| Background:Breast cancer is the most common malignant tumor in the world,it is important to find the potential target of diagnosis and treatment.miRNA(Micro RNA)can bind to m RNA containing MREs(micro RNA response elements),thereby inhibiting the expression of m RNA.lnc RNA(long non-coding RNA)is commonly found in eukaryotic cells.At present,it has been found that MREs exist not only on m RNA,but also on various forms of RNAs such as lnc RNA,circ RNA,etc,which means that miRNA can combine with variety types of RNAs,thus forming a ce RNA(competitive endogenous RNA).Various forms of RNA can competitively bind to the same miRNA,and act like a sponge to reduce the expression of each other,thus achieving a certain regulatory effect.In our study,it was found that miR-330-3p and EREG were differentially expressed in high and low metastatic HCC cells,and the potential binding site between the two were predicted by Target scan.Further studies show that the differentially expressed miR-330-3p and EREG were closely related to the occurrence and development of tumors.lnc021545 was a negatively correlated with miR-330-3p detected in renal cancer solid tissues and leukemia bone marrow mononuclear cell samples by our research group,and a potential binding site between the two was predicted by NON CODE.Since lnc021545 and EREG both have potential binding sites with miR-330-3p,this paper investigated whether miR-330-3p,lnc021545 and EREG affect the regulation of ce RNA in breast cancer and the mechanism of action.Objective:1.To clarify the expression levels and its clinical correlation of miR-330-3p,lnc021545 and EREG in tissue samples of breast cancer patients;2.To investigate the effects of miR-330-3p,lnc021545 and EREG on the proliferation,invasion and metastasis of breast cancer cells;3.To explore the interactions of lnc021545 and EREG with miR-330-3p,and further investigate whether they were in line with the regulatory system of cer NA.4.To investigate the mechanism of miR-330-3p,lnc021545 and EREG regulating invasion and metastasis of breast cancer.Methods:1.The gene expression levels of miR-330-3p,lnc021545 and EREG were detected by q RT-PCR in the tissue samples of 50 breast cancer patients,and the protein expression levels of EREG were detected by immunohistochemistry.SPSS 22.0 and Graph Pad Prism 7.0 were used to analyze the relationship among the three;2.Using immunohistochemistry to detect the expression levels of ER,PR,HER-2,Ki-67,CK5/6,P53 and EGFR in 50 breast cancer patients,and analyze the correlation between the expression levels of miR-330-3p,lnc021545 and EREG by SPSS 22.0 and Graph Pad Prism 7.0 software.3.Using SPSS 22.0 and Graph Pad Prism 7.0 software to analyze the expression levels of miR-330-3p,lnc021545 and EREG in breast cancer tissue samples were analyzed,and analyze the correlation between the expression levels of miR-330-3p,lnc021545 and EREG and their pathological characteristics and clinical parameters;4.Using MTT,wound-healing and Transwell assay to detect the effects of miR-330-3p up-regulation,miR-330-3p down-regulation,lnc021545 down-regulation,EREG up-regulation and EREG down-regulation on the proliferation,migration and invasion of MCF-7 and T47D cell lines;5.Using pmir GLO vector to construct WT-miR-330-3p-EREG,MUT-miR-330-3p-EREG,WT-miR-330-3p-lnc021545 and MUT-miR-330-3p-lnc021545 recombinant expression vector,interaction between miR-330-3p and EREG was detected by dual luciferase reporter experiment;6.The effects of miR-330-3p on the expressions of EREG and lnc021545,the effects of lnc021545 on the expressions of EREG and miR-330-3p were detected by q RT-PCR and western blot in MCF-7 and T47D cell lines;7.The effects of miR-330-3p,lnc021545 and EREG on the expressions of EMT related molecules were detected by western blot in MCF-7 and T47D cell lines;8.To analyze whether