LncRNA-SLC6A9-5:2: A potent sensitizer in 131I-resistant papillary thyroid carcinoma with PARP-1 induction

Abstract

Recent studies have indicated that long non-coding RNAs play crucial roles in numerous cancers, including thyroid cancer, while their function in the mechanism of thyroid cancer 131I resistance has not been elucidated to date. The present study identified a functional long non-coding RNA, SLC6A9-5:2, which was involved in the radioactive therapy resistance of thyroid cancer. We demonstrated that SLC6A9-5:2 was remarkably downregulated in 131I-resistant thyroid cancer cell lines and 131I-insensitive patients and was positively correlated with Poly (ADP-ribose) polymerase 1 (PARP-1) expression and its activation. After downregulating SLC6A9 or blocking PARP-1 artificially, the sensitive thyroid cancer cells mostly displayed a tolerant phenotype under 131I exposure. Furthermore, SLC6A9-5:2 overexpression was positively correlated with PARP-1 mRNA and protein levels, which restored the sensitivity of resistant thyroid cancer cells. The present study further revealed that cancer cell death was primarily caused by ATP exhaustion in excessive DNA repair with high PARP-1 activity. In patients with thyroid cancer, a positive correlation between SLC6A9-5:2 and PARP-1 was identified, and low SLC6A9-5:2 expression was associated with a worse prognosis of papillary thyroid carcinoma. Hence, our data provide a new lncRNA-mediated regulatory mechanism implying that SLC6A9-5:2 can be used as a novel therapeutic target for 131I-resistant thyroid cancer.

Keywords: 131I; PARP-1; SLC6A9-5:2; lncRNA; thyroid cancer.

Figure 1. The long-term sub-lethal dose of ¹³¹I exposure induces the tolerance of thyroid cancer cells

a. Treatment of the sub-lethal dose of ¹³¹I on continuously passaged TPC-1 and BCPAP thyroid cancer cell lines. ¹³¹I radioactivity was calculated with a half-time decay of 8.02 days, and the culture medium was changed every other day. ¹³¹I tolerance of cancer cells was significantly increased after 10 continuous passages. The 10th generation thyroid cancer cells were considered as ¹³¹I-resistant cells. b. Sensitive and resistant cell lines were treated with¹³¹I irradiation for 12 h, and apoptosis was measured by flow cytometry. c. Western blot analysis of the DNA damage marker γH2AX under ¹³¹I exposure. The data were from one representative experiment of three identically performed. The data were expressed as means±SD. * P<0.05;** P<0.01.

Figure 2. SLC6A9 was downregulated in resistant thyroid cancer cells and was correlated with ¹³¹I tolerance

a. Hierarchical clustering analysis around the differentially expressed lncRNAs among ¹³¹I-sensitive and -resistant cells; each group includes two independently cultured TPC-1 cell lines. b. Schematic representation of the SLC6A9 location and five distinct fragments of SLC6A9-1. c. The SLC6A9 expression in sensitive and resistant thyroid cancer cells was analyzed by qRT-PCR. d. TPC-1 growth curves with SLC6A9 RNAsi or blank interference (50 nM) transfection with ¹³¹I treatment (n= 6 wells per group). The cell absorbance was measured every day for 4 continuous days. e. Western blot analysis of γH2AX after SLC6A9 RNAsi transfection for 48h followed by ¹³¹I treatment for 12h. f. The apoptosis rate percentage of TCP-1 and BCPAP cells of SLC6A9 RNAsi-transfected and control groups as measured by flow cytometry. The data are from one representative experiment of three identically performed. The data are expressed as means±SD. ** P<0.01.

Figure 3. SLC6A9 overexpression leads to thyroid cancer cell sensitivity to ¹³¹I treatment

a. TPC-1 growth curves with SLC6A9 plasmid or blank transfection following ¹³¹I treatment, (n= 6 wells per group). The cell absorbance was measured every day for four continuous days. b. Thyroid cancer cell DNA damage marker γH2AX expression after SLC6A9 plasmid transfection by Western blotting. c. Immunofluorescence images showing γH2AX expression (green) in situ after SLC6A9 plasmid transfection under¹³¹I treatment with the quantification displayed on the right. The data are from one representative experiment of three identically performed. The data were expressed as means±SD. *P<0.05;** P<0.01.

