The Effect And Mechanism Of Long Non-coding RNA ANRIL On Radiosensitization Of Lung Cancer In Therapy

Background Since the beginning of the 21 st century,with the increase of industrialization,environmental pollution and smoking and other factors,the age of cancer patients in China tends to be younger.Lung cancer is one of the most common malignant tumors in China.Lung cancer can be divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC),among which NSCLC includes squamous cell carcinoma,adenocarcinoma and large cell lung cancer.World health organization data showed that the incidence and mortality of lung cancer ranks first in the world in terms of malignancies.According to the eighth China lung cancer north and south peak BBS released,it was estimated that by 2020 the number of lung cancer patients in China would reach 800,000 and the death toll would reach 700,000.The treatment of lung cancer mainly includes surgery,radiotherapy,chemotherapy and drug-targeted therapy.Radiotherapy is a very important and critical treatment for non-small cell lung cancer,which accounts for the majority of lung cancer.Although the radiotherapy of lung cancer has been developed and improved to gradually improve the radiotherapy area,the radiation resistance of lung cancer tissue to radiation therapy makes the radiation dose relatively higher,and the normal tissues around lung cancer will inevitably be exposed to radiation damage,which will lead to secondary radiation pneumonitis and other side effects.Therefore,strengthening the study of improving the sensitivity of lung cancer radiotherapy is the key to improving the cure rate of lung cancer.In recent years,long non-coding RNA(Lnc RNA),once considered as "transcription noise",has been reported to be closely related to a variety of cancers,which can promote the occurrence and development of cancers,can play a role similar to that of proto-oncogenes and can affect the sensitivity of cancer radiotherapy.Recent studies have found that the expression of ANRIL(also known as CDKN2B-AS)in lung cancer is significantly higher than that in normal lung tissue,which is involved in the promotion of the formation and evolution of lung cancer,and the expression of ANRIL is increased after radiotherapy in other cancers,playing a role in radiotherapy resistance,but its role in lung cancer radiotherapy has not been studied.Based on the current research results,we suspected that ANRIL can not only promote the development of lung cancer,but also play a role in influencing the sensitivity of lung cancer radiotherapy.Through preliminary experiments,we found that the expression of ANRIL in human lung cancer cells gradually increased with the increase of irradiation dose and the time after irradiation.The increase of ANRIL in lung cancer cells was accompanied by the decrease of irradiation sensitivity,while the radiation sensitivity of lung cancer cells significantly increased after the knockdown of ANRIL.Based on the above questions and preliminary experimental results,we conducted a study on the effect and mechanism of Lnc RNA ANRIL on radiosensitization of lung cancer in therapy.This study mainly includes the following two parts: the effect of ANRIL in the sensitization of lung cancer radiotherapy and the preliminary mechanism exploration of ANRIL in the sensitization of lung cancer radiotherapy.Contents 1.The effect of ANRIL on sensitization of lung cancer radiotherapy(1)The relationship between ANRIL and ionizing radiation in lung cancer First,the basic expression level of ANRIL in four NSCLC cells and normal lung epithelial cells was detected by RT-PCR(Real-time PCR).Then the relationship between ANRIL expression and the time and dose of 60 Co irradiation were detected in lung cancer cells.(2)Effect of ANRIL on radiosensitivity of lung cancer(A)ANRIL knock down(KD)and over expression(OE)stable transgenic cell lines were constructed by lentviral packaging.(B)To study the radiotherapy sensitization effect caused by knockdown of ANRIL in ANRIL-KD cell line;(C)To study the radiotherapy resistance effect induced by overexpression of ANRIL in ANRIL-OE cell line;(D)To verify the radiotherapy resistance effect of ANRIL in the subcutaneous tumor-bearing model of nude mice.2.Preliminary study on the mechanism of ANRIL in radiosensitization of lung cancer(1)The distribution of ANRIL in lung cancer cells;(2)Effects of ANRIL on the number of γH2AX and 53BP1 foci under microscope after 60 Co γ ray irradiation;(3)Influence of ANRIL on DNA damage response pathway after 60 Co γ ray irradiation in lung cancer cells;(4)Search for protein molecules that interact with ANRIL;(5)Screening for micro RNA molecules that interact with ANRIL;(6)To detect the effect of interacting micro RNA molecules on DNA damage response pathway after 60 Co γ ray irradiation in lung cancer cells;(7)Verify the effect of interacting micro RNA molecules on the sensitivity of lung cancer cells to radiotherapy.Methods 1.To study the effect of ANRIL on radiosensitization of lung cancer in therapy(1)To study the relationship between the expression of ANRIL and ionizing radiation in lung cancer(A)RNA was extracted from BEAS-2B normal lung epithelial cells and four NSCLC cells,and the expression level of ANRIL was detected by RT-PCR.