| Objective: It is reported that the worldwide mortality of lung cancer was the highest among all cancers in males.In females,the mortality of lung cancer was also the highest among all cancers in developing countries,and the second highest of all cancers in developed countries.Lung cancer includes small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).NSCLC accounts for about 85% of all lung cancer cases,but the five-year survival rate is less than 20%.The clinical symptoms of most early stage lung cancer patients are not obvious so that lung cancer is difficult to be diagnosed,and the treatment of lung cancer is limited.Therefore,the prognosis remians bad.Epidemiological data and clinical indicators have illustrated the urgency of the research for lung cancer.In our previous study,we have found several long non-coding RNAs(lnc RNAs)which were down-regulated after irradiation.Lnc RNA33 was down-regulated in lung adenocarcinoma cell(A549)after exposure to different types of ionizing radiation.Lnc RNAs are a kind of endogenous RNAs longer than 200 nt without the capability of coding proteins as lack of complete open reading frames.Lnc RNAs could regulate lots of important cellular processes such as cell differentiation,gene activation,histone modification,and so on.An increasing number of literatures show that aberrant expression of lnc RNAs is involved in the tumorigenesis and metastasis of lung cancer.In this study,we have explored the functions of lnc RNA33 in DNA damage and repair of A549 to provide new ideas for the application of lnc RNAs in the clinical treatment of lung cancer.Methods: The main work of this study includes three sections.(1)Functions of lnc RNA33 in regulating DNA double-strand break(DSB)repair induced by ionizing radiation: The expression of lnc RNA33 in normal lung tissues or lung cancer tissues were detected by RT-q PCR.RT-q PCR was also used for detecting the expression of lnc RNA33 at different time points after 2 Gy X-rays or Fe-ion beams in A549 cells.Adenovirus vectors were used for overexpressing lnc RNA33 in A549 cells.DNA damage were detected by neutral commet assay in A549 cells at 0h,12 h after 5 Gy X-ray irradiation withAdv-lnc RNA33,Adv-NC infection or only infected by Adv-lnc RNA33,Adv-NC.(2)The machnism of lnc RNA33 in DNA DSB repair: Adenovirus vectors were used for overexpressing lnc RNA33 in A549 cells.Immunofluorescent staining is used for detecting gH2AX foci in A549 cells after Adv-lnc RNA33/Adv-NC infection.Western blot is used for detecting gH2AX expression in A549 cells after Adv-lnc RNA33/Adv-NC infection or 4 Gy X-ray irradiation.Then we used western blot to detect gH2AX expression in A549 cells at 1h,24 h after 2 Gy X-ray irradiation combined with Adv-lnc RNA33/Adv-NC infection or only Adv-lnc RNA33/Adv-NC infection.RNA immunoprecipitation is used for demonstrating whether Ku80 interacts with lnc RNA33 in A549 cells.Adenovirus vectors were used for overexpressing the expression of lnc RNA33 in A549 cells.Western blot is used for detecting Ku70,Ku80,phosphorylated DNA-PKcs(S2056),DNA-PKcs in A549 cells at 1h,24 h after 2 Gy X-ray irradiation with Adv-lnc RNA33/Adv-NC infection or only infected by Adv-lnc RNA33/Adv-NC.We used immunofluorescent staining to detect co-localization between Ku80 and DNA-PKcs or Ku80 and phosphorylated DNA-PKcs(S2056).(3)The biological effects in A549 cells after overexpressing lnc RNA33: Adenovirus vectors were used for overexpressing lnc RNA33 in A549 cells.Proliferation of A549 cells after Adv-lnc RNA33/Adv-NC infection were detected by cell counting.Clonogenic assay was used for detecting the survival of A549 cells after Adv-lnc RNA33/ Adv-NC infection.We used flow cytometry to detect the cell cycle distribution of A549 cells transfected with Adv-lnc RNA33/Adv-NC at different time points after 5 Gy X-ray irradiation.Results:(1)Functions of lnc RNA33 in regulating DNA double-strand break(DSB)repair induced by ionizing radiation: We found a novel lnc RNA,lnc RNA33,which was down-regulated at 2,12,48 h after X-ray irradiation compared with sham control(P <0.05).It was also down-regulated at 2,12 h after Fe-ion beam irradiation(P <0.05).The expression of lnc RNA33 in normal lung tissues is lower than that in lung cancer tissues(P <0.05).A549 cells were infected with Adv-lnc RNA33(or control adnovirus Adv-NC).The tails moment in A549 cells at 0h after 5 Gy X-ray irradiation combined with Adv-lnc RNA33 infection were shorter than it in A549 cells at 0h after 5 Gy X-ray irradiation with Adv-NC infection(P <0.05).Meanwhile,the tails moment in A549 cells at 12 h after 5 Gy X-ray irradiation with Adv-lnc RNA33 infection were shorter than that inA549 cells at 12 h after 5 Gy X-ray irradiation with Adv-NC infection(P <0.05).(2)The machnism of lnc RNA33 in DNA DSB repair: gH2AX expression and gH2AX foci formation were all significantly increased after over-expressing lnc RNA33,compared with Adv-NC and Ctrl(P <0.05).And gH2AX expression was not decreased at 24 h after irradiation with Adv-lnc RNA33 infection compared with that at 1h after irradiation with Adv-lnc RNA33 infection.Results of RNA immunoprecipitation showed that lnc RNA33 could interact with Ku80(P <0.05).The results of Western blot revealed that expression of phosphorylated DNA-PKcs(Ser2056)was increased after over-expressing lnc RNA33,compared with Adv-NC or Ctrl(P <0.05).Phosphorylated DNA-PKcs(Ser2056)expression was not decreased at 24 h after irradiation with Adv-lnc RNA33 infection compared with that at 1h after irradiation with Adv-lnc RNA33 infection.In the contrary,the expression of total DNA-PKcs was decreased after over-expressing lnc RNA33,compared with Adv-NC or Ctrl(P <0.05).From results of immunofluorescent staining we found that Ku80 didn't co-localize with phosphorylated DNA-PKcs(Ser2056)after over-expressing lnc RNA33 in A549 cells.(3)The biological effects in A549 cells after overexpressing lnc RNA33: A549 cells were infected with Adv-lnc RNA33(or control adnovirus Adv-NC),then we collected and counted the cells at 0h,12 h,24h,36 h,48h after infection to draw growth curves.These curves showed that when over-expressing lnc RNA33,the proliferation of A549 cells were inhibited,compared with Adv-NC and Ctrl(P <0.05).After that,survival of A549 cells was detected by clonogenic assay.From results we can clearly observe that the survival of A549 cells were decreased at 24 h and 48 h after over-expressing lnc RNA33,compared with Adv-NC and Ctrl(P <0.05).The proportion of G2/M phase cells was increased at 24 h and 36 h after over-expressing lnc RNA33,compared with Adv-NC and Ctrl(P <0.05).Conclusions: 1.Over-expressing lnc RNA33 inhibits DNA double-strand breaks in A549 cells induced by ionizing radiation.2.Over-expressing lnc RNA33 up-regulates the expression of gH2AX in A549 cells.Lnc RNA33 interacts with Ku80.Phosphorylation of DNA-PKcs at Ser2056 induced by lnc RNA33 overexpression leads to the dissociation of Ku80-DNA-PKcs complex.3.Over-expressing lnc RNA33 inhibits proliferation,survival and increases the porportion of G2/M phase in A549 cells. |
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