The Effects And Mechanisms Of PARP1 Negatively Regulated MT1M In Ovarian Cancer Metastasis

Background:Ovarian cancer is one of the most common malignant tumors of female reproductive organs,and its morbidity ranks third,which seriously affects human health in China.In recent years,ADP ribose polymerase(Poly ADP-ribose Polymerase,PARP)inhibitors have achieved remarkable success in ovarian cancer treatment,opening the door for small molecule inhibitors to target ovarian cancer.At present,PARP inhibitors are mainly approved for patients with ovarian cancer with BRCA(breast cancer)mutation.However,with the accumulation of clinical trial datas,the researchers found that regardless of whether patients have BRCA mutations,patients with ovarian cancer treated with PARP inhibitors Niraparib and Rucaparib can benefit from this therapy,for example,significantly improving the disease-free survival of patients.Therefore,how to elucidate the clinical phenomenon from the biological function of PARP1(Poly ADP-ribose Polymerase 1)has become a research hot spot in this field.With the deepening of the study of PARP1,its other biological functions also began to receive much attention.Recent studies have found that PARP1 can regulate gene expression by activating transcription factors or inhibiting the activity of transcription factors,also directly acting as transcription factors,to regulate various biological functions of tumor cells.By analyzing the TCGA database(The cancer genome atlas),we found that the high expression of PARP1 m RNA was significantly negatively correlated with the disease-free survival of patients with ovarian cancer,kidney renal papillary cell carcinoma,ER~+(Estrogen receptor-positive)breast cancer,and uterine corpis enfometrial carcinoma.The coincidence analysis of genes related to disease-free survival in these four tumors showed that the expression of 91 genes was negatively correlated with the high expression of PARP1 m RNA.At the same time,knocking down PARP1,overexpressing PARP1 and administering PARP inhibitor Olaparib on ovarian cancer cell-OVCAR8,using a whole gene chip to detect gene changes,we found that 13 genes were significantly negatively correlated with the high expression of PARP1.Furthermore,91 genes obtained from TCGA data analysis and 13 genes obtained from chip analysis were analyzed for coincidence,and surprisingly we found that PARP1 protein expression was significantly negatively correlated with metallothionein 1M(Metallothionein 1M,MT1M)gene transcription,suggesting that MT1M may be the target gene regulated by PARP1.So,does PARP1 negatively regulate MT1M and thus regulate the biological behavior of ovarian cancer?Metallothionein-1M(MT1M)is a small cysteine-rich protein and belongs to the metallothionein(MT-1)family member.It specifically binds to metal ions,plays an important role in metal detoxification and oxidative stress protection.Recent studies have shown that MT1M can significantly inhibit the proliferation and metastasis of various tumors such as hepatocellular carcinoma and papillary thyroid carcinoma,and MT1M is a potential tumor suppressor gene.Considering the role of MT1M in inhibiting tumor cell proliferation and metastasis in tumor cells,we assumed that PARP1 may participate in tumor cell proliferation and metastasis by negatively regulating MT1M.This study will focus on the biological function of PARP1 negative regulation of MT1M expression,and examine the role of PARP1 negative regulation of MT1M in ovarian cancer proliferation and metastasis and find the specific molecular mechanism:(1)analysis of the correlation between high expression of PARP1 m RNA and multiple tumors disease progression-free survival in the TCGA database,combined with the gene chip data of ovarian cancer cell,found that PARP1 negatively regualte MT1M expression in ovarian cancer cells;(2)clarify the effect of MT1M expression on the proliferation and migration of ovarian cancer cells,and look for the molecular mechanism of PARP1regulating MT1M expression;(3)the effect of MT1M expression on the anti-metastasis of ovarian cancer by PARP inhibitors.This study found that PARP1's noval anti-tumor target gene MT1M and also found a new mechanism for regulating ovarian cancer metastasis,that is,PARP1 promotes ovarian