(B)RNA was extracted from H1299 cells at different time points or at different doses after receiving 60 Co γ ray,and the expression level of ANRIL was detected by RT-PCR.(2)To study the effect of ANRIL on radiosensitivity of lung cancer(A)Construction of ANRIL knock down(KD)and over expression(OE)stably transfected cell lines: the ANRIL-KD and ANRIL-OE plasmids were co-transfected with virus packaging plasmids,and the lung cancer cells were infected with viral supernatant.(B)To study the effect of interference with ANRIL on radiotsensitivity of lung cancer cells: in ANRIL-KD and ANRIL-OE cell lines,the survival ability after irradiation was detected by clonal formation assay,the apoptosis rate after 24 h was detected by flow cytometry,the proliferation ability after 24 h and 48 h were detected by CCK-8 assays,and the degree of DNA damage was detected by comet assays.(C)Radiotherapy resistance effect of ANRIL in nude mice with subcutaneous tumor bearing model: the ANRIL-OE cell line was subcutaneously implanted in the left and right thighs of nude mice at a density of 5×106 to form a left-right self-control.10 Gy local irradiation treatment was performed after tumor formation,and the difference in volume and weight after tumor radiotherapy were recorded.2.Preliminary study on the mechanism of ANRIL affecting the sensitivity of lung cancer radiotherapy(1)Distribution of ANRIL in cells: ANRIL-specific labeled FISH kit.(2)Effects of ANRIL on the number of γH2AX and 53BP1 foci under microscope after 60 Co γ ray irradiation: cellular immunofluorescence assay.(3)Influence of ANRIL on DNA damage response pathway after 60 Co γ ray irradiation in lung cancer cells: Western blot.(4)Search for protein molecules that interact with ANRIL: RNA IP.(5)Screening of micro RNA molecules interacting with ANRIL: microrna.org-Targets and Expression database,RT-PCR,luciferase reporter gene assay.(6)Detection of the effects of interacting micro RNA molecules on DNA damage response pathways after 60 Co γ ray irradiation in lung cancer cells: transfection with micro RNA mimic and inhibitor and Western blot.(7)Verify the effect of interacting micro RNA molecules on radiosensitivity of lung cancer cells: transfection with micro RNA mimic and inhibitor and clonal formation assay.Results 1.To study the effect of ANRIL on radiosensitization of lung cancer in therapy(1)The expression level of ANRIL in four lung cancer cells was significantly higher than that in normal lung epithelial cells,and the expression level was significantly correlated with the time and dose of ionizing irradiation.(2)ANRIL can reduce the apoptosis rate and DNA damage of lung cancer cells after 60 Co γ ray irradiation,improve the proliferative ability and independent viability of cells after irradiation,and play a role in enhancing radiotherapy sensitization.After knocking down ANRIL,the radiosensitivity of cells was significantly improved,showing increased apoptosis rate,increased DNA damage,and decreased independent viability and proliferative ability.After subcutaneous tumor-bearing in nude mice,it was found that the tumor volume and weight of the ANRIL over expression group were higher than those of the NC group.After local 60 Co γ ray irradiation treatment,the tumor volume and weight of the NC group decreased significantly,while the ANRIL over expression group decreased slightly,showing the characteristics of radiotherapy resistance.2.Preliminary study on the mechanism of ANRIL affecting the sensitivity of lung cancer radiotherapy(1)Knockdown of ANRIL can significantly inhibit the activation of DNA damage response(DDR)pathway after 60 Co γ ray irradiation,and influence ATR,which is the upstream of homologous recombination repair pathway(HR).Moreover,ANRIL can play a role in DDR pathway activation after ionizing radiation by acting on ATR.(2)The expressions of mi R-7-5p and mi R-122-5p in lung cancer cells were negatively correlated with ANRIL,and the expression of mi R-7-5p and mi R-122-5p were also opposite to ANRIL,showing a significant reduction after irradiation.There is a direct interaction between mi R-7-5p and mi R-122-5p and ANRIL.ANRIL can promote DDR pathway activation and radiotherapy resistance after irradiation by inhibiting the expression and function of mi R-7-5p and mi R-122-5p in lung cancer cells.Conclusion The expression level of ANRIL in lung cancer cells was higher than that in normal lung epithelial cells,and ionizing radiation could promote the expression of ANRIL.Studies on nude mice and in vitro cells showed that ANRIL in lung cancer cells was one of the main causes of radiotherapy resistance of lung cancer.Knockdown of ANRIL in lung cancer cells could reduce the radiotherapy resistance of lung cancer cells and increase the sensitivity of lung cancer cells to radiotherapy.ANRIL may synergistically affect the sensitivity of lung cancer radiotherapy in two ways: 1.Through the interaction with ATR,ANRIL can participate in DNA damage and repair pathways after lung cancer radiotherapy,promote DDR pathway activation,and play a role in radiotherapy resistance;2.In lung cancer cells,ANRIL can reverse the inhibition of DDR pathway by mi R-7-5p and mi R-122-5p through direct interaction with mi R-7-5p and mi R-122-5p,thus reducing the sensitivity of lung cancer radiotherapy.