Expression of cyclic RNA plasmacytoma variant translocation 1
in senescence of nucleus pulposus cells and its regulation mechanism

doi:10.3969/j.issn.2095-4344.2557

Abstract

Abstract:

BACKGROUND: Cyclic RNA plasmacytoma variant translocation 1 (circPVT1) is involved in the senescence of fibroblasts, but the relationship of circPVT1 with nucleus pulposus senescence and its mechanism are still unclear.

OBJECTIVE: To investigate the expression of circPVT1 in nucleus pulposus cell senescence and to explore its possible mechanism.

METHODS: Human nucleus pulposus cells were cultured in vitro, and the senescence of nucleus pulposus cells was induced by ionizing radiation (5 Gy, 6 days). The expression of circPVT1 and let-7 mRNA was detected by real-time quantitative polymerase chain reaction (qRT-PCR). CircPVT1 siRNA and anti-let-7 were transfected into normal nucleus pulposus cells, which were divided into control group, si-NC+anti-NC group, si-circPVT1+anti-NC group, si-NC+anti-let-7 group, and si-circPVT1+anti-let-7 group. The expressions of circPVT1 and let-7 mRNA were detected by qRT-PCR. Cell counting kit-8 assay was used to detect the inhibition of cell proliferation. Plate cell clone formation assay was used to detect colony formation. Cell senescence was detected by SA-β-gal staining. The expressions of p21, p27, let-7 target high mobility group protein A2 (HMGA2) and KRAS were detected by western blot assay. Double luciferase activity assay was used to verify the relationship between let-7 and target regulation of HMGA2 and KRAS.

RESULTS AND CONCLUSION: (1) Compared with normal nucleus pulposus cells, the expression of circPVT1 was decreased, while let-7 expression and the positive rate of SA-β-gal staining were increased in the irradiated cells (P < 0.05). (2) Compared with the control group and si-NC+anti-NC group, the si-circPVT1+anti-NC group appeared to have decreased expression of circPVT1 mRNA, HMGA2 and KRAS proteins and number of clones formed as well as increased let-7 mRNA expression, p21, p27 protein expression, cell inhibition rate and positive rate of SA-β-gal staining (P < 0.05). However, opposite changes were found in the si-NC+anti-let-7 group in relative to the control group (P < 0.05). (3) The expression of circPVT1 mRNA, clone formation, and expressions of HMGA2 and KRAS proteins in the si-circPVT1+anti-let-7 group were higher than those in the si-circPVT1+anti-NC group, and lower than those in the si-NC+anti-let-7 group. Let-7 mRNA expression, cell inhibition rate, positive rate of SA-β-gal staining, and expressions of p21 and p27 proteins in the si-circPVT1+anti-let-7 group were lower than those in the si-circPVT1+anti-NC group, and higher than those in the si-NC+anti-let-7 group (P < 0.05). Double luciferase activity assay showed that HMGA2 and KRAS were the targets of let-7. These findings indicate that inhibition of circPVT1 can inhibit the aging of nucleus pulposus cells. The mechanism may be through binding let-7 to inhibit the targeting of HMGA2 and KRAS proteins.

Key words: nucleus pulposus cells, aging, cyclic RNA plasmacytoma variant translocation 1, let-7, HMGA2, KRAS

CLC Number:

Xie Mingzhong, Li Sen, Zhu Kai, Jiang Huacai, Qi Lisheng. Expression of cyclic RNA plasmacytoma variant translocation 1 in senescence of nucleus pulposus cells and its regulation mechanism[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(17): 2718-2723.

Figures/Tables 11

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