Figure 4. SLC6A9 is positively correlated with PARP-1 expression, and PARP-1 inhibition restored the ¹³¹I tolerance of thyroid cancer cells

a. Illustration of the predicted target region of the PARP-1 promoter sequence with SLC6A9. b. PARP-1 protein was downregulated after transfection with SLC6A9 RNAsi (50nM). c. Survival curve of sensitive and resistant TPC-1 cells for 4 consecutive days of observation with PARP-1 inhibitor 3-AB treatment under ¹³¹I exposure by the MTT assay. 3-AB treatment made sensitive thyroid cancer resistant to ¹³¹I, while no difference was observed in the resistant group. d. DNA repair intensity-related protein PAR and γH2AX expression under ¹³¹I exposure with 3-AB treatment for 24 h. e. DNA repair intensity in the Ctrl, ¹³¹I and ¹³¹I+3-AB groups with double-labeled immunofluorescence of PAR and γH2AX. The nuclei were stained with DAPI. 3-AB significantly weakened DNA repair under ¹³¹I treatment. Co-localization is demonstrated in the merged image. Magnification:×400. The data were expressed as means±SD. *P<0.05;**P<0.01.

Figure 5. Fragment 1 of SLC6A9 (SLC6A9-1) regulates PARP-1 expression and activity

a. Illustration of the SLC6A9 structure and target region where SLC6A9 binds to the PARP-1 promoter. b. Luciferase activity assays for full-length SLC6A9 and its five segments within the PARP-1 promoter region. The wild-type SLC6A9 sequence was co-transfected with the predicted target sequence, respectively. Both SLC6A9-1 and full-length SLC6A9 enhanced the luciferase activity of the targeted reporter. There was no obvious difference in the luciferase activity for SLC6A9-2, -3, -4, and -5, and the randomly built negative control sequence reporter. The graphs represent data from 3 separate experiments and five identical wells. c. The overexpression of SLC6A9-1 sensitized thyroid cancer cells to ¹³¹I treatment (n=6). d. Western blot analysis indicated that the upregulation of SLC6A9-1 resulted in a significant increase of the DNA-repair associated proteins PARP-1, PAR and γH2AX with ¹³¹I treatment. The data were expressed as means±SD. *P<0.05;**P<0.01.

Figure 6. Upregulation of the SLC6A9-PARP-1 pathway enhanced the sensitivity to ¹³¹I treatment through energy exhaustion during excess DNA repair

a. The ATP/ADP ratio in TPC-1 cells under ¹³¹I exposure. The ATP and ADP content was calculated according to the standard curve previously drawn (n=3). b. The ATP/ADP ratio in the TPC-1 cell line treated with 3-AB and ¹³¹I. The energy consuming condition was detected every 12h using a detection kit. c. The ATP/ADP ratio with SLC6A9 and SLC6A9-1 transfection, which protected thyroid cancer cells from energy exhaustion. d. 3-AB reversed the energy exhaustion condition induced by SLC6A9 and SLC6A9-1 in res-TPC-1 cells. e. Supplementation with ATP (2mM) protected thyroid cancer cells from apoptosis in SLC6A9-transfected thyroid cancer cells. f, g. DNA repair was demonstrated in the SLC6A9 and SLC6AP+ATP groups with γH2AX observation. In the SLC6A9 and SLC6AP+ATP groups, DNA repair both remained at a high level and showed no difference. The data were expressed as means±SD. *P<0.05; **P<0.01.

Figure 7. Low SLC6A9 expression predicted a worse prognosis in ¹³¹I-treated patients

a. In a group of 52 patients receiving ¹³¹I treatment, qRT-PCR showed that high SLC6A9 had a higher disease-free rate compared with the low SLC6A9 group by Kaplan-Meier curve analysis. b. SLC6A9 expression was measured by qRT-PCR in thyroid cancer tissues and adjacent normal thyroid tissues. The results were expressed as relative Log² ratios. c. The relationship between PARP-1 and SLC6A9 expression was explored by Spearman's correlation in thyroid cancer specimens (r=0.715).