Exosomal circ_0037104 derived from Hu‐MSCs inhibits cholangiocarcinoma progression by sponging miR‐620 and targeting AFAP1

Exosomes are membrane‐enclosed nanovesicles that shuttle active cargoes, such as circular RNAs (circRNAs) and microRNAs (miRNAs), between different cells. Human umbilical cord‐derived mesenchymal stem cells (Hu‐MSCs) can migrate to tumor sites and exert complex functions throughout tumor progression. In this study, we successfully isolated Hu‐MSCs from human umbilical cords based on their surface marker expression. Hu‐MSC‐derived exosomes significantly reduced the invasion, migration, and proliferation of cholangiocarcinoma (CCA) cells. Furthermore, circ_0037104 was downregulated in CCA and inhibited the proliferation and metastasis of CCA cells. Then, we investigated the effect of Hu‐MSC‐derived exosomal circ_0037104 on CCA. Circ_0037104 mainly regulates miR‐620 and enhances APAF1 expression, inhibiting CCA cell proliferation and metastasis. Overall, Hu‐MSC exosomal circ_0037104 contributes to the progression and stemness of CCA cells via miR‐620/APAF1. In conclusion, Hu‐MSC‐derived exosomal circ_0037104 sponges miR‐620 directly and negatively targets APAF1 to suppress CCA.

survival time of these patients is only 11.7 months. [6]Multitarget kinase inhibitors have been tried for primary liver cancer, including CCA, because of their multitarget inhibitory effect, and have demonstrated some efficacy. [7]Based on the above clinical status, new and effective therapeutic or detection targets for treating CCA continue to be investigated.Circular RNAs (CircRNAs) are a new type of noncoding RNA molecule.Due to the progress made in next-generation sequencing technologies and biological analyses, circRNAs can be revealed and characterized, and the development of RNA-related technologies provides methods for elucidating various functions of circRNAs. [8]rst, the inhibitory function of circRNA on miRNA through adsorption was defined; through this mechanism, circRNA promotes gene translation and increases the corresponding protein level; some circRNAs form functional complexes with proteins, and a small number of circRNAs can be translated into polypeptides or proteins. [9,10]Thus, circRNAs can regulate gene expression and activation/inactivation of signaling pathways at multiple levels.
3][14] Exosomes are small membrane vesicles (30−150 nm) containing many complex RNAs and proteins, and most studies now show that their diameter is generally between 40 and 100 nm and that they are disc-shaped. [15,16]Exosomes are secreted by nearly all cultured cell types and are naturally present in body fluids, including blood, saliva, urine, cerebrospinal fluid, and breast milk. [17]The functions of exosomes are closely related to the cell types from which they are derived and are involved in various processes, such as the immune response, antigen presentation, cell migration, cell differentiation, and tumor invasion. [18]Studies have shown that mesenchymal stem cells (MSC)-derived exosomes play a key inhibitory role in the occurrence and development of tumors, and their mode of action may be related to the secretion of exosomes or inflammatory factors. [19]MSCs are a group of pluripotent stem cells with multidirectional differentiation potential that play an important role in tissue repair and regeneration. [20]3][24] Research suggests that human umbilical cord blood MSC-derived exosomal microRNA-503-3p inhibits the progression of human endometrial cancer cells by downregulating MEST. [25]Recent studies have revealed that MSC have a significant inhibitory effect on tumors, and their mode of action may be related to their inflammatory factors or exosomes. [18]Therefore, studying the molecular mechanism of MSC against cancer has strong scientific significance for the prevention and treatment of tumors.In this study, we investigated the biological role of umbilical cord blood MSC-derived exosomes in the malignant progression of CCA and investigated circ_0037104, which plays a key role in regulating CCA cell proliferation, invasion, metastasis, and progression.
Finally, the molecular mechanism of circ_0037104 in exosomes inhibiting CCA was further elucidated.

| Patient and tissue specimens
Forty CCA patients treated at the General Hospital of Central Theatre Command with surgical resection specimens were selected, and the corresponding tissue cancer specimens with margins of cancer tissue over 5 cm adjacent to the cancer tissue were selected.
CCA and paracancerous tissues specimens were placed in an ultralow temperature freezer at 80°C for cryopreservation.All patients signed informed consent forms consenting to the use of tissue samples in this study.All specimens were collected in accordance with the human specimen collection procedures approved by the Ethical Review Committee of the People's Republic of China and the hospital ethics committee.

