Mechanism Study Of The Subcellular Distribution Of LncRNA CCAT2 And Its Effects On The Function In Regulating Luminal Subtype Of Breast Cancer

ObjectiveTo explore the expression characteristics,distribution of substructure and biological function of long non-coding RNA CCAT2 in Luminal subtype breast cancer,and to clarify the molecular mechanism of CCAT2 regulating the phenotype of Luminal subtype breast cancer,and to reveal the potential of targeting CCAT2 to inhibit the occurrence and development of breast cancer.Methods1)The expression level of CCAT2 in Luminal subtype breast cancer cell lines MCF-7,T47 D and patient samples were detected by qPCR;Western blot was used to detect the protein expression level of related genes.2)By analyzing the clinical sample information of breast cancer patients of Luminal subtype in TCGA database,to verify the expression characteristics of CCAT2 in large samples of breast cancer of Luminal subtype and the correlation between its expression level and patient survival rate.3)The overexpression system of CCAT2 in breast cancer cells was established by transient transfection of pcDNA3.1(+)-CCAT2 vector and stable transfection of pMX-CCAT2 vector.4)The effects of CCAT2 on the proliferation of MCF-7 and T47 D cells were determined by CCK8,cloning formation and other experiments.5)The effect of CCAT2 on breast cancer stem cells was detected by ALDH flow cytometry and mammosphere assay.6)The substructure distribution of CCAT2 in breast cancer cells was determined by cytoplasmic nuclear separation.7)By transplanting breast cancer cells into the fat pad of immune deficient female nude mice,a breast cancer mouse model was established to carry out in vivo research.ResultsIn this study,we found that the expression of long non-coding RNA(Lnc RNA)CCAT2 was down regulated in Luminal subtype breast cancer,mainly distributed in the cytoplasm,and played an anti-tumor effect;However,CCAT2 is mainly distributed in the nucleus of triple negative breast cancer,inducing breast cancer stem cells to play a cancer promoting effect.Using pcDNA3.1(+)-CCAT2 vector to transiently overexpress CCAT2 in Luminal subtype breast cancer cell lines MCF-7 and T47 D,most of the exogenous CCAT2 can be distributed in the cytoplasm.It was found that through RNA sequence complementation,sponge miR-221/222 in the cytoplasm,thereby promoting the expression level of p27 in cancer cells,inhibiting the proliferation of cancer cells in vitro,and transplanting tumor growth in vivo;Using pMX-CCAT2 vector to overexpress CCAT2 in Luminal subtype breast cancer cell line,CCAT2 can be enriched in the nucleus,and it is found that it promotes the expression of OCT4-PG1,a pseudogene of OCT4,and then induces the stemness acquisition of tumor cells,which is manifested by the up-regulated expression of stem genes and the increased proportion of ALDH+ breast cancer stem cells.Conclusions1)Different from triple negative breast cancer,the expression of CCAT2 in breast cancer patients of Luminal subtype decreased,and its expression level was positively correlated with the survival rate of patients.2)The function of CCAT2 in regulating breast cancer is closely related to its subcellular localization in cancer cells.3)Overexpression of CCAT2 in breast cancer cells of Luminal subtype can inhibit the proliferation of cancer cells,and its distribution in the cytoplasm can promote the stemness of cancer cells.4)The cytoplasmic distribution of CCAT2 in breast cancer of Luminal subtype regulates the miR-221-p27 signaling pathway through RNA sponge,and inhibits the proliferation and stemness of breast cancer cells.5)The CCAT2 of Luminal subtype breast cancer cell nuclear distribution promotes breast cancer cell stemness by upregulating OCT4-PG1.