The lncRNA ADAMTS9-AS2 Regulates RPL22 to Modulate TNBC Progression via Controlling the TGF-β Signaling Pathway

Abstract

Background: Long non-coding RNAs (lncRNAs) are key regulators of triple-negative breast cancer (TNBC) progression, but further work is needed to fully understand the functional relevance of these non-coding RNAs in this cancer type. Herein, we explored the functional role of the lncRNA ADAMTS9-AS2 in TNBC.

Methods: Next-generation sequencing was conducted to compare the expression of different lncRNAs in TNBC tumor and paracancerous tissues, after which ADAMTS9-AS2differential expression in these tumor tissues was evaluated via qPCR. The functional role of this lncRNA was assessed by overexpressing it in vitro and in vivo. FISH and PCR were used to assess the localization of ADAMTS9-AS2within cells. Downstream targets of ADAMTS9-AS2 signaling were identified via RNA pulldown assays and transcriptomic sequencing.

Results: The expression ofADAMTS9-AS2 was decreased in TNBC tumor samples (P < 0.05), with such downregulation being correlated with TNM stage, age, and tumor size. Overexpressing ADAMTS9-AS2 promoted the apoptotic death and cell cycle arrest of tumor cells in vitro and inhibited tumor growth in vivo. From a mechanistic perspective, ADAMTS9-AS2 was found to control the expression of RPL22 and to thereby modulate TGF-β signaling to control TNBC progression.

Conclusion: ADAMTS9-AS2 controls the expression of RPL22 and thereby regulates TNBC malignancy via the TGF-β signaling pathway.

Keywords: ADAMTS9-AS2; TGFb (transforming growth factor-beta); TNBC (Triple negative breast cancer); lncRNA; signaling pathway.

Figure 1

ADAMTS9‐AS2 is downregulated and associated with poor prognosis in TNBC patients. (A, B) The cluster heat maps and the volcano plot visualize the expression of lncRNA between TNBC tissues and adjacent non-tumor tissues. The red dots and green dots represent upregulated and downregulated LncRNAs with statistical significance, respectively. (C) GO pathway analysis of TNBC tissues. (D, E) Relative expression of ADAMTS9‐AS2 in 62 TNBC tissues and adjacent normal tissues. (F) Kaplan–Meier survival curve of patients with TNBC downloaded from TCGA database. (G) Relative expression of ADAMTS9‐AS2 in TNBC cell lines and normal breast cell line. (H, I) PCR product in agarose gel electrophoresis and the splicing site verified by DNA sequencing. ***P < 0.001.

Figure 2

ADAMTS9‐AS2 suppresses TNBC cell proliferation. a: qRT-PCR analysis of LncRNA ADAMTS9-AS2 expression in TNBC cells transfected with ADAMTS9-AS2 overexpression vector or NC. (B, C) Growth curves of cells transfected with indicated vectors were evaluated by CCK8 assays. (D, E) EdU assays were conducted in cells after transfection with LV ADAMTS9-AS2 or LV NC. (F, G) The cell cycle progression was analyzed by flow cytometry after indicated transfected. (H, I) Colony formation assays were executed to detect the proliferation of TNBC cells transfected with indicated vectors. (J, K) Western blot was used to detect the influence of ADAMTS9-AS2 on cell cycle markers. *P < 0.05, **P < 0.01.

Figure 3

ADAMTS9‐AS2 regulates TNBC cell invasion, metastasis, and cell cycle progression and inhibited Warburg effect. (A, B) Cell migration capacities were detected by wound healing assays after transfected with indicated vectors. (C, D) Cell migration and invasion abilities were determined by transwell assays after transfection. (E, F) Apoptosis rate of TNBC cells was analyzed by flow cytometry after LV ADAMTS9-AS2. (G) The expression levels of apoptosis-related and epithelial-mesenchymal transition process marker proteins were determined by western blot. (H, I) Production of lactate and ATP were examined in TNBC cells transfected with LV ADAMTS9-AS2 or LV NC as indicated. (J) related LDHA expression was detected by western blot. *P < 0.05.

Figure 4

Overexpression of ADAMTS9‐AS2 suppresses in vivo BC tumor growth. (A, B) Representative images of xenograft tumors of each group. (C) Growth curves of xenograft tumors which were measured every 5 days. (D) Tumor weights from two groups are represented. (E) qRT-PCR detected relative expression in two groups. (F) IHC staining was applied to analyze the protein levels of Ki67, MMP9 and cleaved-caspase 3. **P < 0.01.

Figure 5

ADAMTS9-AS2 interacts with RPL22 in TNBC cells to suppress tumor progression. (A) FISH analysis of the location of ADAMTS9-AS2 in the cytoplasm and nuclear fractions of MDA-MB-231 cells. (B) qRT-PCR was used to detect ADAMTS9-AS2 subcellular fractionation. (C) RNA pull-down assay indicated that ADAMTS9-AS2 interacted with RPL22. (D) Western blot proved the relationship between ADAMTS9-AS2 interacted with RPL22. (E) Relative expression of RPL22 in TNBC tissues (Tumor) compared with normal tissue (normal) was analyzed using TCGA data. (F) Kaplan-Meier survival analysis of overall survival based on TCGA data. (G) Relative expression of RPL22 in TNBC tissues (Tumor) and adjacent non-tumor tissues (Normal) was determined by qRT-PCR (n = 62). (I) IF was used to demonstrate the relative expression of RPL22 in TNBC tissues. (J) Relative expression of RPL22 in xenograft tumors of each group was determined by qRT-PCR. (K) Spearman–Pearson correlation of ADAMTS9-AS2 and RPL22. **P < 0.01, ***P < 0.001.

Figure 6

ADAMTS9-AS2 regulates RPL22 to control TNBC progression through RPL22. (A–D) The cell proliferation was determined after transfection with LV ADAMTS9-AS2, ADAMTS9-AS2+siRPL22 and LV NC by CCK-8 and Colony formation assay. (E, F) The cell migration was determined after transfection by Transwell assay. *P < 0.05, **P < 0.01.

Figure 7

ADAMTS9-AS2 regulates the TGF-β signaling pathway to control TNBC progression. (A) The cluster heat maps displayed the 20 most representative differentially expressed mRNAs with transcriptomic sequencing. (B) KEGG pathway analysis of differentially expressed mRNAs. (C, D) Western blot assay determined the total and active protein level of TGF-β signaling pathway related proteins in MDA-MB231 cell lines. (E) Summary of the mechanism of ADAMTS9-AS2 in TNBC cell lines. **P < 0.01.