| Cell culture
Human ovarian cancer cells TFK-1 and HuCCT1 were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 IU/mL penicillin.The cell culture flasks were placed in a 37°C incubator with 5% CO 2 and 95% humidity.

| Extraction of human umbilical cord-derived MSC (Hu-MSCs)-derived exosomes
Hu-MSCs were cultured to attain 60%−70% confluence, followed by a transfer into serum-free MSC medium (α-MEM; TBD).Post 48 h of incubation, the conditioned medium was harvested and subjected to sequential centrifugation: initially at 300×g for 10 min, and then at 20,000×g for 30 min.Subsequently, the supernatant underwent ultracentrifugation at 120,000×g for 70 min at 4°C using a Type 70Ti rotor (Beckman Coulter L-80 XP).The resultant pellet was resuspended in PBS and further centrifuged at 100,000×g for 70 min.The final exosomal pellet was aliquoted in 200−300 μL of PBS and preserved at −80°C.

| Quantitative real-time PCR (qRT-PCR)
Specific qRT-PCR primers were designed using Primer 5.0.The cDNA was diluted to an appropriate concentration, typically 20-fold, with sterile pure water.Five microliters of qRT-PCR mix, 1 μL of primers, and 4 μL of cDNA were added to a 96-well PCR plate and centrifuged at 2500 rpm for 1 min, and the samples were placed into the qRT-PCR instrument for experimentation.After the reaction was complete, the data were replicated and analyzed.
The logarithmically growing cells were washed three times with precooled PBS, RAPI protein lysis buffer containing protease inhibitors was added, the cells were lysed on ice for 15 min and centrifuged at 12,000 rpm/min for 15 min, and the supernatant was taken, containing the total cell protein.The cellular protein concentration was determined by the BCA method.Protein denaturation was performed at 95°C for 10 min.The proteins were separated by 12% SDS-PAGE, transferred to PVDF membranes at constant pressure, and blocked with 5% nonfat milk powder for 1 h at room temperature.Antibodies targeting Oct-4, Sox2, Nanog, ZEB2, E-cadherin, Vimentin, and VEGFA were diluted to 1:1000, obtained from Proteintech.APAF1 antibodies were diluted to 1:500, procured from Abnova.Furthermore, β-actin antibodies were diluted to 1:5000, also provided by Proteintech.The corresponding antibodies added, refrigerated at 4°C overnight, incubated with TBST, and incubated with HRP-labeled II resistance of the corresponding species for 1 h at room temperature, and ECL was used to develop the chromogenic substrate.

| Transfection
siRNA was used for knockdown, and a plasmid was used for overexpression.Using a six-well plate for transfection of siRNA (RiboBio), 1 μL of siRNA was mixed with 250 μL of Opti-MEM, 1 μL of Lipofectamine 2000, and 250 μL of Opti-MEM and incubated at room temperature for 5 min.Then, the two parts were mixed and mixed.The plate was allowed to stand for 15 min and then samples were added dropwise evenly to well 1 of a sixwell plate.A six-well plate was used to transfect plasmids (RiboBiou); 2 μg of plasmid was mixed with 200 μL Opti-MEM, 3 μL PEI, and 200 μL Opti-MEM, and the two parts were mixed and allowed to stand for 20 min.

| Immunochemistry
CCA tissue specimens were embedded in paraffin and sliced into serial sections (thickness: 4 mm), dewaxed with xylene, dehydrated with different concentrations of ethanol, and heated for antigen retrieval.After inactivation with hydrogen peroxide (3%), the samples were sealed with goat blood.The specific steps were performed strictly in accordance with standard protocols.The kit was used according to the manufacturer's instructions, and the APAF1 antibody was diluted at a ratio of 1:100 (Abnova).The sections were developed with DAB (Abnova), counterstained with hematoxylin and dehydrated in different concentrations of ethanol, mounted on a glass slide with neutral resin and placed under a microscope for observation.

