Function And Regulatory Mechanism Of MiR-199b-5p And MiR-409-3p In The Carcinogenesis And Development Of Breast Cancer

Breast cancer(BC)is the most common malignant tumor that threatening women,s life and the quality of life.China is one of the countries with the fastest growth rate of breast cancer morbidity.The latest data shows number of incident case is 270,000 in 2015.Currently,it‘s widely accepted that breast cancer is a systemic disease.Since the development in molecular biology,cell biology and immunology,molecular targeted therapy is becoming a very promising breast cancer treatment besides surgery,chemotherapy,radiotherapy and endocrine therapy.Especially in recent years the human genome research achieved abundant results further promoted the development of molecular target therapy of cancer.It has become one of the most hopeful and active research fields in seeking for effective targets and targeted therapy.Non-coding RNAs,especially miRNAs,play critical role in the occurrence and development of malignant tumor including BC.In recent years,miRNA has been focused as an important treatment in cancer molecular targeted therapy.Mi RNA is a kind of new and endogenic non-coding RNA,the length is about 17~25 nucleotides.Since Ambros et al.found and reported the first miRNA,huge numbers of miRNAs have been identified,many articles were published about them.Researches show that there is a very tight relation between miRNAs and BC,miRNAs take part in many steps during the occurrence and development of the cancer.Designing for miRNAs targeted therapy has a broad prospect in BC.miR-199b-5p and miR-409-3p are two new members of miRNAs family.With literatures review and miRNA Array,we had found that miR-199b-5p and miR-409-3p were low expression in breast cancer.Which was also confirmed in our pre-experiments.But the research of the relationship between miR-199b-5p and miR-409-3p with BC was still less,and the mechanism was still unclear.We analyzed through bioinformatics and found SIRT1 had a binding site with miR-199b-5p in its 3‘UTR position.ZEB1 had a binding site with miR-409-3p in its 3‘UTR position.These were remind us that SIRT1 was a potential target gene of miR-199b-5p,and ZEB1 was a potential target gene of miR-409-3p in theory.SIRT1(sirtuin type 1),sirtuin(silent mating type information regulation 2 homolog)1,participates in many physiological and pathological process,such as cell senility,life lengthen,antioxidant and tumorigenesis.Our previous studies confirm that SIRT1 played an important role in intestinal ischemia-reperfusion injury and occurrence of malignant tumor.SIRT1 was high expression in breast cancer,and its high expression with poor prognosis.ZEB1(zincfinger E-box binding homeobox 1)belongs to the zinc finger transcription factors,is a member of the zincfinger E-box binding homeobox protein family.It is one of the most necessary transcription factors in embryonic development.Recent studys have found that ZEB1 plays very important role in the development of breast cancer,prostate cancer,colorectal cancer and other malignant tumor.ZEB1 is one of the most important regulatory factor of EMT,it participates in the process of the tumor invasion and metastasis.Through our previous studies and literatures review we could know that miR-199b-5p and miR-409-3p may play important role through different targets in breast cancer.Therefore,we studied the role miR-199b-5p and miR-409-3p in breast cancer occurrence and development process and its regulatory mechanism research on target respectively.We found that miR-199b-5p was lower expressed in breast cancer;it can inhibited tumor biological behaviors such as proliferation,invasion,metastasis;further research reveals miR-199b-5p inhibited BC by targeting SIRT1.The research showed that miR-409-3p was lower expression in breast cancer tissue;it was closely related to tumor size and lymphatic metastasis;miR-409-3p could suppress breast cancer cells proliferation,invasion,metastasis and EMT by targeting ZEB1.Our research reveals pathogenesis of miRNAs on regulating BC partly,meanwhile providing