| Objectives The mortality rate of EOC is the highest among gynecological genital tract malignant tumor, largely due to the disease was hard to early detection and always in a rapid development of disease progress. Distant metastasis and Chemotherapy resistance are the major biological characteristics. Anoikis resistance is an important mechanism of ovarian cancer distant metastasis and chemotherapy resistance. Trk B/BDNF promote ovarian cancer cell anoikis-resistance by activating PI3K/AKT signaling pathway. Research focus on ovarian cancer cell anoikis-resistance for several years, recent studies about micro RNAs in ovarian cancer becomes the hot spot. After screening abnormal micro RNAs expression in EOC tissue, Found the 9q21.12 chromosomal region containing mi R-204 was frequently lost. mi R-204 act as tumor suppressors to inhibit migration and invasion in gastric cancer, endometrioid adenocarcinoma and ovarian cancer. Most target prediction algorithms predicted BDNF to be targeted by mi R-204.However, whether mi R-204 play a role in ovarian cancer anoikis-resistance remained unclear. The aim of this study was to investigate the influence of mi R-204 on EOC cells anoikis sensitive, then to explore the possible mechanism in this process.Methods 1 Choose HO-8910, SKOV-3 cell lines, adhere culture group imitated the status of cancer cells in primary lesions; anoikis group imitated the status of cancer cells in metastatic lesions. mi R-204 expression level was measured by q RT-PCR in adhesive and anoikis of SKOV-3 and HO-8910 cells. 2 Use HO-8910 screened by anoikis pattern in part one. Rate of transfection was measured by transfection reagent; construction of pre-mi R-204 plasmid, transfected HO-8910 cells. mi R-204 expression level was measured by q RTPCR. 3 Establish anoikis pattern,apoptosis rate was measured by FACS. 4 To detect the invasion ability, invasion model in vitro was used. 5 To detect the migration ability, wound-healing assay was used. 6 To detect the level of BDNF m RNA, q RT-PCR was used. 7 To detect p AKT and total AKT expression in protein level, western blot was used.Results 1 Cells in anoikis pattern were suspended, consolidating into multicellular spheroids.mi R-204 was significantly down-expression in anoikis pattern(P<0.05), mi R-204 expression of SKOV-3 cells in adhesive culture and anoikis pattern respectively were 1±0.0082,0.7283±0.0082; 0.6242±0.0024 and 0.2577±0.01122 in HO-8910 cells.2 Transfection rate of HO-8910 cells was almost 90%.The sequencing report showsconstruction of pre-mi R-204 plasmid successfully.After transfection, mi R-204 expression level was significantly raised in mi R-204 plasmid group 10.1±0.016(P<0.05); control, mock and negative group respectively 1 ± 0.0039,1.07±0.014 and 0.929±0.011(P>0.05).3 Cells in anoikis pattern was consolidating into multicellular spheroids. Apoptosis rate was significantly high in mi R-204 plasmid group 73.22±0.70(P<0.05); control, mock and negative group respectively 48.98±0.55,45.16±0.49 and 54.46±0.62(P>0.05).4 Invasion model in vitro assay shows that invasive cells was significantly reduced in mi R-204 plasmid group 86±4.5789(P<0.05);invasive cells in control, mock and negative group respectively 153±15.4973,150±14.1480 and 140±6.0800,(P>0.05).Ovarian cancer cells in mi R-204 plasmid group maintain a lower invasion ability.5 Wound-healing assay shows that migrate distance after 24 h was significantly reduced in mi R-204 plasmid group 7.3±0.126(P<0.05);control, mock and negative group respectively 11.2±0.276, 12.5±0.228 and 10.4±0.335,(P>0.05).migrate distance after 48 h was significantly reduced in mi R-204 plasmid group 10.5±0.252(P<0.05);control, mock and negative group respectively20.3±0.289,19.2±0.288 and 20.6±0276,(P>0.05).Ovarian cancer cells in mi R-204 plasmid group maintain a lower migration ability.6 BDNF m RNA expression level was significantly reduced in mi R-204 plasmid group 0.22±0.0098(P<0.05);control, mock and negative group respectively 1±0.0060, 1.06±0.0139 and 0.96±0.0098,(P>0.05). Ovarian cancer cells in mi R-204 plasmid group BDNF present a low expression in m RNA level.7 Using p AKT/AKT stands for phosphorylation level of AKT. The result shows: p AKT/AKT in mi R-204 plasmid group was 0.068±0.028(P<0.05); control, mock and negative group respectively 0.159±0.046, 0.175±0.037 and 0.164±0.051,(P>0.05).Phosphorylation level of AKT was significantly low in mi R-204 plasmid group.Conclusions 1 mi R-204 mediate anoikis-resistance in EOC cells;2 mi R-204 mediating anoikis-resistance in EOC cells may through Trk B/BDNF-PI3K/AKT signaling pathway. |
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