| Detection of cell proliferation by CCK-8 assay
Cells were trypsinized, counted, and seeded in 96-well plates at a density of 8000 cells per well in six replicate wells.A 96-well plate was removed every 24 h for CCK8 experiments.Ten microliters of CCK-8 solution was added to each well, the plate was incubated in a 37°C cell incubator for 2 h, and the culture was terminated.The microplate reader detects the absorbance value at 490 nm.The proliferation curve was generated from three replicates.

| Cell transfer experiment
TFK-1 and HuCCT1 cells (1 × 10 4 ) resuspended in DMEM were seeded into the upper chamber of an 8 μM Transwell and the lower chamber containing 800 μL of DMEM containing 20% fetal bovine serum.For the invasion test, the upper chamber was treated with boric acid and Matrigel.Twenty-four hours after cell seeding, TFK-1 and HuCCT1 cells were fixed with 4% paraformaldehyde for 10 min at room temperature, stained with 0.1% crystal violet, and incubated for 20 min at room temperature.Five fields of view were selected to count the number of invading cells under a light microscope.

| Statistical processing
The experimental data were analyzed by SPSS 16.0 statistical software and graphed by GraphPad Prism 6.01.The results are presented x ± s of three independent experiments.The comparison of the two samples was performed by t-test; p < 0.05 indicated that the difference was statistically significant.

| Characterization of Hu-MSCs and Hu-MSCderived exosomes
After the umbilical cord blood MSC were isolated and extracted, the marker proteins on their surfaces were detected by flow cytometry.The results suggested that Hu-MSCs did not express cell-surface marker proteins CD45, Hand CD34 and HLA-DR but did express CD73, CD90, and CD105 (Figure 1A).Transmission electron microscopy revealed that the exosomes had a typical cup-shaped double-layer membrane (Figure 1B).The particle size analyzer indicated that the diameter of the vesicles was approximately 96.9 nm, which is within the diameter range of exosomes (Figure 1C).Furthermore, Hu-MSCs were positive for marker proteins commonly used to identify exosomes, namely, TSG101, CD63, and CD9 (Figure 1D).These results demonstrate that Hu-MSCs and their exosomes were successfully extracted.

| Hu-MSC-derived exosomes significantly inhibited the migration, invasion, proliferation, and stemness of CCA cells
Furthermore, we analyzed the effect of Hu-MSC-derived exosomes on the proliferation, invasion, and metastasis of CCA cells.We constructed a coculture system of mesenchymal cells and CCA cells.
After adding the exosome secretion inhibitor GW4869, the transwell results showed that CCA cells' invasion and metastasis ability was significantly enhanced, and the cell proliferation activity was also improved (Figure 1E,F).The exosomes of Hu-MSCs were added to CCA cells, and the Transwell, colony formation, and wound healing experimental results showed that the proliferation, migration, and invasion abilities of CCA cells were significantly decreased (Figure 1G−J).The markers of cancer stem cells were detected by qRT-PCR and western blot analysis experiments, and the results showed that the expression of stem cell markers (Oct4, Nanog, and Sox2) was significantly inhibited after adding Hu-MSC-exosomes (Figure 1K,L).These results indicate that Hu-MSC-derived exosomes inhibit malignant progression and stemness in CCA cells.

| The characteristics and biological role of circ_0037104 in CCA cells
To screen the vital circRNAs in Hu-MSC-derived exosomes, RNA-seq analysis of the differential circRNAs in CCA cells with and without exosomes was performed.The results indicated differentially expressed circRNAs (Figure 2A,B).Then, we confirmed the expression in CCA tissues, and the results indicated that circ_0037104 was significantly downregulated (Figure 2C).Then, we examined the expression of circ_0037104 in CCA cell lines, and the results indicated that circ_0037104 was significantly downregulated in CCA cells compared with HiBEpiC cells (Figure 2D).We found that random hexamer primers could significantly amplify circ_0037104 and the host gene PCSK6, while oligo (dT)18 primers barely amplified circ_0037104 (Figure 2E).The Act D assay showed that circ_0037104 possessed a longer half-life than PCSK6 (Figure 2F).
RNase R assays showed that PCSK6 was digested by RNase R, while circ_0037104 was resistant to RNase R (Figure 2G).After the nucleocytoplasmic separation experiment, the qRT-PCR results indicated that circ_0037104 was mainly localized in the cytoplasm, with almost no expression in the nucleus (Figure 2H).Then, qRT-PCR demonstrated that circ_0037104 was overexpressed in TFK-1 and HuCCT1 cells transfected with circ_0037104-overexpressing lentivirus (Figure 2I).Furthermore, the Transwell, colony formation, and wound healing experimental results showed that the proliferation, migration, and invasion abilities were significantly decreased in circ_0037104-overexpressing CCA cells (Figure 2J-M).Western blot analysis experiments were used to measure the levels of markers of cancer stem cells and EMT, and the results showed that these markers were significantly inhibited in circ_0037104-overexpressing CCA cells (Figure 2N,O).These results indicate that circ_0037104 inhibits malignant progression and stemness in CCA cells.

