| Lung cancer has the highest fatality rate among all types of cancer,and the number of deaths caused by lung cancer in cancer patients in China far exceeds other types of cancer.Radiotherapy is an important means of treating lung cancer and is applicable at various stages of its occurrence and development.Lung cancer is one of the earliest types of cancer to receive heavy ion radiotherapy,with numerous successful cases and experiences.However,there is currently no paradigm that explains the mechanism of heavy ion treatment for cancer.Mitochondrial ribosomal proteins(MRPs)constitute the mitochondrial ribosome,which translates mitochondrial DNA.However,MRPs are transcribed from nuclear DNA,so they are regulated by both the nucleus and the mitochondria.The 13 proteins translated by the mitochondrial ribosome are all assembled onto the respiratory chain complex.Therefore,abnormalities in MRPs can affect mitochondrial ribosomal translation,thereby affecting mitochondrial energy supply.In addition to performing translation functions,MRPs have been identified as biomarkers for the occurrence and development of various tumors and are potential targets for tumor treatment.This study selected A549 cells,a type of non-small cell lung cancer,as the research subject to explore the mechanism of MRPs in irradiation.The biological effects of A549 cells exposed to carbon ion and X-ray irradiation were investigated.Under the same radiation dose,compared to A549 cells exposed to X-ray,the proliferation and survival of cancer cells were significantly more inhibited after carbon ion irradiation,with a longer cell cycle arrest and a higher rate of apoptosis.Therefore,carbon ion irradiation at the same dose had a better inhibitory effect on A549 cells.A quantitative proteomic analysis using TMT was performed on A549 cell samples irradiated with 4 Gy carbon ion and X-ray for 48 h to identify differentially expressed proteins.Compared to the control group,the differentially expressed proteins in A549 cells after carbon ion and X-ray irradiation were found to be concentrated in the mitochondria,with the MRPs showing the closest interaction among the differentially expressed proteins.In comparison to the control group,exposure to carbon ion irradiation resulted in 48 aberrantly expressed MRPs in A549 cells,with 34 down-regulated and 14 up-regulated.In contrast,the X-ray irradiation group showed 7 aberrantly expressed MRPs in comparison to the control group,with2 down-regulated and 5 up-regulated.Furthermore,a comparison between A549 cells exposed to carbon ion and those exposed to X-ray revealed 66 differentially expressed MRPs,indicating that the abnormal expression state induced by carbon ion differed from that induced by X-ray.To determine whether the differential expression of MRPs after irradiation was primarily regulated by the nucleus or the mitochondria,A549 cells were treated with the mitochondrial ribosomal translation inhibitor Doxycycline.The results showed that inhibiting mitochondrial ribosomal translation did not down-regulate the expression of MRPs,confirming that the down-regulation of MRPs induced by irradiation is primarily regulated by the nucleus.The results of the TMT quantitative proteomic analysis revealed that,compared to the control group,the expression of most MRPs was down-regulated after carbon ion irradiation,whereas more MRPs were up-regulated than down-regulated after X-ray irradiation.The protein MRPS28(Mitochondrial ribosomal protein S28)on the mitochondrial ribosomal small subunit was the only MRP that showed down-regulation in expression after both carbon ion and X-ray irradiation.The Cancer Genome Atlas(TCGA)data showed that most MRPs are highly expressed in various tumors,such as MRPS28,which is highly expressed in lung cancer tissues,and patients with high MRPS28 expression have a poorer prognosis.Western blot results demonstrated significantly higher expression of MRPs in A549 cells and large cell lung cancer cells(H460)compared to normal lung epithelial cells(2B).MRPS28 was selected as a key MRP,and si RNA was designed to inhibit its expression in A549 cells.After 48 h of interference,it was found that knocking down MRPS28 suppressed the expression of MRPS16,MRPS36,MRPS18 B,MRPS22,MRPL44,MRPL38,MRPL9,and MRPL40,inhibited cell proliferation and survival,promoted cell apoptosis,significantly increased reactive oxygen species(ROS)levels,and reduced mitochondrial membrane potential.Western blot results indicated that low expression of MRPS28 induced cell apoptosis through the p53-mediated mitochondrial apoptosis pathway.Conversely,overexpression of MRPS28 had no significant effect on the proliferation,survival,or apoptosis of A549 cells,suggesting that MRPS28 may be a potential target for radiotherapy in non-small cell lung cancer.Based on transient transfection results,we established a stable A549 cell line with knocked-down MRPS28 and performed combined interference of MRPS28 expression with radiation.Firstly,A549 cells with MRPS28 knockdown were treated with combined 2,4,and 6 Gy X-ray irradiation.The results showed that compared to the group subjected to radiation alone,the proliferation rate and clone survival rate of the cells treated with combined interference were lower,while the apoptosis rate was higher.The down-regulation of MRPS28 reduced the resistance of A549 cells to X-ray irradiation.Secondly,A549 cells with MRPS28 knockdown were treated with combined 2 and 4 Gy carbon ion irradiation.The results were similar to the MRPS28 knockdown combined with X-ray treatment mentioned earlier,where the combined treatment significantly inhibited the proliferation and survival of A549 cells and increased the apoptosis rate.Conversely,A549 cells treated with combined MRPS28 overexpression and 2,4,and 6 Gy X-ray irradiation did not show significant changes in proliferation,clone survival,or cell apoptosis compared to the group subjected to radiation alone.This demonstrates that down-regulation of MRPS28 reduces the resistance of A549 cells to radiation,while overexpression of MRPS28 does not decrease their resistance.In this study,MRPS28 was selected as the starting point to observe the impact of its expression intervention on the growth status and radiation resistance of A549 cells.It was found that down-regulation of MRPS28 affected cell viability,reduced the resistance of A549 cells to radiation,and promoted cell apoptosis through the p53-mediated mitochondrial apoptosis pathway.Further research is needed to investigate how MRPS28 down-regulation activates p53.Additionally,our experimental results also indicated that MRPS28 down-regulation led to abnormal expression of other MRPs,disrupting the translation function of mitochondrial ribosomes and affecting mitochondrial energy supply.On the other hand,overexpression of MRPS28 did not significantly affect cell viability or reduce cell resistance to radiation.In summary,this study is of great significance in exploring potential targets for treating lung cancer and improving the efficacy of radiation therapy. |
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