PNO1 Promtes Epithelial Ovarian Cancer Cells Proliferation And Inhibite Apoptosis By AMPK Activation

PNO1,homo sapiens partner of NOB1 homolog,is a human novel RNA binding gene Partner of NOB1.Previous research showed that PNO1 is expressed in many tissues,such as liver,lung,kidney,testis,ovary and so on.PNO1 is located in the nucleus,especially in nucleoli.The PNO1 family shows significant conservation in both eukaryotes and prokaryotes.The present study explored the effect of gene PNO1 on proliferation and apoptosis of epithelial ovarian cancer cells.First,we found PNO1 expressed highly in four epithelial ovarian cancer cells.In ovarian cancer cells SKOV3,PNO1 expression was knocked down by a lentiviral short hairpin RNA(Lv-sh RNA)delivery system.The RNA interference-mediated the down regulation of PNO1 expression significantly reduced the proliferation and colony-formation ability of epithelial ovarian cancer cells.Additionally,PNO1 sh RNA-expressing lentivirus-treated SKOV3 were more susceptible to apoptosis.Our present study also showed that PNO1 affected the ability of cells proliferation and apoptosis may through the AMPK activation.These results suggested that PNO1 may act as an oncogenic factor in epithelial ovarian cancer and could be a potential molecular target therapy in the future.Part I.The expression of PNO1 in epithelial ovarian cancerObjective: To detect the expression of PNO1 in epithelial ovarian cancer cell linesMethods: Collected ovarian samples including benign cystadenomas and epithelial carcinoma,and then study PNO1 expression by immumohistochemistry.Human epithelial ovarian cancer cell lines HO8910,HEY,SKOV3,OVCAR3 and the normal ovarian cell line Moody were cultured common method.The m RNA and protein levels of PNO1 were assessed through real-time q PCR and western blotting.Results:(1)Significantly increased PNO1 was observed in epithelial ovarian cancer compared with that observed in benign cystadenomas(P<0.05).(2)PNO1 m RNA levels were increased in cells HO8910,OVCAR3,SKOV3 and HEY,compared with GAPDH.(3)In addition,PNO1 protein levels were increased in HO8910,OVCAR3,SKOV3 and HEY cells compared with Moody cells.Conclusions:PNO1 expressed highly in epithelial ovarian cancer.Part II.The construction of lentivirus-mediated sh RNAObjective: To inhibit PNO1 expression in epithelial ovarian cancer cells effectively.Methods: The lentivirus carrying PNO1 sh RNA and control sh RNA was constructed respectively according to the manufacturers` instructions for the operations.After infected by lentivirus-mediated sh RNA with fluorescent markers,the cells can be observed the situation of transfection and growth.Then,to detect the reduction efficiency of the lentivirus-mediated sh RNA of PNO1 in m RNA and protein level,the real-time q PCR and western blotting experimental technology was applied respectively.Once the reduction efficiency is not up to the standard,the lentivirus-mediated sh RNA should be constucted again.Results: 1.The efficiency of lentivirus-mediated shRNA of PNO1 was above 70 percent,at the same time,the cells after infection were in good shape.The lentivirus could be used for the subsequent experiments.2.The reduced efficiency of PNO1 in epithelial ovarian cancer cells infected by lentivirus-mediated sh RNA reached 68.8%,which was detected by real-time q PCR technology.3.The effect of PNO1 inhibition in epithelial ovarian cancer cells infected by lentivirus-mediated sh RNA was also very significant,which was detected by western blotting technology.Conclusions: The expression of PNO1 can be efficiently inhibited by lentivirus-mediated sh RNA in both m RNA and protein levels.Part III.The effect of PNO1 knockdown on cell proliferationObjective: To detect the effect of PNO1 inhibition on proliferation of epithelial ovarian cancer cells infected by lentivirus-mediated sh RNA.Methods: 1.Epithelial ovarian cancer cells were infected by lentivirus-mediated sh RNA firstly.From the third day after infection,the3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide(MTT)assay was performed for a succession of 5 days and cell growth curves were drawn.All operations were carried out according to the manufacturer`s instructions.2.From the third day after cells and lentivisus-mediated sh RNA inoculation,cells with green fluorescence were taken photos and counted by celigo per day and cell growth curves were drawn for 5 days.3.Epithelial ovarian cancer cells were infected by lentivirus-mediated sh RNA firstly.3 days after infection,cells were seeded in 6-well plates,and were observed after 11 days of incubation under the fluorescence microscope.The total number and shape of colonies stained by Giemsa were recorded by the fluorescence microscope and camera.Results: 1.The results of MTT experimental technology showed that the proliferation ability of cells infected by lentivirus-mediated PNO1 sh RNA was decreased obviously comparing with that of the control group.2.The results of celigo cell counting and growth tracking technology showed that the number of living cells in the experimental group was decreased clearly comparing with that of the control group.3.Clone formation experiment showed that the experimental group compared with control group,the number and the shape of clone cell mass were reduced significantly.Conclusions: PNO1 knockdown can inhibit epithelial ovarian cancer cells proliferation.Part IV.The effect of PNO1 knockdown on cell proliferationObjective: To detect the effect of PNO1 inhibition on apoptosis of epithelial ovarian cancer cells infected by lentivirus-mediated sh RNA.Methods: Epithelial ovarian cancer cells were infected by lentivirus-mediated shRNA firstly.3 days after infection,cells were digested,and 2 days later,cells were stained by Annexin V fluorescent tags and then tested by the flow cytometry instruments.Early-stage apoptotic cells(Ann+)were examined by florescence-activated cell sorting(FACS)analysis.Results: The results of the flow cytometry experimental technology showed that the apoptosis ability of cells infected by lentivirus-mediated PNO1 sh RNA was increased obviously comparing with that of the control group.Conclusions: PNO1 knockdown can promote epithelial ovarian cancer cells apoptosis.Part V.The mechanism of PNO1 inhibition proliferation and promotion apoptosisObjective: In order to explore the possible mechanism of PNO1 inhibition proliferation epithelial ovarian cancer cells and promotion apoptosis.Methods: Epithelial ovarian cancer cells were infected by lentivirus-mediated sh RNA firstly.3 days after infection,the cells were collected and lysed.The Path Scan intracellular signaling array kit was used to detect the activation of intracellular signaling molecules.All operations were carried out according to the manufacturer`s instructions.Results: The results of the Path Scan intracellular signaling array kit showed that the phosphorylation of AMPKα level was reduced after infection of lentivirus-mediated PNO1 sh RNA.Besides,the change had statistical significance through the analysis of grey value.Conclusions: PNO1 plays the role in proliferation and apoptosis by activating AMPK related pathways.