The Role And Mechanism Of Autophagy In The Process Of Cervical Cancer Cells Resisting To Pirarubicin

Background and objective:Cervical cancer is one of the most common cancer and its morbidity ranks the second of women tumors.Recently,clinical data demonstrate that cervical cancer has challenged youth development.On the other hand,the prognosis of cervical cancer is poor and the current treatment has serious side effect.All of the above demonstrate that it's urgent to develop a more effective way for cervical cancer therapy.Among the chemotherapy drugs used to cervical cancer therapy,pirarubicin(THP)is the third generation of anthracyclines.The clinical trials demonstrated that pirarubicin can be used to treat bladder cancer,breast cancer,ovarian cancer,and so on.Compared to the common used drugs,for instance cisplatin,pirarubicin presents stronger anti-tumor effect and triggers limited side effects,such as immunosuppression and gastrointestinal disturbances.At present,pirarubicin is one kind of promising anti-cancer drug.However,the phase?clinical trial has demonstrated that most of cervical cancer patients are not sensitive to pirarubicin.So it's important to investigate the mechanisms of cervical cancer cells resisting to pirarubicin and therefore design associated strategy to sensitize it.Autophagy is a lysosome-mediated intracellular self-catabolic degradation process.An increasing number of investigations indicated that autophagy plays critical role in cancer therapy resistance,either intrinsic or acquired drug resistance.Recent studies have shown that pirarubicin,with its prodrug,induces a cytoprotective autophagy response in bladder cancer and breast cancer cells.All of the above indicated that protective antophagy may contribute to the nonsensitivity of THP in cervical cancer cells.The objective of this study is to investigate the role of autophagy cervical cancer cells resisting to pirarubicin and explore the associated mechanism.Contents and methods:Part I Autophagy promoted the cervical cancer cells resist to pirarubicin1.Validating the effect of pirarubicin on cell viability and cell death in cervical cancer cells.2.Confirming the role of pirarubicin in autophagy induction.3.Combining with autophagy inhibitor,identifying the role of autophagy in pirarubicin-treated cervical cancer cells.Part II miR34c-5p/ATG4 B pathway mediates pirarubicin-induced autophagy in cervical cancer cells1.After treatment with pirarubicin,screening autophagy related genes at both m RNA and protein levels.Through gene knockdown,elevating the role of associated gene in pirarubicin –induced autophagy.2.tection of the role of pirarubicin in ATG4 B transcription activity and RNA stability by luciferase reporter assay and RNA stability assay.3.Screening the micro RNAs target to 3'-UTR of ATG4 B m RNA.Identifying the role of associated miRNAs in pirarubicin-enhanced ATG4 B m RNA stability.Confirming the mechanisms of the miRNA regulating ATG4 B m RNA.4.Validating the role of miR34c-5p-mediated ATG4 B downregulation in pirarubicininduced autophagy,apoptosis and cell death.5.Confirming the change of miR34c-5p and ATG4 B when treated with pirarubicin in vivo.Part III The universality of miR34/ATG4 B pathway in autophagy regulation1.Validating whether epirubicin/doxorubicin induce cytoprotective autophagy in cervical cancer cells and whether sensitize these drugs through targeting miR34c-5p or ATG4 B.2.Identifying the change of miR34 and ATG4 B in rapamycin-induced autophagy and the effect after inhibiting miR34 or ATG4 B.According to the above contents,the main methods used in this study are as follows:1.Cell culture and passage of cervical cancer cell lines C33 A,Si Ha,He La and ovarian cancer cell lines SKOV3 in vitro.2.Identification of growth inhibition with CCK-8 assay.3.Identification of the cell death proportion with trypan blue exclusion assay.4.Detection cell apoptosis with flow cytometry and nucleus stained assay.5.Detection the m RNA and protein levels with Real-time PCR and Western blot respectively.6.Construction of plasmids with molecular cloning technology.7.Transient tranfection to change the gene expression level.8.Observation and calculation of GFP-LC3 transfected cells with GFP punctate.9.Detection of the activity of ATG4 B 5' and 3' flanking region with luciferase reporter asssay.10.Detection of m RNA stability with RNA synthesis inhibition assay.11.Animal experiment to detect the effect triggered by pirarubicin in vivo.Results:1.Pirarubicin induced cytoprotective autophagy and which contributes to pirarubicin resistance in cervical cancer cells.2.In cervical cancer cells,ATG4 B mediated pirarubicin-induced cytoprotective autophagy.3.Pirarubicin enhances the m RNA stability of ATG4 B instead of its transcriptional activity.4.Pirarubicin promotes ATG4 B m RNA stability through decreasing the level of miR34c-5p.5.miR34c-5p inhibits pirarubicin-induced autophagy and enhances the cytotoxicity of pirarubicin in cervical cancer cells.6.Pirarubicin triggers miR34/ATG4B/autophagy signaling pathway in cervical cancer cellsin vivo.7.The role of miR34/ATG4 B pathway in autophagy regulation is universal.Conclusion:In cervical cancer cells,the downregulation of miR34c-5p induced by pirarubicin contributes to the overexpression of ATG4 B,leading to induction of cytoprotective autophagy.The enhanced autophagy protects cervical cancer cells against pirarubicin.Targeting miR34c-5p or ATG4 B sensitize pirarubicin in cervical cancer cells.The role of miR34/ATG4 B pathway in autophagy regulation is universal.