| circ_0037104 sponges miR-620 to regulate CCA cell migration, invasion, and proliferation
We predicted the downstream miRNAs of circ_0037104 and found that miR-620 had binding sequences available in two databases (circInteractome and StarBase) (Figure 3A).The RIP assay indicated that circ_0037104 and miR-620 were significantly enriched in the immunoprecipitated complexes of the anti-Ago2 groups (Figure 3B).RNA pulldown assays showed that bio-miR-620-wt pulled down more circ_0037104 than bio-NC and bio-miR-620-mut controls (Figure 3C).
We used StarBase to predict binding sites between circ_0037104 and miR-620 and mutation binding sequences of circ_0037104 (Figure 3D).
Then, the luciferase reporter assay demonstrated that circ_0037104 could bind with miR-620, while the interaction was inhibited in mutation binding sequences of circ_0037104 (Figure 3E).Subsequent qRT-PCR experiments indicated that miR-620 was downregulated in CCA tissues and cell lines (Figure 3F,G).Furthermore, qRT-PCR revealed that miR-620 was upregulated in TFK-1 and HuCCT1 cells transfected with the miR-620 mimic (Figure 3H).We simultaneously overexpressed miR-620 can significantly inhibit the proliferation, invasion, and metastasis of CCA cells and that miRNA can partially reverse the biological effect (Figure 3I−L).Western blot analysis experiments detected the markers of cancer stem cells and EMT, and the results showed that these markers were significantly inhibited in circ_0037104-overexpressing cells, while the marker expression was partially reversed in miR-620-overexpressing cells (Figure 3M,N).The above results suggest that circ_0037104 inhibits the migration, invasion, and proliferation of CCA by adsorbing miR-620.

| MiR-620 targeting regulated APAF1
The RIP assay indicated that miR-620 and AFAP1 were significantly enriched in the immunoprecipitated complexes of the anti-Ago2 groups (Figure 4A).The RNA pulldown assay showed that bio-miR-620-wt pulled down more AFAP1 than the bio-NC and bio-miR-620-mut controls (Figure 4B).We used StarBase to predict the binding sites between AFAP1 and miR-620 and mutated the binding sequences of AFAP1 (Figure 4C).Then, the luciferase reporter assay demonstrated that miR-620 could bind with AFAP1, while the interaction was inhibited when AFAP1 binding sequences were mutated (Figure 4D).Subsequent qRT-PCR and western blot analysis experiments indicated that miR-620 could downregulate AFAP1 expression in CCA tissues and cell lines (Figure 4E,F).We simultaneously overexpressed miR-620 in circ_0037104-overexpressing CCA cell lines, and qRT-PCR and western blots analysis indicated that AFAP1 was upregulated in circ_0037104-overexpressing cells but partially downregulated in miR-620-overexpressing CCA cell lines (Figure 4G,H).Subsequent qRT-PCR, western blot analysis and IHC indicated that AFAP1 was downregulated in CCA tissues and cell lines (Figure 4I−L).

