The Inhibitory Effect And Mechanism Of TRIM21 On Autophagy And Cell Proliferation In Cervical Cancer Cells

Background and objective:Cervical cancer increases fast in the number of new patients and deaths,according to the latest Global Cancer Statistics report,which seriously threatens the health of women all over the world.Among women tumors,cervical cancer is the top 2 malignant tumor,and its morbidity and mortality are only lower than breast cancer.Recently,cervical cancer has a trend of younger development,and its main pathogenic factor is human papillomavirus?HPV?infection.In the treatment of advanced cervical cancer,there are many problems,such as difficulty to cure and easy to recur,which are related to the high proliferation ability of cervical cancer cells.Therefore,it is urgent to find effective targets and related mechanisms to suppress the proliferation of cervical cancer cells.Tripartite motif 21?TRIM21?,a member of a protein family with highly conserved domains,plays an important biological role in cells.As a cytoplasmic antibody Fc receptor,TRIM21 plays a strong bacteriostatic and antiviral role in the pathogen?such as viruses and bacteria?infection of cells.At the same time,it also has the function of regulating autophagy.Recent studies have shown that the level of TRIM21 is closely related to the occurrence and development of malignancies.Particularly,it plays an important role in regulating the proliferation of tumor cells,but it is unknown whether TRIM21 has a relationship with cervical cancer.Therefore,it will be beneficial to the clinical treatment of cervical cancer by investigating the role and mechanism of TRIM21 in regulating the autophagy and proliferation of cervical cancer cells.The aim of this study is to explore the regulatory effects and molecular mechanism of TRIM21 on the autophagy and proliferation of cervical cancer cells,so as to provide a basis for clinical treatment targeting TRIM21.Contents and methods:Part?TRIM21 inhibits the autophagy of cervical cancer cells1.Detecting the expression of TRIM21 and autophagy marker protein in HPV-positive and HPV-negative cervical cancer cell lines by Western blot.2.Observing the inhibitory effect of TRIM21 on autophagy in cervical cancer cells after overexpression or silencing of TRIM21.Part?TRIM21 inhibits the proliferation of cervical cancer cells1.Detecting the effect of TRIM21 on cervical cancer cell viability with CCK-8 assay.2.Measuring the influence of TRIM21 on apoptosis of cervical cancer cells with flow cytometry.3.Testing the effect of TRIM21 on the proliferation of cervical cancer cells by using plate colony formation assay.4.Determining the role of TRIM21 in cervical cancer cell cycle by flow cytometry.Part?TRIM21/ATG4B complex mediates the proliferation inhibition of cervical cancer cells.1.Analyzing the correlation between the levels of TRIM21 and ATG4B in cervical cancer using TCGA database.2.Observing the effect of TRIM21 on ATG4B expression in cervical cancer cells by Western blot.3.Analyzing the influence of TRIM21 on ATG4B transcription by real-time PCR,and detecting the interaction between TRIM21 and ATG4B by immunoprecipitation assay.4.Determining whether TRIM21 inhibits the proliferation of cervical cancer cells via suppressing ATG4B and autophagy.According to the substances of the above experimentations,the main research methods applied are as follows:1.The cervical cancer cell lines HeLa and C33A were cultured and passaged in vitro.2.The protein level was detected with Western blot.3.The autophagy,apoptosis and cell cycle were measured using flow cytometry.4.The cell viability was tested by CCK-8 assay.5.The target protein was over-expressed by transient transfection in cells.6.The cell proliferation was evaluated by plate colony formation assay.7.The correlation between TRIM21 and ATG4B was analyzed with TCGA database.8.The mRNA level was determined by real-time PCR.9.The TRIM21/ATG4B complex was assayed by immunoprecipitation.Results:1.The level of TRIM21 was higher while the level of autophagy was lower in HPV-positive cervical cancer cells than that in HPV-negative cervical cancer cells.2.Overexpression of TRIM21 inhibited the autophagy,survival and proliferation of cervical cancer cells,while silencing of TRIM21 enhanced the autophagy,survival and proliferation of the cancer cells.3.TRIM21 had no obvious effect on the apoptosis of cervical cancer cells.4.TRIM21 arrested the cell cycle of cervical cancer cells in G₀/G₁ phase.5.TRIM21 downregulated ATG4B and suppressed autophagy in cervical cancer cells.6.TRIM21 downregulated ATG4B by forming TRIM21/ATG4B complex rather than repressing its transcription.7.TRIM21 inhibited the proliferation of cervical cancer cells through downregulating ATG4B.Conclusion:TRIM21 represses the survival of cervical cancer cells.TRIM21 and ATG4B form a complex,leading to the downregulation of ATG4B and the suppression of autophagy.TRIM21 inhibits the proliferation of cervical cancer cells by blocking the ATG4B-mediated cell cycle acceleration and ATG4B-mediated autophagy.These results may pave a way for developing novel strategies to the treatment of cervical cancer by targeting TRIM21.