LncRNA NONHSAT096867 Participates In The Radiation-induced Apoptosis Via Bax And IL-8 In Cervical Cancer Siha Cells

Cervical cancer is the fourth largest female tumor and is the fourth most fatal tumor in women.In 2012,there were about 528,000 new cases and 266,000 deaths.8% of the cases were direct deaths caused by cancer.70% of the new cases occur in developing countries,and in undeveloped areas,cervical cancer is a common cause of cancer death.The early diagnosis,metastasis,recurrence and prognosis of cervical cancer,as well as the individualized treatment scheme,still lack effective and reliable indicators.In recent years,lncRNA as its important role played invepigenetics,transcription and translation level,gradually aroused the attention of scientific workers.LncRNA could control the occurrence and development of tumors by its complicated network.The effects induced by lncRNA affects the therapeutic efficacy and long-term prognosis of tumor patients during the treatments,such as radiotherapy,chemotherapy,endocrine therapy and targeted therapy.Radiotherapy plays an important role in the radical treatment of cervical cancer.It induces cell death through direct and/or indirect DNA damage.Therefore,we are more concerned about the occurrence of death in the radiotherapy of cervical cancer.The occurrence of apoptosis could improve the local control rate and reduce the risk of local recurrence and distant metastasis.In previous researches,apoptosis could be induced by radiation in cervical cancer Siha cells.Elucidating the mechanism of lncRNA in the apoptosis of cervical cancer is very important for the sensitivity of radiotherapy,especially the individualized treatment plan.Objective:The human HPV16(+)cervical squamous carcinoma Siha cell was chosen as the research object to explore the mechanism of lncRNA NONHSAT096867 participate in apoptosis induced by IL-8 and Bax/Bcl-2,so as to provide evidence for individualized treatment of cervical cancer.Methods:1.IL-8,as one of the targets for lncRNA NONHSAT096867,is selected by bioinformatics methods.2.RNA-RIP method is conducted to verify the relationship between lncRNA NONHSAT096867 and IL-8.Bax/Bcl-2 and TNF genes are selected by GO analysis and KEGG pathway analysis according to their relationship with lncRNA NONHSAT096867 and IL-8.And their protein changes are proven by analysis.3.Western blot and RT-qPCR are conducted according to protein and gene test,respectively.The cervical squamous carcinoma Siha cells are selected as its higher expression of lncRNA NONHSAT096867 and IL-8 before and after ionizing radiation compared with other cells.4.LncRNA NONHSAT096867,IL-8 and Bax silencing cells are constructed for further study by technology.5.Irradiation conditions: deep X ray irradiation treatment machine,voltage 180 kV,current of 18 mA,0.25 mm Cu filter plate and 1 mm Al,the source skin distance of 60 cm,the dose rate with 0.40 Gy/min.6.MTT method is refered in survival rate of cells before and after gene silencing,ionizing radiation,and IL-8 antibody applied in Bax gene silencing cells.7.Flow cytometry is conducted for apoptosis before and after radiation,gene silencing,and IL-8 antibody in Bax gene silencing cells.8.CO-IP is detected for the combination in different proteins including IL-8 and Bax,IL-8 and Bcl-2,Bax and Bcl-2 before and after radiation.Results:1.The most likely potential target of lncRNA NONHSAT096867 is IL-8 in bioinformatic detection results.Input NONCODE ID:NONHSAT096867or Ensemble ID: ENST00000483500 in MEM Multi Experiment Matrix.The result shows that the most likely potential target of lncRNA NONHSAT096867 is CXCL 8,which is IL-8.2.LncRNA NONHSAT096867 could combine with IL-8,and the relationship between them is positiveC-33 A cells with HPV(-),adenocarcinoma Hela cells with HPV18(+),and squamous carcinoma Siha cells with HPV16(+)are selected.The expression of IL-8is highest in Siha cells compared with other two