| miR-620 promoted CCA cell progression via AFAP1
First, we found that AFAP1 was significantly upregulated in APAP1-overexpressing cells at the mRNA and protein levels (Figure 5A).Furthermore, in Transwell, plate cloning, and wound healing experiments, we simultaneously overexpressed miR-620 in AFAP1-overexpressing CCA cell lines.The results suggest that AFAP1 could significantly inhibit the proliferation, invasion, and metastasis of CCA cells, and miR-620 can partially reverse the biological effect (Figure 5B−E).Western blot analysis experiments detected the markers of cancer stem cells and EMT, and the results showed that these markers were significantly inhibited in AFAP1-overexpressing cells, while the marker expression was partially reversed in miR-620-overexpressing cells (Figure 5F,G).
The above results suggest that miR-620 promotes the migration, invasion, and proliferation of CCA by targeting AFAP1.

| DISCUSSION
Human MSC have been shown to exert anticancer, antiinflammatory immunomodulatory, and repair effects in various animal models. [26]The bone marrow was found to be the main source of MSC in most investigations, but the collection of Transfected TFK-1 and HuCCT1 cells (5 × 10 6 cells, 100 μL PBS) were injected subcutaneously into the left axilla of male BALB/c nude mice (4 weeks old; Shanghai Experimental Animal Center).At 7, 14, 21, 28, 35, and 42 days after injection, the tumor volumes of mice were examined by the V = 1/2 × ab 2 method (length [a] and width [b] of the tumor length).The mice were killed 42 days after injection, and the subcutaneous tumors were weighed.This study was conducted with the approval of the Institutional Animal Care and Use Committee of General Hospital of Central Theater Command.
in circ_0037104-overexpressing CCA cell lines in Transwell plate cloning and wound healing experiments.The results suggest that circ_0037104 F I G U R E 1 Characterization of Hu-MSCs and Hu-MSC-derived exosomes.(A) Flow cytometry confirmed MSC markers.(B) Electron microscope observation of the morphology of MSC-derived exosomes.(C) Diameter of MSC-derived exosomes detected by particle microscopy.(D) Identification of the expression of exosome marker proteins by immunoblotting.(E) Transwell assay was used to analysis the effect of Hu-MSC-derived exosomes on the invasion and migration of cholangiocarcinoma (CCA) cells treated with exosomes inhibitor GW4869.(F) CCK8 assay was used to analyze the effect of Hu-MSC-derived exosomes on the proliferation of CCA cells.(G, H) Transwell assay was used to analysis the effect of Hu-MSC-derived exosomes on the invasion and migration of CCA cells under coculture conditions.(I) Colony formation assay was used to analyze the effect of Hu-MSC-derived exosomes on the clone formation of CCA cells under coculture conditions.(J) Wound healing assay was used to analysis the effect of Hu-MSC-derived exosomes on the migration of CCA cells.(K, L) PCR and immunoblotting experiments were used to detect the effect of Hu-MSC-derived exosomes on the expression of cancer stem cell markers.**p < 0.01.Hu-MSC, human umbilical cord-derived mesenchymal stem cells.F I G U R E 2 (See caption on next page).

3. 7 |
Hu-MSC-derived exosomes significantly inhibited CCA cell progression via circ_0037104/miR-620/AFAP1 To examine the role of Hu-MSC-derived exosomes in vivo, we constructed a xenograft tumor model.The results indicated that Hu-MSC-derived exosomes could significantly inhibit tumor growth, while the exosome inhibitor GW4869 could partially reverse the biological effect (Figure 5H).The tumor volume growth curve suggested that exosomes could significantly inhibit the volume of tumor growth, and the inhibitor could partially reverse this phenomenon (Figure 5I).qRT-PCR assays indicated that circ_0037104 and AFAP1 were upregulated in tumors treated with exosomes, while miR-620 was downregulated.The exosome inhibitor partially reversed the expression (Figure 5J−L).These results indicated that Hu-MSC-derived exosomes significantly inhibited CCA cell progression via circ_0037104/miR-620/ AFAP1.

