The lncRNA Gm8097 is associated with hypospermatogenesis

Abstract

Objective: To investigate whether long non-coding RNA (lncRNA) Gm8097 (LncGm8097) is associated with male infertility.

Methods: The expression and bilogical role of LncGm8097 were investigated.

Results: LncGm8097 expression was down-regulated in the testis tissues with moderate and severe hypospermatogenesis compared with those with normal spermatogenesis and mild hypospermatogenesis (p<0.05). LncGm8097 down-regulation significantly promoted apoptosis and inhibited proliferation in GC1 and GC2 cells. In addition, LncGm8097 was significantly down-regulated in mouse model of hypospermatogenesis and correlated with cell apoptosis and proliferation. LncGm8097 was located immediately upstream of PRPS2, and correlated with Bcl-2/P53/caspase 6/caspase 9 signal pathway.

Conclusion: LncGm8097 down-regulation correlates with hypospermatogenesis, which may offer new insights into the pathogenesis of male infertility.

Keywords: Infertility, male; Oligospermia; PRPS2; RNA, long noncoding; Spermatogenesis.

Figure 1.

Long non-coding RNA (lncRNA) Gm8097 (lncGm8097) was identified as a nearby lncRNA of phosphoribosyl-pyrophosphate synthetase 2 (PRPS2) associated with male infertility. (A) An lncRNA expression microarray was performed in GC1 cells with PRPS2 overexpression and controls. (B) The expression of lncGm8097 is shown by a heat map. (C) Quantitative real-time polymerase chain reaction (qRT-PCR) indicated that lncGm8097 was downregulated in human testicular biopsy tissues with hypospermatogenesis. (D) The expression of lncGm8097 was detected in mouse germ cells by qRT-PCR. ^a)p<0.05 compared with normal spermatogenesis and mild hypospermatogenesis group; ^b)p<0.01 compared with normal spermatogenesis and mild hypospermatogenesis; ^c)p<0.05 compared with the GC1 group.

Figure 2.

Long non-coding RNA (lncRNA) Gm8097 (lncGm8097) was found to regulate cell apoptosis and proliferation. (A) LncGm8097 knockdown and overexpression were validated in GC1 and GC2 cells by quantitative real-time polymerase chain reaction. (B) Flow cytometry assay showing that lncGm8097 downregulation promoted cell apoptosis. (C) 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay showing that lncGm8097 downregulation inhibited proliferation in GC1 and GC2 cells. NC, negative control; PI-A, propidium iodide-apoptosis; FITC-A, fluorescein isothiocyanate-apoptosis. ^a)p<0.05 compared with the NC group.

Figure 3.

Long non-coding RNA (lncRNA) Gm8097 (lncGm8097) downregulation was correlated with hypospermatogenesis. (A) Increased apoptosis and proliferation inhibition were observed in testis tissues with hypospermatogenesis. Caspase 3 and Ki-67 is immunohistochemical staining and each panel's magnification is ×40. (B) LncGm8097 downregulation was observed in a mouse model of hypospermatogenesis by quantitative real-time polymerase chain reaction. (C) Statistical analysis of the immune expression levels of caspase 3 and Ki-67 in testis tissues. ^a)p<0.05 compared with the normal spermatogenesis group; ^b)p<0.01 compared with the normal spermatogenesis group.

Figure 4.

Long non-coding RNA (lncRNA) Gm8097 (lncGm8097) regulated phosphoribosyl-pyrophosphate synthetase 2 (PRPS2) and was correlated with the B-cell lymphoma 2 (Bcl-2)/P53/caspase 6/caspase 9 signal pathway. (A) LncGm8097 positively regulated PRPS2 by quantitative real-time polymerase chain reaction. (B) The luciferase activity of PRPS2 was significantly higher in cells with lncGm8097 overexpression according to a dual luciferase reporter gene assay. (C) The expression of PRPS2, Bcl-2, P53, caspase 6, and caspase 9 was determined using Western blot assays. (D) Statistical analysis of the expression levels of PRPS2, Bcl-2, P53, caspase 6, and caspase 9. NC, negative control; WT, wild-type; MUT, mutant; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ^a)p<0.05 compared with the NC group.