| Objective:(1)To screen and identify the growth kinetics of seminoma to cisplatin,which will provide new directions for the study of hypersensitivity to chemotherapy of seminoma;(2)to study the mechanism of cisplatin-induced senescence in seminoma by highthroughput sequencing method.Methods:(1)After treating TCam-2 with different concentrations of cisplatin,CCK8 and colony formation assay were used to examine the viability of TCam-2 to cisplatin.Later,the Incucyte S3 live cell imaging system was used to monitor growth kinetics and morphological changes of TCam-2 cells after treatment with different concentrations of cisplatin.(2)According to the results of Incucyte S3,flow cytometry was applied to analyze apoptosis and cell cycle distribution about the cells treated with different concentrations of cisplatin at different time.The expression and localization of related proteins were detected by Western blotting and immunofluorescence,and SA-β-gal staining was used to identify senescent cells.The levels of ROS were determined by flow cytometer and fluorescence microscope after the cells were stained with DCFH-DA probe.(3)The expression of p21 in testicular germ cell tumors(TGCTs)and normal testis were detected by immunohistochemistry,and further verified by querying the TCGA database.(4)According to the aforementioned results,a certain concentration of cisplatin was used to induce senescence of TCam-2 cells,and the total RNA of experimental groups and control groups were extracted.Then,the extracted RNAs were subjected to high-throughput sequencing analysis.Results:(1)With the concentration of cisplatin increasing,the proliferation activity of TCam-2 cells gradually weakened.(2)The cell viability of TCam-2 cells was significantly inhibited by cisplatin treatment for 6 hours.Flow cytometry analysis showed that arrests in S phase were induced by cisplatin in the early stage.(3)Images taken from Incucyte S3 live-cell imaging system showed that sparse shrinkage spindle-shaped cells and worse cell adhesion were observed compared to the control group after treatment with high-dose cisplatin for 24 h.At 48 h,many cells detached from the plate slip and floated in the culture medium.To gain insight into the effect of high-dose cisplatin on TCam-2,flow cytometry analysis was used and detected that the apoptosis was induced by high-dose cisplatin.Western blotting showed that high dose cisplatin also induced the up-regulation of cell membrane-mediated extrinsic apoptosis-related proteins Fas L,Fas,Caspase8 and Cleaved-caspase3.(4)Compared with the high-dose group,the apoptotic ratio of TCam-2 cells was significantly reduced in the lower-dose group.After 96 hours of treatment with the lower dose of cisplatin,the cells showed the characteristics of senescence.Incucyte S3 found that the cells became larger and flatter,and the SA-β-gal staining was obviously positive.Western blotting and immunofluorescence detection showed that the senescence-related proteins p53,p21 and p16 were significantly upregulated,and flow analysis showed that cisplatin-induced senescent cells exhibited irreversible G2 M arrest.(5)Different concentrations of cisplatin caused different levels of γ-H2 AX and ROS production,and the amount of the two was positively correlated with the dose of cisplatin.(6)Immunohistochemical analysis found that no p21 staining was seen in all 26 testicular seminomas.In non-seminomas,4 of 29(13.8%)nonseminomas showed positive p21 staining.However,9 out of 10(90%)cancer adjacent normal testis tissues had positive p21 staining,and positive staining was found in 4 of 5(80%)normal testes,which is consistent with the result that p21 m RNA expression in TCGA database was significantly down-regulated in TGCTs.(7)We performed transcriptome sequencing after induction of senescence in TCam-2 cells with 2 μ g/ml cisplatin.Compared with the control group,131 genes expressed differentially were enriched in the senescence pathway.KEGGmodule analysis found that the above 131 genes were mainly enriched in G1/S transition,G2/M transition,PI3K-Akt,TGF-beta and MAPK(ERK1/2,P38,JNK)pathways.And further analysis of differential expression profiles found that p21 and its regulatory network were significantly enriched in the regulation of senescence process.(8)Compared with the control group,1832 genes were up-regulated and 752 genes were down-regulated in the Lnc RNA expression profile.Among them,451 were enriched in the mitochondrial autophagy pathway,and452 were enriched in the autophagy pathway.The Lnc RNA related to cell growth regulation were mainly enriched in senescence,mitochondrial autophagy and autophagy regulation.(9)Compared with the control group,122 mi RNAs were up-regulated and 88 mi RNAs were downregulated in the mi RNA expression profile.A large number of mi RNAs expressed differentially target genes related to the senescence process.Target analysis revealed a variety of novel mi RNAs that can regulate p53,p21 and p16,which are key genes in the regulation of senescence.Conclusion:(1)TCam-2 cells are time and dose-dependent to cisplatin.High and lower concentrations of cisplatin induce extrinsic apoptosis and senescence,respectively.These results may provide some new hints for the hypersensitivity research of seminoma in the future.(2)A certain concentration of cisplatin can stably induce the senescence of TCam-2cells,which provides a good cell model for the study of chemotherapyinduced senescence.(3)p21 is down-regulated in TGCTs(especially in seminoma),which may be related to the pathogenesis of TGCTs as a tumor suppressor gene.(4)p21-mediated regulatory network plays an important role in the senescence induced by cisplatin.(5)Lnc RNAmediated mitophagy and autophagy may play an important role in cisplatin-induced senescence.(6)A large number of mi RNAs mediate cisplatin-induced senescence by regulating senescence-related genes. |
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