786-O cells [American Type Culture Collection (ATCC)] underwent a 72-h culture using Dulbecco's modified Eagle's medium (DMEM) containing 10% EV-free fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.) and 1% penicillin streptomycin. The supernatant was collected and subjected to 5-min (at 1,100 × g) and then 30-min (at 40,000 × g) centrifugation regimens to eliminate the cells and cellular debris, and large vesicles, respectively. After filtration of the supernatant using a 0.2-µm filter and centrifugation (at 1,000,000 × g) for 2 h, the EVs underwent a phosphate-buffered saline (PBS) rinse and a 2-h regimen of centrifugation (at 100,000 × g), followed by resuspension in PBS. An EV isolation reagent (Thermo Fisher Scientific, Inc.) was used for EV isolation. Nanoparticle tracking analysis (NTA) was applied for EV analysis using the NanoSight NS300 (Malvern Panalytical Co., Ltd.). The obtained EVs were allocated into the EV group and the blank group [supplemented with 1 µM GW4869 (MedChemExpress Co., Ltd.)] (19). The small-interfering (si)-MALAT1-1, si-MALAT1-2 and si-negative control (NC) (Shanghai GenePharma Co., Ltd.) were transfected into the 786-O cells, respectively, using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) to attain a final transfection concentration of 50 nM. The EVs (EVs/si-MALAT1-1, EVs/si-MALAT1-2 and EVs/si-NC) were extracted respectively by ultracentrifugation and identified with the preceding protocol. The morphology of the EVs was observed under transmission electron microscopy (TEM). The protein determination by the bicinchoninic acid (BCA) method was performed on all the aforementioned EVs and the result was 2.2 mg/ml. Subsequent experiments were conducted using the protein concentration as the concentration of EVs.

Bioinformatics analysis. The lncDisease database (http://www.cuilab.cn/lncrnadisease) was applied to search for lncRNAs associated with RCC. The expression of the candidate lncRNAs in the tumor-EVs was searched through the exoRBase database (http://www.exorbase.org/). The lncMAP database (http://bio-bigdata.hrbmu.edu.cn/LncMAP/) was used to predict the downstream regulatory transcription factors in kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP). Next, the genes related to RCC were searched through the DisGeNET database (https://www.disgenet.org/). Gene interaction network diagram was constructed using the STRING database (https://string-db.org/) and Cytoscape V3.7.1 software (www.cytoscape.org). RCC microarray GSE16441 (20) including 17 normal samples and 17 RCC samples was obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). With the normal samples as the control, the R language ‘limma’ package (21) was applied for differential analysis. TheP-value was corrected using the false discovery rate (FDR) method, and |logFC|>2 and FDR <0.05 were used as the screening criteria for the differentially expressed genes. JASPAR database (http://jaspar.genereg.net/) was used to predict the E26 transformation specific-1 (ETS1) downstream regulatory genes, and to obtain the existing binding site between transcription factor CP2-like 1 (TFCP2L1) promoter and ETS1.

RCC cell lines A498 and ACHN supplied by ATCC were cultured in minimal essential medium (MEM; Gibco Co.; Thermo Fisher Scientific, Inc.) with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in an incubator at 37°C under saturated conditions with 5% CO₂. The A498 and ACHN cells were co-cultured with 10 µg/ml of the above-mentioned 786-O-EVs (22) for 24 h, grouped and named similarly as the aforementioned EVs.

The 786-O-EVs were labeled by a PKH26 red fluorescence labeling kit (Sigma-Aldrich; Merck KGaA) in compliance with the provided instructions. Briefly, the EVs underwent a 4-min incubation regimen with the PKH26 dye, which was terminated by the addition of EV-free FBS. The EVs were rinsed three times and the excess PKH26 dye was removed with an Amicon Ultra-4 (100-kDa, Merck Millipore Corp.), followed by a 4-h incubation regimen with the RCC cells. The internalization of EVs by RCC cells was observed under confocal microscopy.

The total RNA content from the cells in each group was extracted using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and the RNA concentration was determined using a NanoDrop 2000 (Thermo Fisher Scientific, Inc.). The PrimeScript RT reagent kit and gDNA Eraser kit (Takara Bio Inc.) were used to transcribe the total RNA (1 mg) content into cDNA. Genomic DNA termination was induced by the addition of 5X DNA Eraser buffer and gDNA Eraser at 42°C for 2 min. Next, the cDNA was synthesized with the reaction condition as 37°C for 15 min and 85°C for 5 sec. Next, qPCR was conducted using a SYBR Premix Ex Taq (Tli RNaseH Plus) kit (Takara) on a ABI 7500 real-time PCR instrument (Thermo Fisher Scientific, Inc.). Data of each gene were analyzed based on the 2^−ΔΔCq method (23). The primer sequences are listed in Table I.