cancer metastasis by inhibiting MT1M expression,to provide a new theoretical basis for the application of PARP inhibitors in the treatment of ovarian cancer.Section 1 The Role of PARP1 in Ovarian Cancer Metastasis Promotion by Regulating MT1M Transcriptional Activity.Methods(1)Using TCGA database to analyze the correlation between the expression of PARP1m RNA and the correlation ovarian cancer,papillary renal cell carcinoma,ER~+(Estrogen receptor-positive)breast cancer,uterine body cancer,bladder cancer,testicular germ cell tumor,head and neck squamous cell carcinoma and pancreatic ductal adenocarcinoma;(2)Using Kmplot database to find genes that are highly correlated with PARP1 m RNA expression;(3)By using small-interfering RNA to knock down PARP1,and overexpression of PARP1 and administration of PARP inhibitor Olaparib on ovarian cells OVCAR8,using gene chip to detect gene changes to look for genes negatively correlated with PARP1 expression;(4)By analysis of TCGA database for genes negatively correlated with PARP1 expression,and analysis of chip genes with negatively correlation with PARP1 expression in OVCAR8,we found that PARP1 regulates a noval target gene MT1M;(5)Small-interfering RNA was used against PARP1 in non-BRCA mutation ovarian cancer cells SKOV3,Ca OV3 and OVCAR8,and Western blotting,real-time quantitative PCR and Elisa were used to verify the effect of PARP1 on MT1M expression;(6)MT1M plasmid was constructed to overexpression MT1M in ovarian cancer cells SKOV3 and Ca OV3,and the SRB experiment was assigned to investigate the effect of overexpression of MT1M on ovarian cancer cell proliferation;(7)By overexpression MT1M or knock down MT1M to change MT1M expression,Transwell experiment and scratch experiment were used to investigate the effect of MT1M expression on ovarian cancer cell migration;(8)Overexpress MT1M in ovarian cancer cell SKOV3,and collected supernatant as conditioned medium to investigate the effect of MT1M secretion on ovarian cancer cell migration;(9)PARP1 plasmid was used to construct in ovarian cancer cell OVCAR8,use scratch experiments to investigate the effect of high expression of PARP1 on the migration of ovarian cancer cells;meanwhile,knock down PARP1 in SKOV3 and Ca OV3,and Transwell experiment was conducted to investigate the effect PARP1 on ovarian cancer cell migration.Results(1)The high expression of PARP1 m RNA is negatively correlated with the disease-free survival of ovarian cancer,papillary renal cell carcinoma,ER~+(Estrogen receptor-positive)breast cancer,and uterine body cancer.The PARP1 expression has no significant correlation with the disease-free survival of patients with bladder cancer,testicular germ cell tumor,head and neck squamous cell carcinoma,and pancreatic ductal adenocarcinoma.(2)The Kmplot database was used to analyze ovarian cancer,uterine body cancer,papillary renal cell carcinoma,and ER~+(Estrogen receptor-positive)breast cancer.Among these four tumors,genes negatively correlated with PARP1 expression were found.91 genes were discovered to be associated negatively with PARP1expression.(3)By analysis gene chip data in different treatment group in non-BRCA mutation OVCAR8 ovarian cells,and 13 genes were detected,which had a negative correlation with the expression of PARP1.(4)Compare 91 genes obtained from the TCGA database with 13 genes obtained from gene chip of OVCAR8 ovarian cancer cell,and a target gene-MT1M that was negatively correlated with PARP1 expression was found.(5)Knock down PARP1 in ovarian cancers SKOV3,Ca OV3 and OVCAR8,and the transcriptional expression and protein expression of MT1M significantly increased.At the same time,knocking down PARP1 could promote the secretion of MT1M.(6)The overexpression of MT1M does not affect the proliferation of ovarian cancer cells.By using SRB assay,MT1M overexpression had little effects on cell proliferation of ovarian cells SKOV3 and Ca OV3.(7)The overexpression of MT1M can affect the migration of ovarian cells.MT1M overexpression could significantly inhibit the migration of SKOV3 and Ca OV3,while knocking out MT1M,the migration ability of SKOV3 and Ca OV3 was significantly increased.