclabeled NEDD4L in cells. As displayed in (Fig. 8D), Myc-NEDD4L could be co-precipitated with anti-CEP55 antibody but not with control immunoglobulin IgG. It reveals the interaction of NEDD4L with endogenous CEP55 in RCC cells.
To validate the direct interaction between the two, a GST-pull-down assay was conducted with in vitro synthetic His-CEP55 and GST-NEDD4L (Fig. 8E). The effect of NEDD4L overexpression on CEP55 ubiquitination was then explored. As shown in (Fig. 8F), NEDD4L could promote the ubiquitination and degradation of CEP55, while such effect was reduced when mutations occurred in NEDD4L. This implies that CEP55 ubiquitination and degradation can be modulated by E3 ubiquitin ligase NEDD4L.
Altogether indicates that M2-EVs inhibit the E3 ubiquitin ligase NEDD4L from preventing the ubiquitination and degradation of CEP55 and activating the PI3K/AKT/mTOR signaling pathway.

Discussion
It is well established that the formation and maintenance of a cancer niche profoundly depend on its surrounding microenvironment, which consists of a network of reciprocal cell types such as endothelial cells, inflammatory cells, fibroblasts, stem cells, and immune cells [12,27]. TAMs are the most prominent tumor-infiltrating immune cells within the TME, and despite growing research, the regulatory crosstalk between TAM and RCC cells, especially how macrophages modulate various hallmarks of RCC during tumor progression remains not completely understood. Here, we found that elevated exogenous miR-342-3p within M2-EVs effectively disrupted NEDD4L expression and thus prevented the ubiquitination and degradation of CEP55. The up-regulated CEP55 subsequently activated the PI3K/AKT/mTOR pathway and strongly induced the proliferative, migratory and invasive properties of RCC cells.
Dysregulated miRNAs play a critical role in carcinogenesis [28]. miR-342-3p, localized to 14q32, has become as an important cancer-related miRNA in human cancers [29]. This miRNA is frequently down-regulated in hepatocellular carcinoma, non-small cell lung cancer, and gallbladder cancer, and acts as a tumor suppressor [22,30,31]. However, its functional significance in RCC is still poorly elucidated. In this study, we intriguingly verified a significant up-regulated expression of miR-342-3p in RCC tissues compared with normal controls, revealing that miR-342-3p was a carcinogenic factor rather than a suppressor implicated in the development and progression of RCC. In addition, we uncovered the highly expressed miR-342-3p in M2 macrophage-derived EVs. Accumulating evidence has supported the important roles of EV-derived miRNAs in RCC [20][21]32]. For instance, serum exosomal miR-210 originating from tumor tissue has been regarded as a novel diagnostic marker and prognostic predictor for ccRCC progression [21]. Additionally, urinary exosomal miR-30c-5p targets heat-shock protein 5 and inhibits ccRCC progression, which has considerable potential as a diagnostic biomarker for early-stage ccRCC [20]. Moreover, EVs shuttled miR-31-5p can transfer resistance information and promote sorafenib resistance in RCC by directly targeting Measurement data were expressed in the form of mean ± standard deviation (n = 3). One-way ANOVA (followed by Tukey posthoc test) was electively used for data comparison between multiple groups. Data were compared between groups at different time points using repeated measures ANOVA and Tukey posthoc test.
MutL homolog 1, thereby both miR-31-5p and its target gene are likely to be predictive biomarkers and therapeutic targets for sorafenib resistance [32]. In current work, we demonstrated that M2-EVs could carry and transmit miR-342-3p into RCC cells, thereafter driving RCC proliferative, migratory and invasive capacities in vitro and in vivo. This observation indicated that miR-342-3p appeared to be a novel and effective therapeutic target gene for RCC.
Another important observation in this study was that miR-342-3p could bind to NEDD4L and down-regulate its expression. NEDD4L is an E3 ubiquitin ligase that regulates channel internalization and turnover [33]. It appears to possess roles in multiple cell processes, such as transporter modulation, autophagy and signal transduction [33]. NEDD4L level is significantly changed, and it exhibits distinct functions in different carcinomas by regulating certain major pathways (such as TGF-β, WNT and EGFR signaling pathways) [34,35]. In gallbladder cancer, NEDD4L is significantly up-regulated and exerted a pro-oncogenic role through regulation of matrix metallopeptidase 1 and 13 genes transcription [36], whereas in prostate cancer, downregulation or loss of function of NEDD4L (that can exert an antitumor activity via regulating the TGFβ1 signaling) is observed and proposed to be associated with the malignancy [37]. Also, decreased NEDD4L expression in NSCLC is found to be more tumor aggressive and can predict poorer survival time [34,38]. Here, we showed that NEDD4L expression was decreased in RCC cell lines by miR-342-3p delivered in M2-EVs, thus consequently promoting the RCC cell migration and invasion. Our observation was inconsistent with other reports of the low expression of NEDD4L in ccRCC [37,39]. All these elucidated the tumor ; (E) RT-qPCR assay for miR-342-3p expression in lung tissue in each treatment group in nude mouse model of lung metastasis; (F) Western blot analysis for NEDD4L and CEP55 protein expression in lung tissue in each treatment group in nude mouse model of lung metastasis. Ã indicates p < 0.05 compared to the Control group, and # indicates p < 0.05 compared to the M2-EVs group. Measurement data were expressed in the form of mean ± standard deviation (n = 3). One-way ANOVA (followed by Tukey posthoc test) was electively used for data comparison between multiple groups. Data were compared between groups at different time points using repeated measures ANOVA and Tukey posthoc test.
