| Objective:To reveal the expression levels of MIR22HG in benign ovarian tissues and ovarian cancer(OC)tissues at different stages and the expression distribution in OC cell lines,and to analyze its correlation with the clinicopathological features of ovarian cancer.To investigate the effects of MIR22HG on the proliferation and metastasis of OC cells in vitro and tumorigenic growth in vivo,to screen and identify the proteins that specifically interact with MIR22HG,and to clarify the biological functions of the interaction between the two and the effects on the downstream signaling pathways.Methods:1.RNA in situ hybridization(RNA-ISH)was used to detect the expression of MIR22HG in benign ovarian tissues and ovarian cancer tissues of different stages;RNA fluorescence in situ hybridization(RNA-FISH)and qRT-PCR after nucleoplasmic separation were used to detect the expression of MIR22HG in human Ovarian Surface Epithelial Cells(HOSEpiC)and cell lines CAOV3,OVCAR3 and SKOV3.2.The efficiency of knockdown or exogenous overexpression of MIR22HG lentivirus and plasmid was verified by qRT-PCR;the effect of the above operation on the malignant proliferation ability of ovarian cancer cells in vitro was examined by CCK-8 and plate cloning assay;the effect of the above operation on the metastatic ability of ovarian cancer cells in vitro was examined by Transwell cell migration and invasion assay;the effect of the above operation on the apoptosis of ovarian cancer cells in vitro was examined by Annexin V-FITC/PI method;a naked subcutaneous xenograft tumor model was constructed and the effect of the above operation on the growth of ovarian cancer cells in vivo was observed at regular intervals.3.RNA pull-down combined with protein profiling(LC-MS/MS)was used to screen the candidate interacting proteins of MIR22HG in ovarian cancer cell lines;the interaction between MIR22HG and the target protein DHX9 was verified by Western blot and RNA immunoprecipitation(RIP),respectively;Immunohistochemistry was performed to detect DHX9 protein expression in benign ovarian tissues and ovarian cancer tissues at different stages;DHX9 expression changes were detected at mRNA and protein levels after knockdown or exogenous overexpression of MIR22HG lentivirus and plasmid,respectively;immunofluorescence,qRT-PCR after nucleoplasmic separation was performed to detect DHX9 expression in HOSEpiC and OC cell lines CAOV3,OVCAR3 and SKOV3.4.The proteasome inhibitor MG132(20uM)and the protein synthesis inhibitor Cycloheximide(CHX;25ug/mL)were used to pretreat the corresponding ovarian cancer cells,and the effect of knockdown or exogenous overexpression of MIR22HG on the expression level and half-life of DHX9 protein was examined by Western blot.In vivo ubiquitination assay was performed to detect the effect of these operations on the total ubiquitination modification level of DHX9 protein;coimmunoprecipitation(Co-IP)was used to screen and identify MARCH6 as a candidate protein downstream of the MIR22HG and DHX9;after 6h pretreatment with MG132,Co-IP was used to study the effect of MIR22HG on MARCH6-DHX9 interaction.5.The efficiency of knockdown or exogenous overexpression of DHX9 lentivirus and plasmid was verified by qRT-PCR;the effect of these operations on the malignant proliferation of ovarian cancer cells in vitro was examined by CCK-8 and plate cloning assays;the effect of these operations on the metastatic ability of ovarian cancer cells in vitro was examined by Transwell cell migration and invasion assays;finally,the interaction between MIR22HG and DHX9 was again verified by functional reversion assays,and the effect of altered expression of both on the levels of epithelial mesenchymal transition(EMT)-related proteins and phosphorylation of PI3K/AKT pathway marker proteins was examined.Results:1.Expression and subcellular localization of LncRNA MIR22HG in ovarian cancerRNA-ISH results showed that the expression level of MIR22HG was significantly lower in OC tissues compared to benign ovarian tissues(P<0.0001).MIR22HG tended to be more highly expressed in low-grade