The total protein content was extracted from cells in each group, and the protein concentration was measured using a BCA kit (Thermo Fisher Scientific, Inc.). Total proteins (30 mg) underwent 35-min (at 80 V) and 45-min (at 120 V) polyacrylamide gel electrophoresis successively. Next, the proteins were loaded onto polyvinylidene fluoride membranes (Amersham Pharmacia), blocked for 1 h using 5% skim milk, and probed with the corresponding primary antibodies [cluster of differentiation (CD)63 (dilution 1:1,000, ab134045), CD81 (dilution 1:1,000, ab109201) and TFCP2L1 (dilution 1:1,000, ab140197) (all from Abcam Inc.)] at 4°C. Following 3 rinses (10 min each) with PBS and 0.1% Tween-20 (PBST), the membranes underwent a 1-h incubation regimen with the horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) H&L (dilution 1:2,000, ab205718; Abcam Inc.), and rinsed 3 times with PBST (10 min each). An optical illuminator (General Electric) was utilized for membrane visualization. Image-Pro Plus 6.0 (Media Cybernetics) was applied for protein band gray value analysis.

RCC cell proliferation was detected using the CCK-8 kit (Dojindo Molecular Technologies) in strict accordance with the provided instructions. RCC cells were seeded into 96-well plates and each well was supplemented with EVs based on cell grouping and incubation for 24 h. Then approximately 10 µl CCK-8 reagent was added to each well for a 2-h regimen of incubation at 37°C. The optical density (OD) at the wavelength of 490 nm was determined using a microplate reader (Thermo Fisher Scientific, Inc.). Relative cell viability of each group was determined.

RCC cell invasion was assessed according to the passage of the number of transfected cells through Transwell chambers (8-micron chamber; Corning Inc., Life Sciences). The Transwell chambers were pre-coated with 100 µl Matrigel (BD Biosciences) at 37°C for 5 h until gelling was visible. Next, 1×10⁵ A498 and ACHN cells that underwent a 24-h starvation in 500 µl serum-free MEM medium were seeded in the apical chamber, respectively (3 technical replicates for each group). The basolateral chamber was filled with 700 µl MEM with 10% FBS. Following a 48-h incubation at 37°C, Matrigel and non-invaded cells were removed from the upper surface of the filters. Cells that adhered to the lower surface of filters were fixed using 4% paraformaldehyde and stained with crystal violet. Finally, the cells were counted and photographed using a Nikon digital camera (magnification, ×100). Three independent experiments were repeatedly set. The method for the migration experiment was the same as that of the invasion experiment, without utilization of Matrigel.

The cells were seeded on 24-well plates and received different treatments. Next, the cells were fixed for 15 min in 4% paraformaldehyde, permeated for 10 min with PBS containing 0.1% Triton X-100, and sealed for 30 min using the 3% plasma blocking solution (all performed at room temperature). Subsequently, the cells were subjected to overnight incubation at 4°C with the diluted primary antibodies E-cadherin (dilution 1/200, ab1416, Abcam) and N-cadherin (dilution 1/200, ab76057, Abcam), followed by a 1-h incubation regimen with the secondary antibody goat anti-mouse IgG H&L (Alexa Fluor^® 488) (dilution 1/200, ab150117, Abcam) in conditions devoid of light at room temperature. Finally, 4′,6-diamidino-2-phenylindole (DAPI) was added for nuclear staining. After resting at room temperature for 1 min, the cells were observed under a fluorescence microscope (Olympus Optical Co., Ltd.).

MALAT1 subcellular localization was observed using FISH assay in strict accordance with the provided instructions of the Ribo™ lncRNA FISH Probe Mix (Red) (C10920, RiboBio Co., Ltd.). The cells (6×10⁴ cells/well) were seeded into 24-well plates. Upon achieving 60–70% confluence, the cells were fixed using 4% paraformaldehyde, rinsed and permeabilized. The plates were sealed using the pre-hybridization solution. After elimination of the pre-hybridization solution, the cells were subjected to overnight hybridization at 37°C using the probe hybridization solution supplemented with anti-MALAT1 nucleotide (Wuhan Genecreate Bioengineering Co., Ltd.) in conditions devoid of light. Next, the cells were eluted, stained with DAPI, rinsed, and fixed using nail polish for observation under fluorescence microscopy (Olympus). Five different fields were selected, and the cells in these fields were observed and documented.

