| Objective:Breast cancer is the most common female malignant tumor all over the world which threatens the health of women seriously.According to the latest data of ASCO,there are 240,000 women diagnosed as breast cancer in 2016,and 40,000 died because of the disease.The Chinese epidemiologic data shows that 272,400 are diagnosed and 70,700 are dead in 2015.Although there are plenty of methods for breast,such as operation,chemical therapy,radiotherapy,endocrinotherapy and targeted therapy,prolonging the disease free survival(DFS)and overall survival(OS),the problems remains on disease progression,relapse and drug resistance.Further researches on the molecular mechanism of relapse and metastasis of breast cancer,the cause of drug resistance and searching a valuable marker for predicting relapse,metastasis and resistance are very important.Flap endonuclease-1 is a structure specific nuclease which has 5'-flap endonuclease,Gap dependence endonuclease(GEN)and 5'-flap exonuclease activity(EXO)activity.It plays important roles in the process of DNA replication in Okazaki fragment maturation,base excision repair,maintaining telomere stability,inducing DNA degradation,trinucleotide repeat and preventing restart of replication forks.A recent study demonstrated that gene polymorphsim of FEN1 and gene mutation may cause deficiency and incapacitation in biological function.Consequently,it result in instability of genome and cancer.Previous studies found that the expression of FEN1 was higher in cancer tissues than that in paracanerous tissues in different organs.And it was also found that the over-expression of FEN1 altered the normal breast epithelial cells to tumor phenotype cells,stimulated the proliferation of gastric and breast cells,which was in accordance with the histological grade of breast cancer and Gleason score of prostate cancer.These studies illustrated the important role of FEN1 in the process of canceration and proliferation of tumor.And there are multiple retrospective study from single centers showed that over-expression of FEN1 was relative to cancer T-stage of breast,ovary and stomach,regional lymph node metastasis,poor differentiation and shorter DFS/OS,which illustrated the role of FEN1 in the metastasis and prognosis.The mechanism of FEN1 influencing the processes of tumor invasion and metastasis is still unclear.The worth of FEN1 in prognosis is not demonstrated by large-sample and multiple-center studies.Thus it is necessary for further researches on FEN1 influencing the invasion and metastasis of breast cancer and on its worth for prognosis..Drug resistance is a common trouble in the process of tumor treatment.The resistance on endocrinotherapy is the main cause of poor prognosis of hormone receptor-positive breast cancer patients.Studies showed the increasing expression of FEN1 was most distinct in estrogen-relative breast and uterus cancer,and FEN1 could interact with ER?,enhancing the DNA transcription of ER?and ERE,meanwhile the estrogen could regulate FEN1.Interactions between FEN1,estrogen,ER?and ERE were implied.On the other,studies found that the over-expression of FEN1 was related to the resistance on temozolomide,cis-platinum,taxol and fluorouracil,Inhibition of FEN1 expression could increase the sensitivity of anticancer drugs.However these studies were restricted to phenomenal level,such as MTT and cellular cycles,lacking of further discuss on mechanism of drug resistance.Studies on FEN1 influencing tamoxifen resistance are not reported yet.Our study analysized the data on Oncomine data platform and Kaplan Meimer plotter survival analysis database to identify the relationship between FEN1 mRNA and characteristics of breast cancer patients,and to assess the value of FEN1 mRNA.Then we analysized the tissue chip of our center(280 breast cancer)for validation.Furthermore,in vitro experiment,we studied whether FEN1 expression influenced the invasion and matastasis of triple-negative breast cancer.Then we studied the molecular mechanism how FEN1 promoted the invasion and matastasis of triple-negative breast cancer.We found thatthe over-expression of FEN1 was a biomarker for poor prognosis by Kaplan Meier-plotter..Methods:1.Inverted microscope was used for observing cell morphological change;2.Construction of si RNA targeting FEN1 and overexpression FEN1 plasmids which were stably transfected cells;3.Construction FEN1 silencing and overexpression lentivirus plasmids which were stably transfected into HCC-1937,MDA-MB-231,MCF7 and T47 D cells;4.Migration ability were determined by Transwell Assay;5.Cell proliferation was measured using MTT assay;6.Cell proliferation ability was observed with Colony-Formation Assay;7.Animal model was established to test the influence of lung metastasis which depend on FEN1 expression;8.HE and IHC staining of loci of lung metastasis;9.Expression of FEN1,AKT,p-AKT,ERK,p-ERK,AKT,p-AKT and GAPDH