EREG is regulatory protein of miR-330-3p that affects the EMT and migration and invasion of breast cancer cells,MCF-7 cell lines that were co-transfected miR-330-3p mimics and PCDH-EREG were tested by Western blot and Transwell method.Results:1.In 50 breast cancer tissue samples,compared with paracancerous tissues,miR-330-3p was highly expression(P<0.0001),EREG(P<0.0001)and lnc021545(P<0.0001)were low expression in 50 breast cancer specimens,The relative expression level of miR-330-3p was negatively correlated with that of lnc021545(R~2=0.4480,P<0.0001),the relative expression level of miR-330-3p was negatively correlated with the relative expression level of EREG(R~2=0.2014,P<0.0011),and the relative expression of lnc021545 was positively correlated with the relative expression level of EREG(R~2=0.1572,P<0.0044);The relative expression of miR-330-3p was correlated with the protein expression of EREG in the cancer tissue(P=0.003),and the relative expression of lnc RNA was correlated with the protein expression of EREG in the cancer tissue(P=0.003);2.The expression of miR-330-3p was correlated with HER-2 amplification(P=0.001)and the expression levels of EGFR(P=0.005);The expression of lnc021545was correlated HER-2 amplification(P=0.012);The expression of EREG was correlated with HER-2 amplification(P=0.001)and the expression levels of ER(P=0.023);3.The expressions of miR-330-3p was correlated with tumor size(P<0.0001),TNM stage(P<0.0001),lymphatic metastasis(P=0.001)and DFS(P=0.0016);The expressions of lnc021545 was correlated with tumor size(P=0.025),TNM stage(P=0.003),lymphatic metastasis(P=0.01)and DFS(P=0.0085);The expressions of EREG was correlated with tumor size(P<0.0001),TNM stage(P=0.003),lymphatic metastasis(P=0.045)and DFS(P=0.0099);4.24 h after miR-330-3p mimic was transfected,miR-330-3p was up-regulated by4190(P=0.0002)in MCF-7 cells,13966(P=0.0043)in T47D cells;24 h after miR-330-3p inhibitor was transfected,miR-330-3p was down-regulated by 92.2%(P<0.0001)in MCF-7 cells,90.8%(P<0.0001)in T47D cells;24 h after si-lnc021545was transfected,lnc021545 was down-regulated by 62.3%(P=0.0003)in MCF-7 cells,85.7%(P=0.0005)in T47D cells;24 h after PCDH-EREG was transfected,the protein expression of EREG was up-regulated by 162.7%(P=0.0177)in MCF-7 cells,146.0%(P=0.0166)in T47D cells;24 h after si-EREG was transfected,the protein expression of EREG was down-regulated by 63.9%(P=0.0023)in MCF-7 cells,73.3%(P=0.0019);5.MTT results showed that there were no significant changes in the proliferation ability of the cells transfected with miR-330-3p mimic,miR-330-3p inhibitors,si-lnc021545,PCDH-EREG,si-EREG in MCF-7 and T47D cells;6.The results of wound-healing in MCF-7 cell lines showed that compared with the NC groups,miR-330-3p mimic made the scratch healing wider 48.3±5.5μm(50.1%,P=0.0052),the up-regulation of miR-330-3p promoted cell motility;miR-330-3p inhibitor made the scratch healing narrower 12.8±2.7μm(48.0%,P=0.0047),the down-regulation of miR-330-3p inhibited cell motility;si-lnc021545 made the scratch healing wider 54.0±1.7μm(48.4%,P=0.0009),the down-regulation of lnc021545 promoted cell motility;PCDH-EREG made the scratch healing narrower 9.4±0.6μm(59.3%,P=0.0009),the up-regulation of EREG inhibited cell motility,si-EREG made the scratch healing wider 41.8±2.3μm(46.7%,P=0.0008),the down-regulation of EREG promoted cell motility;7.Compared with the NC groups,the number of migrating MCF-7 and T47D cell increased 53.9%(P=0.0083),44.8%(P=0.0086)in the miR-330-3p mimic group;Compared with the NC groups,the number of invading MCF-7 and T47D cell increased53.6%(P=0.0002),39.1%(P=0.0091)in the miR-330-3p mimic group;Compared with the NC groups,the number of migrating MCF-7 and T47D cell decreased 32.4%(P=0.0069),49.6%(P=0.0071)in the miR-330-3p inhibitor group;Compared with the NC groups,the number