F I G U R E 2
Hu-MSC-derived exosomes significantly inhibited the migration, invasion, proliferation, and stemness of cholangiocarcinoma (CCA) cells.(A, B) RNA-seq analysis of the differentially expressed circRNAs in CCA cells with and without exosomes.(C) qRT-PCR analysis of the expression of differentially expressed circRNAs in CCA.(D) qRT-PCR analysis of the expression of circ_000037104 in CCA.(E) The existence of circ_0037104 in the reverse transcription products.(F) using random hexamer primers and oligo (dT)18 primers.(G) The levels of circ_0037104 and PCSK6 in TFK-1 and HuCCT1 cells treated with Act D were examined with a qRT-PCR assay.(H) qRT-PCR assay was used to detect the levels of circ_0037104 and PCSK6 in TFK-1 and HuCCT1 cells treated with or without RNase R. (I) Subcellular fraction analysis of the expression of circ_ 0037104 in the cytoplasm and nucleus of TFK-1 and HuCCT1 cells.(J, K) Transwell assays were used to analyze the migration and invasion ability of circ_0037104 on TFK-1 and HuCCT1 cells.(L) Colony formation assay was used to analyze the clone formation of circ_0037104 on TFK-1 and HuCCT1 cells.(M) Wound healing assay was used to analyze the effect of circ_0037104 on TFK-1 and HuCCT1 cell migration.(N, O) Western blot analysis of cancer stem cell and EMT marker expression in control and circ_0037104 overexpressing cells.*p < 0.05.**p < 0.01.circRNAs, circular RNAs; Hu-MSC, human umbilical cord-derived mesenchymal stem cells; qRT-PCR, quantitative real-time PCR.

F
I G U R E 4 MiR-620 promoted cholangiocarcinoma (CCA) cell progression via AFAP1.(A) RIP assay analysis of the interaction between miR-620 and APAF1.(B) RNA pulldown assay was performed to analyze the interaction between miR-620 and APAF1.(C) Bioinformation predicted the binding sites between APAF1 and miR-620.(D) Luciferase reporter analysis of the interaction with miR-620 and APAF1.(E, F) qRT-PCR and western blot assays were used to analyze APAF1 expression in miR-620-overexpressing CCA cells.(G, H) qRT-PCR and western blot assays were used to analyze APAF1 expression in miR-620-and circ_0037104-overexpressing CCA cells.(I, J) qRT-PCR and IHC analysis of APAF1 expression in CCA tissues.(K, L) qRT-PCR and western blots analysis of the APAF1 expression in CCA cell lines.*p < 0.05.**p < 0.01.F I G U R E 3 MiR-620 targeting regulated APAF1.(A) Intersection analysis of downstream miRNAs of circ_0037104 based on two databases (circinteractome and StarBase).(B) RIP assay analysis of the interaction between miR-620 and circ_0037104.(C) An RNA pulldown assay was performed to analyze the interaction between miR-620 and circ_0037104.(D) Bioinformatic analysis predicted the binding sites between circ_0037104 and miR-620.(E) Luciferase reporter analysis of the interaction with miR-620 and circ_0037104.(F) qRT-PCR was performed to analyze the expression of miR-620 in cholangiocarcinoma (CCA) tissues.(G) qRT-PCR was performed to analyze the expression of miR-620 in CCA cell lines.(H) qRT-PCR confirmed miR-620 upregulation in CCA cells.(I−L) Functional assay analysis confirmed that miR-620 reversed migration, invasion, and proliferation in circ_0037104-overexpressing CCA cells.(M, N) Western blot analysis confirmed that miR-620 reversed cancer stem cell and EMT marker expression in circ_0037104-overexpressing CCA cells.*p < 0.05.**p < 0.01.miRNAs, microRNAs.

F
I G U R E 5 Hu-MSC-derived exosomes significantly inhibited cholangiocarcinoma (CCA) cell progression via circ_0037104/miR-620/AFAP1.(A) Western blot analysis of APAF1 expression in APAF1-overexpressing CCA cells.(B−E) Functional assay analysis confirmed that APAF1 reversed migration, invasion, and proliferation in miR-620-overexpressing CCA cells.(F, G) Western blot analysis confirmed that miR-620 reversed cancer stem cell and EMT marker expression in circ_0037104-overexpressing CCA cells.(H) In vivo assay analysis showed that Hu-MSC-derived exosomes significantly inhibited CCA cell progression via circ_0037104.(I) Growth curve analysis of the volume of subcutaneous tumors in the indicated groups.(J−L) qRT-PCR analysis of circ_0037104/miR-620/AFAP1 expression in subcutaneous tumors in the indicated groups.*p < 0.05.**p < 0.01.Hu-MSC, human umbilical cord-derived mesenchymal stem cells.