ovarian cancers(FIGO Ⅰ-Ⅱ)compared to ovarian cancers with high FIGO staging(FIGO Ⅲ-Ⅳ).RNA-FISH results showed that MIR22HG fluorescence signal was stronger in the cytoplasm than in the nucleus in the OC cell line CAOV3,OVCAR3.Analysis of qRTPCR after nucleoplasmic separation suggested that MIR22HG was mainly expressed in the cytoplasm,with a small amount in the nucleus.2.Effect of overexpression and knockdown of MIR22HG on the malignant progression of ovarian cancerThe qRT-PCR assay showed that lentiviruses targeting MIR22HG effectively downregulated MIR22HG in OVCAR3 and the exogenous overexpression plasmid significantly up-regulated MIR22HG in CAOV3.Functional assays showed that knockdown of MIR22HG significantly promoted proliferation,migration and inhibited apoptosis of OC cells.Overexpression of MIR22HG inhibited the proliferation,migration and invasion of OC cells,and the growth of OC cells in vivo was significantly delayed by the establishment of a subcutaneous tumorigenic model and regular observation and weighing of tumor size.3.Screening and identification of RNA-binding protein DHX9 interacting with MIR22HG in ovarian cancer cellsThe RNA pull-down combined with LC-MS/MS method screening identified DHX9 protein as a candidate reciprocal protein for MIR22HG,and the results of RIP experiments further confirmed the binding of both.In addition,exogenous regulation of MIR22HG expression did not affect DHX9 mRNA levels and affected its protein levels.Western blot results showed that exogenous regulation of MIR22HG expression was consistent with the trend of changes in DHX9 protein expression,and there was a positive correlation between MIR22HG and DHX9 protein levels in ovarian cancer patient samples.RNA-FISHconjugated protein immunofluorescence showed that MIR22HG-DHX9 was co-localized in the cell plasma.4.Effect of MIR22HG on the stability of DHX9 protein and the regulatory mechanismCo-IP screening identified MARCH6 as a candidate protein downstream of the MIR22HGDHX9 complex,which functions to mediate the ubiquitinated degradation of DHX9.Further studies revealed that knockdown of MIR22HG reduced DHX9 protein levels without affecting DHX9 mRNA expression,but returned to normal with MG132 treatment,suggesting that MIR22HG may be involved in inhibiting the proteasomal-ubiquitination degradation of DHX9 protein,as confirmed by cycloheximide chase assay and in vivo ubiquitination assay.The results showed that overexpression of MIR22HG decreased the interaction of MARCH6 with DHX9,and knockdown decreased the opposite.5.Effect of the interaction between MIR22HG and DHX9 protein on the malignant progression of ovarian cancerFunctional assays showed that knockdown of DHX9 significantly promoted proliferation,migration and invasion of OC cells,and overexpression of DHX9 inhibited proliferation,migration and invasion of OC cells;overexpression of MIR22HG resulted in reduced proliferation of OC cells in vitro,which was enhanced by knockdown of DHX9;Western Blot showed that knockdown of DHX9 reversed the inhibitory effect of MIR22HG on Ncadherin and Vimentin protein expression and promoted E-cadherin protein expression,while reversing the inhibition of PI3K/AKT pathway phosphorylation activation by overexpression of MIR22HG.Conclusion:1.MIR22HG is lowly expressed in ovarian cancer and correlates with late FIGO staging.2.MIR22HG inhibits tumor cell proliferation,migration,invasion and cell cycle transition,promotes tumor cell apoptosis and suppresses malignant biological behavior of OC.3.MIR22HG binds to DHX9,a key downstream effector molecule,to exert oncogenic effects.4.MIR22HG attenuates the binding of DHX9 to MARCH6 and reduces the ubiquitin-proteasome pathway degradation of DHX9 protein,thereby enhancing its protein stability.5.MIR22HG inhibits the activation of phosphorylation of the PI3K/AKT pathway by regulating DHX9 protein expression and inhibits the epithelial mesenchymal transition of OC cells,thereby suppressing malignant proliferation and metastasis of OC cells. |
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