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. 2022 Jun 7;20(1):258.
doi: 10.1186/s12967-022-03422-7.

miR-18a-5p derived from mesenchymal stem cells-extracellular vesicles inhibits ovarian cancer cell proliferation, migration, invasion, and chemotherapy resistance

Xiaoying Wang  1 , Lili Jiang  1 , Qifang Liu  2
Affiliations

miR-18a-5p derived from mesenchymal stem cells-extracellular vesicles inhibits ovarian cancer cell proliferation, migration, invasion, and chemotherapy resistance

Xiaoying Wang et al. J Transl Med. .

Abstract

Objective: Ovarian cancer (OC) is a major threat to women's health. Mesenchymal stem cells (MSCs) are key regulators in cellular communication by secreting extracellular vesicles (EVs) that are involved in OC. This study probed into the mechanism of human MSCs derived-EVs (hMSC-EVs) in regulating OC cell growth and chemotherapy resistance.

Methods: hMSCs and EVs were isolated and identified. After adding EVs, the uptake of EVs by OC CAOV3/ES2 cells (for in vitro studies), and cell proliferation, migration, and invasion were detected. Downregulated miRNAs in hMSC-EVs were screened and miR-18a-5p expression in OC patients was detected. The prognosis of OC patients was analyzed. Binding sites of miR-18a-5p and NACC1 were predicted and validated. NACC1 expression in OC tissues was measured by RT-qPCR, and its correlation with miR-18a-5p was analyzed by Pearson method. AKT/mTOR pathway activation was assessed by WB. The cisplatin sensitivity of EVs-treated CAOV3 cells was evaluated via MTT assay and tested by tumor formation assay in nude mice.

Results: hMSC-EVs suppressed OC cell proliferation, migration, and invasion. miR-18a-5p was downregulated in OC and miR-18a-5p low expression was associated with a poor prognosis. EV-encapsulated miR-18a-5p targeted NACC1. NACC1 was upregulated in OC tissues. miR-18a-5p knockdown and NACC1 overexpression both annulled the inhibition of hMSC-EVs on OC cell growth. AKT and mTOR were elevated in OC and NACC1 activated the AKT/mTOR pathway in OC cells. hMSC-EVs promoted cisplatin sensitivity of OC cells by carrying miR-18a-5p.

Conclusion: hMSC-EVs-derived miR-18a-5p inhibits OC cell proliferation, migration, invasion, and chemotherapy resistance.