To explore the effect of MALAT1 on TFCP2L1 promoter activity, overexpressed (oe)-NC, oe-MALAT1, short hairpin (sh)-NC, and sh-MALAT1 were co-transfected with the TFCP2L1-2 kb luciferase reporter plasmid respectively into 293T cells. After 48 h of transfection, the cells were collected and lysed. The dual-luciferase reporter gene assay was performed using a luciferase assay kit (K801-200, BioVision Inc.) and a dual-luciferase reporter gene analysis system (Promega Corp.). Renilla luciferase was adopted for internal reference. The activation degree of the target reporter gene was measured using the ratio of the relative unit of firefly luciferase to that of Renilla luciferase.

A RIP kit (Millipore Corp.) was utilized to assess the binding of MALAT1 to the ETS1 protein. The cells were lysed in an ice bath, and then subjected to centrifugation. Next, the supernatant was collected. The cell extract was incubated with the corresponding antibody for co-precipitation. Following a rinse and re-suspension in RIP wash buffer, the magnetic beads were supplemented with the appropriate antibody for binding. After a rinse, the magnetic bead-antibody complex was resuspended in RIP wash buffer, incubated overnight at 4°C with the cell extract, and harvested. The RNA content was extracted from the sample for subsequent PCR detection after detachment with proteinase K. The antibody used above was ETS1 (dilution 1:200, ab225868, Abcam), with IgG (dilution 1:100, ab172730, Abcam) as the control.

Biotinylated RNA sequence probe in the TFCP2L1 promoter region and its negative control (NC) probe were dissolved in washing/binding buffer at room temperature and incubated with the streptavidin-coupled magnetic beads for 2 h. Then, the cell lysate was added and incubated for 2 h. The protein complex conjugated to magnetic beads was washed. ETS1 content in the complex was determined by western blot analysis.

The experimental procedures were approved by the Ethics Committee of The Second Affiliated Hospital of Harbin Medical University. Significant efforts were made in order to minimize both the number of animals used as well as their respective suffering.

A total of 48 BALB/C nude mice (male, aged 4 weeks, weighing 15–18 g; 6 each per group) used for animal experiments and xenograft collection were supplied by Beijing Wantai BioPharm Co., Ltd. [Beijing, China; SYXK (Jing) 2017-0041]. Each nude mouse was housed in a separate cage and provided with free access to food and water. All mice were fed with clean grade maintenance feed and kept at a temperature of 26–28°C and humidity at 40–60% with 10/14 h light/dark cycle. The nude mice were subcutaneously injected with the 786-O cell suspension (5×10⁶ cells), and treated with the blank, EVs, EVs/si-NC or EVs/si-MALAT1-1 (20 nM) using Entranster™-in vivo Transfection Reagent (Engreen Biosystem Co., Ltd.) after acquiring a tumor volume greater than 100 mm³ (n=6) in each nude mouse. Xenografts were determined weekly. The nude mice were euthanized via intraperitoneal injection of 800 mg/kg pentobarbital sodium after 5 weeks and the tumor was dissected and weighed. The Ki67-positive rate was observed and counted.

The grouping of the lung metastasis model was the same as the subcutaneous tumorigenesis. Then, 6 nude mice from each group were injected with the ACHN cells (2×10⁶ cells) via tail vein. After 42 days, the mice were euthanized by an intraperitoneal injection with 800 mg/kg pentobarbital sodium. The lung tissues were extracted and stained using hematoxylin and eosin (H&E) (JRDUN Biotechnology Co., Ltd.).

The removed tumor sample was fixed using 4% paraformaldehyde and embedded in paraffin, which was then sectioned, dewaxed, and hydrated independently. The Ki67 antibody (dilution 1:500, ab15580, Abcam) was used for immunohistochemical staining. The Ki67-positive rate was observed and counted under a microscope (BX53M, Olympus; magnification, ×200).

The extracted lung tissue was fixed using 4% paraformaldehyde and embedded in paraffin. Then, the tissue was sectioned, dewaxed and hydrated independently. The sections were stained using the hematoxylin solution for 10 min and decolorized in 70 and 90% ethanol after a rinse with distilled water. Subsequently, the sections were stained with the eosin solution for 2–3 min, dehydrated with pure ethanol, cleared with xylene, sealed with neutral gum and then observed under a microscope (BX53M, Olympus; magnification, ×200).

SPSS 21.0 (IBM Corp.) software was used for data analysis. The experimental data were in normal distribution as verified by Kolmogorov-Smirnov test, and expressed as mean ± standard deviation. Comparison between two groups was conducted with the independent sample t-test. The comparison among groups was analyzed using one-way analysis of variance (ANOVA), followed by the Tukey's multiple comparisons test. The P-value was obtained from a two-sided test and a value of P<0.05 was indicative of statistical significance.

