The Mechanism Of HEATR1 Promoting The Progression Of Triple-negative Breast Cancer By Regulating NLRP3/caspase-1/GSDMD Cell Pyroptosis Pathway Through Nrf2

Objective:Compared with Luminal A breast cancer,triple negative breast cancer(TNBC)has unique biological characteristics such as high invasion,high recurrence and high metastasis,and has fewer effective treatment means and poor prognosis.Chemotherapy is an effective means to treat TNBC.However,if chemotherapeutic drug resistance or dose tolerance occurs,relapse and metastasis often occur quickly,which is life-threatening.It is urgent to find specific therapeutic targets for TNBC or reverse the occurrence of drug resistance.As the executor of biological function,it plays a key role in biological processes associated with breast cancer.Differential protein expression can affect cell signaling in breast cancer.As a result,different subtypes of breast cancer show different biological characteristics.At present,a large number of breast cancer proteomics studies have shown differentially expressed proteins.These studies are helpful to promote the development of individualized treatment for breast cancer and increase the understanding of recurrence after treatment.However,due to the limitation of proteomics technology at that time,some low abundance differentially expressed proteins(LADEPs)may be ignored.The biological effects of these LADEPs are still unclear,which is a blind area.In this study,DIA proteomics technology was used to comprehensively identify and analyze the proteomics of TNBC and Luminal A-type breast cancer.The differentially expressed proteins(DEPs)were screened out and further analyzed by bioinformatics analysis based on IPA database.It will help us to deepen our understanding of the heterogeneity of TNBC,find the root cause of heterogeneity,and explore new targets for TNBC treatment.The results were verified by in vivo and in vitro experimental studies.It is hoped that this study can change the current dilemma of TNBC after chemotherapy drug resistance,and provide information and reference for future related research.Methods:1.Samples from 52 breast cancer tissues and 3 paracancerous tissues were selected to construct DDA database.On this basis,another 3 cases of TNBC,3 cases of Luminal A breast cancer and 3 cases of paracancerous tissue samples were quantitatively identified based on DIA proteomics technology.The classical pathway analysis of DEPs based on IPA was performed among the groups.The SUGT1,CARS2,HEATR1,ILKAP,MACROD1,PSME4,PLOD3 and SRPRB genes up-regulated in the TN vs LA group were screened by HCS and Transwell to determine the target genes.2.Western Blot and qRT-PCR were performed to verify the high expression of HEATR1 in five breast cancer cell lines.MDA-MB-468 cells and MDA-MB-231 cells with the highest and the lowest expression level were selected as the cells for subsequent study.40 pairs of TNBC and adjacent tissues were selected for IHC to verify the high expression of HEATR1 in TNBC tissues.Western Blot and q RT-PCR showed that the expression levels of HEATR1 and Nrf2 in shHEATR1 group were significantly decreased.It was confirmed that HEATR1 gene was successfully knocked down and Nrf2 expression was inhibited.The CCK-8 assay was used to detect the activity of cells.The Flow cytometry showed a significant increase in the proportion of double-positive cells in the shHEATR1 group.The scratch test showed that the mobility of shHEATR1 group was significantly decreased.3.The xenograft tumor model of nude mice was established.Tumor formation time and tumor volume changes of nude mice were recorded,tumor weight was weighed,and the influence of HEATR1 reduction on tumor growth was observed.Western Blot and q RT-PCR were used to detect the expression of HEATR1 and Nrf2 in tumor tissues.IHC detected the expression of HEATR1 and Nrf2 in each group.The influence of HEATR1 knocked down on Nrf2 signaling pathway was analyzed.4.MDA-MB-468 cells and MDA-MB-231 cells were infected with lentivirus to knock down Nrf2.Western blot and q RT-PCR were used to detect the expression of Nrf2 in the cells of each group to verify the knockdown effect of Nrf2.Reset the grouping shControl group,shHEATR1 group,shNrf2 group,shHEATR1+shNrf2 group.q RT-PCR was used to detect the gene expression of HEATR1 and Nrf2.Western blot was used to detect the expression of HEATR1,Nrf2 and pyroptosis-related indicators.The levels of IL-1β and Il-18 in the supernatants were detected by ELISA.5.DNA damage and morphological changes were detected by TUNEL.Annexin V and PI double staining cells were detected by Flow cytometry.The content of LDH in cell culture supernatant was detected by automatic biochemical analyzer.CCK-8 assay was used to detect the activity of cells in each group.Scratch test and Transwell were used to detect the migration and invasion of the cells.6.The xenograft tumor model of nude mice was established.Tumor formation time and tumor volume changes of nude mice were recorded,tumor weight was weighed,and the influence of HEATR1 reduction on tumor growth was observed.q RT-PCR was used to detect the expressions of HEATR1 and Nrf2 in tumor tissues of nude mice.IHC detected the expression of HEATR1,Nrf2,Ki67 and PCNA in tumor tissues of nude mice,and observed the changes of tumor proliferation ability.The expression levels of HEATR1,Nrf2 and NLRP3,GSDMD-FL,GSDMD-N,caspase1,cleaved caspase1,IL-1β,and IL-18 in tumor tissues of nude mice were detected by Western blot.TUNEL was used to detect pyrodeath of tumor cells in each group..Results:1.Based on DIA proteomics technology,a total of 533 DEPs were found in TN vs LA group,of which 207 were up-regulated and 326 were down-regulated.Functional screening