Mechanism of Exosomal LncRNA PART1 in Esophageal Cancer Angiogenesis by Targeting miR-302a-3p/CDC25A Axis

Abstract

Objective

LncRNA PART1 has been confirmed related to multiple cancer bioactivities mediated with vascular endothelial growth factor signaling. Nevertheless, the role of LncRNA PART1 in esophageal cancer induced angiogenesis remains unclear. The present work focused on assessing LncRNA PART1 effects on esophageal cancer–induced angiogenesis and exploring possible mechanisms.

Methods

Western blot and immunofluorescence were conducted for identifying EC9706 exosomes. MiR-302a-3p and LncRNA PART1 levels were assessed by real-time quantitative polymerase chain reaction. Cell Counting Kit-8, EdU, wound healing, transwell, and tubule information were adopted for detecting human umbilical vein endothelial cell viability, proliferation, migration, invasion, and tubule information, respectively. Starbase software and dual-luciferase reporter were conducted for predicting and judging the expression interrelation of LncRNA PART1 and its potential target-miR-302a-3p. The same methods were carried out for verifying the inhibiting influences of miR-302a-3p upregulation and its potential target-cell division cycle 25 A.

Results

LncRNA PART1 levels were upregulated and related to the overall survival of patients in esophageal cancer. EC9706-Exos accelerated human umbilical vein endothelial cell proliferation, migration, invasion, and tubule formation via LncRNA PART1. LncRNA PART1 served as a sponge of miR-302a-3p, then miR-302a-3p targeted cell division cycle 25 A, and EC9706-Exos accelerated human umbilical vein endothelial cell angiogenesis via LncRNA PART1/ miR-302a-3p/cell division cycle 25 A axis.

Conclusion

EC9706-Exos accelerates human umbilical vein endothelial cell angiogenesis via LncRNA PART1/miR-302a-3p/ cell division cycle 25 A axis, indicating EC9706-Exos may act as a promoter of angiogenesis. Our research will contribute to clarify the mechanism of tumor angiogenesis.

Keywords

LncPART1 exosome HUVECs esophagus cancer tumor angiogenesis

Introduction

Esophageal squamous cell carcinoma (ESCC) has a high morbidity and mortality rate on a global scale,¹ and the 5-year survival rate is poor due to tumor recurrence.² Therefore, it is essential to explore the molecular mechanisms and highly sensitive and specific biomarkers in ESCC progression and metastasis. Tumor microenvironment is a dynamic environment composed of tumor cells, stromal cells, and extracellular matrix.³ Tumor cells can exhibit their bioactivities through interacting with microenvironment.⁴ Thus, investigating tumor microenvironment reveals a new approach to cancer diagnosis and treatment.

As a bridge between tumor and microenvironment, exosomes exert an essential effects on the information exchange between tumor and its microenvironment.⁴ Exosomes (50-100 nm) are small membrane vesicles deriving from endocytosis, containing long non-coding RNAs (lncRNAs), microRNAs (miRNAs), mRNAs, DNA fragments and proteins, meanwhile they shuttle from the donor cell to the target cell.⁵ Exosomes are released into the extracellular environment following fusing with the cell membrane, which not only exist in the supernatant of tumor cell but also in body fluids.⁶ There are a variety of biologically active molecules in exosomes, which can be delivered to target cells via fusing with them.⁷ As one of these substances, lncRNA has a long transcript of more than 200 nt, with few or limited protein coding potential, which exist in the nucleus or cytoplasm, and interact with DNA, RNA, or proteins.⁸ What's more, LncRNA is a new type of regulatory RNA that can be selectively entered into exosomes. Exosomes lncRNA plays a crucial role in cancer progression.⁹ It is worth noting that the dysregulation of exosome lncRNAs may affect the tumor microenvironment and regulate crucial oncology behaviors.¹⁰ Thus, the differential expression of lncRNA in exosomes might be a potential marker for cancer diagnosis.

