MCRS1 Regulates The Aging Of Endometrial Cancer Cells And Its Mechanism

ObjectiveMicroglobulin 1(MCRS1)is involved in many important cellular activities,and studies have shown that it is closely related to the occurrence and development of a variety of tumors,but no correlation between MCRS1 and endometrial cancer cells has been reported.Protein kinase membraneassociated tyrosine/threonine kinase 1(PKMYT1)is a regulator of the cell cycle,and previous studies in our group have confirmed that there is an interaction between MCRS1 and PKMYT1 and is related to the occurrence and development of gastric cancer.In this study,the purpose of this study was to verify the regulatory effect of MCRS1 on the aging of endometrial cancer cells and explore the regulatory mechanism by constructing an endometrial cancer cell senescence model and then upregulating or down-regulating the expression of MCRS1 gene in endometrial cancer cells.Methods1.Cell culture and screening: in vitro culture of four endometrial cancer cell lines Ishikawa,RL95-2 and AN3CA,HEC.The basal expression of MCRS1 protein was detected by Western blotting experiments.Subsequent overexpression experiments were carried out on cell lines with low protein ontology expression,and down-regulation experiments were performed on cell lines with high ontology expression.2.Preparation of cell senescence model: etoposide(ETO)was used to induce and construct an endometrial cancer cell aging model.Etoposide at different concentrations of 0,2,4,8 and 16 μmol was used to induce the cells for aging,and the cell viability rate was measured by CCK8 method and IC50 was calculated after dosing,and the degree of cell senescence was observed by SA-β-gal staining,and the drug concentration and action time of the aging model with better cell state were screened.Under the concentration and action time of the screened drug,the expression level of P53 was further detected.3.Gene transfection and cell grouping: pcDNA3.1-MCRS1-myc plasmid was transfected into Ishikawa,RL95,HEC cells for overexpression experiments.MCRS1-siRNA was transfected into AN3CA cells for interference assays.The groups were as follows: control group(normal cancer cells),control siRNA transfection group,MCRS1-siRNA transfection group(down-regulation group),model group(ETO induced aging group),control siRNA transfection model group,MCRS1-siRNA transfection model group.4.CCK8 experiment: to detect the changes of cell activity after overexpression or downregulation of MCRS1 in four endometrial cancer cells.5.Western blotting experiment: detection of the ontology expression of MCRS1 protein in four endometrial cancer cells;The expression of MCRS1 in AN3CA cell senescence models induced by different concentrations of ETO was detected.Detect the transfection efficiency of MCRS1 down-regulated by cells;Detection of P53 expression in AN3CA cell senescence models;The expression of MCRS1 protein in each group was downregulated by the AN3CA cell senescence model MCRS1.The expression of PKMYT1 protein in each group was detected in the AN3CA cell senescence model MCRS1.6.Immunofluorescence experiment: verify the changes of MCRS1 expression in the down-regulated MCRS1 group,model group and downregulated MCRS1 model group.To verify the colocalization of MCRS1 and PKMYT1 in endometrial cancer cells.7.Bioinformatics analysis: analyze the correlation between MCRS1 and PKMYT1 through bioinformatics.Results1.Cell growth state: In AN3CA cells,with the increase of ETO concentration,cell density decreases,cell volume gradually increases,and the morphology shows signs of aging.After down-regulating MCRS1,cell density increased and the number of senescent cells decreased.2.Expression results of MCRS1 in four endometrial cancer cells:Western blotting results showed that MCRS1 expression was higher in AN3CA cells(P<0.05)and lower in Ishikawa,RL95 and HEC cells(P <0.05)。 Therefore,Ishikawa,RL95 and HEC cell lines were subsequently used for MCRS1 overexpression experiments,and AN3CA cell lines were used for interference experiments.3.Cell viability detection results: CCK8 experimental results showed that in the four endometrial cancer cells of AN3CA,Ishikawa,RL95 and HEC,the cell activity was reduced after overexpression of MCRS1(P<0.05);After downregulation of MCRS1 in AN3CA cells,cell viability was enhanced(P<0.05).4.ETO induced successful cell senescence: CCK8 was used to detect cell activity at various drug concentrations at different times and calculate IC50.ETO-induced AN3CA cells have an IC50 of 17.0 μmol for 48 h and an IC50 of13.7 μmol for 72 h.It was found that adding 2 μmol ETO culture to AN3CA cells for 48 h induced a stable endometrial cancer cell aging model.The cytostatic rate in this state is about 13%.The results of SA-β-gal staining showed that the positive rate of cell staining after 2 μmol ETO induction increased significantly,but then with the increase of ETO concentration,the staining positive rate did not change much,and the cell state was poor,and the cell activity was in an inhibited state.AN3CA cell senescence was induced by 2 μmol concentration ETO for 48 h,and the expression of cancer cell aging marker P53 was significantly increased.5.MCRS1 regulates the aging of endometrial cancer cells: Western blotting results show that when ETO induces AN3CA cells for 48 h at a concentration of 0-2μmol,the expression of MCRS1 gradually increases,while the positive rate of staining of senescent cells gradually increases,while the expression of MCRS1 decreases after 4μmol,and the positive rate of staining of senescent cells no longer increases.Compared with the control group,the expression of MCRS1 was significantly increased in the ETO-induced aging model group(P<0.05),but the expression of MCRS1 in the downregulated group of MCRS1 gene in AN3CA cells was significantly reduced(P<0.05).Compared with the MCRS1 gene down-regulated group,the expression of MCRS1 in the down-regulated MCRS1 gene + ETO induction model group was restored(P<0.05).6.Immunofluorescence results: Compared with the control group,the cell fluorescence intensity of the down-regulated MCRS1 group was weakened,while the cell fluorescence intensity induced by ETO was enhanced.MCRS1 and PKMYT1 are colocalized in endometrial cancer cells.7.Correlation analysis of MCRS1 and PKMYT1The results of bioinformatics analysis showed that MCRS1 was strongly correlated with PKMYT1 expression in endometrial cancer tissues R=0.578(P<0.05).The Western blotting results showed that compared with the control group,the expression of PKMYT1 in the MCRS1 gene down-regulated group decreased significantly,but the PKMYT1 expression in the ETO-induced aging model group was significantly enhanced,while the expression of PKMYT1 in the downregulated MCRS1 gene + ETO induction model group was increased compared with the MCRS1 gene down-regulated group.Meanwhile,immunofluorescence showed that MCRS1 and PKMYT1 were colocalized in endometrial cancer cells.It was suggested that MCRS1 gene had a regulatory effect on PKMYT1 expression.ConclusionsThe experimental results show that MCRS1 is related to the development of endometrial cancer,and the mechanism may be that MCRS1 jointly promotes the process of cellular aging by regulating the expression of PKMYT1.This study suggests that further in-depth research on MCRS1 may lay the foundation for the development of new clinical diagnostic and therapeutic targets for endometrial cancer.