(8)MT1M secretion can affect the migration of ovarian cells.First,MT1M was induced overexpression in SKOV3,and cultured in serum-free medium,then the cell supernatant was collected as conditioned medium.Transwell was determined that conditioned medium that overexpressed MT1M could significantly inhibit the migration of SKOV3 cells.(9)The expression of PARP1 can affect the migration of tumor cells.High expression of PARP1 can significantly inhibit the migration of ovarian cell OVCAR8 by the scratch test method;while knocking out PARP1 significantly inhibited the migration ability of ovarian cancer cells SKOV3 or Ca OV3.Section 2 The Mechanism of PARP1 Inhibiting MT1M Transcription in Ovarian CancerMethods(1)Applying prediction database to analyze the potential transcription factors regulating MT1M expression,using q RT-PCR experiments to investigate the role of predicted transcription factors invoveled in the mechanism that PARP1 downregulating MT1M transcription expression,and found transcription factor FOXP3;(2)Using q RT-PCR and Western blotting experiment to detect the effects of FOXP3 on MT1M transcription and protein expression;(3)Immunoprecipitation was used to investigate the binding between PARP1 and FOXP3;(4)Detecting PAR modification to confirm the ribosylation modification of FOXP3 by PARP1;(5)Detecting PAR modification to investigate the effect of Olaparib on PARP1 ribosylation FOXP3;(6)Plasmid with ribosylation site mutation in PARP1(Flag-PARP1-E988K)was constructed to investigate the site mutation Flag-PARP1-E988K effect on FOXP3 ribosylation modification;(7)Dual luciferase reporter gene method was applied to found the fuction of PARP1 and Flag-PARP1-E988K on FOXP3-mediated MT1M transcription expression;(8)Western blotting experiment was used to examine the protein expression of FOXP3 on ovarian cells OVCAR8 after given Olaparib and knocking down PARP1;(9)The scratch healing experiment was used to investigate the effect of Flag-PARP1-E988K on the migration of ovarian cancer cell SKOV3.Results(1)PARP1 regulates the transcription of MT1M through FOXP3First,the database was used to predict and analyze the potential transcription factors of MT1M.It was found that transcription factors such as STAT1,STAT3,FOXP3,and E2F1 may be transcription factors that regulate the expression of MT1M.Further,q RT-PCR experiments were used to investigate these transcription factors whether were invoveled in the mechanism that PARP1 downregulating MT1M transcription expression,and we found transcription factor FOXP3.(2)FOXP3 regulates the transcription and protein expression of MT1MAfter overexpression of FOXP3 and knocking down FOXP3 in SKOV3 cells,it was found that overexpression of FOXP3 can significantly increase the MT1M transcription level and protein levels;while knocking down FOXP3 significantly reduced the transcription and protein levels of MT1M through Western blotting and q RT-PCR experiments.(3)PARP1 was found directly combined with FOXP3.In 293T cells,after overexpression of Flag-PARP1 and HA-FOXP3,it was found that there was binding between PARP1 and FOXP3 by immunoprecipitation.(4)FOXP3 was PARylated by PARP1In 293T cells,after overexpression of Flag-PARP1 and HA-FOXP3,results indicated that FOXP3 was PARylated by PARP1 through PARP1 ribosylation activity.(5)Olaprib can inhibit the ribosylation modification of FOXP3 mediated by PARP1Flag-PARP1 and HA-FOXP3 were constructed in 293T cells and Olaparib was given for 6 h.Using methods to detect PAR modification,it showed that the administration of PARP inhibitor Olaparib inhibited the activity of PARP1,also blocking the ADP-ribosylation of FOXP3 and suppressed PARP1 inducing FOXP3 ribosylation modification.(6)The ribosylation site mutation Flag-PARP1-E988K couldn't PARylated FOXP3After Flag-PARP1-E988K and HA-FOXP3 were overexpressed on 293T cells,the PAR modification results indicated that Flag-PARP1-E988K could not promote FOXP3to undergo ribosylation modification.(7)PARP1 regulates the transcriptional expression of MT1M by regulating the ribosylation modification of FOXP3.Overexpressed Vector,HA-FOXP3,Flag-PARP1,Flag-PARP1-E988K,HA-FOXP3+Flag-PARP and HA-FOXP3+Flag-PARP1-E988K on ovarian cancer cell SKOV3,the luciferase assay results showed that HA-FOXP3 promotes the transcriptional expression of MT1M,while PARP1 inhibits the transcriptional expression of MT1M mediated by FOXP3;PARP1-E988K cannot affect the transcriptional expression of MT1M mediated by FOXP3 due to its lack of ribosylation function.