Long non-coding RNA BRM promotes proliferation and invasion of papillary thyroid carcinoma by regulating the microRNA-331-3p/SLC25A1 axis

Abstract

Long non-coding RNA BRM (lncBRM) was first identified in liver cancer stem cells and was reported to promote multiple cancer types. However, the function of lncBRM in papillary thyroid carcinoma (PTC) remains unclear. The primary focus of the present study was to determine the biological role of lncBRM in PTC. Reverse transcription-quantitative PCR assays revealed that lncBRM was upregulated in PTC tissues and cells. Cell Counting Kit-8, Transwell invasion and colony-formation assays were performed to assess cell proliferation, invasion and migration, respectively. Furthermore, high expression of lncBRM was associated with poor overall survival time in patients with PTC. lncBRM knockout significantly suppressed cell proliferation, migration and invasion. lncBRM was predicted to bind to microRNA (miR)-331-3p and targets SLC25A1. Overexpression of miR-331-3p or inhibition of SLC25A1 resulted in significantly suppressed proliferation, migration and invasion of PTC cells. Rescue assays demonstrated that inhibition of miR-331-3p significantly abrogated the effects of lncBRM knockout on PTC cell proliferation, migration and invasion. In conclusion, the present study suggests that lncBRM promotes PTC by regulating miR-331-3p and targeting SLC25A1.

Keywords: SLC25A1; long non-coding RNA BRM; micoRNA-331-3p; papillary thyroid carcinoma.

Figure 1.

lncBRM is upregulated in PTC tissues and cell lines and is associated with prognosis. (A) RT-qPCR was performed to examine the expression level of lncBRM in 90 pairs of PTC tissues and adjacent normal tissues. (B) RT-qPCR was performed to examine the relative expression levels of lncBRM in normal Nthy-ori3-1 cell lines and TPC-1 and SW1736 PTC cell lines. (C) Patients with PTC were divided into two groups, lncBRM high level (n=45) and lncBRM low level (n=45), to perform Kaplan-Meier analysis of overall survival time, followed by a log-rank test. (D) The expression levels of lncBRM in PTC tissues with metastasis (n=45) or no metastasis (n=45) were examined by RT-qPCR. (E) Expression levels of lncBRM in stage I–II (n=45) or stage III–IV (n=45) PTC tissues were determined using RT-qPCR. Fold changes were normalized to 18S in all cases. *P<0.05. RT-qPCR, revere transcription-quantitative PCR; lnc, long non-coding; PTC, papillary thyroid carcinoma.

Figure 2.

lncBRM knockout impairs the proliferative, migratory and invasive abilities of TPC-1 cells and SW1736 cells. (A) Expression levels of lncBRM were examined by RT-qPCR. Knockout efficiency of lncBRM was determined in TPC-1 and SW1736 cells. Fold changes were normalized to 18S. (B) Cell Counting Kit-8 assay was performed to examine the proliferation curves of TPC-1 and SW1736 cells following transfection with the negative control or shlncBRM. (C) Transwell assay was performed to determine the migration and invasive abilities of TPC-1 cells and SW1736 cells, following transfection with the negative control or shlncBRM. (D) Colony-formation assay was performed to determine the proliferative abilities of TPC-1 and SW1736 cells, transfected with negative control or shlncBRM. *P<0.05. RT-qPCR, revere transcription-quantitative PCR; lnc, long non-coding; PTC, papillary thyroid carcinoma; sh, short hairpin.

Figure 3.

lncBRM interacts with miR-331-3p. (A) Schematic representation of the predicted target site for miR-331-3p and lncBRM. (B) Luciferase reporter assay was performed in TPC-1 cells. Cells were co-transfected with the reporter plasmid (or the corresponding mutant reporter) and the indicated miRs. RT-qPCR was performed to examine the relative expression of miR-331-3p, following transfection with (C) negative control or shlncBRM plasmids and (D) negative control, miR-331-3p mimic or miR-331-3p inhibitor. (E) RT-qPCR was performed to examine the relative expression of lncBRM in cells transfected with negative control or ectopic miR-331-3p expression or inhibition of miR-331-3p. Fold changes were normalized to 18S for all RT-qPCR experiments. (F) Proliferative ability of TPC-1 and SW1736 cells, following transfection with the negative control, shlncBRM alone or with miR inhibitor, were determined through CCK-8 assays. *P<0.05. RT-qPCR, reverse transcription-quantitative PCR; lnc, long non-coding; sh, short hairpin; CCK-8, Cell Counting Kit-8; WT, wild type; MUT, mutant; miR, microRNA.

Figure 4.

lncBRM regulates PTC progression through sponging miR-331-3p and targeting SLC25A1. (A) Schematic representation of the predicted target site for miR-331-3p and SLC25A1. (B) Luciferase activity assay in the WT or MUT reporter containing SLC25A1 3′UTR when transfected with different vectors in TPC-1 cells. Cells were co-transfected with the reporter plasmid (or the corresponding mutant reporter) and the indicated miRs. The relative expression of SLC25A1 following (C) ectopic miR-331-3p expression or inhibition of miR-331-3p in TPC-1 cells and (D) ectopic miR-331-3p expression or knockout of lncBRM and inhibition of miR-331-3p in TPC-1 cells, examined by RT-qPCR. Samples were normalized to 18S. (E) The protein expression of SLC25A1 was examined by western blot analysis. Samples were normalized to β-actin. (F) The relative expression of SLC25A1 following knockout or overexpression of SLC25A1. Samples were normalized to 18S. (G and H) Migratory abilities of TPC-1 cells and SW1736 cells transfected with control and different plasmids, examined using transwell assays. *P<0.05. RT-qPCR, revere transcription-quantitative PCR; lnc, long non-coding; sh, short hairpin; WT, wild type; MUT, mutant; miR, microRNA; UTR, untranslated region, NS, not significant.