miR-21 Induces Chemoresistance in Ovarian Cancer Cells via Mediating the Expression and Interaction of CD44v6 and P-gp

Abstract

Background: Ovarian cancer (OC), a representative female reproductive system tumor, is one of the most malignant tumors in female. The most important reason for its poor prognosis is because of its high rate of chemotherapy resistance.

Results: This study aims to explore the effects of miR-21 on the chemotherapy resistance of OC cells. The functions of miR-21 on proliferation, migration and invasion of OC cells were assessed by transwell, clonal formation and CCK8 assay. Expression levels of miR-21, P-gp and CD44v6 in SKOV3 (cisplatin sensitive) cells and SKOV3/DDP (cisplatin resistant) cells were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting. Si-CD44v6 was transfected into OC cells to detect the influence on P-glycoprotein (P-gp) expression. Immunofluorescence was used to detect the localization of CD44v6 and P-gp in cell. Co-immunoprecipitation was used to detect the relationship between CD44v6 and P-gp. Results showed that miR-21 expression in cisplatin-resistant SKOV3/DDP cells was significantly higher than that in SKOV3 cells, at the same time, cells proliferation, as well as invasion and migration ability were enhanced after the miR-21 mimics transfected into SKOV3 cisplatin-sensitive cells. Furthermore, miR-21 expression level affected the CD44v6 and P-gp expression. Immunofluorescence and co-immunoprecipitation showed that CD44v6 and P-gp protein could interact.

Conclusion: In conclusion, the high miR-21 expression level could increase the proliferation, invasion, and migration ability of OC cells. And the interaction of CD44v6 and P-gp may mediate miR-21 involvement in chemotherapy resistance of OC cells.

Keywords: CD44v6; P-glycoprotein; chemotherapy resistance; ovarian cancer.

Figure 1

(A) The IC₅₀ value of different cells. (B) miR-21 expression level of A2780 cells transfected in miR21 mimics/inhibitors/NC by qRT-PCR. (C) miR-21 expression level of SKOV3 cells transfected in miR21 mimics/inhibitors NC by qRT-PCR. *P<0.05; **P<0.01.

Figure 2

CCK8 assay detected cell proliferation of (A) SKOV3 and (B) A2780 at 0 h, 2 h, 4 h, 12 h, 24 h. *P<0.05; **P<0.01.

Figure 3

(A) Clonal proliferation assay of SKOV3 cells. a: SKOV3+NC mimics group; b: SKOV3/DDP+ NC inhibitors group; c: SKOV3+ miR21 mimics group; d. SKOV3/DDP+ miR21 inhibitors group. (B) Quantitative analysis of cell clone proliferation rate. **P <0.01.

Figure 4

(A) Transwell assay detect the cell migration ability. a: SKOV3+NC mimics group; b: SKOV3/DDP+ NC inhibitors group; c: SKOV3+ miR21 mimics group; d: SKOV3/DDP+ miR21 inhibitors group. (B) Transwell assay detects the cell invasion ability. a: SKOV3+NC mimics group; b: SKOV3/DDP+ NC inhibitors group; c: SKOV3+ miR21 mimics group; d: SKOV3/DDP+ miR21 inhibitors group. (C) Quantitative analysis of migratory cells. (D) Quantitative analysis of invading cells. **P <0.01.

Figure 5

(A) qRT-PCR detected the P-gp relative mRNA level of SKOV3 cells transfected in miR21 mimics/inhibitors/NC/PBS. (B) qRT-PCR detected the CD44v6 relative mRNA level of SKOV3 cells transfected in miR21 mimics/inhibitors/NC/PBS. (C) The bland of P-gp and CD44v6 protein in different cells. (D) and (E) Gray value analysis of P-gp and CD44v6 protein bands. **P<0.01.

Figure 6

siRNA knockdown of CD44v6 expression in SKOV3/DDP cells. (A) Migration and invasion of SKOV3/DDP cells, a: Control group, b: si-CD44v6 group; (B) and (C)Quantification of migrating and invading cells, c: Control group, d: si-CD44v6 group; qRT-PCR and Western blot detected (D) miR-21 expression level, (E) P-gp mRNA expression level, (F) CD44v6 mRNA expression level, (G) P-gp and CD44v6 Protein expression levels. (H) and (I) Gray value analysis of CD44v6 and P-gp protein bands. *P<0.05; **P<0.01.

Figure 7

Immunofluorescence co-localization to detect the expression of CD44v6 and P-gp. Blue represent Nuclear, Green represent P-gp, Red represent CD44v6.

Figure 8

Co-immunoprecipitation method detected the interaction between CD44v6 and P-gp.