The Effect And Mechanism Of PRMT2 And PRMT2? Regulated Autophagy In Breast Cancer

Breast cancer,a highly heterogeneous disease,is classified into multiple molecular subtypes according to the different expression of the biomarkers including estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER 2).Autophagy is a lysosomal degradation pathway that exists in cells and is necessary to maintain cell survival,growth,differentiation,and homeostasis.The most recent studies have shown that autophagy is closely related to tumors,and may serve as a double-edged sword in both promoting and suppressing tumors in the development of tumors.Protein arginine methyltransferases(PRMTs)mainly contain 9 family members.As common enzymes in mammals,they play a vital role in many biological processes such as signal transduction,gene transcription,DNA repair,and m RNA splicing.Recent studies have shown that members of PRMTs are closely related to tumor occurrence and metastasis.As a member of the PRMTs family,PRMT2 acts as a coactivator of ERa,directly binds to ERa in an estrogen-independent manner,and improves the transcriptional activity of ERa in an estrogen-dependent manner.Subsequent studies have revealed that PRMT2 is clearly involved in a variety of cellular processes,including cell cycle regulation,inflammation,lung tissue function,leptin signaling pathway,and Wnt signaling regulation.However,the function of PRMT2 in breast cancer remains unclear.In this study,we used immunohistochemistry(IHC)to detect the expression of PRMT2 protein and autophagy-related proteins LC3,P62,and Beclin1 in breast cancer tissues on tissue microarrays,and we also analyzed its expression differences in different tissue types and molecular types.Based on this,the stable cell lines MCF7-PRMT2 and MDA-MB-231-PRMT2 overexpressing Tet-on-PRMT2 were constructed.The autophagy in MCF7 cells and MDA-MB-231 cells after stable over expression of PRMT2 was observed,and to preliminary explore the molecular mechanism of PRMT2 affecting autophagy in MCF7 cells and MDA-MB-231 cells.Meanwhile,a tissue microarray was used to further identify the association of PRMT2? with breast cancer progression.Stable cell lines with PRMT2?-3Flag or PRMT2-3Flag were established to explore the role of PRMT2? in the cell proliferation,apoptosis,cell cycle and autophagy in breast cancer cells,and to explore the molecular mechanism of PRMT2? in MCF7 cells.Part 1 The effect of PRMT2 on autophagy of breast cancer cellsObjective: We aimed to investigate the expression and clinical significance of PRMT2 and autophagy-related proteins LC3,Beclin1,P62 in breast cancer,to observe the effect of PRMT2 overexpression on autophagy in breast cancer cells,and to explore its possible mechanism.Methods: We used IHC to detect the expression of PRMT2 protein and autophagy-related proteins LC3 B,Beclin1 and P62 in tissues microarray of breast cancer,which consisting of 159 breast malignant tumor case,and we also analyzed the expression differences in different differentiation and different molecular types of breast cancer.To address the cellular and molecular mechanisms of PRMT2 in breast cancer cells,a tetracycline(doxycycline hyclate,Dox)-inducible lentiviral system was established to overexpress PRMT2.To investigate the effect of stable overexpression of PRMT2 on autophagy in MCF7 and MDA-MB-231,the autophagic vesicles were observed by transmission electron microscopy,and the changes of LC3 immunofluorescence were detected by laser confocal microscopy.The changes of autophagy-related proteins LC3,Beclin1,P62 were detected by WB and WES in MCF7 and MDA-MB-231.The process of autophagosome formation was observed by laser confocal microscope with m RFP-EGFP-LC3 dual fluorescence.Phosphorylated antibody chip detected the possible signal pathways that the stable overexpression of PRMT2 in MCF7 cells and MDA-MB-231 cells affects cells.WB was used to detect the expression of autophagy pathway related proteins in MCF7 cells and MDA-MB-231 cells after stable overexpression of PRMT2.Results: The results showed that PRMT2 and Beclin1 expression in breast carcinoma did not exhibit a statistically significant