GRSF1-Mediated MiR-G-1 Promotes Malignant Behavior And Nuclear Autophagy By Directly Up-Regulating TMED5 And LMNB1 In Cervical Cancer Cells

[Objective]GRSF1 is originally identified as a RNA-binding protein with high affinity for G-rich sequences,which plays key roles in all steps of post-transcriptional regulation of RNAs,including RNA transport and localization,RNA stability,RNA splicing,and translation by binding with the unique mRNAs via RNA-binding domains in a sequence-and structure-specific manner.Recently,Noh et al reported that GRSF1 can interact with the lnc-RMRP and facilitate the localization of lnc-RMRP into the mitochondrial matrix;And that,lnc-RMRP was best known for being a component of the nuclear RNase MRP complex,which participates in the processing of ribosomal RNA in yeast.These data identified that GRSF1 mediates the non-coding RNAs to regulate the process and expression of ribosomal RNA,indicating that GRSF1 may also mediate the non-coding RNAs to regulate the protein expression.[Methods]A Flag-GRSF1-RIP-small RNA library was constructed and sequenced and found many novel miRNAs-riched in this complex.RT-qPCR assay identified their expression levels of miRNAs.Northern blot assay was used to identify the novel miR-G-1.RT-qPCR assay detected the expression levels of miR-G-1 in cervical cancer.We used MTT assay,colony formation assay,EdU assay,cell apoptosis and cell cycle assay,Transwell migration and invasion assay,nude mice model assay,IF assay,IHC assay,Western blot assay and cell adhesion assay to investigate the function of miR-G-1.Then,we predicted the target genes and used EGFP reporter assay to identify the directly interaction of miR-G-1 and its' target genes.We used RT-qPCR assay,Western blot assay,IF assay and IHC assay to detect the expression levels of target genes.Next,we used MTT assay,colony formation assay,EdU assay,cell apoptosis and cell cycle assay,Transwell migration and invasion assay,IF assay,IHC assay,Western blot assay to investigate the function of its target genes.We used String software,IF assay and IP assay to identify the interaction protein.Finally,we used Western blot,IF assay and dual-luciferase reporter assay to verify the regulation of WNT/?-catenin pathway.[Results]We found a novel miRNA named miR-G-1 by GRSF1-RIP-Deep sequences in HeLa cells.The expression level of miR-G-1 in cervical cancer tissues and serum and cervical cancer cell lines was up-regulated compared to the control groups.miR-G-1 overexpression promoted cell proliferation,cell migration/invasion,accelerated cell cycle and EMT progress,inhibited cell apoptosis and anoikis,enhanced the resistivity for cis-platinum by upregulating TMED5 in cervical cancer cells.miR-G-1 overexpression in vivo promoted the tumor growth at the growth rate and the tumor size.In addition,we found that TMED5 could interact with WNT7B and thus activated WNT/?-catenin pathway.miR-G-1 mediated the activation of this pathway.miR-G-1 overexpression promoted the serum starvation induced nuclear autophagy,accelerated the TAX induced DNA-damage repair in cervical cancer cells.Furthermore,we demonstrated that miR-G-1 upregulates TMED5 and LMNB1 in a GRSF1-dependent manner in the shR-GRSF1 cells.[Conclusions]In conclusion,we identified that ectopic miR-G-1 promoted nuclear autophagy and malignant behavior in cervical cancer by up-regulating LMNB1 and TMED5 in a GRSF1-dependent manner.Collectively,these findings may provide a new insight into the up-regulation mechanism mediated by miRNAs and a potential biomarker for cervical cancer.