MicroRNA is an evolutionarily conserved noncoding small RNA with a length of 18 to 20 nt, which is involved in gene expression regulation and protein translation, and accumulating evidence has indicated that miRNAs act as oncogenes or tumor suppressors during tumorigenesis of different types of cancer.^11,12 As expected, miR-302a-3p has been confirmed to participate in multiple disease progression. For example, miR-302a-3p modulates the proliferation and migration of pancreatic ductal adenocarcinoma cells.¹³ What's more, Yang et al research shows that miR-302a-3p inhibits gastric cancer cell proliferation through regulation of vascular endothelial growth factor.¹⁴ This indicated that miR-302a-3p regulated cancer progression by regulating angiogenesis. We focused on exploring the underlying mechanisms by which miR-302a-3p participates in angiogenesis.

In summary, exosomes adjusted the tumor microenvironment by changing the physiological state of target cells. Therefore, exosomes were vital messengers in tumor progression. In this article, we focused on investigating the roles of EC9706-Exos in angiogenesis, and the underlying mechanism. Our research will contribute to clarify the mechanism of tumor angiogenesis.

Materials and Methods

Human Tissue and Specimens

This study collected 30 pairs of ESCC patient tissues and adjacent nontumor tissue samples from Jiangsu Cancer Hospital from December 2020 to May 2021. All tissue samples were stored at −80 °C. All patients in the study were initially diagnosed with ESCC and had not received any chemotherapy or radiotherapy before. The study was approved by the Ethics Committee of Jiangsu Cancer Hospital (ethical approval number: 2020 ke-052). Each participant provided written informed consent for participation.

EC9706-Exos Isolation and Identification

EC9706 cells were provided by ATCC. EC9706-Exos were isolated from EC9706 cells using Ribo™ Exosome Isolation Reagent (RiboBio). Transmission electron microscope (BD Biosciences) was adopted for observing EC9706-Exo morphology; the concentration particle size of EC9706-Exos was analyzed by nanoparticle tracking analysis; Western blot was conducted for identifying the EC9706-Exos surface markers (CD63 and tumor susceptibility gene 101 [TSG101]), and immunofluorescence was used to observe the internalization of PKH-26-labeled EC9706-Exos by human umbilical vein endothelial cells (HUVECs), according to the previous description.¹⁵

Cell Culture

Human umbilical vein endothelial cells and ESCC cell lines, including HET-1A, EC9706, KYSE150, and YES2, were provided by ATCC and cultivated within DMEM (Thermo Fisher Scientific) that contained 10% fetal bovine serum (FBS; Gibco) under 37 °C and 5% CO₂.

Cell Transfection

Genechem was responsible for constructing miR-302a-3p mimics/mimics NC, miR-302a-3p inhibitors/inhibitors NC, sh-NC, sh-LncPART1, sh- cell division cycle 25 (CDC25A), pcDNA3.1, pcDNA3.1-LncPART1, and pcDNA3.1-CDC25A plasmids. According to the instruction, each of the above plasmids was individually transfected in cells via Lipotransfectamine 6000 (Thermo Fisher Scientific).

Real-Time Quantitative Polymerase Chain Reaction

After extracting total RNA from esophageal cancer (EC) tumor tissues, EC9706-derived exosomes, HUVECs transfected with corresponding plasmids and EC cell lines including EC9706, KYSE150, and YES2 according to the manufacturer's protocol. cDNA was prepared with the extracted total RNA by the RNeasy plus micro kit through reverse transcription in line with specific instructions, as the starting material of real-time quantitative polymerase chain reaction (RT-qPCR) carried out using Step One System (Life Technologies Corp). 2^−ΔΔCT approach normalized U6 and GAPDH. Primers used in this research are displayed in Table 1.

Table 1.

Primer Sequences.