of some genes by HCS and Transwell revealed that HEATR1 played a role in the proliferation and metastasis of MDA-MB-231 cells.Nrf2 Signaling was significantly activated in TN vs LA group by IPA analysis..2.Western Blot and qRT-PCR detection of HEATR1 in breast cancer cell lines found that MDA-MB-468 cell line and MDA-MB-231 cells had higher HEATR1 expression,which was selected for subsequent research.IHC detection of 40 pairs of TNBC and adjacent tissues verified that HEATR1 was highly expressed in TNBC tissues.Western Blot and q RT-PCR showed that the expression levels of HEATR1 and Nrf2 in the shHEATR1 group were significantly decreased,which confirmed that HEATR1 gene knockout was successful and Nrf2 expression was inhibited.CCK-8 experiment showed that the optical density of shHEATR1 group decreased and the cell proliferation activity decreased.Flow cytometry results showed that the proportion of double-positive cells in shHEATR1 group increased significantly.Scratches showed that the mobility of shHEATR1 group decreased significantly.3.The tumor formation experiments in nude mice indicated that the average tumor formation time in the shHEATR1 group was prolonged,and the tumor volume and weight were less than those in the Uninfected and shControl groups.The results showed that the tumor growth in nude mice was inhibited after HEATR1 reduction.Western Blot and q RT-PCR detection of nude mice showed that the expression levels of HEATR1 and Nrf2 decreased in the shHEATR1 group.The IHC results indicate that the optical density values of HEATR1 and Nrf2 in the Sh HEATR1 group decrease significantly.This indicates that Nrf2 expression is inhibited after HEATR1 knockdown.4.Western Blot and qRT-PCR indicated that Nrf2 expression in shNrf2 group decreased significantly,which confirmed that Nrf2 gene knockdown in MDA-MB-468 cells and MDA-MB-231 cells were successful.After regrouping,the detection results of Western Blot and q RT-PCR showed that Nrf2 expression levels in shHEATR1 group,shNrf2 group and shHEATR1+shNrf2 group were all down-regulated.However,NLRP3,GSDMD-N and cleaved caspase1 were significantly up-regulated,while GSDMD-FL and caspase1 had no significant changes.ELISA detection showed that IL-18 and IL-1βin the supernatant of cell culture in shHEATR1 group,shNrf2 group and shHEATR1+shNrf2 group were significantly increased.This indicates that down-regulated HEATR1 can activate the NLRP3/caspase-1/ GSDMD pyroptosis pathway by inhibiting Nrf2.5.TUNEL experiment results indicated that the shHEATR1 group,shNrf2 group and shHEATR1+shNrf2 group all showed intracellular chromatin aggregation imaging,corresponding to cell swelling and deformation.Flow cytometry results showed that the proportion of double-positive cells in shHEATR1 group,shNrf2 group and shHEATR1+shNrf2 group increased significantly.Automatic biochemical detector found that LDH concentration increased in the cell culture supernatant of shHEATR1 group,shNrf2 group and shHEATR1+shNrf2 group.CCK-8 experiment results showed that the optical density of shHEATR1 group,shNrf2 group and shHEATR1+shNrf2 group all decreased,and cell proliferation activity decreased.These results indicate that after HEATR1 reduction,MDA-MB-468 cells and MDA-MB-231 cells showed pyrodeath and decreased proliferative activity.Scratch test results show that the mobility of shHEATR1 group,shNrf2 group and shHEATR1+shNrf2 group decreases obviously.Transwell experiment results showed that the number of cells in the micromembrane pore at the bottom of the chamber in shHEATR1,shNrf2 and shHEATR1+shNrf2 groups was significantly less than that in shControl group.This indicates that the migration and invasion ability of MDA-MB-468 cells and MDA-MB-231 cells decreased after HEATR1 depletion.6.The results of tumor formation experiment in nude mice show that the tumor formation time in shHEATR1 group,shNrf2 group and shHEATR1+shNrf2 group is prolonged,and the tumor volume and weight are less than those in shControl group,which is most obvious in shHEATR1+shNrf2 group.IHC results suggest that Nrf2,Ki67 and PCNA expressions in shHEATR1 group,shNrf2 group and shHEATR1+shNrf2 group are down-regulated.It was proved that the proliferation activity of tumor cells decreased after HEATR1 and Nrf2 knockdown.Western blot and q RT-PCR results indicated that Nrf2 expression in shHEATR1 group,Nrf2 group and shHEATR1+shNrf2 group decreased significantly.Protein levels of NLRP3,GSDMD-N,cleaved caspase1,IL-18,and IL-1β were up-regulated in the cytokinesia pathway,while GSDMD-FL and caspase1 had no significant changes.TUNEL experiment results show that a large number of pyrogenic cells appear in shHEATR1 group,shNrf2 group and shHEATR1+shNrf2 group under the fluorescence microscope,and shHEATR1+shNrf2 group is the most significant.It is proved that down-regulated HEATR1 can activate the NLRP3/caspase-1/GSDMD pyrogenic pathway by inhibiting Nrf2 expression,and promote pyroptosis of cells.Conclusion:1.HEATR1 expression was up-regulated in the proteomic comparison between TNBC and Luminal A-type breast cancer,which promoted the proliferation and migration of MDA-MB-231 cells.2.Nrf2 Signaling pathway was significantly activated in TNBC compared with Luminal A breast cancer.3.HEATR1 is highly expressed in TNBC cell lines(MDA-MB-468 and MDA-MB-231)and TNBC tissues.Down-regulation of HEATR1 expression can reduce the expression of Nrf2 and inhibit the growth of MDA-MB-468 cell-derived TNBC.4.HEATR1 can mediate the NLRP3/caspase-1/GSDMD pyroptosis pathway by regulating Nrf2 in TNBC.Down-regulating HEATR1 to inhibit Nrf2 can activate the NLRP3/caspase-1/GSDMD pathway and inhibit tumor growth.5.HEATR1 is a promising anti-TNBC individualized therapeutic target.