The Functions Of Nucleophosmin In Chromosome Polarization Of Lung Cancer Cells

Purpose:With the development of China’s society and economy,under the influence of the aging population,environmental pollution,and irrational diet,malignant tumors have become one of the major problems that seriously threaten the health of the Chinese population,and they are becoming younger and younger.According to reports,the morbility and mortality of cancer are continuously rising and the annual medical expense caused by malignant tumors exceeds more than 220 billion,which becomes a huge financial burden to our country.In 2015,it is reported that 787,000 new cases have suffered from lung cancer and 631,000 patients dead in China.Lung cancer is the most common malignant tumor and the leading cause of cancer deaths in our countryThere are several ways to classify lung cancer.According to its histopathological classification,it is mainly divided into two categories,small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).Nearly 20%of lung cancer are SCLC and about 80%are NSCLC.The main treatment methods for lung cancer include surgical treatment,chemotherapy,radiation therapy,molecular targeted therapy,immunotherapy,and traditional Chinese medicine treatment,etc.Combination therapy is recommended clinically.However,despite the current widespread use of these treatments in clinical practice,the 5-year survival rate of lung cancer,especially NSCLC,has not improved significantly and remains about 15～20%.Therefore,it is of great significance to search for more effective diagnostic and therapeutic targets from the molecular mechanism of lung cancer.Nucleophosmin(NPM),a nucleolar protein,plays an important role in a series of cell survival and proliferation pathways,including ribosomal biosynthesis,apoptosis,cell cycle,and response to stress.NPM has been confirmed to be closely related to tumorigenesis and tumor progression.NPM is also related to biological processes such as CENP-A-containing nucleosome assembly and cell proliferation.Our preliminary results also show that NPM can inhibit cell proliferation and increase cell G2-/-M phase blockage,suggesting that NPM may become one of the therapeutic targets of lung cancer.Methods:This study was divided into four sections.(1)Establishing cell lines.Vector and virus were constructed and then stable cell lines were established.Western-blot method was used to detect the expression of NPM in the stable cell line,to identify their transfection efficiency.(2)Response of NPM to ionizing radiation.In this section,we used the Western-blot to detect the time-and dose-dependent induction of NPM in cells after radiation.(3)Biological effects of NPM on cell cycle,cell proliferation,and chromosomal polarization.The effects of NPM on cell proliferation and survival were detected by CCK8 and cell colony-forming assay.The effect of NPM on cell cycle was detected by PI staining and flow cytometry.The impact of NPM on chromosome polarization was investigated by immunofluorescence assay and Western blot.Results:Firstly,stable cell lines with knockdown and overexpression of NPM were established by packaging lentivirus respectively and its working efficiency was verified by Western-blot method.Then,we used the Western-blot method to verify the expression of NPM protein in lung cancer cells after radiation,and the results showed that radiation suppressed the expression of NPM(p<0.05).Cell proliferation experiments showed that the proliferation rate of A549 cells in the overexpressed NPM group was significantly reduced(p<0.05),but the change in the knockdown group was not obvious.We used cell cloning experiments to detect the formation of cell clones in each group,calculated the cell survival score,and simulated the cell survival curve by software.The results showed that the cell clone formation rate of the overexpressed NPM group was significantly reduced(p<0.05).We used flow cytometry to detect the cell cycle distribution of cells in each group at 0,10,and 24 hours after 2 Gy irradiation.The results showed that G2/M occurred in A549 cells in the overexpressed NPM group at 10 and 24 hours after receiving 2Gy X-ray irradiation.Phase block(p<0.05),and the proportion of cells in the G2/M phase increased at 10 hours after irradiation and did not decrease after 24 hours(p<0.05).NPM expression reduced the proliferation rate of A549 cells and cell survival.Thirdly,at 10 and 24 hours after receiving 2 Gy X-ray irradiation,A549 cells were blocked at the G2/M stage and the proportion of G2/M stage cells increased at 10 hours after irradiation while the proportion of G2/M stage cells did not decrease at 24 hours.Finally,by detecting the expression of NPM in A549 cells after applying radiation and overexpressing LNC CRYBG3,we found that there was a certain relationship between NPM and LNC CRYBG3.Overexpression of LNC CRYBG3 had no significant effect on the expression of NPM;however,when over-expressed LNC CRYBG3 increased radiation-induced expression of NPM(p<0.05).Finally,we used immunofluorescence to detect centrosomes and tubulin proteins to characterize cell division.The experimental results indicate that NPM overexpression increased the abnormal polarization of A549 cells,resulting in increased genomic instability(p<0.05).Conclusions:Overexpression of NPM leads to suppressed proliferation and reduced cell survival of lung cancer cells.It also promotes the G2/M phase block of 3,and then leads to the genomic instability and promoted cell de lung cancer cells caused by X-ray irradiation,increases the proportion of abnormal cell polarization through the interaction with LNC CRYBGath of lung cancer cells.In summary,this study illustrates the important role of NPM in the cellular response of lung cancer cells to ionizing radiation,which may inspire a new strategy for the treatment of lung cancer.