different with patient age,TNM stage,pathological grade,lymph node metastasis,or molecular typing(P >0.05).The number of cases in the LC3 B high expression group was significantly higher than that in the low expression group in histopathological grade III(P=0.0332),but the number of cases in the LC3 B high expression group was significantly less than that in the low expression group in Luminal breast cancer(P=0.0068).There was no significant difference between the two groups with patient age,TNM stage,or lymph node metastasis(P > 0.05).The number of cases of the P62 high expression group was significantly higher than that of the P62 low expression group in the lymph node metastasis(P<0.0001).There were no significant differences between the two groups with patient age,TNM stage,pathological grade or molecular typing(P > 0.05).Transmission electron microscopy observed that the stable overexpression of PRMT2 reduced the area of autophagic vesicles in MCF-7 cells.The autophagic vesicles were reduced at 12 h after Dox treatment compared with those without Dox,and the autophagic vesicles were significantly reduced in the 24 h and 48 h groups.The area of autophagic vesicles in MDA-MB-231 cells was increased.The autophagic vesicles increased after 12 h of Dox treatment compared with the group without Dox,and the autophagic vesicles increased significantly in the 24 h and 48 h groups.Laser confocal microscopy observed the decrease of LC3 fluorescence in MCF7 cells after stable overexpression of PRMT2,and the increase of LC3 fluorescence in MDA-MB-231 cells.WB detected the expression of autophagy-related proteins Beclin1 and LC3II/I in MCF7 cells after the stable overexpression of PRMT2 gradually decreased with time,and the changes were more obvious in the 12 h,24h,and 48 h groups.The expression of autophagy-related proteins Beclin1 and LC3II/I in MDA-MB-231 cells gradually increased with time,and the changes were more obvious in the 12 h,24h,and 48 h groups.After the stable overexpression of PRMT2 was detected by WES,the expression of autophagy-related protein Beclin1 in MCF7 cells gradually decreased with time,and the expression of P62 gradually increased with time.The expression of autophagy-related protein Beclin1 in MDA-MB-231 cells gradually increased with time,while the expression of P62 protein gradually decreased with time.Knockdown of PRMT2 can up-regulate the protein expression levels of autophagy-related proteins Beclin1 and LC3II/I in MCF7.Observation of the autophagosome formation process in MCF7 cells and MDA-MB-231 cells with m RFP-EGFP-LC3 dual fluorescence after stable overexpression of PRMT2 by laser confocal microscope showed that in MCF7 cells,after the stable overexpression of PRMT2,autophagosomes(Yellow spots)and autophagy lysosomes(red spots)are reduced,which indicating that the autophagy is inhibited.In MDA-MB-231 cells,after PRMT2 was stably overexpressed,autophagosomes(yellow spots)and autophagolysosomes(red spots)increased,indicating that the autophagy was promoted.The results of phosphorylation antibody chip detection showed that in MCF7 cells,PRMT2 up-regulated the phosphorylation levels of m TOR,P53,and Caspase 9,and down-regulated the phosphorylation levels of AMPK,Akt,GSK3?,NF?B,MAPK,PTEN,and STAT1.In MDA-MB-231 cells,PRMT2 up-regulated the phosphorylation levels of AMPK,GSK3?,NF?B,MAPK,P53,STAT1,and down-regulated the phosphorylation levels of Akt,Caspase 9,m TOR,MAPK,and PTEN.Western blot results showed that in MCF7 cells,after inducing the PRMT2 overexpression,the level of p-m TOR protein was increased,and the level of ULK1,p-AMPK,PI3 K,PTEN,and p-AKT protein was decreased.In MDA-MB-231 cells,after inducing the PRMT2 overexpression,the expression levels of p-m TOR,PI3 K,PTEN,and p-AKT proteins was decreased,and the expression levels of ULK1 and p-AMPK proteins was increased.Among them,in MCF7 and MDA-MB-231 cells,after induction of PRMT2 overexpression,the change trends of p-m TOR,ULK1 and p-AMPK proteins were opposite,while the PI3 K,PTEN,and p-AKT proteins maintain the same change trend.Conclusion: LC3 B protein expression level varies in tumor pathological grading and molecular