The Role And Mechanism Of Circular RNA CircPLEKHM3 In Ovarian Cancer

BackgroundCircular RNAs(circRNA)are a class of non-coding RNAs in plant and animal cells and protected from degradation by exonuclease RNase R treatment due to its loop structure.In the late 1970s,scientists firstly discovered that RNA can exist in the cytoplasm of eukaryotic cells in a circular form through electron microscopy.Then,Arnberg et al.found the presence of circRNA in the mitochondria of yeast.However,circRNA had been regarded as a meaningless by-product in the cell and a useless substance produced during RNA splicing for a long time.In the 1990s,scientists discovered some circRNA molecules spliced by exons,but due to their low expression,they were still thought to be caused by incorrect splicing of exons.But with the development of next generation sequencing,a large number of circRNA molecules had been identified.Studies have shown that circRNAs occupy a considerable proportion in the transcriptome.Most of the circRNA molecules are derived from exon splicing,a few are composed of introns or introns and exons,and most of the circRNAs are conservative.And the distribution of circRNAs is tissue specific.So far,more and more studies have shown that circRNAs play an important role in the occurrence and development of human diseases.Ovarian cancer is one of the common malignant tumors of female reproductive organs.The incidence of ovarian cancer is currently only less than that of endometrial cancer and cervical cancer.Ovarian cancer is the leading cause of death from gynecological malignancy worldwide,the mortality exceeds the sum of endometrial and cervical cancer.The 5-year survival rate of patients with ovarian cancer is directly correlated to stage.And the survial rate of stage Ⅰ and Ⅱ is about 90%and 80%,respectively,and stage Ⅲ(15%to 20%)and stage Ⅳ(5%)drop sharply.Ovarian cancer in early stage is often curable but without distinct symptoms.These challenges make it urgent to identify potential diagnostic and prognostic biomarkers and new therapeutic targets.So far,numerous abnormal expressed lncRNAs and miRNAs have been identified for their diagnostic and prognostic value in ovarian cancer.Compared to lncRNA,circRNA is more stable and conserved.Previous studies also showed that circRNAs have diagnostic and therapeutic potentials in hepatocellular carcinoma.However,the expression profiles and underlying mechanisms of circRNAs in ovarian cancer require further investigation.MK2206 is a highly selective AKT1/2/3 inhibitor and allosteric inhibitor,which is activated by the pleckstrin homology domain.MK2206 can inhibit autophosphorylation of the threonine 308 and serine 473 sites of AKT.In addition,MK2206 blocks the phosphorylation of Akt-regulated downstream signaling molecules,including TSC2,PRAS40,and ribosomal S6 proteins.At present,MK2206 is the first small molecule allosteric inhibitor of the AKT kinase that entered the clinical research stage and has entered the second phase of clinical practice.Studies have shown that MK2206 has a broader prospect of clinical combination therapy.ObjectiveThe aim of this study is to explore the potential functions and mechanisms of circRNAs in ovarian cancer,and to explore further new diagnostic and therapeutic circRNA biomarkers for ovarian cancer.Materials and methods5 tumor tissues from ovarian cancer patients and 5 normal ovarian tissues from patients with benign gynaecologic diseases were collected,and performed RNA sequencing.A large number of circRNA molecules that are significantly differentially expressed in ovarian cancer tissue samples were identified.Base-scope assays were performed to assess the association of circPLEKHM3 expressions with clinical outcomes using formalin-fixed paraffin-embedded(FFPE)tissues from 88 ovarian cancer patients.The Kaplan-Meier curves revealed that the expression of circPLEKHM3 was associated with overall survival and recurrence-free survival in ovarian cancer patients.Subsequently,we explored the function and mechanism of circPLEKHM3 by knockdown and overexpression of circPLEKHM3 in ovarian cancer cells.To further confirm the role of circPLEKHM3 in vivo,a nude mice xenograft model by subcutaneous inoculation of ovarian cancer cells stably transfected with sh-circPLEKHM3 and sh-NC were used.In addition,ovarian cancer cells scramble and sh-circPLEKHM3 were treated with the combination of Akt inhibitor MK-2206 and taxol.Results1.The expression level of circPLEKHM3 in ovarian cancer tissues is significantly lower than that in ovarian normal tissues.2.The Kaplan-Meier curves revealed that the downregulated circPLEKHM3 was associated with poor overall survival and recurrence-free survival in ovarian cancer patients.3.Functionally,overexpression of circPLEKHM3 significantly inhibited cell proliferation,migration and epithelial-mesenchymal transition(EMT),but its knockdown reversed these effects.4.circPLEKHM3 acted as ceRNA for miR-9 to regulate the expression of BRCA1,DNAJB6 and KLF4,and inactive AKT1 in ovarian cancer cell.5.AKT inhibitor MK-2206 blocked the tumor-promoting effect of circPLEKHM3 knockdown,and potentiated taxol induced growth inhibition of ovarian cancer cells.Conclusion1.circPLEKHM3 is a valuable diagnostic and prognostic biomarker and its downregulation is associated with poor prognosis in ovarian cancer patients.2.circPLEKHM3 could play a tumor suppressor role in vitro and vivo.3.circPLEKHM3 acted as a ceRNA for miR-9 to regulate the expression of BRCA1,DNAJB6 and KLF4,and inactive AKT1 in ovarian cancer cells.4.MK-2206 has potential value in the clinical combination of ovarian cancer,this may because of the low expression of circPLEKHM3 in ovarian cancer tissue.