new therapeutic targets and lay the foundation for further clinical translational research(small chemical molecular manufacture;design antineoplastic agents specially for miR-199b-5p,miR-409-3p,SIRT1 and ZEB1).Part ?Function and regulatory mechanism of miR-199b-5p in the arcinogenesis and development of breast cancer y targeting SIRT1Objective: This study aimed to investigate the expression of miR-199b-5p and SIRT1 in breast cancer,and their clinical significance,reveal the targeted relationship between miR-199b-5p and SIRT1,expound the function of miR-199b-5p during this process.Methods:Chapter 1: We used 5 breast cell lines(MCF-7,BT-474,MDA-MB-231,SK-BR-3,MCF-10A)and 35 pairs breast cancer tissues from patients as research objects.Using q RT-PCR,cell transfection,CCK-8,colony formation assay,transwell,wound healing to test the expression of miR-199b-5p and its inhibition role to proliferation,invasion and metastasis.Chapter 2: Searching for the predicted targets in databases such as miRanda,Target Scan,miRDB and miRWalk.We detected the regulation of miR-199b-5p to SIRT1 on protein and m RNA level via Western-blot and q RT-PCR in MDA-MB-231 cell line.Chapter 3: Testing SIRT1 expression in human breast cancer tissues through IHC and Western-Blot for further analysis on the relation between SIRT1 level and BC clinical pathological characteristics and prognosis.Results: Chapter 1:(1)miR-199b-5p level was lower in cancer tissues than in NATs significantly(P<0.001).Expression in LN-negative cases was higher than in LN-positive ones(P <0.001).Different TNM stage cases had different miR-199b-5p expression with statistical significance.(2)miR-199b-5p was down-regulated in breast cancer cell lines obviously(P <0.05),lowest in MDA-MB-231.(3)miR-199b-5p inhibited proliferation of MDA-MB-231(P <0.05).(4)miR-199b-5p inhibited invasion of MDA-MB-231(P <0.05).(5)miR-199b-5p inhibited migration of MDA-MB-231(P <0.05).Chapter 2:(1)Four databases(miRanda,Target Scan,miRDB and miRWalk)predict SIRT1 as a target of miR-199b-5p.(2)In the cells transfected with miR-199b-5p mimics,the expression of SIRT1 and its message RNA was down-regulated.As higher transfection concentration as lower protein level(P<0.05).Chapter 3:(1)SIRT1 is obviously higher in breast cancer tissue compared with NATs as the positive rate was 41.2%(P<0.05).(2)SIRT1 level was closely related with histology,lymphatic metastasis and molecular type,P value was 0.001,0.006,0.001 correspondingly.(3)SIRT1 level was tightly combine with prognosis of BC.Kaplan-Meier survival analysis showed: SIRT1 negative cases could be better than positive ones in prognosis,P<0.05(calculated by log-rank test).Conclusion: 1.In BC tissue and cell lines miR-199b-5p stay in a lower level relating with TNM stage,lymphatic metastasis and molecular type.2.miR-199b-5p could inhibit proliferation,invasion and migration prompting,it owns the function of anti-oncogene.3.miR-199b-5p suppressed expression of SIRT1,and SIRT1 was its direct target.4.SIRT1 was up-regulated in cancer compared with NATs.Its level was closely related with histology,lymphatic metastasis,molecular type and prognosis.Part ? Function and regulatory mechanism of miR-409-3p in the carcinogenesis and development of breast cancer by targeting ZEB1Objective: This study aimed to investigate differential expression of miRNAs in breast cancer,the expression of miR-409-3p and ZEB1 together with the clinical significance,reveal the targeted relationship between miR-409-3p and ZEB1,expound the function of miR-409-3p during this process.Methods: Chapter 1: Using miRNA micro array(Exiqon,Denmark)to test the expression of miRNA in different cell lines including MCF-10A(normal),MCF-7(luminal A),BT-474(luminal B),MDA-MB-231(triple negative)and SK-BR-3(Her-2 Overexpression).Then we explored the expression of miR-409-3p in these cell lines,111 cases of cancer tissues and 45 cases of normal ones.Chapter 2: We used MDA-MB-231 and laboratory BALAB/c-nu nude mice(SPF)as research objects.Using q RT-PCR,cell transfection,WST-1,colony formation assay,transwell,wound healing to test expression