ABSTRACT
Objective:To explore the effect of miR-3607-3p on the malignant phenotype of cervical cancer cells and related mechanisms.Methods:The OncoLnc bioinformatics website was used to analyze the relationship between the expression of miR-3607-3p and the prognosis of cervical cancer patients. Real time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-3607-3p in human normal cervical epithelial cells (H8) and cervical cancer cell lines (Hela, HCC94, SiHa, C33A). SiHa cells transfected with negative control nucleotide sequence in vitro were defined as negative control (NC) group, and SiHa cells transfected with miR-3607-3p mimicked sequence was defined as miR-3607-3p group. qRT-PCR was used to detect the expression changes of miR-3607-3p in SiHa cells. Cell counting kit-8 (CCK-8) method and scratch test were used to detect the malignant biological behavior changes of SiHa cells. qRT-PCR and Western blot were used to detect the expression changes of profilin 2 (PFN2) gene, and the dual luciferase reporter gene experiment was used to detect the targeting relationship between miR-3607-3p and PFN2. Results:The survival time of patients with high expression of miR-3607-3p was significantly higher than that of patients with low expression of miR-3607-3p ( P<0.01). Compared with H8 cells, the expression of miR-3607-3p was abnormally low in human cervical cancer cell lines ( P<0.05), and the expression of miR-3607-3p was the lowest in the SiHa cell line ( P<0.01). The expression of miR-3607-3p in the NC group and miR-3607-3p group was (1.04±0.31) and (9.28±1.76), respectively. The expression level of miR-3607-3p in SiHa cells transfected with miR-3607-3p mimic sequence was significantly higher than that in NC group, and the proliferation activity and scratch healing rate were significantly lower than that in NC group (all P<0.01). Dual-luciferase reporter gene assay confirmed that miR-3607-3p directly targeted and binded to PFN2 ( P<0.01). qRT-PCR and Western blot results showed that the expression of PFN2 mRNA and protein in miR-3607-3p group was lower than that in NC group; Western blot results showed the expression of proliferating proteins CDK3 and CDK2 in miR-3607-3p group was lower than that in NC group (all P<0.05), and the expression of epithelial phenotype related proteins Claudin-1 and ZO-1 in miR-3607-3p group was higher than that in NC group (all P<0.05). Conclusions:miR-3607-3p is positively correlated with the survival of cervical cancer patients. Up-regulating the expression of miR-3607-3p can inhibit the proliferation and migration of cervical cancer SiHa cells. The mechanism may be related to the targeted inhibition of PFN2.
ABSTRACT
Objective:To explore the effects of scutellarein on the cell proliferation and migration of cervical cancer cell line HCC94 through miRNA-496 (miR-496) and stromal cell-derived factor 1 (SDF-1).Methods:HCC94 cells in the logarithmic growth phase were taken as objects, and 20 μmol/L scutellarin solution (scutellarin group) and dimethyl sulfoxide (DMSO) (control group) were added respectively. The CCK-8 method and Transwell experiment were used to detect the proliferation and migration ability of the two groups of HCC94 cells. The relative expression levels of miR-496 and SDF-1 mRNA were detected by real-time quantitative polymerase chain reaction (qRT-PCR). The target gene of miR-496 was predicted by the bioinformatics software TarBase and verified by the dual-luciferase reporter gene assay. Western blot was used to detect the expression of related proteins.Results:Compared with the control group, the proliferation ability of HCC94 cells in the scutellarin group was decreased on the 3rd, 4th and 5th day of culture (all P < 0.05). The number of HCC94 cells in the scutellarin group and the control group were 25±5 and 134±19, respectively, and the cell migration ability of the scutellarin group was lower than that of the control group ( t = 5.61, P < 0.01). The relative expressions of miR-496 in the control group and the scutellarin group were 1.07±0.12 and 11.24±2.75, respectively, and the difference was statistically significant ( t = 3.68, P < 0.01). The dual-luciferase reporter gene assay confirmed that SDF-1 was the downstream targeted gene of miR-496. The relative expressions of SDF-1 mRNA in the scutellarin group and the control group were 0.29±0.05 and 1.01±0.07, respectively, and the difference was statistically significant ( t = 7.22, P < 0.01). Compared with the control group, after scutellarin promoted the expression of miR-496, the expressions of SDF-1 protein, the cell proliferation protein cyclin-dependent kinase 3 (CDK3) and the cell migration proteins Slug and Zeb-2 were decreased. Conclusions:Scutellarin could inhibit the proliferation and migration of cervical cancer HCC94 cells through the miR-496-SDF-1 axis.
ABSTRACT
Objective:To investigate the expression of miRNA-4766-5p (miR-4766-5p) in ovarian cancer tissues, the effect of miR-4766-5p on the proliferation and invasion of ovarian cancer cells in vitro and its related mechanism.Methods:The cancerous tissues and adjacent tissues of 32 patients with ovarian cancer who underwent surgical treatment in the Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology from February 2019 to December 2020 were selected, as well as the ovarian cancer cell lines (A2780, OC3, SKOV-3. HO-8910, and OVCAR-3) and normal ovarian epithelial cell line IOSE80 were selected for subsequent experiments. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the relative expression of miR-4766-5p in each cell line and ovarian cancer tissues. The cell line with the lowest relative expression of miR-4766-5p was taken as the experimental subject, and the negative control plasmid (control group) and the plasmid expressing miR-4766-5p (miR-4766-5p group) were transfected respectively into the ovarian cancer cells. CCK-8 method and Transwell experiment were used to detect the effect of overexpression of miR-4766-5p on the proliferation and invasion ability of the selected cells. PITA and starBase V2.0 softwares were used to predict the target genes of miR-4766-5p, and the dual luciferase reporter gene method was used for verification. qRT-PCR and Western blot were used to detect the effect of overexpression of miR-4766-5p on target gene expression of the selected cell lines.Results:The relative expressions of miR-4766-5p in ovarian cancer tissues and adjacent tissues were 1.06±0.17 and 5.25±0.70, respectively, and the difference was statistically significant ( t = 5.86, P < 0.01). Compared with IOSE80 cells, the relative expression of miR-4766-5p in all ovarian cancer cell lines decreased (all P < 0.01), and the relative expression of miR-4766-5p in OC3 cells was the lowest, so this cell line was used for subsequent experiments. The result of CCK-8 method showed that the absorbances of OC3 cells in the miR-4766-5p group were lower than those in the control group after 1 d, 2 d and 3 d of cell culture (all P < 0.05). The result of Transwell experiment showed that the number of penetrating cells in OC3 cells of the miR-4766-5p group and the control group were 25±6 and 86±11, respectively, and the difference was statistically significant ( t = 4.77, P < 0.01). PITA and starBase V2.0 softwares predicted that miR-4766-5p may have binding sites with microtubule unstable protein 1 (STMN1) mRNA; the result of dual luciferase reporter gene showed that the target gene of miR-4766-5p may be STMN1. The relative expression of STMN1 mRNA in OC3 cells of the miR-4766-5p group and the control group were 0.28±0.05 and 1.00±0.05, respectively, and the difference was statistically significant ( t = 10.47, P < 0.01). Compared the control group, the expression of STMN1 protein in the miR-4766-5p group decreased, the expression of epithelial cell phenotype protein β-catenin increased, the expression of mesenchymal cell phenotype protein Snail decreased, and the expressions of cyclin CDK2 and cyclin E decreased. Conclusion:miR-4766-5p is under-expressed in ovarian cancer tissues and ovarian cancer cell lines, and miR-4766-5p may inhibit the proliferation and invasion of ovarian cancer cells by down-regulating the expression of its target gene STMN1.