The Expression Of GRK4 In Human Breast Cancer And Its Effect On The Proliferation And Migration Of Breast Cancer Cells

Objective: To investigate the expression of G protein-coupled receptor kinases 4(GRK4)in breast cancer and its effects on the proliferation and migration of breast cancer cells.Methods: 1.p EGFP-GRK4α/β/γ/δ plasmids were constructed and transfected into HEK-293 T cells.GRK4(+)and GRK4(-)cells were separated by flow cytometry.The effects of GRK4 splicing variants on cell proliferation were detected by MTT assay.q RT-PCR was used to detect the differential expression of genes related to proliferation regulation in 78 genes of different GRK4 subtypes after sorting.2.The expression and prognostic relationship of GRK4 in human breast cancer and adjacent tissues were searched in the database.Western blot and immunohistochemistry were used to detect the expression and distribution of GRK4 in breast cancer and adjacent tissues.Total RNA was extracted from the tissue and reversed into c DNA,PCR amplified the c DNA into DNA and then sequenced.3.The expression of GRK4 in human breast cancer cells MCF-7 and MDA-MB-231 was detected.Silencing GRK4 gene in MCF-7 cells with high GRK4 expression and lentivirusmediated GRK4 overexpression experiments were performed on MDA-MB-231 cells with low GRK4 expression.MTT and clonogenesis assay were used to detect the effect of GRK4 on cell proliferation,and scratch assay was used to detect the effect of GRK4 on cell migration.4.The expression of GRK4 in MDA-MB-231 cells was observed after treated with 5-aza for 72 h.The methylation status of GRK4 promoter region in MDA-MB-231 cells was observed by MSP assay.Results:1.p EGFP-N1-GRK4α/β/γ/δ plasmids were successfully constructed and transfected into HEK-293 T cells.Fluorescence expression was observed 24 h later.HEK-293T-GRK4(+)and HEK-293T-GRK4(-)cells were sorted by flow cytometry.Over expression of GRK4α/β/γ/δ inhibited the proliferation of HEK-293 T cells(P<0.05).q RT-PCR results showed that compared with the negative control group,the m RNA levels of 11 growth-related genes CITED2,COL1A1,COL3A1,ING1,MAPK14,MORC3,PIK3 CA,PTEN,SPARC,TGFB1 and TP63 in GRK4α(+)cells were significantly increased(P<0.05).The m RNA levels of Cyclin A2,EGR1 and MYC were significantly decreased(P<0.05).The m RNA levels of CD44,CITED2,COL1A1 and PLAU were significantly increased in GRK4β(+)cells(P<0.05).The m RNA levels of Cyclin E1,CDK2,CDK4,CDK6,p21,EGR1,ETS2,HPRT1,MAP2K1,MAP2K3,MYC and TWIST1 were significantly decreased(P<0.05).The m RNA levels of ATM,CD44,p15,CITED2,COL1A1,GADD45 A,IGFBP3,IGFBP7 and SPARC genes in GRK4γ(+)cells were significantly increased(P<0.05).The m RNA levels of p57,EGR1,ETS2 and PCNA were significantly decreased(P<0.05).The m RNA levels of CALR,p19,IGFBP3,MDM2,MORC3,SPARC,TBX2,TERF2 and THBS1 in GRK4δ(+)cells were significantly increased(P<0.05).Cyclin B1,CD44,p21,p57,CHEK1,COL1A1,CREG1,EGR1,GSK3 B,HPRT1,MAP2K6,NBN,TP53BP1,TWIST1 the m RNA levels of 14 genes were significantly decreased(P<0.05).2.Database and Western Blot showed that the expression of GRK4 was significantly lower in breast cancer tissues(P<0.05).Immunohistochemical showed that GRK4 was expressed in the cytoplasm(membrane).GRK4δ was expressed in breast cancer and adjacent tissues by PCR amplification and sequencing.3.MTT and clonogenesis experiments showed that silencing GRK4 inhibited cell proliferation in MCF-7cells(P<0.05),overexpression of GRK4 in MDA-MB-231 cells inhibited cell proliferation(P<0.05),scratch test showed that overexpression of GRK4 inhibited cell migration(P<0.05).4.The expression level of GRK4 in MDAMB-231 cells increased with the increase of 5-aza concentration(P<0.05).MSP experiment showed that there were bands in all amplification with methylated primers,but no bands in non-methylated primers.Conclusions: 1.The four isoforms of GRK4 all had biological effects on inhibiting the proliferation of HEK293 T cells in vitro.2.GRK4 expression was significantly low in human breast cancer tissues,and it expressed GRK4δ subtype in breast cancer tissues.3.GRK4 promotes the proliferation of breast cancer MCF-7 cells(Luminal A type),but inhibits the proliferation of MDA-MB-231 cells(Triple negative type).4.Hypermethylation of the promoter region of GRK4 gene leads to its low expression in breast cancer MDA-MB 231 cells.This study provided valuable information for revealing the mechanism of GRK4 inhibiting the proliferation and migration of breast cancer cells and for further research on GRK4 anti-tumor.