of miR-409-3p and its inhibition to proliferation,invasion and migration.Chapter 3: Searching for the predicted target genes of miR-409-3p in databases such as miRanda,Target Scan,miRDB.Then we confirmed the targeted relation between miR-409-3p and ZEB1 through dual-luciferase report in MDA-MB-231.Chapter 4: Testing the expression of ZEB1 in cell and tissue via Western-Blot;obvserve changes of invasion after transfecting si RNA to interfere ZEB1;detect regulation of miR-409-3p to ZEB1 on protein and m RNA level via Western-blot and q RT-PCR;check the effect of miR-409-3p to EMT corresponding protein(E-cadherin,Vimentin)in the down-stream of ZEB1.Results: Chapter 1:(1)Micro Array showed: all these cancer cell lines have an significant lower level of miR-409-3p compared with normal ones(P<0.05).(2)miR-409-3p level in MDA-MB-231 was the lowest via q RT-PCR the same as in tissues(P <0.001).(3)Further researches in 111 BC cases,suppression of miR-409-3p was tightly related to tumor size(P =0.018),lymphatic metastasis(P =0.001)and Ki67 expression ratio(P =0.033).None with age,ER,PR and Her-2(P >0.05).Cox multivariate analysis revealed miR-409-3p positive expression,bigger tumor size,lymphatic metastasis and higher TNM stage were the independent risk factors influence BC remote migration after surgery.Specific results: miR-409-3p(Exp(B)1.639,95%CI 1.373-3.573,P =0.001),tumor size Exp(B)2.131,95%CI 1.540-4.913,P = 0.001),lymphatic metastasis(Exp(B)1.378,95%CI 1.031-2.713,P =0.003).Chapter 2:(1)miR-409-3p inhibited proliferation of MDA-MB-231(P <0.05).(2)miR-409-3p inhibited invasion of MDA-MB-231(P <0.05).(3)miR-409-3p inhibited migration of MDA-MB-231(P <0.05).(4)miR-409-3p inhibited subcutaneous tumorigenesis in nude mice.All mice were transplanted human breast cancer cells MDA-MB-231-NC and MDA-MB-231-409-3p successfully.NC group grew faster than miR-409-3p group.3 weeks later,the average diameter(weight)of miR-409-3p group and NC group are 2.5 cm(0.70 ± 0.07 g)and 3.0 cm(1.60 ± 0.12 g)(P <0.05).Chapter 3:(1)3 databases(miRanda,Target Scan and miRDB)predict SIRT1 as a target of miR-199b-5p.There were complementary sequences between ZEB1 3‘UTR and miR-409-3p 5‘region.(2)Dual-luciferase report demonstrates: we used psicheck 2.0 system to found 2 types of plasmids.One is ZEB1 wild-type(Luc-ZEB1-WT 3‘UTR,W-T),another was mutant type(Luc-ZEB1-MT 3‘UTR,M-T).Result revealed that after co-transfecting,the luciferase activity decreased obviously in W-T group(P<0.05),rarely detected changes in M-T group.Chapter 4:(1)ZEB1 expression was much higher in cancer tissues than in NATs collected from 35 patients via Western-Blot method.(2)In the cells transfected with ZEB1 si RNA,the ability of invasion is downregulated.As higher transfection concentration as lower potentiality(P <0.05).(3)In the cells transfected with miR-409-3p mimics,the expression of ZEB1 and its m RNA was down-regulated.As higher transfection concentration as lower protein level(P <0.05).(4)We tested the influence of miR-409-3p to ZEB1 down-stream corresponding protein,E-cadherin and vimentin.Result indicates: in the cells transfected with miR-409-3p mimics,the expression of E-cadherin was increased compared with NC group.As higher transfection concentration as lower protein level(P <0.01).Meanwhile,Vimentin showed a completely opposite tendency.Conclusion:1.miR-409-3p was lower expressed in BC,which prompting that it plays the role of anti-oncogene.2.Suppression of miR-409-3p was tightly related with tumor size,lymphatic metastasis and Ki67 expression ratio.miR-409-3p positive expression is one of the independent risk factors influence BC remote migration after surgery.3.miR-409-3p inhibited proliferation,invasion,migration and tumorigenesis in vivo.4.ZEB1 was a potential target of miR-409-3p based on databases.5.Dual-luciferase report proves ZEB1 was a direct target of miR-409-3p.6.ZEB1 was up-regulated in BC acting as oncogene;miR-409-3p can decrease its expression.7.miR-409-3p increased E-cadherin and decreased Vimentin,this hinted that miR-409-3p suppresses EMT through targeting ZEB1 possibly.