MiR-224-3p Inhibits Autophagy In Cervical Cancer Cells By Targeting FIP200

[Objective] Micro RNA(mi RNA)is a non-coding small RNA of about 21-25 nucleotides that acts as a post-transcriptional regulator capable of gene expression.It has been found that these small molecules regulate genes involved in different cell physiological processes such as regulatory cell proliferation,development,differentiation,apoptosis,and the like.Cervical cancer(CC)is a malignant solid tumor,which is one of the main causes of morbidity and mortality in women.Persistent High-risk human papillomavirus(hr HPV)infection is closely related to cervical cancer and autophagy has been suggested to inhibit viral infections.Mi RNAs can participate in the regulation of autophagy by regulating their target genes and play a function similar to oncogenes or tumor suppressor genes.The aim of this study was to investigate whether mi R-224-3p was involved in the regulation of autophagy in cervical cancer cells caused by HPV infection.To predict and determine the target gene and target protein that direct action of mi R-224-3p.To further elucidate the molecular mechanism of mi R-224-3p in the development and progression of cervical cancer.[Methods] In this study,we performed a mi RNA microarray analysis on CC tissues and found that a large number of mi RNAs with differential expressions in hr HPV-infected tissues.We identified mi R-224-3p as a candidate mi RNA selectively up regulated in HPV-infected tissues and cell lines.Further analysis revealed that mi R-224-3p regulates autophagy in cervical cancer tissues and cell lines.The expression of mi R-224-3p was detected by RT-PCR in cervical lesions.Western blot was used to validate the expression of autophagy-related protein P62 and LC3 in cervical tissues.The autophagy mechanism was verified by transfection of the mi R-224-3p plasmid and its inhibitor in different HPV-infected cell lines.And the expression levels of autophagy-related proteins P62 in the above transfected cell lines were examined by immunofluorescence.The mi R-224-3p target gene was predicted by Target Scan7.0 and the candidate gene FIP200 of mi R-224-3p was predicted and screened by two-factor luciferase assay.The effect of mi R-224-3p on m RNA and protein levels of FIP200 in cervical cancer cell lines was examined by RT-PCR and Western methods.The effect of mi R-224-3p on FIP200 expression was examined by the same method as above in overexpression or knockdown of mi R-224-3p in cervical cancer cell lines.[Results] The expression of mi R-224-3p in HPV(+)cervical tissue was significantly higher than that in HPV(-)cervical tissue.The expression of mi R-224-3p in the cervical tissue of different stages was significantly increased with the increase of lesion level.The expression of autophagy-related protein LC3 in HPV(+)cervical tissue was significantly decreased,and the expression of P62 protein was significantly increased,indicating that autophagy was inhibited in HPV(+)tissue.In HPV(-),HPV16(+),HPV18(+)cell lines,the expression of P62 protein was significantly increased after transfection with mi R-224-3p.The expression of P62 protein was significantly decreased after transfection with mi R-224-3p-inhibitor.The changes above in HPV18(+)cell line was the most obvious.Genetic prediction showed that FIP200 was mi R-224-3p candidate target gene,and the expression of FIP200 was significantly decreased after transfection with mi R-224-3p.The expression of FIP200 was significantly increased after transfection with mi R-224-3p-inhibitor.The above results were statistically significant(P <0.05).[Conclusions] HPV infection can cause increased expression of mi R-224-3p and inhibition of autophagy in cervical lesions,the same results in different cell lines were verified.Overexpression of mi R-224-3p inhibits autophagy in HPV-infected cells,but knockdown of endogenous mi R-224-3p increases autophagic activity in the same cell.In addition,we found that mir-224-3p directly inhibits the expression of autophagy related gene,FAK family-interacting protein of 200 k Da(FIP200).In summary,we found that mi R-224-3p regulates autophagy in hr HPV-induced cervical cancer cells through targeting FIP200 expression.