Effects Of GRK4-mediated LncRNA CTD-2270P14.5 On Proliferation And Invasion Of Human Breast Cancer Cells

Objective: In previous studies,we found a competing endogenous RNAs(ce RNA)network mediated by G Protein-coupled Receptor Kinase 4(GRK4)associated with human NASopharyngeal carcinoma CNE2 cell proliferation.The ce RNA network including seven long noncoding RNA(lnc RNA)and Cyclin-dependent Kinases 6(CDK6).The purpose of this study was to verify and screen lnc RNAs molecules in the ce RNA network model.GRK4 mediated the expression regulation of lnc RNA CTD-2270P14.5.To investigate the effect of CTD-2270P14.5 on proliferation and invasion of breast cancer cells and its potential mechanism.Methods: 1.The expression of GRK4 in human breast cancer tissues and its prognostic value were analyzed by bioinformatics.2.CNE2 cells were transfected with p EGFP-GRK4 plasmid,and GRK4(+)/(-)cells were sorted by flow cytometry.Quantitative real-time PCR(q PCR)was used to verify the expression of lnc RNAs in GRK4-mediated ce RNA network model.3.Six fresh clinical breast cancer and adjacent tissue samples and cultured human breast cancer MDA-MB-231 and MCF-7 cells were collected,and the expression of lnc RNAs in GRK4 and its mediated ce RNA network was detected by Western Blot and q PCR.SPSS statistical software was used to analyze the key lnc RNAs with the most differentially expressed.4.Breast cancer MCF-7cells with high expression of GRK4 were transfected with small interfering RNA(si RNA)of GRK4,and si-GRK4 knockout cell line and control cell line(NC)were constructed.Breast cancer MDA-MB-231 cells with low GRK4 expression were transfected with p EGFP-GRK4 plasmid,and GRK4 overexpression test group and control group were constructed.Western Blot and q PCR were used to verify the expression of GRK4 after transfection,and the expression level of CTD-2270P14.5 was detected.5.MCF-7 cell line was transfected with short hairpin RNA(sh RNA)of CTD-2270P14.5 and MDA-MB-231 cell line was transfected with CTD-2270P14.5 expression plasmid.The expression of CTD-2270P14.5 after transfection was verified by q PCR,and the proliferation ability of breast cancer cells was detected by MTT colorimetry and clonogenesis assay.Transwell assay was used to detect the invasive ability of breast cancer cells.The senescence phenotype of breast cancer cells was detected by SA-β-Gal staining.The cell cycle distribution was detected by Flow Cyto Metry(FCM).6.q PCR and Western Blot were used to detect CDK6 expression levels in CTD-2270P14.5 overexpression group,interference group and control group.Results: 1.Gene Expression Profilling Interactive Analysis(GEPIA)database Analysis indicated that GRK4 was significantly lower in breast cancer tissues(p < 0.05).Kaplan-meier database suggested that breast cancer patients with high expression of GRK4 had better prognosis(p < 0.05).2.In the GRK4-mediated ce RNA network model,seven lnc RNAs were differentially expressed in GRK4(+)-CNE2 and GRK4(-)-CNE2 cells,among which the expression difference of CTD-2270P14.5 was most significant(p < 0.001).3.The expression level of GRK4 in breast cancer tissues was significantly lower than that in adjacent tissues(p < 0.01);The expression level of GRK4 in MCF-7 cells was significantly higher than that in MDA-MB-231 cells(p < 0.01).In ce RNA network model,seven lnc RNAs were differentially expressed in MCF-7 and MDA-MB-231 cell lines.The expression level of CTD-2270P14.5 in MCF-7 cells was significantly higher than that in MDA-MB-231 cells(p < 0.001).4.The interference efficiency of GRK4 in MCF-7 cells was about 70%(p < 0.001).The expression level of GRK4 in MDA-MB-231 cells of the overexpressed test group was significantly increased,about 3 times that of the control group(p < 0.001).q PCR results showed that compared with the control group,the expression level of CTD-2270P14.5 in MCF-7 cells in interference group(si-GRK4)was significantly decreased(p <0.001).The expression level of CTD-2270P14.5 in MDA-MB-231 cells of GRK4 overexpression group was significantly increased(p < 0.001).5.The knockdown efficiency of CTD-2270P14.5 in MCF-7 cells was about 58%(p <0.001).The expression level of CTD-2270P14.5 in the overexpressed test group was about 1.63 times of that in the control group(p < 0.001).Compared with the control group,the proliferation ability of MDA-MB-231 cells in CTD-2270P14.5 overexpression group was significantly decreased.The proliferation ability of MCF-7 cells was significantly enhanced in CTD-2270P14.5 interference group.The difference was statistically significant.Cell cycle results showed that overexpression of CTD-2270P14.5 resulted in increased percentage of G1 phase cells and cell cycle arrest.SA-β-Gal staining indicated that CTD-2270P14.5 induced phenotypic changes in MDA-MB-231 cells.The results of transwell invasion assay showed that overexpression of CTD-2270P14.5 inhibited the invasion ability of MDA-MB-231 cells.6.Compared with the control group,the m RNA and protein levels of CDK6 in CTD-2270P14.5 interference group were significantly up-regulated;The expression level of CDK6 in CTD-2270P14.5 overexpression group was significantly lower than that in control group.The difference was statistically significant.Conclusions: 1.GRK4 is differentially expressed in human breast cancer,and GRK4 positively regulates the expression of lnc RNA CTD-2270P14.5.2.CTD-2270P14.5 inhibited the proliferation and invasion of breast cancer cells and induced senescent phenotypic changes in breast cancer cells.3.GRK4 may regulate the proliferation and invasion ability of breast cancer cells through CTD-2270P14.5-CDK6 signaling pathway.