Primer name	（5′-3′）Primer sequences
F-LncRNA PART1	5′-AAGGCCGTGTCAGAACTCAA-3′
R-LncRNA PART1	5′-GTTTTCCATCTCAGCCTGGA-3′
F-miR-302a-3p	5′-AATAAGTGCTTCCATGTTTTGGTGA-3′
R-miR-302a-3p	5′-GCCGCATCTTCTTTTGCGTCGC-3′
F-U6	5′-CTCGCTTCGGCAGCACATATACT-3′
R-U6	5′-ACGCTTCACGAATTTGCGTGTC-3′
F-GAPDH	5′-GAGTCAACGGATTTGGTCGT-3′
R-GAPDH	5′-TTGATTTTGGAGGGATCTCG-3′

Western Blot

Protein from EC9706-Exos was extracted and measured with BCA kit (Beyotime Biotechnology). Protein was transferred to PVDF membrane after 10% SDS-PAGE gel electrophoresis. After enclosing with 5% skimmed milk, the antibodies including anti-CD63 (bs-1523R, 1: 2, 000, Bioss), anti-TSG101 (bs-1365R, 1: 2, 000, Bioss), and anti-β-actin (bs-0061R, 1: 2, 000, Bioss) were incubated overnight. Secondary resistance (1: 4,000, SA00004-10, ProteinTech) was subsequently incubated at 37 °C. Finally, ECL was utilized to detect protein blots, whereas ImageJ software (NIH, version 4.3) was adopted for quantification (Table 2).

Table 2.

Acronyms/Symbol Description.

Acronyms	Full written
EC	Esophagus cancer
VEGF	Vascular endothelial growth factor
LncPART1	LncRNA PART1
HUVECs	Human umbilical vein endothelial cells
LncRNAs	Long non-coding RNAs
MiRNAs	MicroRNAs
SDS-PAGE	Sodium dodecyl sulfate-polyacrylamide gels
PVDF	Polyvinylidene fluoride
TEM	Transmission electron microscopy
CDC25A	Cell division cycle 25 A

Cell Counting Kit-8 Assay

Human umbilical vein endothelial cells that were treated with EC9706-Exos (1 μg/μL, 200 μL) or transfected with corresponding plasmids were inoculated into 96-well plates. After incubated for indicated times, CCK-8 kit solution (Sigma) was utilized. Absorbance (OD) (450 nm) was measured using a spectrophotometer (Molecular Devices).

EdU Assay

Transfected HUVECs (4 × 10⁴/well) from different groups were inoculated into the 24-well plates for 48 h. After being fixed with 4% paraformaldehyde, and incubated with EdU medium. Goat serum was used to block cells for another 1 h. Furthermore, cells were stained in line with specific protocols.

Wound Healing Assay

Human umbilical vein endothelial cells (5 × 10⁵/well) from different groups were placed into 6-well plates at the appropriate density. After cells reached 80% confluence, EC9706-Exos (1 μg/μL) were added, and corresponding plasmids were transfected. Wound was scratched. Cell images at 0 and 48 h were photoed with a light microscope (Nikon, Japan) (200×).

Transwell Assay

For transwell migration assay, HUVECs (1 × 10⁵/well) treated with EC9706-Exos (1 μg/μL) or transfected with corresponding plasmids were seeded in the upper chamber in serum-free medium. Medium containing 10% FBS was added into lower chambers. After 24-h incubation at room temperature, migrated and invaded cells were stained with crystal violet (0.1%) and photoed (Nikon) (200×).

Tubule Formation Assay

Human umbilical vein endothelial cells (2 × 10⁴/well) from different groups were seeded on Matrigel pretreated 96-well plates. Following 12-h culturing, the formation of tubules was observed under phase-contrast microscopy (Olympus).

Bioinformatics and Dual Luciferase Reporter Gene Assay

This study subcloned lncPART1 or CDC25A WT/MUT for generating pmirGLO-lncPART1 or pmirGLO-CDC25A WT/MUT to cotransfect into HUVECs cells with NC mimics or miR-302a-3p mimics. After cotransfected for 48 h, the luciferase activity was measured (Promega).

