Cancer stem cell-like cells-derived exosomal lncRNA CDKN2B-AS1 promotes biological characteristics in thyroid cancer via miR-122-5p/P4HA1 axis

Abstract

Introduction: Here, the discussion focused on the function and possible mechanism of cancer stem cell-like cells (CSCs)-derived exosomal CDKN2B-AS1 in thyroid cancer.

Methods: Specifically, the bioinformatics analysis, dual-luciferase reporter assay and RT-qPCR were conducted to obtain the expression and regulation of CDKN2B-AS1, and the downstream miR-122-5p/P4HA1 axis. Exosomes were identified by transmission electron microscopy. The uptake of exosome by recipient cells was observed by PKH67 labeling. Functional experiments and western blot were adopted to detect the effects of exosomal CDKN2B-AS1/miR-122-5p/P4HA1 axis on thyroid cancer cells. Tumor xenograft and in vivo metastasis model combined with RT-qPCR, western blot and hematoxylin-eosin staining verified the role of CDKN2B-AS1.

Results: Exosomal CDKN2B-AS1 up-regulated P4HA1 expression through miR-122-5p. CDKN2B-AS1 and P4HA1 expressions were up-regulated, and miR-122-5p expression was down-regulated in thyroid cancer. Silent CDKN2B-AS1 reduced cell viability and stemness. CDKN2B-AS1 was found to be abundant in CSCs and CSCs-derived exosomes. Exosomal CDKN2B-AS1 silencing could transfer to thyroid cancer cells to elevate E-cadherin level, and diminish P4HA1, N-cadherin and Vimentin levels, thus impeding cell migration and invasion. MiR-122-5p inhibitor reversed the function of exosomal CDKN2B-AS1, while P4HA1 silencing attenuated the effect of miR-122-5p inhibitor. Exosomal CDKN2B-AS1 affected the growth and metastasis of thyroid cancer through the miR-122-5p/P4HA1 axis.

Conclusion: CSCs-derived exosomal CDKN2B-AS1 acts as an oncogene in thyroid cancer through miR-122-5p/P4HA1 axis.

Keywords: CDKN2B-AS1; Cancer stem cell-like cells; Exosomes; Long non-coding RNA; Thyroid cancer.

Fig. 1

CDKN2B-AS1 was highly expressed in thyroid cancer and affected cell viability and tumor sphere formation (A) StarBase (http://www.sysu.edu.cn) analyzed the expression of CDKN2B-AS1 in thyroid cancer.(B) The level of CDKN2B-AS1 in thyroid cancer cells was assessed by RT-qPCR.(C–D) Short hairpin RNA (shRNA)-targeted CDKN2B-AS1 plasmid was transfected into TPC-1 and SW579 cells, and the level of CDKN2B-AS1 was detected by RT-qPCR.(E–F) The effect of CDKN2B-AS1 silencing on the viability of thyroid cancer cells was tested by cell counting kit-8 (CCK-8).(G–H) Sphere-forming assay was performed to detect the effect of CDKN2B-AS1 silencing on the stemness of thyroid cancer cells. ∗∗∗p < 0.001 vs. Nthy-ori3-1; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. shRNA targeted NC (sh-NC).

Fig. 2

The characterization of exosomes and the expression of CDKN2B-AS1 in cancer stem cell-like cells (CSCs) and their exosomes.(A) Observation and identification of exosomes under a transmission electron microscope (TEM).(B–C) The expression of CDKN2B-AS1 in CSCs and their parental cells.(D–E) The expression of CDKN2B-AS1 in exosomes derived from CSCs.(F–G) CSCs-derived exosomes were co-cultured with TPC-1 and SW579 cells respectively to observe the localization of PKH67 (green fluorescent label) in the cells. ^ˆˆˆp < 0.001 vs. Control (TPC-1 or SW579 cells); ^&&&p < 0.001 vs. Control-EXO (exosomes derived from TPC-1 or SW579 cells).

Fig. 3

The effects of exosomes derived from CSCs with CDKN2B-AS1 silencing on the migration and invasion of thyroid cancer cells.(A–B) RT-qPCR detection found that sh-CDKN2B-AS1 reduced the level of CDKN2B-AS1 in exosomes.(C–D) RT-qPCR was performed to examine the effects of exosomes derived from CSCs with CDKN2B-AS1 silencing on the level of CDKN2B-AS1 in thyroid cancer cells.(E–F) Exosomes from CSCs transfected with sh-CDKN2B-AS1 reduced the migration of parental cells, which was tested by wound healing experiments.(G–H) Transwell assay tested the effects of exosomes derived from CSCs with CDKN2B-AS1 silencing on cell invasion in each group. ∗∗p < 0.01, ∗∗∗p < 0.001 vs. sh-NC-EXO (exosomes derived from TPC-1 or SW579 cells-CSCs transfected with sh-NC).

