MiR-101 inhibits ovarian carcinogenesis by repressing the expression of brain-derived neurotrophic factor

Abstract

Ovarian cancer is one of the most lethal malignant gynecological tumors as a result of difficulties in early-stage detection and a lack of effective treatments for patients with advanced or recurrent cancer. In the present study, we aimed to explore whether some of the microRNA (miRNA) content of serum might be related to ovarian cancer, as well as the role of these miRNAs and their intercellular transport via exosomes in ovarian cancer. We first detected the expression of six candidate miRNAs in ovarian cancer tissues and adjacent nontumor ovarian samples from 36 patients and confirmed the altered expression of four miRNAs. The level of these six candidate miRNAs was also examined in exosomes from patient serum samples. Only the level of miR-101 was altered in both ovarian tissue samples and serum exosomes. After prediction using online bioinformatics tools and confirmation by dual-luciferase assay and immunoblotting, we identified that miR-101 can repress the expression of brain-derived neurotrophic factor by targeting its 3'-UTR. Using Transwell assays, we examined the effect of miR-101 on migration and invasion capacity of ovarian cancer cells. The results indicated that the reduction of miR-101 is mostly related to significant enhanced ovarian cancer cell migration. Thus, the results of the present study indicate that miR-101 content in serum exosomes has potential as a marker for diagnosis of ovarian cancer and that miR-101 mimics are potential therapeutic drugs for the treatment of ovarian cancer.

Keywords: exosome; invasion; miR‐101; migration; ovarian cancer.

Figure 1

miRNA levels in ovarian cancer and adjacent nontumor control samples. Total RNA was extracted from ovarian cancer and adjacent nontumor control samples from 36 ovarian patients. The miRNA level was determined by quantitative RT‐PCR. The results were analyzed by Student's t‐test and P < 0.05 was considered statistically significant. *P < 0.05; **P < 0.01.

Figure 2

miRNA levels in exosomes from serum samples from ovarian cancer patients and healthy controls. Total RNA was extracted from the serum exosomes from 20 ovarian cancer patients and age paired controls. The miRNAs level was determined by quantitative RT‐PCR. The results were analyzed by Student's t‐test and P < 0.05 was considered statistically significant. **P < 0.01.

Figure 3

BDNF is a direct target of miR‐101. (A) Schematic diagram of the predicted interaction between miR‐101 and BDNF miRNA. (B,C) Dual luciferase assay to examine the direct interaction between and miR‐101 and BDNF. The results were analyzed by Student's t‐test and P < 0.05 was considered statistically significant. *P < 0.05; **P < 0.01. (D) Western blotting. SKOV3 cells were transfected by miR‐101 mimic or inhibitor. Forty‐eight hours after transfection, the cells were lysed and the protein level of BDNF was determined by immunoblotting. (E) SKOV3 cells were transfected with miR‐101 mimic or inhibitor for 48 h. Total RNA was extracted and the BDNF mRNA level was examined by quantitative RT‐PCR. The results were analyzed by Student's t‐test and P < 0.05 was considered statistically significant. *P < 0.05; **P < 0.01. (F) The level of BDNF mRNA in the tumor samples from 36 patients with OA was detected by quantitative RT‐PCR, and the correlation between BDNF and miR‐101 was analyzed using a chi‐squared test.

Figure 4

BDNF repression reduces cell migration and invasion ability. SKOV3 cells were transfected with Si‐1 or Si‐2 targeting BDNF for 48 h, with sequence scrambled short RNA oligo as a control. A typical Transwell assay was used to detect migration (A) and invasion (B) capacity of SKOV3 cells. The cells on the lower surface were stained with hematoxylin and eosin. Ten random fields were selected to determine the average number of cells per view field. The results were analyzed by Student's t‐test and P < 0.05 was considered statistically significant. *P < 0.05. The protein level of BDNF was examined by immunoblotting (C).

Figure 5

BDNF overexpression increases cell migration and invasion ability. SKOV3 cells were transfected with BDNF overexpression vector for 48 h, with empty vector as a control. A typical Transwell assay was used to detect migration (A) and invasion (B) capacity of SKOV3 cells. The cells on the lower surface were stained with hematoxylin and eosin. Ten random fields were selected to determine the average number of cells per view field. The results were analyzed by Student's t‐test and P < 0.05 was considered statistically significant. *P < 0.05 (A, B). The protein level of BDNF was examined by immunoblotting (C).

Figure 6

miR‐101 represses migration and invasion capacity of ovarian cancer cells. A typical Transwell assay was used to detect the function of miR‐101 with respect to migration (A) and invasion (B) capacity of SKOV3 cells. The cells on the lower surface were stained with hematoxylin and eosin. Ten random fields were selected to determine the average number of cells per view field. The results were analyzed by Student's t‐test and P < 0.05 was considered statistically significant. *P < 0.05; **P < 0.01.