Study On The Role Of Noncoding RNAs In Cancer Cells

Objective:(1)Micro RNAs(miRNAs)bind to the 3?-untranslated region of target m RNAs in a sequence-specific manner and subsequently repress gene translation.Human miR-26 a has been studied extensively,but the target transcripts are far from complete.(2)Hypoxia is a physiologically important endoplasmic reticulum(ER)stress that is present in all solid tumors including ovarian tumor that is the leading cause of death among the gynecological malignancies.The previous study showed miR-199 a arising from dynamin-2(DNM2)intron 15 was downregulated under hypoxia and decreased HIF-1? and HIF-2?,cell migration and metastasis.It suggests that miRNA arising from DNM family may play a key role in the response of ovarian cancer cells to hypoxia.Method:(1)We first employed the CRISPR/Cas9 system to generate an miR-26a-knockout line in human cervical cancer He La cells.The miR-26a-knockout line showed increased cell growth and altered proliferation.Proteomics technology of sequential window acquisition of all theoretical mass spectra(SWATH-MS)was utilized to compare the protein abundance between the wild-type and the knockout lines,with an attempt to identify transcripts whose translation was influenced by miR-26 a.Several proteins in the cell cycle/proliferation signaling pathway were chosen to be validated by western blotting and parallel reaction monitoring(PRM).The predicted targets were confirmed by luciferase assays and Western blotting.(2)To evaluate this relationship,we first used q PCR to determine global changes of miR-3120-5p arising from DNM3 family in the different ovarian cancer cells.And then we used q PCR\Western blotting and Confocol to investigate the role of miR-3120-5p in ER stress and acute inflammatory response.Result:(1)Functional classification of the proteins with significant changes revealed their function in stress response,proliferation,localization,development,signaling,etc.Several proteins in the cell cycle/proliferation signaling pathway were chosen to be validated by western blot and parallel reaction monitoring(PRM).The satisfactory consistency among the three approaches indicated the reliability of the SWATH-MS quantification.Among the computationally predicted targets,a subset of the targets was directly regulated by miR-26 a,as demonstrated by luciferase assays and Western blotting.(2)We found that miR-3120-5p arising from DNM3 intron 14 is ubiquitously upregulated under hypoxic conditions in different ovarian cancer cells.Therefore,we investigated the role of miR-3120-5p in ER stress and acute inflammatory response.Our studies show that miR-3120-5p targeted CREBH to destabilize the CRP levels,thereby regulates acute inflammatory response.Furthermore,miR-3120-5p decreased IL-6 expression.Conclusion:(1)This study creates an inventory of miR-26a-targeted transcripts in He La cells and provides fundamental knowledge to further explore the functions of miR-26 a in human cancer.(2)Our study suggests that miRNA-3120-5p may be a new way to regulate the levels of CREBH and ER stress.It is of great practical significance to explore the role of micro RNA-3120-5p in the development of ovarian cancer.