Long non-coding RNA LINC00616 promotes ferroptosis of periodontal ligament stem cells via the microRNA-370 / transferrin receptor axis

Figures & data

Table 1. Primer sequences

Gene	Primer
DPP10-A1	5’-CAGCCCTGATAGGCAGTAGAC-3’
	5’-TCATTGCCTTTGATCCTGCTGT-3’
RP11-1C8.7	5’-TGTTTTTCCCCAAGTGGCAC-3’
	5’-AGAGACATATCAGATTCCCCAATT-3’
GSE61474_XLOC_022835	5’-ACTTTTATGTGAAGCCCAGAGT-3’
	5’-TCCTGGTTGTTGCGGCTT-3’
RP11-142A22.4	5’-CTCTCCTGATGGTTGCACGA-3’
	5’-TGACAAAGCCAAGGCCAGAA-3’
G009665	5’-AGAACACCCCGATGACGGA-3’
	5’-GGCATCATTATGTACCCGGAAT-3’
MAFA-AS1	5’-GTGAATGAGGGCACAGCTCT-3’
	5’-CCCCAACTCCTCCCACTTTC-3’
LINC00616	5’-TGCAGCAGGCCACTATTTGA-3’
	5’-AGCCACTGAGATCAACTGCC-3’
G083982	5’-AGGTGAAGAGCACTACGAAGA-3’
	5’-CTACTATGACGCCACATGCCT-3’
RP4-660H19.1	5’-TAGGACCCTCCAAGCCAGG-3’
	5’-TTTCACAGGGAATGGTGGCC-3’
TRHDE-AS1	5’-CCTGAGAGCTTACTGCCACA-3’
	5’-CAACGCGCTCATCGAGAATG-3’
miR-370	5’-CTCACGGCCTGCTGGGGTG-3’
	5’-ACCTCAAGAACAGTATTTCCAGG-3’
TFRC	5’-TCGTGAGGCTGGATCTCAAAA-3’
	5’-CAATAGCCCAAGTAGCCAATCAT-3’
GAPDH	5’-AGCCACATCGCTCAGACAC-3’
	5’-GCCCAATACGACCAAATCC-3’
U6	5’-CTCGCTTCGGCAGCACA-3’
	5’-AACGCTTCACGAATTTGCGT-3’

Figure 1. LINC00616 was highly expressed in LPS-PG-treated PDLSCs with ferroptosis. (a) Expression of 10 lncRNAs in PDLSCs before and after LPS-PG treatment measured with qRT-PCR. (b) qRT-PCR analyses of LINC00616 expression in patients with periodontitis. (c) qRT-PCR analyses of LINC00616 expression in PDLSCs. (d-g) Levels of ROS, Fe²⁺, GSH, and MDA in PDLSCs.

*P < 0.05, **P < 0.01, ***P < 0.001, compared to the control and healthy groups. ^#P < 0.05, ^##P < 0.01, compared with the LPS-PG group.

Figure 2. Knockdown of LINC00616 promoted cell viability and inhibited ferroptosis of PDLSCs. (a) LINC00616 expression levels were detected by qRT-PCR in PDLSCs after transfection. (b) Cell viability is measured using an MTT assay after transfection in PDLSCs treated with LPS-PG. (c-f) Levels of ROS, Fe²⁺, GSH, and MDA in PDLSCs treated with LPS-PG after transfection. (g) Images and quantized bar chart of TUNEL stained cells. (h) Expression of GPX4, SLC7A11, and ACSL4 proteins detected by western blotting.

*P < 0.05, **P < 0.01, compared with si-nc and the control group. ^#P < 0.05, ^##P < 0.01, compared with the LPS-PG group. ^&P < 0.05, ^&&P < 0.01, compared with the LPS-PG + Ferrostatin-1 + si-nc group. The scale length is 200 μm.

Figure 3. LINC00616 sponged miR-370 in PDLSCs. (a) The binding sites between miR-370 and LINC00616 are predicted by StarBase. (b) The dual luciferase reporter assay confirmed that miR-370 is a target of LINC00616 in PDLSCs. (c-e) qRT-PCR analysis for miR-370 expression in periodontal ligament tissues and PDLSCs.

**P < 0.01, compared with the nc mimic, si-nc, healthy group, and control group.

Figure 4. Suppression of miR-370 reversed the effects of LINC00616 on cell viability, and ferroptosis in PDLSCs treated with LPS-PG. (a) qRT-PCR analyses of miR-370 expression levels. (b) Cell viability is measured using an MTT assay after cotransfection in LPS-PG treated PDLSCs. (c-f) Levels of ROS, Fe²⁺, GSH, and MDA in PDLSCs treated with LPS-PG after cotransfection. (g) Images and quantized bar chart of TUNEL stained cells. (h) Expression of GPX4, SLC7A11, and ACSL4 proteins detected by western blotting.

**P < 0.01, compared with nc inhibitor and control group. ^#P < 0.05, ^##P < 0.01, compared with the LPS-PG + si-LINC00616 + nc inhibitor group. The scale length is 200 μm.

Figure 5. TFRC was the target gene for miR-370. (a) Bioinformatics predicted the binding sites between miR-370 and TFRC. (b) A dual-luciferase reporter assay was performed to confirm the association between TFRC and miR-370. (c-e) qRT-PCR analysis for the expression of TFRC in periodontal ligament tissues and PDLSCs.

**P < 0.01, compared with nc mimic, si-nc, Healthy, and control group. ^##P < 0.01, compared with the nc mimic group.

Figure 6. Upregulated TFRC inhibited the effects of downregulated miR-370. (a) qRT-PCR analyses of TFRC expression levels. (b) Cell viability is measured using an MTT assay after cotransfection in PDLSCs treated with LPS-PG. (c-f) Levels of ROS, Fe²⁺, GSH, and MDA in PDLSCs treated with LPS-PG after cotransfection. (g) Images and quantized bar chart of TUNEL stained cells. (h) Expression of GPX4, SLC7A11, and ACSL4 proteins detected by western blotting.

**P < 0.01, compared with oe-nc and control group. ^#P < 0.05, ^##P < 0.01, compared with the LPS-PG + mimic + oe-nc group. The scale length is 200 μm.