Statistical Analysis

The average ± standard deviation represents data from 3 repetitions. GraphPad Prism 8.0 Software (GraphPad Software, Inc.). Each experiment was repeated 3 times. The comparison between the 2 groups used t test, the group comparison used 1-way ANOVA and 2-way ANOVA with Tukey posttest, P < .05, the difference is statistically significant.

Results

Isolation and Identification of EC9706-Exos

We extracted exosomes from EC9706 cells to evaluate exosomes’ effects on angiogenesis of EC, TEM revealed that exosomes were round vesicles with typical cup-shaped structure and a diameter of around 100 nm (Figure 1A and B). Western blot analysis indicated the enrichment of well-recognized exosome markers CD63 and TSG101 (Figure 1C). What's more, confocal microscope observed that there was a higher absorption rate after HUVECs were cocultured with PKH-26-labeled EC9706-Exos, suggesting HUVECs successfully internalized EC9706-Exos (Figure 1D). Taken together, EC9706-Exos were successfully isolated and were internalized by HUVECs.

Figure 1.

Isolation and identification of EC9706-exos. A, Electron microscope was applied to observe EC9706-Exos (Scale bar, 100 nm). (B) Nanoparticle tracking analysis was performed to assess EC9706-Exo NTA. (C) Western blot was performed to assess the levels of CD63 and tumor susceptibility gene 101 (TSG101) proteins. (D) Confocal microscope was used to observe human umbilical vein endothelial cells (HUVECs) that were co-cultured with PKH-26-labeled EC9706-Exos.

LncRNA PART1 Is Upregulated and Concerns Overall Survival of Patients in EC

It was reported that LncRNA PART1 (LncPART1) participated in the progression of various diseases.¹⁶ Real-time quantitative polymerase chain reaction was conducted for detecting LncPART1 levels within EC tumor tissues and EC cells. Findings showed that relative to the control group, LncPART1 was increased within EC tumor tissues, and the expression became higher when the degree rose (P < .01; Figure 2A and B), indicating LncPART1 exerted a positive role in EC progression. Subsequently, the analysis of the overall survival rate in the bioinformatics database (http://gepia.cancer-pku.cn/; http://starbase.sysu.edu.cn/panCancer.php) showed that LncPART1 levels were negatively correlated with the overall survival rate. Consistently, compared with control, LncPART1 was upregulated in EC cell lines, including EC9706, KYSE150, and YES2 (P < .01; Figure 2D). What's more, LncPART1 levels were negatively correlated with the overall survival rate of patients (P = .0365, R = .4269). These findings revealed that LncPART1 was upregulated and concerned the overall survival of patients in EC.

Figure 2.

LncRNA PART1 (LncPART1) is upregulated and concerns overall survival of patients in esophagus cancer. A, Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to detect the levels of LncPART1 in esophageal cancer (EC) tissues, para-carcinoma tissues serve as the negative control for EC tissues (n = 30). (B) LncPART1 expression in different degree of EC tissues (n = 15). (C) The analysis of overall survival rate in bioinformatics database (http://gepia.cancer-pku.cn/; http://starbase.sysu.edu.cn/panCancer.php). (D) RT-qPCR was performed to assess the levels of LncPART1 in EC cell lines, including EC9706, KYSE150, and YES2, HET-1A served as the negative control for EC cells (n = 3). Student t test or 1-way ANOVA is used for comparison between 2 groups, and Tukey posttest is used for comparison between multiple groups. **P < .01 versus control group. Error bars represent SD. Data represent 3 independent experiments.

EC9706-Exos Accelerate HUVEC Proliferation, Migration, Invasion, and Tubule Formation

EC9706-Exos (1 μg/μL, 200 μL) were cocultured with HUVECs to determine the biological role of exosomes in HUVECs. An inverted fluorescence microscope was applied to observe internalization (Figure 3A). Cell Counting Kit-8 (CCK-8), EdU, wound healing, transwell, and tubule information were adopted for detecting HUVEC viability, proliferation, migration, invasion, and tubule information, respectively. Data revealed that compared with control, EC9706-Exos significantly accelerated cell viability, proliferation, migration, invasion, and tubule information of HUVECs (P < .01; Figure 3B-F). What's more, RT-qPCR analysis suggested EC9706-Exos significantly promoted LncPART1 levels (P < .01; Figure 3G).