proteins were analyzed by Western blot;10.Relative levels of p85,PTEN,micro RNA-26b-5p and mRNA were measured by Real-time PCR.11.Relative changes of mRNA and micro RNAs were measured by Gene Chip mRNA and mi RNA Array after silence-FEN1 in MDA-MB-231;12.Transient transfection was performed using Lipofectamine 2000 reagent;13.Immunoblot analysis of expression of FEN1 protein,p AKT,p ERK in breast cancer tissue microarray and prognosis;14.Download data from GEO and run differential gene expression analysis;15.Statistical analysis.All values are expressed as means ± SD.The differences of the results between two groups were evaluated by Student's t-test.P<0.05 was considered to be statistically significant..Results:1.FEN1 was related to relapse and metastasis of breast cancer and the triple-negative molecular subtype.The results from Oncomine database analysis showed that the expression of FEN1 mRNA was higher in breast cancer tissue than that in normal tissue.The analysis on the relationship between FEN1 mRNA expression and clinicopathologic parameters of breast cancer showed a positive correlationship between FEN1 expression level and histilogical grade.The expression level was higher on higher histilogical grade and on the patients with lymph node metastasis(N+)and distant metastasis(M+).The analysis on the relationship between FEN1 mRNA expression and clinical outcome showed FEN1 mRNA expressed higher in patients with tumor recurrence for 3 or 5 years than without recurrence.The FEN1 mRNA expression was higher in patients with metastasis in the 1st,3rd or 5th year after operation.The expression was higher in patients dead in the first year after operation than in survivors.Meta analysis of mutiple datasets showed FEN1 mRNA expression level was higher in triple-negative molecular subtype than other subtypes.Above mentioned results,the overexpression of FEN1 was closely related to the poor prognosis,clinicopathologic parameter,recurrence,metastasis and the triple-negative molecular subtype.2.The over-expression of FEN1 mRNA was relative to poor prognosis in breast cancer.The data analysis from online database Kaplan Meier plotter showed that the RFS DMFS and OS were shortened in patients with over-expression of FEN1.The influence of FEN1 over-expression on RFS,DMFS and OS was more distinct in ER(+)patients while there was no significant differences in triple-negative breast cancer.The results demonstrated over-expression of FEN1 mRNA was relative to poor prognosis of breast cancer,especially in ER(+)subtype.3.The overexpression of FEN1 protein was relative to the lymphatic metastasis and distant metastasis.Immunohistochemical staining of FEN1 protein from our own breast cancer tissue microarray was done and found that FEN1 protein was expressed higher in triple-negative than in others.The subtype analaysis showed a distinct correlationship between FEN1 protein overexpression and lymphatic metastasis,shorter DMFS and OS.The results implied that FEN1 might a biomarker for recurrence and death in triple-negative breast cancer.4.FEN1 promoted the invasion and migration of triple-negative breast cancer cells.MDA-MB-231 and HCC1937 cell line were treated with silencing and over-expression FEN1 gene by lentivirus vector.The migration capacity of the cells with silent FEN1 gene decreased via Transwell assay.The mean value of migration rate of MDA-MB-231 cells decreased to 59.4 ± 5.11%(p<0.05),while the HCC1937 did to 44.33 ± 9.97%(p<0.05).In over-expressed FEN1 cells,the mean value of migration capacity increased to 127.1± 11.41%(p<0.05)in MDA-MB-231 and to 206.31 ± 16.27%(p<0.05)in HCC1937.We built an animal model of mouse breast cancer with lung metastasis.We injected the silencing-FEN1 cell and over-expressed-FEN1 cell into nude mice's caudal vein by intravenous injection.After a 8-week circle,the number of the lung metastasis lesions were counted.The results showed that,the mean counting number of lung metastastic lesions in knockout FEN1 control group(NC-KD)was 18.8±4.87,while in knockout FEN1 group(KD-FEN1)it was 5.6±2.3(p<0.05).In over-expressed FEN1 control group(NC-OE),the number is 29.8±2.68,while in over-expressed FEN1 group(OE-FEN1)was 67.8±13.16(P<0.05).Experiments in vivo and in vitro indicated silencing FEN1 could decrease the capacity of migration and metastasis of breast cancer cell.5.FEN1 promoted the migration of triple-negative breast cancer cells by activating PTEN/PI3K/AKT pathway.In order to research the molecular mechanisms of FEN1 promoting the invasion and metastasis of triple-negative breast cancer cell,we tested the mRNA microarray before and after knocking out FEN1 gene in MDA-MB-231.We found a significant difference before and after knocking out FNE1 in MAPK and p ERK pathways.Analysis of data from 77 triple-negative breast cancer tissue microarrays was done for the correlation between FEN1,p AKT and p ERK by immunohistochemistry.The