tsRNA-3043a intensifies apoptosis and senescence of ovarian granulosa cells to drive premature ovarian failure by targeting FLT1

Abstract

Premature ovarian failure (POF) represents the pathological aging of the ovary. The tRNA-derived small fragments (tsRNAs) play significant roles in diseases; however, whether tsRNAs are involved in POF remains unknown. The cell and mice models of POF were established, and the tsRNAs profile in the ovarian tissues of POF mice was revealed through sequencing. The functions of tsRNA-3043a and its target gene FLT1 in POF cells and mice were detected. POF mice were characterized by a decreased number of normal follicles, ovarian weight, SOD level, and serum contents of E2, LH, and FSH. A total of 81 tsRNAs were aberrantly expressed in the ovarian tissue of POF mice. The expression of tsRNA-3043a was up-regulated in POF mice. tsRNA-3043a mimics inhibited the proliferation and promoted apoptosis, lipid accumulation, and cellular senescence of ovarian granulosa KGN cells, as well as altered the transcriptome. tsRNA-3043a inhibitor had the opposite effect. tsRNA-3043a targets and binds to FLT1. Overexpression of FLT1 protected KGN cells from pathological aging. tsRNA-3043a promotes the progression of POF by inhibiting FLT1 in vitro and in vivo. tsRNA-3043a targets FLT1 and promotes apoptosis and senescence of ovarian granulosa cells, leading to the progression of POF. This study provides a new target for pharmacological intervention in POF.

Keywords: FLT1; Apoptosis and senescence; Oxidative stress; Premature ovarian failure; tsRNA-3043a.

Fig. 1

Ovarian pathological in POF mice. (A) The animal experimental flowchart. (B) HE staining was performed on ovarian tissues (n = 3) and the number of normal follicles was counted. Scale bar: 50 μm. (C) Ovarian weight (n = 8) determination. (D) Concentration of LH, E2, and FSH in serum of mice were detected by ELISA (n = 8). (E) SOD activity in ovarian tissue (n = 8) was detected by SOD activity detection kit. Scale bar: 100 μm

Fig. 2

Dysregulation of tsRNA expression profile in ovarian of POF mice. (A) Volcano plot of differentially expressed tsRNAs of mice between POF vs. NC. (B) The predicted target gene of differentially expressed tsRNAs (C) GO analysis of target gene of differentially expressed tsRNAs (D) KEGG pathway analysis of target gene of differentially expressed tsRNAs

Fig. 3

tsRNA-3043a promotes POF progression. (A) Detection of candidate tsRNA expression by qRT-PCR. (B) The overexpression of tsRNA-3043a in KGN cells was detected by qRT-PCR. (C) CCK8 assay was used to detect the effect of tsRNA-3043a mimics on the viability of POF model KGN cells. (D) Flow cytometry was utilized to assess the effect of tsRNA-3043a mimics on apoptosis of POF model KGN cells. (E) Oil red O and β-galactosidase staining was used to detect the effect of tsRNA-3043a on lipid accumulation and cell senescence in POF model KGN cells, respectively. Scale bar: 100 μm

Fig. 4

tsRNA-3043a inhibitor suppresses POF progression. (A) The knockdown of tsRNA-3043a in KGN cells by using tsRNA-3043a inhibitor was detected by qRT-PCR. (B) CCK8 assay was used to detect the effect of tsRNA-3043a inhibitor on the viability of POF model KGN cells. (C) Flow cytometry was utilized to assess the effect of tsRNA-3043a inhibitor on apoptosis of POF model KGN cells. (D) Oil red O and β-galactosidase staining was used to detect the effect of tsRNA-3043a inhibitor on lipid accumulation and cell senescence in POF model KGN cells, respectively. Scale bar: 100 μm

Fig. 5

tsRNA-3043a overexpression alters transcriptome of KGN cells. (A) Volcano map of DEGs in POF model KGN cells between tsRNA-3043a mimics and mimics NC groups. (B) Cluster heatmap of DEGs. (C) Bubble plot of GO enrichment of DEGs. (D) Bubble plot of KEGG enrichment of DEGs. (E) The network of tsRNA-3043a – target gene – pathways

Fig. 6

FLT1 inhibits POF progression. (A) qRT-PCR was used to validate the expression of DEGs. (B) Dual-luciferase reporter assay verified tsRNA-3043a binding to target gene FLT1. (C) The mRNA expression of FLT1 was detected by qRT-PCR. (D) The protein expression of FLT1 was detected by western blot. (E) Cell proliferation of KGN cells was detected by CCK8. (F) Apoptosis of KGN cells was detected by flow cytometry. (G) Oil red O staining was used to detect cellular lipid deposition. Cell senescence was detected by SA-β-Gal staining. Scale bar: 100 μm

Fig. 7

tsRNA-3043a induces POF by targeting FLT1 in KGN cells. (A) The mRNA expression of FLT1 in KGN cells was detected by qRT-PCR. (B) The protein expression of FLT1 in KGN cells was detected by western blot. (C) CCK8 was used to detect proliferation of KGN cells. (D) Apoptosis of KGN cells was detected by flow cytometry. (E) Oil red O staining was used to detect cellular lipid deposition. Cell senescence was detected by SA-β-Gal staining. Scale bar: 100 μm

Fig. 8

Rescue experiments using FLT1 knockdown in cell POF model upon tsRNA-3043a inhibitor. The mRNA (A) and protein (B) expression of FLT1 in KGN cells was detected by qRT-PCR and western blot, respectively. (C) CCK8 was used to detect proliferation of KGN cells. (D) Apoptosis of KGN cells was detected by flow cytometry. (E) Oil red O staining was used to detect cellular lipid deposition. Cell senescence was detected by SA-β-Gal staining. Scale bar: 100 μm

Fig. 9

tsRNA-3043a facilitates POF by inhibiting FLT1 in mice. (A) The animal experimental flowchart. (B) HE staining (n = 3) was used to observe ovarian tissue morphology and follicles. (C) Weighing of ovarian tissue (n = 5). (D) The level of SOD in mouse ovarian tissue (n = 5) was detected. (E) The levels of E2, FSH, and LH in serum of mice (n = 5) were detected by ELISA. (F) The expression of FLT1 in mouse ovarian tissue was detected by immunohistochemistry. Scale bar = 20 μm

Fig. 10

Diagram of the hypothesis mechanism of tsRNA-3043a facilitates POF by inhibiting FLT1