After a 48-h culture regimen of 786-O cells in EV-free FBS, the EVs were separated by ultracentrifugation. NTA confirmed the size of the EVs as ranging between 30–150 nm (Fig. 1A). Consistently, TEM revealed that the EVs were oval with a diameter of about 100 nm (Fig. 1B). Next, according to the WB results, the EV symbolic markers (CD63 and CD81) were positively expressed in the 786-O-EVs (Fig. 1C). From the aforementioned findings, the separated granules were identified as EVs.

An existing study reported that the tumor-derived EVs elicit negative effects on habitual cellular functions (24). Therefore, we speculated that RCC cell-derived EVs may have similar effects on the biological behaviors of RCC cells. To ascertain this speculation, we labeled the EVs with PKH26 red fluorescence dye after which these EVs were co-cultured (10 µg/1×10⁵ cells) with the A498 and ACHN cells for 48 h. Under fluorescence microscopy the A498 and ACHN cells elicited red fluorescence in their cytoplasm (Fig. 2A), indicating the internalization of 786-O-EVs by the RCC cells. Next, the A498 and ACHN cell viability was investigated to explore the effect of 786-O-EVs on A498 and ACHN cell functions. CCK-8 assay verified that the A498 and ACHN cell viability increased significantly after treatment with the 786-O-EVs (Fig. 2B) (both P<0.05). Meanwhile, Transwell assays revealed that 786-O-EV treatment significantly increased A498 and ACHN cell migration and invasion (Fig. 2C) (all P<0.05). Next, the A498 and ACHN cell epithelial mesenchymal transition (EMT) was detected by immunofluorescence, which was evidently promoted after 786-O-EV treatment (Fig. 2D). Briefly, 786-O-EVs promoted RCC cell invasion and metastasis.

From the preceding results, it is evident that the 786-O-EVs promoted RCC cell invasion and metastasis. To explore the underlying mechanism, we referred to previous studies. For instance, EVs were found to release and carry lncRNAs into the target cells to manipulate cellular functions (25). MALAT1 was also found to promote RCC cell malignant biological behaviors (26). Therefore, we searched RCC-related lncRNAs through the lncDisease database (http://www.cuilab.cn/lncrnadisease), and found the involvement of lncRNAs such as MALAT1 in the regulation of RCC (Table SI). Furthermore, the expression of these candidate lncRNAs in the EVs was searched through the exoRBase database (http://www.exorbase.org/) (Fig. 3A), which revealed that MALAT1 was expressed in various tumor-EVs, suggesting that MALAT1 may also exist in RCC cell-derived EVs. Hence, we speculated that 786-O-EVs carried MALAT1 and perhaps were internalized by the A498 and ACHN cells, thus inducing RCC cell malignant biological behaviors. MALAT1 expression pattern in the A498, ACHN and 786-O cells was determined by qPCR with no distinctive difference indicated by the results. Subsequently, the MALAT1 expression pattern in the A498 and ACHN cells before and after EV treatment was initially detected, which revealed a significant increase after EV treatment (Fig. 3B) (both P<0.01). RNase treatment did not affect the MALAT1 expression pattern in the culture medium of ACHN and A498 cells; while RNase and Triton X-100 treatment showed a significant decreased MALAT1 expression pattern in the culture medium of ACHN and A498 cells (Fig. 3C) (all P<0.01). These results indicated that MALAT1 was membrane-encapsulated over direct release.

From the preceding results, we established that 786-O-EVs were the communication medium of MALAT1 between RCC cells. To further validate the mechanism, we silenced the MALAT1 expression pattern in 786-O cells, and then extracted the EVs (EVs/si-MALAT1-1/2). We found that the MALAT1 expression pattern in the 786-O-EVs was significantly decreased after inhibition of MALAT1 in the 786-O cells (Fig. 4A and B). Similar reduction in the MALAT1 expression pattern was evident in RCC cells treated with 786-O-EVs after treatment with EVs/si-MALAT1-1/2 (Fig. 4C) (all P<0.01). In addition, the biological behavior changes of cells in each group were evaluated. EVs/si-MALAT1-1/2 weakened the explicit effect of simple 786-O-EVs on RCC cell viability, invasion and migration and EMT capacity (Fig. 4D-F) (all P<0.01). Collectively, 786-O-EVs transferred MALAT1 to induce RCC cell vitality, invasion and migration and EMT.