results showed a significant positive correlation between FEN1 and p AKT(r=0.46,p=0.011),while the correlation between FEN1 and p ERK was not significant(r=0.02,p=0.722).We tested the relative signal expression by Western blot.Compared with the control group,after knocking out FEN1,PI3K/AKT pathway activity was significantly down-regulated/decreased.While after over-expressed FEN1,PI3K/AKT pathway activity increased distinctly.But MAPK/ERK pathway was effected slightly after knocking out and over-expressed FEN1.Above all,we speculate that FEN1 may influence the metastasis of triple-negative breast cancer through PI3K/AKT pathway.Furthermore,we tested expression of PTEN which could influence AKT phosphorylation.Compared with control group,after silence FEN1,the expression of PTEN increased.While after over-expressed FEN1,results were opposite.Andrealtime PCR after knocking out and over-expressed FEN1,we found that the mRNA expression of PTEN was not changed.We speculared that FEN1 would influenced PTEN expression after transcriptional level.6.FEN1 promoted migration of triple-negative breast cancer cells thought mi R-26b-5p/PTEN/AKT pathway.By analysizing the mi RNAs microarray before and after knocking out FEN1 and predicting the mRNAs regulating PTEN through mi RDB,targetscan and mi RWalk websits,and mi RDB website and Targetscan,we suggested that FEN1 might promote the migration of triple-negative breast cancer by target-regulating PTEN though mi R-26b-5p.We tested mi R-26b-5p expression level was down-regulated 3.52 ± 0.24 times(p<0.05)in knocking out FEN1 cells,while its expression level was up-regulated 2.26 ± 0.34times(p<0.05)in over-expressed FEN1 cells.The two results were in accordance with each other.In order to confirm the effect of mi R-26b-5p to triple-negative breast cancer cell migration,we used mimics regulating the expression of mi R-26b-5p.The results showed that when up-regulating mi R-26b-5p,PTEN which is the target protein of mi R-26b-5p was suppressed,and p AKT signal which is a downstream molecus of mi R-26b-5p was activated.The mean migration rate of mi R-26b-5p-mimics in MDA-MB-231 was increased to 227.25 ± 29.7%(P<0.05).While down-regulating mi R-26b-5p,the phenomenon was opposite and the mean migration rate was decreased to 68.65 ± 14.3%(P<0.05).In order to demonstrate that FEN1 could down-regulate PTEN through mi R-26b-5p,and promote the migration of triple-negative breast cancer,we over-expressed mi R-26b-5p in silenced FEN1 cells,the Western bolt showed PTEN the target protein of mi R-26b-5p was down-regulated and the p AKT signal was activated,which was partially reversed the suppression of p AKT by silencing FEN1.The results of Transwell suggested that over-expressed mi R-26b-5p promoted the migration capacity of triple-negative breast cancer and partially reversed the decreasing of migration capacity caused by silencing FEN1.The results demonstrated mi R-26b-5p played an important role in the process of FEN1 mediated triple-negative breast cancer migration.FEN1 down-regulated PTEN to mediate the migration of triple-negative breast cancer via mi R-26b-5p.7.FEN1 may take part in tamoxifen resistance.Download the tamoxifen resistant breast cancer database from GEO.We found that both in vitro and in vivo on the cell level,the expression of FEN1 mRNA in tamoxifen resistant cell lines were higher than tamoxifen sensitive group,it showed the potential effect of FEN1 on tamoxifen resistance.In order to clarify the effect of FEN1 on tamoxifen sensitivity,we over-expressed FEN1 in tamoxifen sensitive and ER positive breast cancer cell lines MCF7 and T47 D,the proliferation rates of the two cell lines were tested by MTT assay on different concentrations of tamoxifen.And the influence on the proliferation rates(between original cell group,tamoxifen group,over-expressed FEN1 group and over-expressed FEN1 coupled with tamoxifen group)were tested by colony-forming assay.The results showed that tamoxifen could not suppress the over-expressed FEN1 breast cancer cell.To clarify whether suppressed FEN1 expression could increase the sensitivity,we deleted FEN1 in MCF7 and T47 D cell lines,and tested the proliferation rates by MTT and colony-forming assay.The results showed the sensitivity of tamoxifen was increased after knocking out FEN1 in ER positive breast cancer.The results implied that FEN1 took part in the process of tamoxifen resistance.Conclusion: 1.The high expression of FEN1 is associated with a higher risk of recurrence / metastasis and the triple negative phenotype of breast cancer.2.FEN1 promotes invasion and metastasis in triple-negative breast cancer cells.3.FEN1 promotes invasion and metastasis of TNBC by mi R26b-5p/PTEN/AKT and PI3K-p85/AKT pathway.4.FEN1 participates in tamoxifen resistance.5.High expression of FEN1 is a predictive marker of tamoxifen. |
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