Abstract:

Objective

To explore the impacts and mechanisms of salvianolic acid A （SalA）on the biological behavior of cervical cancer cells.

Methods

Human cervical cancer SiHa cells were cultured in vitro and divided into different groups based on different treatment as and named control group； SalA-L group， SalA-M group， SalA-H group （untreated cervical cancer SiHa cells were treated with SalA at concentrations of 10， 25， and 50 μmol/L for 48 hours）； anti- miR- NC group， anti- miR-212-5p group， miR-NC group， miR-212-5p group （cells were transfected with anti- miR- NC， anti-miR-212-5p， miR-NC， and miR-212-5p respectively）； SalA-H+anti- miR-N group、SalA- H+anti- miR-212-5p group、SalA-H+anti-miR- 212-5p+si-PRR11 group （cells were transfected with anti-miR-NC， anti-miR-212-5p， and co-transfected with anti-miR-212-5p and si- PRR11， followed by treatment with 50 μmol/L SalA for 48 hours）.The expression levels of miRNA- 212- 5p （miR- 212-5p） and proline-rich protein 11 （PRR11） mRNA were detected by RT- qPCR.The expression level of PRR11 protein was detected by Western blot.The targeting relationship between miR-212-5p and PRR11 was verified using a dual-luciferase reporter assay.Cell proliferation activity was detected using the MTT method.Cell colony formation was assessed by cell colony formation assay.The apoptosis rate was determined by flow cytometry.Cell migration was evaluated by scratch assay.Cell invasion was assessed by Transwell assay.Comparisons between two groups were performed by t-test， comparisons among multiple groups were performed by one- way ANOVA， and pairwise comparisons between groups were performed by LSD-t test.

Results

Compared with the control group， the number of the invasive cells， cell colony formation，OD values， wound healing rates， and the expression levels of PRR11 mRNA and protein （0.49 ± 0.04，0.35 ± 0.03， 0.21 ± 0.03 vs 0.61 ± 0.05， P ＜ 0.05） were reduced in the SalA-L， SalA-M， and SalA-H groups （at 24， 48， and 72 hours）， while the apoptosis rate and expression level of miR-212-5p were increased （1.75 ± 0.14， 2.89 ± 0.23， 4.12 ± 0.35 vs 1.00 ± 0.06， P ＜ 0.05）.Compared with the control group and the miR-NC group， the expression levels of PRR11 protein （0.23 ± 0.03 vs 0.57 ± 0.05，0.58 ± 0.06， P ＜ 0.05）， the number of invasive cells， cell colony formation， OD values （at 24， 48， and 72 hours）， and wound healing rates were decreased in the miR-212-5p group， while the apoptosis rate was increased.Compared with the SalA-H group and the SalA-H+anti-miR-NC group， the expression levels of PRR11 protein （0.68 ± 0.07 vs 0.20 ± 0.02， 0.22 ± 0.03， P ＜ 0.05）， cell colony formation，OD values （at 24， 48， and 72 hours）， the number of invasive cells， and wound healing rates in SalA- H+anti- miR- 212-5p group were increased， while the apoptosis rate was decreased； Compared with the SalA-H+anti-miR-212- 5p group， the PRR11 protein expression levels （0.29 ± 0.03 vs 0.68 ±0.07， P ＜ 0.05）， cell colony formation， OD values （at 24， 48， and 72 hours）， invasive cell numbers，and cell scratch healing rates in SalA-H+anti-miR-212-5p+si-PRR11 group were reduced， while the apoptosis rate was increased.

Conclusion

SalA may regulate the biological behavior of cervical cancer SiHa cells through the miR-212-5p/PRR11 pathway.

Key words: Salvianolic acid A, miR-212-5p, PRR11, Cervical cancer cells, Proliferation, Migration, Invasion, Apoptosis