For a comprehensive understanding of the regulatory mechanism of MALAT1, we predicted the downstream regulatory transcription factors of MALAT1 in KIRC and KIRP through the lncMAP database (Table SII). Intersection of the predicted transcription factors in the two diseases was evidently intersected (Fig. 5A), from which four candidate transcription factors were chosen. Subsequently, the genes relevant to RCC were retrieved through the DisGeNET database (https://www.disgenet.org/) (Table SIII), where the genes with scores higher than 0.3 were selected for subsequent analyses. The interaction between the retrieved known genes and the predicted four transcription factors was analyzed, after which the gene interaction network diagram was constructed (Fig. 5B). It was evident that ETS1 had the most interactions with known genes, suggesting the superior vitality of ETS1 relative to others. In a previous work, ETS1 served as an oncogene in RCC with intimate relations with a low survival rate in a large number of RCC samples (27). Meanwhile, a RCC microarray GSE16441 was obtained through the GEO database (https://www.ncbi.nlm.nih.gov/geo/), where 406 differentially downregulated genes were identified by differential analysis of the GSE16441 chip. Concurrently, the ETS1 downstream regulatory factors were predicted through the JASPAR database (http://jaspar.genereg.net/), where the intersection between the downregulated genes in the chip and JASPAR prediction results was engaged, and five genes were identified to be present in the respective intersection (Fig. 5C). Their differential expression levels in the GSE16441 chip were further studied, where TFCP2L1 was regarded as the most notably downregulated gene (Table II). As previously highlighted, TFCP2L1 is a critical developmental transcription factor in normal kidney development (28). Meanwhile, JASPAR database predicted a number of binding sites between ETS1 and the TFCP2L1 promoter (Table SIV). Therefore, we speculated that 786-O-EVs transferred MALAT1, to subsequently regulate the transcription factor ETS1 to modulate TFCP2L1 expression, thereby affecting RCC development.

Subsequently, FISH revealed nuclear subcellular localization of MALAT1 in the cell (Fig. 5D). Dual-luciferase reporter gene assay verified that the MALAT1 overexpression remarkably reduced the TFCP2L1 promoter activity, while MALAT1 knockdown dramatically increased the TFCP2L1 promoter activity, indicating the negative regulatory effect of MALAT1 on the TFCP2L1 gene expression pattern (Fig. 5E) (both P<0.01). Next, RIP was applied to further elucidate the underlying mechanism of MALAT1 to negatively regulate the TFCP2L1 gene expression pattern, and we observed that ETS1 would bind to more MALAT1 than IgG (Fig. 5F) (P<0.01), indicating that the ETS1 protein could specifically bind to MALAT1. RNA pull-down showed that ETS1 could bind to the TFCP2L1 promoter region (Fig. 5G). The binding of ETS1 and the TFCP2L1 promoter in the A498 and ACHN cells treated with EVs was enhanced whereas the binding of ETS1 and TFCP2L1 promoter was weakened after silencing of MALAT1 in EVs (Fig. 5H). As shown by qPCR results, 786-O-EV treatment significantly decreased the TFCP2L1 mRNA expression pattern. Following knockdown of MALAT1 in the 786-O cells, the downregulation effect of EVs on TFCP2L1 was alleviated (Fig. 5I). The above results indicated that lncRNA MALAT1 would bind to the transcription factor ETS1 and identify the fundamental complex in the sequence of TFCP2L1 promoter to inhibit transcription. Namely, MALAT1 could specifically bind to ETS1 and inhibit TFCP2L1 expression.

To confirm that 786-O-EVs promote RCC cell invasion and migration through MALAT1 in vivo, we conducted tumor xenograft formation assay, and measured the tumor volume weekly for 5 weeks. We observed that 786-O-EVs had notably promoted tumor growth in vivo (Fig. 6A). Five weeks after EV injection, the mice were euthanized, and the xenografts were dissected and weighed. The results revealed that 786-O-EV treatment apparently increased the average tumor weight (Fig. 6B) (both P<0.01). In addition, immunohistochemical staining revealed that the 786-O-EVs resulted in noticeably increased Ki67-positive rates, while the tumor growth-promoting effect of 786-O-EVs was weakened after inhibition of MALAT1 in the 786-O cells (Fig. 6C) (both P<0.01). Moreover, the lung metastasis model was established via tail vein injection. According to H&E staining results, nude mice exhibited increased lung tissue lesions after 786-O-EV treatment, while the lung tissue lesions were reduced after silencing of MALAT1 in the 786-O cells (Fig. 6D). In conclusion, 786-O-EVs released MALAT1 to promote RCC cell growth and metastasis in vivo.