Figure 3.

EC9706-Exos accelerates human umbilical vein endothelial cell (HUVEC) proliferation, migration, invasion, and tubule formation. A, EC9706-Exos (1 μg/μL, 200 μL) were added into HUVECs for co-culturing 24 h, 200 μL 1 ×PBS served as the negative control for EC9706-Exos (n = 3). (B) Cell Counting Kit (CCK)-assay was performed to evaluate cell viability. (C) EdU assay was conducted to assess cell proliferation (n = 3). D, Wound healing assay was carried out to assess cell migration (n = 3). (E) Transwell assay was performed to evaluate cell invasion (n = 3). (F) Tubule formation assay was conducted to assess tubule formation ability (n = 3). (G) Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to detect LncRNA PART1 (LncPART1) expression (n = 3). Student t test or 1-way ANOVA is used for comparison between 2 groups, and Tukey posttest is used for comparison between multiple groups. **P < .01 versus control group. Error bars represent SD. Data represent 3 independent experiments.

EC9706-Exos Accelerate HUVEC Proliferation, Migration, Invasion, and Tubule Formation via LncPART1

Given that EC9706-Exos was internalized by HUVECs, we inhibited LncPART1 expression in EC9706 cells, exosomes were cocultured with HUVECs to testify LncPART1 effects on HUVECs. Real-time quantitative polymerase chain reaction analysis indicated LncPART1 levels were suppressed following sh-LncPART1 plasmid transfecting (P < .01; Figure 4A). Subsequently, HUVEC bioactivities were analyzed by a series of experiments. Findings revealed that compared with blank, EC9706-Exos accelerated HUVECs cell viability, proliferation, migration, invasion, and tubule information, whereas the accelerative effect was antagonized by LncPART1 silence (P < .01; Figure 4B-F), suggesting that EC9706-Exos might regulate HUVECs bioactivity via LncPART1.

Figure 4.

EC9706-Exos accelerates human umbilical vein endothelial cell (HUVEC) proliferation, migration, invasion, and tubule formation via LncRNA PART1. sh-LncPART1 was transfected into EC9706-Exos-internalized HUVECs for LncRNA PART1 (LncPART1) silence, sh-NC served as the negative control for sh-LncPART1. A, Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to detect the levels of LncRNA PART1 (n = 3). (B) Cell Counting Kit-8 (CCK-8) assay was conducted to assess cell viability (n = 3). (C) EdU assay was carried to evaluate cell proliferation (n = 3). (D) Wound healing assay was performed to detect cell migration (n = 3). (E,) Transwell assay was conducted to evaluate cell invasion (n = 3). (F) Tubule formation assay was performed to assess tubule formation ability (n = 3). Student t test or 1-way ANOVA is used for comparison between 2 groups, and Tukey posttest is used for comparison between multiple groups. **P < .01 versus blank group, ##P < .01 versus sh-NC group. Error bars represent SD. Data represent 3 independent experiments.