Fig. 4

CDKN2B-AS1 in exosomes targeted miR-122-5p/P4HA1 axis in recipient thyroid cancer cells.(A) The starBase and GSE113629 data were applied to analyze the miRNAs targeted by CDKN2B-AS1 and acting on thyroid cancer, the results of which showed the targeted binding sites of CDKN2B-AS1 and miR-122-5p.(B) The miRDB (http://mirdb.org/mirdb/index.html) and TargetScan database (http://www.targetscan.org/vert_72/) predicted the target genes of miR-122-5p, and the TargetScan database predicted the targeted binding sites of miR-122-5p and P4HA1.(C–D) The binding relationship between miR-122-5p with CDKN2B-AS1 and P4HA1 was verified by dual-luciferase reporter assay.(E–F) The expressions of miR-122-5p and P4HA1 in thyroid cancer cells were detected by RT-qPCR.(G–J) RT-qPCR was performed to detect the effects of exosomes derived from CSCs with CDKN2B-AS1 silencing on the expressions of miR-122-5p and P4HA1. ∗∗∗p < 0.001 vs. Nthy-ori3-1; ^###p < 0.001 vs. MC (mimic control); ^ˆˆˆp < 0.001 vs. sh-NC-EXO.

Fig. 5

The effects of CDKN2B-AS1/miR-122-5p/P4HA1 axis on the viability and migration of thyroid cancer cells.(A–D) RT-qPCR was used to detect the transfection efficiency of miR-122-5p inhibitor (I) and shRNA-targeted P4HA1 (sh-P4HA1), with U6 and GAPDH serving as internal controls.(E–F) After 24 or 48 h of co-culture of exosomes and recipient cells, the effect of CDKN2B-AS1/miR-122-5p/P4HA1 on cell viability was assessed by CCK-8 assay.(G–H) Cell migration in each group was measured by wound healing experiment. ∗∗∗p < 0.001 vs. IC (inhibitor control) + sh-NC; ^###p < 0.001 vs. IC + sh-P4HA1; ^ˆˆˆp < 0.001 vs. I + sh-NC; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.001 vs. control (No transfection); ^θp < 0.05, ^θθθp < 0.001 vs. sh-CDKN2B-AS1 (CSCs) (transfected into CSCs); ^△p < 0.05, ^△△p < 0.01, ^△△△p < 0.001 vs. I (transfected into recipient thyroid cancer cells); ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 vs. sh-P4HA1 (transfected into recipient thyroid cancer cells).

Fig. 6

CDKN2B-AS1/miR-122-5p/P4HA1 axis altered the thyroid cancer cell invasion and epithelial-mesenchymal transition (EMT)-related proteins.(A1, A2, B1, B2) MiR-122-5p inhibitor reversed the invasion of thyroid cancer cells inhibited by exosomes derived from CSCs with CDKN2B-AS1 silencing, and P4HA1 silencing reversed miR-122-5p inhibitor-promoted cell invasion as determined by Transwell.(C, C1-6) The expression changes of epithelial markers E-cadherin, mesenchymal markers N-cadherin and Vimentin were detected by western blot, with GADPH serving as the internal control. ^&&p < 0.01, ^&&&p < 0.001 vs. control (No transfection); ^θθθp < 0.001 vs. sh-CDKN2B-AS1 (CSCs); ^△△p < 0.01, ^△△△p < 0.001 vs. I; ^†††p < 0.001 vs. sh-P4HA1.

Fig. 7

The effect of CDKN2B-AS1 derived from CSCs in thyroid cancer was verified in vivo.(A–C) In vivo transplantation tumor experiment determined the effects of exosomes extracted from CSCs transfected with or without sh-CDKN2B-AS1 on the volume and weight of transplanted tumors.(D–E) The expressions of CDKN2B-AS1 and miR-122-5p in tumor tissues were evaluated by RT-qPCR.(F) An in vivo metastasis model was constructed, and hematoxylin and eosin (H&E)-stained lung tissues were used to detect the effect of CDKN2B-AS1 on metastatic nodules.(G) The expressions of P4HA1 and EMT-related proteins in tumor tissues of each group were detected by western blot. ∗∗∗p < 0.001 vs. Sham; ^##p < 0.01, ^###p < 0.001 vs. CSCs-shNC-EXO (exosomes derived from TPC-1 or SW579 cells-CSCs transfected with sh-NC).