An Experimental Study Of PKMYT1 Expression In Breast Carcinoma Based On Bioinformatics Analysis To Investigate Its Effect On The Biological Behavior Of Breast Carcinoma Cells

Objective: Bioinformatics analysis was performed to predict PKMYT1 expression in breast carcinoma and its impact on breast carcinoma biology.The character of PKMYT1 in breast carcinoma and its influence on breast carcinoma development were investigated by comprehensive clinical correlation analysis,GSEA functional enrichment analysis,and survival analysis.We also investigated the impact of PKMYT1 expression on the malignant biological properties of breast carcinoma cell lines by upregulating PKMYT1 expression.Methods:1.Bioinformatics: the gene expressed level of PKMYT1 in breast carcinoma patients were investigated using various bioinformatics analysis databases,such as TCGA,GEPIA 2 and TIMER2.0,and the correlation of clinical characteristics and functional enrichment associated with PKMYT1 expression was analyzed.The influence of PKMYT1 on the malignant biological properties of breast carcinoma was forecast using the Cancersea database,and the connection between PKMYT1 expression and the prognosis of breast carcinoma patients was analyzed using the Kaplan-Meier Plotter database.The protein interaction network of PKMYT1 was analyzed using the PINA database to understand its mechanism of action in breast carcinoma progression and to predict cancer-related factor targets and drug targets.2.PKMYT1 was detected by real-time fluorescence quantitative q RT-PCT analysis in one human epithelial cell line MCF10 A and four human breast carcinoma cell lines MCF-7,MDA-MB-231,MDA-MB-468,HCC1937 and four murine breast carcinoma cell lines C127,4T1,EMT6,E0771 to determine the relative expression levels.3 Using the RNA interference-liposomal transfection technique,specific si-RNA and negative control sequence si-Negative Control were transfected into MCF-7 and MDA-MB-231 cells to construct si-PKMYT1 and si-NC cell sets with specific functions to achieve efficient inhibition of PKMYT1 gene expression.4.Perform q RT-PCR analysis to verify the influence of si-PKMYT1 electransfection.5.Western blot was used to test the gene expressed level of PKMYT1 protein after MCF-7 and MDA-MB-231 electransfection.6.The influence of PKMYT1 on the proliferation of breast carcinoma cells was detected by cell counting kit.7.The influence of PKMYT1 on the colony forming ability and proliferation of breast carcinoma cells was tested by plate cloning test.8.The change of cell migration and healing ability after transfection was detected by scratch damage test.9.Transwell cell invasion test was conducted to examine the influence of si RNA electransfection on the invasion ability of breast carcinoma cells.10.Flow cytometry was performed to detect the effect of decreased PKMYT1 expression on the cell cycle of breast carcinoma cells.11.The influence of PKMYT1 on apoptosis in breast carcinoma cells was examined by flow cytometry.12.Statistical analysis of the data was performed using SPSS25.0.Results: 1.Through bioinformatics analysis,the expression level of PKMYT1 in breast carcinoma tissue was significantly higher than that in normal tissue,P<0.001;For the same sample,the expression level of PKMYT1 in cancer tissue is much higher than that in paracancerous tissue,P=<2e-16 has statistical difference;GEPIA2 and TIMER2.0 online analysis tools also further confirmed that PKMYT1 in breast carcinoma the results showed that PKMYT1 was highly expressed in breast carcinoma.Clinical correlation analysis showed that the expression of PKMYT1 in breast carcinoma patient age,tumor stage(I-II and I-III)and primary tumor size(T,T1～2,T1～3,T1～4)with statistically significant P<0.05,while there was no association with gender,distant movement(M)and lymph node metastasis(N)with no statistical difference at P>0.05.By GSEA functional enrichment analysis,the correlation with cell cycle,DNA replication,homologous recombination and other functions was found to be higher when PKMYT1 was highly expressed,indicating,that PKMYT1 may be related to cancer cell growth and proliferation in breast carcinoma,and the above P values are less than0.05,which are The influence of PKMYT1 on the biological behavior of breast carcinoma was predicted by the Cancersea database,and it was found that PKMYT1 had different degrees of influence on cell cycle,DNA damage and repair,proliferation,invasion,metastasis,EMT and apoptosis in breast carcinoma development,and had a positive correlation;Analysis of the Kaplan-Meier Plotter database from the study found that HR>1 indicates that PKMYT1 is a high risk factor for breast carcinoma and that high PKMYT1 expression results in a poor prognosis for breast carcinoma patients.Protein interaction network analysis revealed that proteins significantly associated with PKMYT1 are closely related to cell cycle mechanisms and cell proliferation in breast carcinoma development,and PKMYT1-related tumor drug targets include MAPK10,MAPK8,CDK1,BCL2,and PLK1.2.The expression level of PKMYT1 in human breast cancer cell line was determined by the following methods:q RT-PCR PKMYT1 expression levels were MDA-MB-231,MDA-MB-468,MCF-7 and HCC1937 in descending order,and 4T1,E0771,C127 and EMT6 in descending order in murine breast carcinoma cell lines.3 In the experiment of measuring the electransfection efficiency,it was found that compared with the control group and si-NC group,the transfection of si-PKMYT1-2 group showed a significant decrease in the expression level of PKMYT1 in si-PKMYT1-2 group,P<0.05.4.The expression level of PKMYT1 protein in si-PKMYT1-2 group decreased significantly decreased by Western Blot analysis P<0.05.5.Cell Counting Kit assay results revealed that compared with MCF-7,MDA-MB-231 and both cell groups and si-NC cell group,the cell proliferation ability was significantly suppressed in the transfected si-PKMYT1-2 cell group P<0.05.6.Platelet clone formation assay revealed that colony formation ability and proliferation were the electransfection si-PKMYT1-2 cell group was compared with the control group and si-NC cell group.7.The results of scratch injury test showed that the migration rate of the transfected si-PKMYT1-2cells increased within 48 hours.8.Transwell cell invasion test confirmed that the number of invasive cells in the transfected si-PKMYT1-2 cell group was significantly reduced,with a statistically significant difference,P<0.05.9.Through flow cytometry analysis,the proliferation ability of cells transfected by si-PKMYT1-2 group decreased.The analysis results showed that the proportion of G0/G1 phase cells was more,and the proportion of S+G2/M phase cells was less,indicating that cell proliferation was inhibited.10.Flow cytometry analysis showed that the apoptosis rate increased significantly after the PKMYT1 expression decreased,P<0.05 was statistically different.Conclusions: 1.Bioinformatics analysis shows that PKMYT1 is highly expressed and is a high risk factor in breast carcinoma samples and is associated with a poor prognosis for breast carcinoma.2.The decrease of PKMYT1 expression significantly inhibited the proliferation,invasion and apoptosis of MCF-7 and MDA-MB-231 cells.