typing,while P62 protein expression level is correlated with lymph node metastasis.PRMT2 overexpression can inhibit autophagy in ER-positive breast cancer MCF7 cells and promote autophagy in ER-negative breast cancer MDA-MB-231 cells in a time-dependent manner.PRMT2 may inhibit the autophagy in MCF7 cells through down-regulation of AMPK-m TOR-ULK1 signaling pathway and Beclin1 signaling pathway.While PRMT2 promotes autophagy in MDA-MB-231 cells may be achieved by up-regulating AMPK-m TOR-ULK1 signaling pathway and Beclin1 signaling pathway.The effect of PRMT2 on autophagy of breast cancer cells may not be related to the PI3K-Akt signaling pathway.Part 2 Expression and significance of PRMT2 and its splice variant PRMT2? protein in breast cancer tissuesObjective: To investigate the expression and clinical significance of PRMT2 and its splice variant PRMT2? protein in breast cancer.Methods:The expression of PRMT2 and PRMT2? protein was detected using IHC in 138 cases of breast cancer,6 cases of normal breast tissue,and 6 cases of benign breast tissue with a tissue microarray,and was further analyzed to investigate its correlation with clinicopathological parameters.Results:The tissue microarray analysis revealed that the PRMT2? protein expression exhibited a decreased tendency in different tissue types:66.7% in normal breast tissue,50.0% in breast benign tumors and 29.0%in breast malignant tumors,although the statistical analysis revealed no significant difference(P=0.096).Conversely,the percentage of cases with PRMT2 protein expression increased from normal breast tissue to breast malignant tumors: 33.3% in normal breast tissue,83.3% in breast benign tumors and 95.7% in breast malignant tumors,and the difference was significant(P=0.0002).Furthermore,statistical analysis confirmed that the PRMT2? expression was negatively correlated with HER2(P=0.033),when analyzed regardless of breast tumor types.PRMT2?expression in breast carcinoma did not exhibit a statistically significant correlation with androgen receptor(AR),ER,PR,Ki-67 and the p53 status of the tumor(P>0.05).PRMT2 expression in breast carcinoma also exhibited a statistically significant correlation with the PR and HER2status(P=0.039 and 0.019,respectively).Conclusion: The expression of PRMT2? protein in normal breast tissue and benign breast tumor tissue is higher than that in breast cancer tissue.The expression of PRMT2?protein in breast cancer tissue is negatively correlated with HER2.The expression of PRMT2 protein in breast cancer tissues was significantly higher than that of normal breast tissues and benign breast tumor tissues.The expression of PRMT2 protein in breast cancer tissues is negatively correlated with HER2 and PR.Part 3 PRMT2 splice variant PRMT2? inhibits the growth and autophagy of breast cancer MCF7 cellsObjective: To investigate the effect of PRMT2 splice variant PRMT2?on breast cancer cell proliferation,apoptosis,and autophagy.Methods:The MCF7 cell line stably overexpressing PRMT2? was constructed.The roles of PRMT2? in breast cancer cells through MTT,clone formation,cell cycle and apoptosis were analyzed.And the effect of PRMT2? on the transcriptional activity of the cell cycle protein CCND1 promoter and the influence of Akt signaling pathway was investigated through luciferase activity detection and WB.The effect of PRMT2? on autophagy of breast cancer cells was investigated by transmission electron microscopy and WB.Results: The PRMT2?-Tet-on stable overexpression breast cancer cell line was successfully constructed.PRMT2? overexpression significantly suppresses the cell proliferation and the clone formation,induces cell cycle arrest and apoptosis,and inhibits autophagy of breast cancer MCF7 cells.PRMT2? antagonizes the transcription suppressive activity of PRMT2 on the CCND1 promoter,and inhibits CCND1 expression via the suppression of Akt/GSK-3? signaling in breast cancer cells.Conclusion: As a novel splice variant of PRMT2,PRMT2? has a potential antitumor effect through the suppression of CCND1 expression and inhibition of Akt signaling activity,which will also open a broader horizon for the future breast cancer therapy.