| Background:Cervical cancer is a common malignancy of the female reproductive system.Its incidence is the second highest among female malignancies in the world,and its mortality is the first among women in developing countries.The occurrence of cervical cancer is closely related to the continuous infection of Human papillomavirus(HPV).The early protein E7 encoded by HPV is a key oncogenic protein,which is mainly involved in the carcinogenesis process by degrading retinoblastoma protein(pRB).However,only the infection of HPV cannot lead to malignant transformation of cells.A series of changes in molecular signaling pathways in host cells induced by HPV infection of cervical epithelium eventually led to canceration of cells.At present,the mechanism of cervical epithelial carcinogenesis remains to be further elucidated.The abnormality of autophagy plays an important role in the carcinogenesis of cervical epithelium.The so-called "autophagy" refers to the process in which the components in the cytoplasm are wrapped in bilayer membrane vesicles to form autophagosomes and then transferred to lysosomes for degradation.In cervical cancer,autophagy can significantly inhibit the malignant growth of cells,the self-renewal ability of tumor stem cells and the tumorigenesis ability in mice,and the Akt/mTOR signaling pathway is a classic way to regulate autophagy in cervical cancer cells.However,what other unknown proteins in host cells are involved in the regulation of autophagy?What is the mechanism of its regulation?These questions remain unclear.NCAPH(Non-SMC Condensin I Complex Subunit H)gene,also known as BRRN1,the NCAPH protein encoded is a subunit of the condensed protein complex I,which plays an important role in maintaining the stability of the complex and the separation of sister chromatids.Recent studies have found that the expression of NCAPH is significantly increased in patients with malignant melanoma,non-small cell lung cancer and prostate cancer,and is associated with poor prognosis of patients.NCAPH promoted the proliferation,migration and tumorigenesis of colon cancer,liver cancer and breast cancer.In addition,NCAPH promotes cell cycle and reduces DNA damage by regulating chromatin agglutination and maintaining chromosome stability in pancreatic cancer cells.Thus,NCAPH is a novel key oncogenic protein,which can affect many aspects of tumor cell growth and thus promote tumor progression.However,there are few studies on NCAPH,and the related molecular mechanisms have not been reported.Our previous study found that NCAPH promotes cervical carcinogenesis,which can significantly promote the survival,invasion,migration and EMT ability of cervical cancer cells,while HPV E7 can promote the transcriptional expression of NCAPH.However,three problems have not been clarified in previous studies:1)There is a lack of comprehensive understanding of the effect of NCAPH on the growth of cervical cancer cells;2)What is the molecular mechanism of NCAPH promoting the occurrence and development of cervical cancer?3)Could NCAPH transregulate E7?Objectives:This study intends to verify the effect of NCAPH on the growth of cervical cancer cells in vitro experiments,reveal the regulation of NCAPH on autophagy of cervical cancer cells and its preliminary mechanism,and clarify the trans-regulation of NCAPH on HPV E7 and its mechanism.Methods:(1)Cervical cancer cell lines(HeLa and Siha)were cultured and transfected with targeted NCAPH siRNA.The effect of NCAPH on cell cycle was detected by flow cytometry.Annexin V-FITC/PI double-dye flow cytometry and TUNEL staining were used to detect the effect of NCAPH knockdown on cell apoptosis.β-galactosidase staining was used to detect the effect of NCAPH on cell senescence.(2)Western blot was used to detect the protein expression levels of LC3Ⅱ/Ⅰ and P62 in HeLa and Siha cells after the down-regulation of NCAPH expression.Then,the number of autophagosomes in HeLa and SiHa cells was detected by electron microscopy.The expression of autophagy index LC3 and P62 was detected by immunofluorescence and the process of autophagy flow in cervical cancer cells after interference with NCAPH was monitored by confocal microscopy.(3)The changes of LC3Ⅱ/Ⅰ and P62 proteins under the action of rapamycin(mTOR inhibitor)and Bafilomycin(autophagosomal-lysosomal fusion inhibitor)were detected by Western Blot assay.The effect of NCAPH on lysosomes was detected by AO staining and Lyso-Tracker Red staining and the expression changes of key autophagy proteins in the process of autophagy were assessed.(4)The possible target genes downstream of NCAPH were screened by biochemistry analysis,and the expression changes of key molecules in the PDK1/Akt/mTOR pathway in HeLa and SiHa cells were detected by Western Blot after the down-regulation of NCAPH expression.(5)NCAPH siRNA was transfected into HeLa and SiHa,and the expressions of E7 and pRb were detected by Western Blot and real-time quantitative PCR.The dual luciferase assay combined with AP-1 binding site mutation was used to verify whether NCAPH was involved in the transcriptional regulation of E7 expression through AP-1 transcription factor Western Blot and real-time quantitative PCR were used to detect the expression of AP-1 related componentsResults:(1)After interfering with the expression of NCAPH in cervical cancer cells,flow cytometry,tunel staining and β-galactosidase staining were used to confirm that NCAPH had no significant effect on cell cycle,apoptosis and senescence.(2)After interfering with NCAPH in cervical cancer cells,LC3Ⅱ/Ⅰ ratio was increased and the protein expression of P62 was decreased,the number of autophagosomes was increased under electron microscopy,and the level of autophagy was significantly upregulated by cellular immunofluorescence.The changes of autophagy flux were monitored by the mRFP-GFP-LC3 dual fluorescence autophagy indicator system,the results showed that interference with NCAPH promoted the process of autophagy flux.(3)After interference with NCAPH,mTOR expression was inhibited to induce autophagy and promote the formation of autophagosomes,but there was no significant effect on autophagosome-lysosome fusion stage.The results of AO staining and Lyso-Tracker Red staining showed that the regulation of autophagy by NCAPH was independent of lysosomal function.After NCAPH interference,the expression levels of Beclin-1,ATG5 and ATG7 proteins increased,suggesting that NCAPH regulates autophagy process by affecting autophagosome formation.(4)Genetic analysis showed that NCAPH was significantly correlated with PDK1 expression,suggesting that NCAPH might regulate PDK1/AKT/mTOR signaling pathway.After interfering with the expression of NCAPH in cervical cancer cells,the expression of PDK1 decreased significantly,while the total AKT and mTOR had no significant changes,but the phosphorylated AKT and mTOR proteins were significantly reduced.The protein expression levels of p70S6K and S6 remained unchanged,while the levels of p-p70S6K and p-S6 decreased significantly.(5)After interference with NCAPH,the mRNA level of HPV E7 was significantly decreased,and the protein level of pRb was significantly increased.The transcription factor AP-1 can bind to the promoter region of E7 and up-regulate the transcription of E7.Silencing NCAPH can induce the conversion of AP-1 heterodimer from c-Fos/c-Jun to Fra-1/c-Jun,thus its binding activity with HPV LCR was reduced and HPV E7 transcription was down-regulated.Conclusions:(1)NCAPH has no significant effect on cell cycle,apoptosis and senescence of cervical cancer cells,but it has an important regulatory effect on autophagy.NCAPH may affect the formation of autophagosomes by promoting the activity of mTOR,and then inhibit autophagy.(2)NCAPH regulates the PDK1/Akt/mTOR signaling pathway.(3)NCAPH can transregulate HPV E7 transcription by regulating components of the AP-1 complex. |
