The Molecular Mechanism Of LncRNA PVT1 Involved In Breast Cancer Proliferation And Metastasis

Objective: The purpose of this study was to investigate the relative expression levels of long non-coding RNA PVT1 in the plasma and cell lines of breast cancer,the effect of long non-coding RNA PVT1 on the proliferation and the metastasis of breast cancer,as well as the possible molecular mechanism of long non-coding RNA PVT1 involved in proliferation and metastasis of breast cancer.Methods: 1.The plasma level of PVT1 was measured in 80 breast cancer patients,70 patients with breast fibroadenoma and 70 healthy volunteers by q RT-PCR.And the correlation between the clinical data and the expression level of PVT1 was analyzed;2.The expressions of PVT1 in 6 human breast cancer cell lines and human normal breast epithelial cell line HBL-100 were detected respectively by q RT-PCR,and the two cell lines with highest expression levels of PVT1 were selected for further study;3.We knocked down PVT1 expression by small interfering RNA(si RNA),the breast cancer cell proliferation and metastasis capability were detected by colony formation assay,wound healing assay and transwell assay;4.The effects of PVT1 knockdown on the regulation of the tumor cell proliferation marker PCNA,the apoptosis associated protein Bax,the epithelial associated protein E-cadherin and the interstitial marker Vimentin was detected by western blot assay;5.Bioinformatics software or websites were used to analyze and predict the potential mi RNA targets of PVT1,and its downstream target gene.And the luciferase reporter assay was performed to prove the targeting relationship between them;6.Bioinformatics software or websites were used to predict the protein interacting with PVT1,and the RNA immunoprecipitation assay was used to verify the biding relationship;7.We knocked down PVT1,the expression of target mi RNA and protein were detected by q RT-PCR and western blot respectively;8.Co-transfection assay verified whether PVT1 regulated breast cancer cell behavior and related protein expression by targeting its target mi RNA and its interacting protein.Results: 1.PVT1 expression levels were significantly up-regulated in breast cancer compared with breast fibroids patients and healthy volunteers,but there was no significant difference between breast fibroids patients and healthy volunteers.Furthermore,the high expression level of PVT1 was correlated with the clinical stage of breast cancer;2. PVT1 expression in 6 breast cancer cell lines was also significantly higher than that in normal breast epithelial cell HBL-100,among which Hs578 t and MCF-7 were the most highly expressed;3.The proliferation and metastasis ability of the 2 breast cancer cells were inhibited after knockdown the PVT1 expression;4.After knockdown of PVT1,the expressions of PCNA and Vimentin were decreased,while the Bax and E-cadherin expression levels were increased,indicating that PVT1 promoted the proliferation and metastasis of breast cancer cells;5.According to the analysis of bioinformatics prediction website,PVT1 can play the role of ce RNA by competitively binding mi R-128-3p,and mi R-128-3p could directly target FOXQ1.The luciferase reporter assay further proved its targeting relationship;6.According to the analysis of bioinformatics prediction website,PVT1 interacted with the protein UPF1,and the RNA immunoprecipitation assay also confirmed this prediction;7.After knockdown of PVT1,the expressions of mi R-128-3p and UPF1 were both increased,which once again verified the fact that mi R-128-3p and UPF1 were the downsteam target genes of PVT1;8.Co-transfection assay showed that PVT1 did directly target mi R-128-3p and UPF1 to regulate the expression of downstream proteins related to proliferation and metastasis.Conclution: In summary,this study indicated that long non-coding RNA PVT1 was up-regulated in breast cancer patients’ plasma as well as breast cancer cell lines.In breast cancer,long non-coding RNA PVT1,as an oncogene,promotes tumor proliferation and metastasis by competitive binding of mi R-128-3p to promote FOXQ1 expression and inhibiting UPF1 expression.Long non-coding RNA PVT1 plays a crucial regulatory role in the process of breast cancer and may be a potential molecular target for breast cancer treatment.