(8)The function of Olaparib administration and silencing PARP1 on the expression of FOXP3 in ovarian cancer cellsIn the ovarian cells OVCAR8,the administration of Olaparib resulted in the increase in FOXP3 expression in Olaparib-dependent manner;while knocking down PARP1 on SKOV3 and Ca OV3,the protein expression of FOXP3 was increased.Section 3 PARP inhibitors promoted MT1M expression against ovarian cancer metastasisMethods(1)The ovarian cancer cells SKOV3 and OVCAR8 were administrated a series concentrations of Olaparib,and Western blotting,q RT-PCR and Elisa experiments were used to investigate the expression and ecretion of MT1M;(2)The different PARP inhibitors Olaparib,Veliparib and Niraparib were given to ovarian cells SKOV3 and Ca OV3,and the effect of different PARP inhibitors on the migration of ovarian cells was investigated by using the scratch test and Transwell assay;(3)By using lentivirus to constructovarian cancer cell with stable knockout of MT1M(SKOV3-sh MT1M),and then PARP inhibitor Olaparib was used to investigate the role of MT1M in PARP inhibitor in inhibiting migration of ovarian cancer cells;(4)Construct the metastatic model of ovarian cancer,the ovarian cells Ca OV3 were inoculated through tail vein of nude mice,then Olaparib was given to investigate the effect of PARP inhibitors on the metastasis of ovarian cancer cells;(5)The humanized high metastasis PDX model was made to assess the effect of Olaparib on ovarian cancer metastasis,and detected MT1M expression in the role of PARP inhibitors in anti-tumor metastasis.Results(1)PARP inhibitors could upregulate the expression and secretion of MT1M in ovarian cancer cellsWestern blotting and q RT-PCR were used to detect the expression and secretion of MT1M in ovarian cancer cells.And the results indicated that different PARP inhibitors could promote MT1M transcription expression and secretion.(2)PARP inhibitors could significantly inhibit the migration of ovarian cancer cellsBy using scratch test and Transwell assay,we found that different PARP inhibitors could significantly inhibit the migration of ovarian cancer cells.(3)Knockout of MT1M significantly inhibited the ability of PARP inhibitors to inhibit the migration of ovarian cancer cellsIn order to investigate the role of MT1M in PARP inhibitor-mediated migration,ovarian cancer cell with stable knockout of MT1M(SKOV3-sh MT1M)was constructed.The results showed that when knocking out MT1M,the ability to inihibit cell migration of Olaparib was significantly impaired.(4)In vivo animal experiments showed that olaparib could significantly inhibit ovarian cancer metastasis and promote MT1M expressionIn the model of ovarian cancer metastasis by in vein inoculation of Ca OV3,the results of H&E staining showed that Olaparib obviously inhibited the formation of lung tumor metastasis.And MT1M secretion was increased in Olaparib-treatemnt group compared to control group.In the humanized high metastasis PDX model,Olaparib had little effect on tumor growth.Boin's staining and H&E staining showed that Olaparib could inhibit lung metastasis of ovarian cells.By using Western blotting,q RT-PCR and Elisa assay to dectect MT1M expression.All these data showed that Olaparib could up-regulate MT1M transcription level and promoted MT1M secretion.Conclusion:In this study,we found that a noval target gene of PARP1,MT1M,negatively regulated by PARP1 in ovarian cancer cells,and MT1M high expression could inhibit the migration of ovarian cancer cells but not affect the proliferation of tumor cells;further study found that FOXP3 regulated the expression of MT1M and participated in PARP1-mediated negatively regulation of MT1M transcription expression.At the same time,FOXP3 was PARylated by PARP1,which inhibiting the transcriptional activity of FOXP3 to reduce MT1M expression.In vivo animal experiments showed that Olaparib,a PARP inhibitor,could inhibit the metastasis of ovarian cancer cells and promote the expression of MT1M in tumor cells.This study not only found a new mechanism of regulating ovarian cancer metastasis,but also provided a new theoretical basis for the application of PARP inhibitors in the treatment of ovarian cancer.