Keywords: AKT/mTOR; Drug resistance; Extracellular vesicles; Mesenchymal stem cells; NACC1; Ovarian cancer.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Identification of hMSCs and EVs. The hMSCs were passage-cultured. A Morphology of the fourth-passage hMSCs was observed under a microscope; B Expression of specific molecular markers CD29, CD34, CD45, CD73, CD90, CDl05, CD117 and HLA-DR was detected using flow cytometry; C Osteogenic differentiation was observed by Alizarin red staining; D Adipogenic differentiation was observed via Oil Red O staining; E Cartilage differentiation was observed through Alcian blue; F Morphology of hMSC-EVs was observed using TEM after the extraction of EVs; G Size distribution of EVs was analyzed by NTA; H Expression of CD63, CD9, CD81, HSP70 and Calnexin in EVs was measured using WB
Fig. 2
Fig. 2
hMSC-EVs suppressed OC cell proliferation, migration, and invasion. OC CAOV3/ES2 cells were treated with hMSC-EVs. A, B Immunofluorescence assay was used to detect the uptake of EVs by OC cells; C MTT assay was performed to detect the cell viability; D, E Transwell assays were employed to analyze OC cell migration and invasion. Cell experiments were repeated 3 times. Data were expressed as mean ± SD and processed by two-way (panel C) or one-way ANOVA (panels D/E). Tukey's multiple comparisons test was adopted for the post hoc test. ***P < 0.001
Fig. 3
Fig. 3
miR-18a-5p was downregulated in OC. A Heatmap of significantly downregulated miRNAs in OC microarray GSE161784 (abscissa: sample name; ordinate: miRNA name; small square: the expression level of a miRNA in a sample; upper right histogram: color scale); B Intersection of significantly reduced miRNAs in chemotherapy-resistant samples and miRNAs expressed in hMSC-EVs in the EVmiRNA database was obtained, with the middle as the intersection; C, D Expression of miR-18a-5p was determined by RT-qPCR; E Prognosis of OC patients with low and high miR-18a-5p expression was assessed using Kaplan–Meier survival analysis. Data analysis was performed using one-way ANOVA (panel C) with Tukey's multiple comparisons test for the post hoc test. The independent sample t test was used for comparisons between the two groups (panel D). ***P < 0.001
Fig. 4
Fig. 4
miR-18a-5p knockdown partially annulled the inhibition of EVs on OC cell proliferation, migration and invasion. A EVs were treated differently and miR-18a-5p expression in EVs was detected by RT-qPCR; B Size distribution of EVs was analyzed by NTA; C–E miR-18a-5p expression was measured via RT-qPCR; F Cell viability was assessed using MTT assay; G, H Migration and invasion of OC cells were evaluated by Transwell assays. Cell experiments were replicated 3 times. Data were presented as mean ± SD. Data were analyzed by two-way (panel F) or one-way ANOVA (panels A/C/D/E/G/H).One-way ANOVA was employed for comparisons between groups and Tukey's multiple comparisons test was performed for the post hoc test. **P < 0.01, ***P < 0.001
Fig. 5
Fig. 5
miR-18a-5p targeted and inhibited NACC1 expression. A Chromosome expression maps of significantly differentially expressed genes in OC included in TCGA and GTEx (green and red lines: evidently differentially expressed genes; position: the location in chromosome; red: upregulated genes; green: downregulated genes); B Prediction of target genes of miR-18a-5p: 3 circles respectively represented the prediction results of Starbase database, TargetScan databases and significantly upregulated genes in OC, and the middle part indicated the intersection of the 3 groups of data; C NACC1 expression in OC and normal samples included in TCGA and GTEx (abscissa: sample type; ordinate: expression level; red: tumor sample; gray: normal sample); D Expression of NACC1 mRNA in 76 early/advanced OC tissues and 36 benign ovarian tumor tissues was measured by RT-qPCR; E Correlation analysis of miR-18a-5p and NACC1; F Binding sites of miR-18a-5p and NACC1 was predicted via Starbase database; G Targeted binding of miR-18a-5p and NACC1 was validated using dual-luciferase assay; H Expression of NACC1 mRNA in CAOV3/ES2 cells and normal ovarian epithelial cells (IOSE80) was detected by RT-qPCR; I, NACC1 expression in OC cells was determined by WB. Cell experiments were repeated 3 times. Data were exhibited as mean ± SD. One-way ANOVA was used for comparisons between groups and Tukey's multiple comparisons test was employed for the post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 6
Fig. 6
NACC1 overexpression reversed the inhibition of hMSC-EVs on OC cell proliferation, migration and invasion. NACC1 overexpression was accomplished in EVs-treated CAOV3/ES2 cells. A WB was used to detect the expression of NACC1; B MTT assay was adopted to assess cell viability; C, D Transwell assays were performed to detect OC cell migration and invasion. Cell experiments were replicated 3 times. Data were showed as mean ± SD. One-way ANOVA was performed for comparisons between groups and Tukey's multiple comparisons test was adopted for the post hoc test. ***P < 0.001
Fig. 7
Fig. 7
NACC1 activated the AKT/mTOR pathway in OC cells. A, B RT-qPCR was used to detect the expression of AKT and mTOR in OC tissues and benign ovarian tumor tissues; C–F Pearson analysis was used to assess the correlation of miR-18a-5p/NACC1 with the AKT/mTOR pathway; G, H WB was employed to measure the levels of p-AKT, AKT, p-mTOR, and mTOR in cells. Cell experiments were repeated 3 times. Data were expressed as mean ± SD. One-way ANOVA was adopted for comparisons between groups and Tukey's multiple comparisons test was performed for the post hoc test. *P < 0.05,**P < 0.01, ***P < 0.001
Fig. 8
Fig. 8
hMSC-EVs enhanced OC cell sensitivity to chemotherapy drugs by transferring miR-18a-5p. CAOV3 drug-resistant cell lines were constructed. A The cisplatin sensitivity of resistant cells and parental cells was compared via MTT assay; B The IC50 value of drug-resistant cells was assessed; C The cisplatin sensitivity of cells was compared using colony formation assay; D Schematic diagram of tumorigenesis test in mice; E, F RT-qPCR was used to detect the expression of miR-18a-5p and NACC1 mRNA; G WB was employed to measure the levels of p-AKT, AKT, p-mTOR, and mTOR in cells; H Tumor volume of nude mice was detected; I, J Tumors in nude mice were separated, photographed and weighed; K HE staining was used to detect tumor nodules in lung tissues of nude mice. Independent test was repeated 3 times. Data were presented as mean ± SD. One-way ANOVA was used for comparisons between groups and Tukey's multiple comparisons test was adopted for the post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001
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