| Background and purpose:about 20%of all cancer patients known sooner or later there will be a brain metastasis,Primary focal is given priority to with lung cancer,breast cancer and melanoma,brain metastases are the common characteristics i5 considered by circulating tumor cells to spread to the brain microvascular happen,in this unique environment,tumor growth by promoting(that is,the more malignant),there is no good treatment to improve prognosis.The malignant progression of metastatic brain cancer is still one of the important factors in the death of patients with advanced cancer.Therefore,it is of great interest to understand the molecular mechanism driving brain metastasis and the molecular characteristics of brain metastasis relative to the primary tumor,so as to provide information for the selection of targeted therapy.Based on proteomics and always cDNA array is different,can be directly from the thousands of proteins,instead of mRNA,in the form of interaction network diagram is meet the visual requirements,and he was able to filter to the key target protein from many proteins,so the purpose of this article transfer screening and optimization of simulation,the actual is artificial(vaccinations)growth in nude mouse indoor uveitis black lump key proteins,and used in the study of targeted therapy.Experimental methods:(1)92.1 cell 1 ×105 in stereotactic apparatus aided by inoculation in green fluoreseent nude mouse right lateral ventricle,after tumorigenic anatomical tumor tissue to the original generation of culture,the purification of monoclonal tumor cells named 92.1-A,with parents of 92.1 and is associated with proliferation and malignant degree of cell and molecular biology detection,the malignant phenotype was observed.(2)two strains of cells,92.1 and 92.1-a,were sent to hangzhou jingjie biotechnology co.,ltd.for the establishment of proteomics database(protein chip).The GO,KEGG and interaction network diagrams in high-throughput molecular bioinformatics were used to optimize screening of 92.1 malignant progression into 92.1-a target protein.(3)Wersten Blot was used to verify the expression of the target protein in 92.1 and 92.1-a cells cultured in vitro,and the knockdown and overexpression methods were used respectively to demonstrate that p62 plays A key role in the malignant progression of uveal melanoma.Results and analysis:(1)p62 was an important research target:proteomic analysis showed that SQSTM1(p62),CDK1 and CASP3 were the target proteins.p62,also known as autophagy adaptor protein,plays a regulatory role in autophagosomes that play a double-edged sword role in tumor tissue,which is an important research object of this paper.(2)92.1-A stronger than 92.1 proliferation ability:92.1-A drawing according to the method of CCK8 cell proliferation curve,you can see that in 96-well plate,2000/100/hole initial cells(including 1 the number of 92.1 and 92.1-A cell culture medium with the passage of time,the two cell proliferation curve separation is more and more obvious,maintained A significant differences during 4-9 d(4-9 days of P values were 0.0122,0.0155,0.0041,0.0391,0.0017,0.0164);In the logarithmic phase of 92.1 and 92.l-a,5 105 cells were taken,and the cell cycle was detected by flow cytometry.The percentage of cells in S phase and g2-m phase of 92.1-a was significantly higher than that of 92.1(P=0.001 and p=0.0213),while the percentage of cells in g0-g1 phase was significantly lower than that of 92.1(p<0.0001).(3)4.92.1-a had stronger migration and invasion ability than 92.1:the cell migration ratio was analyzed by scratch healing test,and the healing rate of 92.1-a cells was significantly higher than 92.1,with statistical difference(P<0.01).In Traswell,direct counting method was used to compare the cells of the two kinds of cells attached to the lower surface of the polycarbonate membrane and it was found that 92.1-a was more than 92.1,with statistical difference(P<0.01).Three sgRNA targets(4)design,built into the carrier pLenti respectively-U6-sgRNA-SFFV Cas9-2-A-Puro,plasmid type low p62 expression,tumors appear benign transformation trend:western blot is shown in the knock the chain of low 2(sip62#2)p62 expression decreased obviously(P<0.01),then choose sip62#2 with liposome transfection tumor cells,the results show that the control group significantly higher than the 92.1-92.1-A proliferation rate A-sip62#2 groups;The migration(24h p=0.032,48h p=0.041)and invasion ability of group 92.1-a-sip62#2 were also significantly lower than that of group 92.1-a(p=0.004),with statistical significance.(5)western blot was used to verify that the p62 expression level of 92.1-over-p62 was significantly higher than that of the 92.1 group(P=O,017).Further detection of its biological characteristics revealed enhanced migration(24h:P=0.003)and invasion(P=0.0083)of 92.1-over-p62,with statistically significant results.Conclusion and discussion:(l)in situ growth within the orbit of uveal melanoma,artificial inoculation in mude mouse indoor,part of the cells in the ventricle choroid plexus engraftment,other cells to continue along the cerebrospinal fluid circulation flow,the cerebral pia mater and engraftment in many place,although not spontaneous brain with extensive metastasis,but as the seed cells metastasize to brain tumor after engraftment and subsequent malignant progress,can think and spontaneous transfer is similar,because of its subsequent malignant progression and spontaneous transfer,1s the relationship between the"seed and soil",Therefore,92.1-a and 92.1 can be used to study the proteomics of tUmor cells representing metastatic and primary lesions respectively,and the established database can be used to study the molecular mechanism of melanoma brain metastasis.(2)it has been reported that CDKN2A and PIK3CA mutations,PTEN deletion,ERBB2 amplification and KRAS activation were not found in the primary lesions in metastatic lesions,all of which had an impact on the treatment.SQSTM1 is reported in this paper(p62)in 92.1 A(metastases)expression is significantly higher than 92.1(primary tumors),may have A different biological significance,this paper has proved that hitting low and slow virus with plasmid expressing p62,demonstrated that the expression of p62 positively correlated with the malignant degree of uveal melanoma,may play A key role in the progress of malignant tumor.In summary,we proposed for the first the that taking p62 as the target molecule for treatment has therapeutic effects on the primary and metastatic tumor cells of brain metastatic carcinoma represented by uveal melanoma,which will provide scientific basis for further clinical promotion. |
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