LncRNA PART1 Serves as a Sponge of miR-302a-3p

To investigate the underlying molecular mechanism of LncPART1 in angiogenesis, StarBase V2.0 was used for predicting the effective target miRNAs of LncPART1 downstream. Subsequently, the levels of 5 miRNAs in HUVECs that were transfected with pcDNA-LncPART1 plasmid were analyzed by RT-qPCR. Results revealed that except for miR-302a-3p, there were no changes in miRNAs expression, including miR-3529-3p, miR-6842-3p, miR-4797-3p, and miR-4653-3p (P < .01; Figure 5A). Subsequently, potential binding sites of LncPART1 and miR-302a-3p were obtained (Figure 5B). Further, dual-luciferase reporter was conducted to further evaluate the relationship between these 2 factors.MiR-302a-3p overexpression inhibited the luciferase activities of LncPART1-Luc, while no significant difference occurred when the putative binding sites were mutated (P < .01; Figure 5C). What's more, RT-qPCR analysis showed that the miR-302a-3p overexpression inhibited LncPART1 levels within HUVECs (P < .01; Figure 5D). Based on these, it was inferred that LncPART1 worked as a sponge of miR-302a-3p. Therefore, we predicted that LncPART1 functioned as a ceRNA to sponge miR-302a-3p, thus inhibiting the biological function of targets.

Figure 5.

LncRNA PART1 (LncPART1) serves as a sponge of miR-302a-3p. A, Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to detect the levels of microRNAs (miRNAs), including miR-3529-3p, miR-6842-3p, miR-302a-3p, miR-4797-3p, and miR-4653-3p (n = 3). (B) The potential binding site between LncPART1 and miR-302a-3p was predicted by Starbase software. MiR-302a-3p mimics were transfected into human umbilical vein endothelial cells (HUVECs) for miR-302a-3p overexpression, NC mimics served as the negative control for miR-302a-3p mimics. (C) The dual luciferase reporter gene assay was performed to confirm the direct binding relationship between LncPART1 and miR-302a-3p (n = 3). (D) RT-qPCR was conducted to assess the levels of LncPART1 (n = 3). Student t test or 1-way ANOVA is used for comparison between 2 groups, and Tukey posttest is used for comparison between multiple groups. **P < .01 versus control group. Error bars represent SD. Data represent 3 independent experiments.

MiR-302a-3p Targets CDC25A

MiRNAs participated in multiple disease progression through modulating targets.¹⁷ Bioinformatics tools were jointly utilized for selecting possible miR-302a-3p targets, and a total of 131 targets were detected (Figure 6A). Real-time quantitative polymerase chain reaction was conducted for detecting miRNA levels within HUVECs that were transfected with miR-302a-3p mimics. Results revealed that CDC25A was downregulated by overexpressed miR-302a-3p instead of LCOR, ZKSCAN1, LHX6, and E2F7 (P < .01; Figure 6B). Subsequently, potential binding sites of miR-302a-3p and CDC25A were obtained (Figure 6C). Further, dual-luciferase reporter was conducted to further evaluate the relationship between these 2 factors. MiR-302a-3p overexpression inhibited the luciferase activities of CDC25A-Luc, while no significant difference occurred when the putative binding sites were mutated (P < .01; Figure 6D). What's more, RT-qPCR analysis showed that the miR-302a-3p overexpression inhibited CDC25A levels within HUVECs. What's more, RT-qPCR was performed to assess the levels of miR-302a-3p in HUVECs, following CDC25A overexpression. Results showed that miR-302a-3p expression was inhibited by CDC25A overexpression (P < .01; Figure 6E).

Figure 6.

MiR-302a-3p targets cell division cycle 25 A (CDC25A). A, Targetscan software was applied to predict the potential targets of miR-302a-3p downstream. MiR-302a-3p mimics were transfected into human umbilical vein endothelial cells (HUVECs) for miR-302a-3p overexpression, NC mimics served as the negative control for miR-302a-3p mimics. (B) Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to detect the mRNA levels of genes, including LCOR, CDC25A, ZKSCAN1, LHX6, and F2F7 genes (n = 3). (C) The potential binding site between miR-302a-3p and CDC25A. (D) The dual luciferase reporter gene assay was conducted to confirm the direct binding relationship between miR-302a-3p and CDC25A (n = 3). pcDNA-CDC25A plasmids were transfected into HUVECs for CDC25A overexpression, pcDNA-NC served as the negative control for pcDNA-CDC25A plasmids. (E) RT-qPCR was conducted to assess the levels of miR-302a-3p (n = 3). Student t test or 1-way ANOVA is used for comparison between 2 groups, and Tukey posttest is used for comparison between multiple groups. **P < .01 versus control group. Error bars represent SD. Data represent 3 independent experiments.