kinds of cells,and in Hela cells arehigher than in C-33 A cells with significant stastistical difference before and after 4Gy irradiation by ELISA detection.IL-8 mRNA and lncRNA NONHSAT096867 are significantly higher after 4Gy irradiation than before by RT-qPCR with significant stastistical difference.Siha cells are selected for the following related research.The combinations between lncRNA NONHSAT096867 and IL-8 are showed in RNA RIP before and after irradiation.The construction of lncRNA NONHSAT096867-siRNA and IL-8 siRNA cells are successful.In Siha cells,the expression of IL-8 is decreased and lncRNA NONHSAT096867 is increased after irradiation.In lncRNA NONHSAT096867-siRNA cells,the expression of IL-8 is decreased before and after irradiation.In IL-8 si RNA cells,the expression of lncRNA NONHSAT096867 is decreased compared with control and increased after irradiation.3.Both lncRNA NONHSAT096867 and IL-8 suppress apoptosis in Siha cells.Cell viability of IL-8siRNA cells is lower than control in 24 h after 4Gy radiation by MTT detection with significant stastistical difference.Cell viability of IL-8siRNA and lncRNA NONHSAT096867 si RNA are lower than control in 48 h and 72 h after radiation with significant stastistical difference.The percentage of apoptosis increased significantly in lncRNA NONHSAT096867 siRNA and IL-8siRNA cells by flow cytometry after 4Gy radiation.4.lncRNA NONHSAT096867 participates in the radiation-induced apoptosis via Bax and IL-8 in cervical cancer Siha cells.GO analysis and KGG pathway analysis are conducted for all the genes according to result 1 in DAVID website.Network diagram are drawn according to lncRNA NONHSAT096867(ENST00000483500)and its target genes.Potential related genes with IL-8 are screened by Biogrid website.The final genes related to apoptosis and IL-8 are confirmed with TNF,Bax and Bcl-2.The expression of Bax/Bcl-2 changed obviously in lncRNA NONHSAT096867 siRNA and IL-8 siRNA cells,and the expression of TNF does not change significantly through Western blot tests.After irradiation,the expression of Bax is increased,IL-8 is decreased and their combination is less than before irradiation according to CO-IP results.The expression of Bcl-2 is decreased,IL-8 is decreased and their combination is less than before irradiation.The expression of Bcl-2 is decreased,Bax is increased and their combination is less than before irradiation.5.After Bax silencing,apoptosis is suppressed but revised after IL-8 antibody.The construction of Bax-Ri cells is successful.The apoptosis of Bax-Ri cells is reduced before and after irradion compared with control by flow cytometry.However,the apoptosis for Bax-Ri cells could be equal to control after the application of IL-8antibody.Cell viability in Bax-Ri cells is equal to control after irradiation in 24 h,48h and 72 h by MTT tests.Cell viability after the application of IL-8 antibody in Bax-Ri cell is higher to control and Bax-Ri cells after irradiation with significant stastistical difference.Conclusion:1.lncRNA NONHSAT096867 and IL-8 inhibits the occurrence of apoptosis induced by irradiation in cervical Siha cells.After lncRNA NONHSAT096867 or IL-8silencing,the apoptosis could be enhanced after radiation for Siha cells.2.The relationship between lncRNA NONHSAT096867 and IL-8 is positive.After lncRNA NONHSAT096867 or IL-8 silencing,the expression of IL-8 or lncRNA NONHSAT096867 is reduced.3.LncRNA NONHSAT096867 regulate apoptosis induced by radiation through the Bax/Bcl-2 pathway.After lncRNA NONHSAT096867 silencing,the expression of Bax is increased,Bcl-2 is suppressed,and apoptosis is enhanced.4.IL-8 regulate apoptosis induced by radiation through the Bax/Bcl-2 pathway.After lncRNA NONHSAT096867 silencing,the expression of Bax is increased,Bcl-2is suppressed,and apoptosis is enhanced.5.According to Bax mutant Siha cells,the rate of apoptosis could be enhanced by the application of IL-8 antibody after irradiation.