of invading MCF-7 and T47D cell decreased 33.1%(P=0.01),45.3%(P=0.0038)in the miR-330-3p inhibitor group;Compared with the NC groups,the number of migrating MCF-7 and T47D cell increased 43.3%(P=0.0042),38.0%(P=0.001)in the si-lnc021545 group;Compared with the NC groups,the number of invading MCF-7 and T47D cell increased 43.1%(P=0.003),43.1%(P=0.0018)in the si-lnc021545 group;Compared with the NC groups,the number of migrating MCF-7and T47D cell decreased 44.3%(P=0.0034),34.7%(P=0.0038)in the PCDH-EREG group;Compared with the NC groups,the number of invading MCF-7 and T47D cell decreased 55.0%(P=0.0013),28.7%(P=0.008)in the PCDH-EREG group;Compared with the NC groups,the number of migrating MCF-7 and T47D cell increased 26.1%(P=0.0042),48.7%(P=0.0012)in the EREG group;Compared with the NC groups,the number of invading MCF-7 and T47D cell increased 29.3%(P=0.0032),41.2%(P=0.0027)in the EREG group;8.The dual luciferase reporter assay showed that the activity of luciferase decreased by 42.3%(P=0.0017),after co-transfection of miR-330-3p mimic and lnc021545-miR-330-3p-WT recombinant plasmid,and the activity of luciferase decreased by 32.7%(P=0.002),after co-transfection of luciferase and EREG-miR-330-3p-WT recombinant plasmid;9.When the expression of miR-330-3p was up-regulated,the expression of lnc021545 and EREG were both reduced in MCF-7 and T47D cell;When the expression of miR-330-3p was down-regulated,the expression of lnc021545 and EREG were both raised in MCF-7 and T47D cell;When the expression of lnc021545 was down-regulated,the expression of miR-330-3p was both raised,the expression of EREG was both reduced in MCF-7 and T47D cell;10.The results of western blot showed that 48 h after the miR-330-3p mimic,si-lnc021545 and si-EREG were transfered into MCF-7 and T47D cells lines,the expression of E-cadherin were significantly reduced,and the expressions of Vimentin,Snail,N-cadherin and Slug were significantly raised;48 h after the miR-330-3p inhibitor and PCDH-EREG were transfered into MCF-7 and T47D cells line,the expression of E-cadherin were significantly raised,and the expressions of Vimentin,Snail,N-cadherin and Slug were significantly reduced;11.48 h after miR-330-3p mimic and PCDH-EREG were co-transfected into MCF-7 cells line,the expression of E-cadherin was significantly raised,the expression of Vimentin,Snail,N-cadherin and Slug were reduced,and the migratory and invasive ability of MCF-7 cell lines was significantly reduced,comparing with that the miR-330-3p mimic was transfected;48 h after miR-330-3p mimic and PCDH-EREG were co-transfected into MCF-7 cells line,the expression of E-cadherin was significantly reduced,the expression of Vimentin,Snail,N-cadherin and Slug were raised,and the migratory and invasive ability of MCF-7 cell lines was significantly raised,comparing with that the PCDH-EREG was transfected.Conclusion:1.In breast cancer specimens,it was found that miR-330-3p was highly expressed,while lnc021545 and EREG were low expressed.The relative expression levels of miR-330-3p,lnc021545 and EREG were significantly correlated,and the changes of the three were related to the progression of breast cancer;2.lnc021545,miR-330-3p,EREG affected the migration and invasion ability of MCF-7 and T47D cell lines,but had no effect on proliferation ability;3.miR-330-3p targeted binding to lnc021545 and EREG,respectively,and the expression levels of miR-330-3p and lnc021545 affected the expression levels of EREG and regulated the regulatory system of breast cancer ce RNA;4.The miR-330-3p,lnc021545,EREG regulated the expression of EREG through the ce RNA regulatory system,which leads to the change of the expression of EMT related molecules,and then regulates the migration and invasion ability of breast cancer cells.Finally,it can affect the metastasis of breast cancer. |
PDF Full Text Request