MicroRNA-375 regulates the anti-tumor effect of CIK cells against cervical cancer SiHa cells by inhibiting the expression of YAP gene
10.3781/j.issn.1000-7431.2016.11.671
- Author:
Jinhang LI
1
Author Information
1. Department of Gynecology, Qingdao Municipal Hospital Affiliated to Qingdao University Medical College
- Publication Type:Journal Article
- Keywords:
Cytokine-induced killer cells;
Gene expression regulation;
MicroRNAs;
MiRNA-375;
Uterine cervical neoplasms;
Yes-associated protein
- From:
Tumor
2016;36(1):43-51
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To investigate the regulatory role of microRNA-375 (miR-375) in the process of cytokine-induced killer (CIK) cells killing cervical cancer SiHa cells, and to determine the target genes of miR-375 and their molecular mechanisms. Methods: The peripheral blood mononuclear cells (PBMC) were isolated from the healthy people, and cultured in vitro with different cytokines for 14 d to induce into CIK cells. CIK cells were stimulated as the effector cells. The phenotype of CIK cells was analyzed by FCM, and the killing effect of CIK cells on cervical cancer SiHa cells was detected by CCK-8 method. Real-time fluorescent quantitative PCR was used to detect the change of miR-375 expression level in SiHa cells after co-culture with CIK cells. CCK-8 method was used to detect the proliferation of SiHa cells transfected with miR-375 mimic and the inhibitory effect of CIK cells on proliferation of SiHa cells transfected miR-375 inhibitor. The expression of Yes-associated protein (YAP) in SiHa cells transfected with miR-375 inhibitor and co-cultured with CIK cells was detected by immunofluorescence assay and Western blotting, respectively. Results: After co-culture with CIK cells for 24, 48 and 72 h, the growth inhibitory rates of SiHa cells were (22.97±3.54)%, (37.48±3.64)% and (54.32±4.25)%, respectively. The relative expression levels of miR-375 in SiHa cells after co-culture with CIK cells for 24, 48 and 72 h were about 1.39, 1.57 and 2.68 times higher than those before co-culture, respectively. The viability of SiHa cells transfected with miR-375 mimic was significantly decreased (P < 0.001). After co-culture with CIK cells, the suppression effect on the growth of SiHa cells transfected with miR-375 inhibitor was significantly decreased (P < 0.001). YAP protein was expressed abundantly in SiHa cells, and mainly concentrated in cell nucleus. After co-culture with CIK cells, the expression of YAP protein in SiHa cells transfected with miR-375 inhibitor was more significantly up-regulated (P < 0.01). Conclusion: CIK cells can effectively kill cervical cancer SiHa cells. As a tumor-suppressor factor, miR-375 may play an important role in the process of CIK cells killing SiHa cells through down-regulating the expression of YAP gene.
