A Novel Identified Long Intergenic Noncoding RNA, LINC01574, Contributes to Breast Cancer Deterioration via the Regulation of miR-6745/TTYH3 Axis

Abstract

Objective: Compelling evidence suggested that lncRNAs performed vital functions in the development of breast cancer (BC). The study intended to mine the functional roles of LINC01574 in BC and further excavated its underlying regulatory mechanism.

Methods: The expression and prognosis of LINC01574 in BC were detected by integrating analysis of data mining, bioinformatics, and RT-qPCR. Then, the effect of LINC01574 knockdown on BC cell growth and metastasis was evaluated in vitro and in vivo. Interactions between miR-6745 and LINC01574 or TTYH3 were revealed by both target prediction and dual luciferase reporter assay.

Results: Our data found that LINC01574 was markedly elevated in BC tissues and cells and was an independent prognostic risk factor for patients with BC. Further functional studies revealed that knockdown of LINC01574 remarkably inhibited the growth and metastasis of BC cells in vitro and in vivo. Mechanistically, LINC01574 competitively binds with miR-6745 to prevent the degradation of TTYH3, thereby promoting the development of BC.

Conclusion: Our results unmasked a novel LINC01574/miR-6745/TTYH3 regulatory axis in BC progression and suggested that LINC01574 might be a promising prognostic indicator and therapeutic target for patients with BC.

Figure 1

LINC01574 expression was elevated in BC and was positively related to adverse outcomes. (a) Volcano plots showed the differentially expressed lncRNAs between BC tumor and normal tissues. (b) Heat maps showed the OS-related differentially expressed lncRNAs. OS: overall survival; BC: breast cancer. (c) The transcription level of LINC01574 was tested via RT-PCR. (d) The quantitative determination of LINC01574 mRNA level in BC tumor tissues through using qRT-PCR. (e) Receiver operative characteristic of LINC01574. (f) The levels of LINC01574 in different BC tumor sizes, histological stages, and lymph node metastasis. (g) The distributions of LINC01574 in BC cells were analyzed by subcellular localization analysis. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 2

Knockdown of LINC01574 inhibited BC tumor growth in vitro and in vivo. (a) The mRNA levels of LINC01574 after transfection were tested by qRT-PCR. (b, c) The effects of shLINC01574 on the proliferation of BC cells were examined. (d, e) In the tumorigenesis model, the body weight and tumor size were detected. (f) Tumor volume and weight in the shNC group and shLINC01574 group were assessment. (g, h) H&E and Ki-67 staining in the tumor. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 3

Knockdown of LINC01574 inhibited BC tumor metastasis in vitro and in vivo. (a, b) After being transfected with shNC or shLINC01574, the migration and invasion capabilities were assessed. (c) EMT-related proteins were detected by western blot. (d, e) In the lung metastasis model, the body weight and tumor weight of nude mice were measured. (f, g) Lungs H&E staining.^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 4

miR-6745 regulated by LINC01574 inhibited BC cell proliferation, migration, and invasion. (a) The mRNA level of miR-6745, miR-633-5p, and miR-4291 was detected by qRT-PCR. (b, c) Relative expression of miR-6745 in BC cell tumor tissues. (d) After WT-LINC01574 or MUT-LINC01574 were cotransfected with miR-6745 mimics, the relative luciferase activity was evaluated. (e–i) After corresponding transfection, the proliferation, migration, and invasion abilities were detected. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 5

LINC01574 affected TTYH3 expression by regulation of miR-6745. (a) miRWalk, TargetScan7, and miRDB algorithm were used to predicted downstream target mRNAs with potential binding sites for miR-6745. (b) TCGA and CPTAC databases were used to analyze the expression of TTYH3 and survival in BC patients. (c) QRT-PCR quantitatively detected the transcriptional expression of TTYH3 in BC tumor tissues. (d) After corresponding transfection, the mRNA level of miR-6745 and TTYH3 and the protein level of TTYH3 were analyzed. (e) The interaction between miR-6745 and TTYH3 was verified through luciferase reporter assay. (f, g) After corresponding transfection, the mRNA level of miR-6745 and TTYH3 and the protein level of TTYH3 were analyzed. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 6

LINC01574 regulated BC cell proliferation, migration, and invasion by targeting miR-6745. (a, b) After corresponding transfection, the OD value and cloning numbers of BC cells were evaluated. (c, e) After corresponding transfection, the wound closure rates of BC cells were tested. (d, f) After corresponding transfection, the number of invasive BC cells was tested.^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 7

LINC01574 regulated BC cell proliferation, migration, and invasion by targeting TTYH3. (a, b) The transcription and translation levels of TTYH3 were quantitatively detected in BC cells. (c, d) After corresponding transfection, the OD value and cloning numbers of BC cells in different groups (shNC+mock, shLINC01574+mock, and shLINC01574+TTYH3) were evaluated. (e–h) After corresponding transfection, the migration and invasion capabilities of BC cells in different groups (shNC+mock, shLINC01574+mock, and shLINC01574+TTYH3) were evaluated. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.