suppressive role of NEDD4 in RCC carcinogenesis. Meanwhile, we found that transfer of M2-EVs-drived miR-342-3p into RCC cells to target NEDD4L diminished the degradation of CEP55. NEDD4L has been widely reported to modulate multiple signaling pathways in tumor cells through the ubiquitination and degradation pathways [40,41]. For example, ERBB3 (HER3), one of the EGFR family proteins, can undergo NEDD4L-mediated ubiquitination and degradation to down-regulate the signal transduction pathways of occurrence and development in cancers [42]. Additionally, NEDD4L is involved in ULK1 ubiquitination and degradation, thereby playing vital roles in initiating autophagy and maintaining redox homeostasis [43,44]. Our study verified the negative regulatory relationship FIGURE 8. M2-EVs regulates E3 ubiquitin ligase NEDD4L to affect CEP55 ubiquitination and degration and active PI3K/AKT/mTOR signaling pathway. (A) Changes of CEP55 level in ACHN cells treated with CHX or CHX-MG132; (B) Western blot analysis for CEP55 expression after overexpressing ubiquitin ligases Smurf1, Smurf2, Itch and NEDD4L; (C) Western blot analysis for CEP55 expression after overexpressing NEDD4L with or without treatment of MG132; (D) Co-IP assay to verify the interaction between NEDD4L and CEP55 in ACHN cells (IgG as negative control and Input as positive control); (E) In vitro GST-pull down assay to verify the interaction between GST-NEDD4L and His-CEP55 in ACHN cells (GST as negative control and Input as positive control); (F) Ubiquitination of CEP55 by NEDD4L as detected by IP with anti-Flag antibody followed by immunoblotting with anti-CEP55 or anti-HA antibody; (G) Protein levels of NEDD4L, CEP55, PI3K/AKT/mTOR signaling pathway markers (PI3K, p-AKT-Ser473 and p-mTOR-ser2448) in ACHN cells after treatment with sh-CEP55 or sh-CEP55+M2-EVs. Ã indicates p < 0.05, compared to the DMSO, oe-NC, oe-NC+DMSO or sh-NC groups; # indicates p < 0.05, compared to oe-NC+MG132 or sh-CEP55 groups. Measurement data were expressed in the form of mean ± standard deviation (n = 3). One-way ANOVA (followed by Tukey posthoc test) was electively used for data comparison between multiple groups. Data were compared between groups at different time points using repeated measures ANOVA and Tukey posthoc test. between NEDD4L and CEP55; thus, the above reports would support our hypothesis that NEDD4L exerts the ubiquitination regulation effect on CEP55. CEP55, a centrosome-and midbody-associated protein, is critical for cell cycle progression and cytokinesis [45]. Many studies have revealed that CEP55 serves a pivotal role in the cell cycle and survival through the regulation of the PI3K/AKT pathway [25,45,46]. For instance, Li et al. has shown that CEP55 can promote proliferation and inhibit apoptosis via the PI3K/AKT/p21 signal pathways, further attributing to the carcinogenesis and progression of glioma cells [46]. Additionally, Chen et al. have demonstrated that the PI3K/AKT/mTOR pathway is capable of regulating the effects of CEP55 on the migration, invasion and EMT of RCC cells and might be used as an effective prognostic marker [25]. In consistent with this, our study also verified that upregulated CEP55 could sustain RCC growth, proliferation and metabolism through activation of the PI3K/AKT/mTOR pathway. In conjunction with existing evidence, we preliminarily indicated that M2-EVs encapsulated miR-342-3p could promote the CEP55 expression by targeting NEDD4L and inhibiting NEDD4L expression, thus consequently promoting the proliferative, migratory and invading properties of RCC cells via activation of the PI3K/AKT/mTOR pathway.
In fact, PI3K/AKT pathway is modestly mutated but highly activated in RCC [47]. Recent studies have evidenced that classical activation of the PI3K/AKT network is not always triggered by extracellular stimuli and transmembrane protein receptors [47]. Currently, miRNAs are emerging as a new class of important regulators of the PI3K/AKT pathway. For example, miR-122 is proved to be a positive modulator of the PI3K/AKT signaling, that can promote proliferation, invasion, and migration of RCC cells [48]. Additionally, miR-182-5p is in a down-regulated expression pattern of AKT, and its reduction results in AKT activation and subsequent RCC proliferation [49]. These results partially supported that miR-342-3p could be identified to be a novel regulator that contributed to aberrant activation of the PI3K/AKT pathway in RCC, representing a promising target for RCC therapy. Moreover, a previous study has reported that in the presence of tumors, EV-enrichment may represent an epigenetic silencing mechanism whereby ccRCC maintains tumor development and growth by activating the PI3K/AKT pathway [32]. The overall activation of the PI3K/AKT pathway in ccRCC is higher than in other cancers, suggesting that dysregulation of the PI3K/AKT pathway in RCC may be a consequence of EV-mediated epigenetic mechanisms [47,50].
Altogether, findings obtained in our study concluded that M2-EVs-drived miR-342-3p could play an important role in the invasion and migration process of RCC, and had the potential to be used as a therapeutic biomarker. However, we recognize that additional mechanisms may be involved in such pathways, which warrants further exploration.

Conclusion
To sum up, a preliminary conclusion can be obtained in this research that M2 macrophages may carry miR-342-3p through EVs to target NEDD4L in RCC cells, inhibit the ubiquitination degradation of CEP55 and activate the PI3K/AKT/mTOR signaling pathway, thereby promoting the growth and metastasis of RCC (Fig. 9). This finding provides a novel therapeutic target in future RCC treatment.