EC9706-Exos Accelerate EC Progression via LncPART1/miR-302a-3p/CDC25A Axis

According to the above findings, whether EC9706-Exos exerts its function in angiogenesis via miR-302a-3p/CDC25A axis was confirmed through rescue assays. For further research, miR-302a-3p inhibitor was transfected for miR-302a-3p inhibition, and sh-CDC25A plasmid was transfected for CDC25A silence (P < .01; Figure 7A). Functionally, CCK-8, EdU, wound healing, transwell, and tubule information revealed that the LncPART1 silence-EC9706-Exos-induced reduction in cell viability, proliferation, migration, invasion, and tubule information abilities of HUVECs was promoted by miR-302a-3p inhibitor, whereas the impact of miR-302a-3p inhibitor was subsequently recovered by CDC25A depletion (P < .05; Figure 7B-F). Based on these findings, it was inferred that EC9706-Exos accelerated angiogenesis via LncPART1/ miR-302a-3p/CDC25A axis.

Figure 7.

EC9706-Exos accelerated esophageal cancer (EC) progression via long non-coding RNA (lncRNA) PART1/miR-302a-3p/cell division cycle 25 A (CDC25A) axis. sh-LncPART1, sh-CDC25A, or miR-302a-3p inhibitor was transfected into human umbilical vein endothelial cells (HUVECs) for LncRNA PART1 (LncPART1) silence or CDC25A silence or miR-302a-3p inhibition, sh-NC served as the negative control for sh-LncPART1 and sh-CDC25A, and NC inhibitor served as the negative control for miR-302a-3p inhibitor. A, Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to assess the levels of miR-302a-3p and CDC25A (n = 3). (B) Cell Counting Kit-8 (CCK-8) assay was performed to detect cell viability. (C) EdU assay was performed to evaluate cell proliferation (n = 3). (D) Wound healing assay was conducted to evaluate cell migration (n = 3). (E) Transwell assay was performed to assess cell invasion (n = 3). (F) Tubule formation assay was conducted to detect tubule formation ability (n = 3). Student t test or 1-way ANOVA is used for comparison between 2 groups, and Tukey posttest is used for comparison between multiple groups. **P < .01 versus control group, #P < .05, ##P < .01 versus control group, miR-302a-3p mimics + pcDNA-NC group. Error bars represent SD. Data represent 3 independent experiments.

Discussion

Cancer cells and surrounding tumor mechanisms constitute the malignant tumor microenvironment.¹⁸ Evidence shows that exosomes exert a pivotal role in the local and systemic intercellular communication of cancer.¹⁹ Our findings illustrated that EC9706-Exos promotes HUVECs angiogenesis, and the specific mechanism may be realized via LncPART1/miR-302a-3p/CDC25A axis, indicating EC9706-Exos may act as a promoter of angiogenesis. Our research will contribute to clarify the mechanism of tumor angiogenesis.

Exosomes are a medium for information exchange between cells and play an important regulatory role in the tumor microenvironment.²⁰ Tumor-derived exosomes promote tumor progression by enhancing cell biological activity, angiogenesis, and regulating the immune system.²¹ Increasing evidence confirm lncRNA exert a vital role in regulating tumor microenvironment and tumor progression.²² However, it is still essential to explore the physiological and pathological roles of EC9706-Exos, LncPART1-EC9706-Exos in EC microenvironment, and its underlying molecular mechanism. Our finding showed that LncPART1 was increased within EC tumor tissues, and the levels became higher when the degree rose, indicating that LncPART1 acted as a cancer factor to promote EC progression. Further functional assay is essential for verification. Given that EC9706-Exos was internalized by HUVECs, we inhibited LncPART1 expression in EC9706 cells, from which exosomes were subsequently extracted to coculture with HUVECs to testify the biological roles of LncPART1 in HUVECs. Results showed that EC9706-Exos accelerated HUVEC proliferation, migration, invasion, and tubule formation, which exerted via LncPART1. In a word, EC9706-Exos might regulate EC angiogenesis through LncPART1.

