TMPO-AS1 promotes cell proliferation of thyroid cancer via sponging miR-498 to modulate TMPO

Abstract

Background: Thyroid cancer (TC) is the most frequent endocrine malignancy. Long noncoding RNAs (lncRNAs) have been confirmed to act as significant roles in tumor development. The role of lncRNA TMPO-AS1 in TC is still unclear, so it remains to be explored. The aim of the research is to investigate the role and regulatory mechanism of TMPO-AS1 in TC.

Methods: TMPO-AS1 and TMPO expression in TC tumors and cells was detected by TCGA database and QRT-PCR assay respectively. CCK-8, EDU, TUNEL and western blot assays were conducted to identify the biological functions of TMPO-AS1 in TC. Luciferase reporter and RNA pull down assays were conducted to measure the interaction among TMPO-AS1, TMPO and miR-498.

Results: TMPO-AS1 was overexpressed in TC tissues and cell lines. Knockdown of TMPO-AS1 suppressed cell growth and accelerated cell apoptosis in TC. Furthermore, downregulation of TMPO-AS1 suppressed TMPO expression in TC. The data suggested that TMPO expression was upregulated in TC tissues and cell lines and was positively correlated with TMPO-AS1 expression in TC. Furthermore, the expression of miR-498 presented low expression in TC cells. And miR-498 expression was negatively regulated by TMPO-AS1, meanwhile, TMPO expression was negatively regulated by miR-498 in TC cells. Besides, it was confirmed that TMPO-AS1 could bind with miR-498 and TMPO in TC cells. In addition, it was validated that TMPO-AS1 elevated the levels of TMPO via sponging miR-498 in TC cells.

Conclusions: TMPO-AS1 promotes cell proliferation in TC via sponging miR-498 to modulate TMPO.

Keywords: TMPO; TMPO-AS1; Thyroid cancer; miR-498.

Fig. 1

The expression of TMPO-AS1 is significantly upregulated in TC tissues and cells and facilitates TC cellular processes. a TMPO-AS1 expression was upregulated in TC tissues from TCGA database. b QRT-PCR assay was utilized to detect the expression of TMPO-AS1 in TC cell lines (A-PTC, CUTC5, TPC-1 and IHH-4) and human normal thyroid epithelial cell line (Nthy-ori3-1). c The knockdown efficiency of TMPO-AS1 was assessed by QRT-PCR assay in TPC-1 and IHH-4 cells transfected with sh-TMPO-AS1#1/2 or sh-NC. d, e The proliferation ability of transfected cells was measured by EdU assays and colony formation assay. f, g TUNEL and cytometry analysis was conducted to evaluate cell apoptosis rate in transfected cells. h, i Migration and invasion were examined in different groups in transwell assays. **p < 0.01

Fig. 2

TMPO-AS1 positively regulates TMPO in TC. a The location of TMPO-AS1 in chromatin was displayed. b TMPO was upregulated in TC tissues from TCGA database. c There was a positive correlation between TMPO-AS1 and TMPO from GEPIA database. d The expression of TMPO in TC cell lines and human normal thyroid epithelial cell line was detected by QRT-PCR. e QRT-PCR was performed to examine the mRNA expression of TMPO in transfected cells. f Luciferase reporter assay was used to detect the interaction between TMPO-AS1 and TMPO promoter. g It was tested by FISH and subcellular fractionation assays that TMPO-AS1 mainly located in cytoplasm. h RIP assays demonstrated TMPO-AS1 and TMPO were enriched in Ago2 antibody. *p < 0.05, **p < 0.01

Fig. 3

MiR-498 is downregulated and negatively regulated by TMPO-AS1 as well as represses the growth of TC. a Target genes that could bind with TMPO-AS1 were obtained from starBase and DIANA tools database. b RNA pull down assays selected out miRNAs to bind with TMPO-AS1. c MiR-498 had a binding site for TMPO-AS1. d MiR-498 had a binding site for TMPO. e The expression of miR-498 in TC cell lines and human normal thyroid epithelial cell line was detected by QRT-PCR. f QRT-PCR was used to examine the expression of TMPO in TPC-1 and IHH-4 cells transfected with NC mimics or miR-498 mimics. g QRT-PCR assays were applied to measure the mRNA expression of TMPO in transfected cells. h–m The effects of miR-498 on TC growth were assessed by functional assays. **p < 0.01

Fig. 4

TMPO facilitates the biological functions of TC cells. a The interaction among miR-498, TMPO-AS1 and TMPO was testified by RIP assay. b Luciferase reporter assay was used to confirm that miR-498 bound with TMPO-AS1 or TMPO. c RNA pull-down assay was utilized to validate that miR-498 could bind with TMPO-AS1 or TMPO. d The mRNA expression of TMPO was detected by QRT-PCR in transfected cells. e The efficiency of sh-TMPO#1/2 was appraised by QRT-PCR. f–k Functional assays evaluated the influence of TMPO. **p < 0.01. n.s.: no significance

Fig. 5

TMPO-AS1 contributes to the progression of TC via sponging miR-498 to upregulate TMPO. a TMPO expression was assessed by QRT-PCR in cells with pcDNA3.1/TMPO. b, c EdU assays and colony formation assays were used to evaluate the proliferative ability in transfected cells. d, e The apoptosis of transfected cells was measured by TUNEL assay and flow cytometry analysis. f, g Migration and invasion were appraised in cells transfected with sh-TMPO-AS1#1. **p < 0.01. n.s.: no significance

Fig. 6

TMPO-AS1 promotes TC cell growth in vivo. a Tumors resected from mice injected with TPC-1 cells stably transfected with sh-NC or sh-TMPO-AS1#1. b Tumor volume and weigh in two different groups were shown. c TMPO-AS1 expression in 40 pairs of TC tissues and adjacent normal tissues. d TMPO expression in paired tissues collected from 40 TC patients. e Pearson correlation test about TMPO-AS1 and TMPO expression in TC tissues. *p < 0.05, **p < 0.01