MiRNAs have been confirmed related to tumor progression regulation, including cell proliferation and angiogenesis, and may serve as new and effective therapeutic targets.²³ It's worth noting that miR-302a-3p is involved in the regulation of various disease progression via regulating cell bioactivity. For example, miR-302a-3p inhibits cancer progression by repressing liver cell proliferation and migration.²⁴ For most tumors, miR-302a-3p may be a tumor suppressor.²⁵ What's more, miR-302a-3p is targeted regulation by sponging molecules lncRNAs. In endometrial carcinoma, LINC01016 promotes tumor cell proliferation and metastasis through modulating miR-302a-3p.²⁶ Zhu et al research shows that lncRNA MEG3 promotes hepatocyte proliferation and invasion through modulating miR-302a-3p.²⁷ LncRNAs may be served as endogenous competitive RNA of miR-302a-3p to participate in the disease process. Our results revealed that LncPART1-EC9706-Exo promoted angiogenesis via miR-302a-3p/CDC25A. Further functional assays were needed to prove our point.

Cell cycle disorder is one of the main reasons for the vicious proliferation of tumor cells, and cell cycle may be regulated by phosphorylated cyclin-dependent kinase (CDK).²⁸ As a dual-specific protein phosphatase, CDC25A belongs to the CDC25A bisphosphonate family, which can phosphorylate CDK into a cell cycle checkpoint kinase to exert its regulatory function.²⁹ It has been reported that CDC25A exhibits higher expression in a variety of tumor tissues or cell lines and is associated with poor tumor prognosis.³⁰ These findings indicated that CDC25A acted as a cell cycle regulator to participate in tumor progression. Our findings revealed that CDC25A served as the miR-302a-3p target, and their levels were negatively correlated. What's more, further functional studies have indicated that EC9706-Exos accelerates angiogenesis progression via LncPART1/miR-302a-3p/CDC25A axis, indicating that EC9706-Exos may regulate cell cycle to regulate cell proliferation, migration, invasion, and tubule information, resulting in accelerating angiogenesis progression. Thus, CDC25A acted as a biomarker for angiogenesis.

However, there are still some limitations regarding this study. This study only investigated the molecular mechanism of EC9706-Exos in tumor angiogenesis through the LncPART1/miR-302a-3p/CDC25A axis at the in vitro cellular level. Therefore, further validation from in vivo animal models is still needed based on these preliminary results.

Conclusion

In summary, our findings revealed that LncPART1 was upregulated and concerned overall survival of patients in EC; EC9706-Exos accelerated HUVEC proliferation, migration, invasion, and tubule formation via LncPART1; LncPART1 served as a sponge of miR-302a-3p which targeted to CDC25A, and EC9706-Exos accelerated angiogenesis progression via LncPART1/miR-302a-3p/CDC25A axis. These results indicated that EC9706-Exos may act as a promoter of angiogenesis. Our research will help elucidate the mechanism of tumor angiogenesis and provide potential therapeutic targets for the clinical treatment of EC.

Footnotes

Abbreviations

Authors’ Note

All procedures performed in studies involving human participants were carried out in accordance with the Helsinki Declaration. The study was approved by the ethics committee of Jiangsu Cancer Hospital on December 31, 2020 (ethics approval number: 2020 ke-052). Each participant provided the written informed consent for participation. The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Ethics Committee of the Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research &The Affiliated Cancer Hospital of Nanjing Medical University (approval no. 2020ke-052). All patients provided written informed consent prior to enrollment in the study. All participants agreed with the publication in the Journal. The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Jiangsu Institute of Cancer Research, (grant number ZM201914).

ORCID iD

Xia He

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