Effect of lncRNA MALAT1 on the Granulosa Cell Proliferation and Pregnancy Outcome in Patients With PCOS

Figures

• 

  Figure 1. LncRNA MALAT1 is upregulated in granulosa cells of women with PCOS. (A) Graph showing the biotypes of RNA transcriptome sequencing profile. (B, C) Graph showing the whole differentially expressed genes. Among them, 48 were downregulated and 216 were upregulated. Red: upregulated genes; blue: downregulated genes. (D) Ten differentially expressed genes (DEGs) in RNA-seq. (E) Graph showing the DEG expression level in granulosa cells of women with or without PCOS (13 control patients and 16 PCOS patients) (*p < 0.05,**p < 0.05, ***p < 0.001). (F) Heatmap showing the hierarchical clustering of the most striking differentially expressed genes in patients with and without PCOS. Red: upregulated genes; green: downregulated genes. (G) Graph showing the expression levels of MALAT1 in a larger cohort (48 control patients and 48 PCOS patients) using RT-qPCR (***p < 0.001).

• 

  Figure 2. Knockdown of MALAT1 promoted KGN cell proliferation. (A) Graph showing the expression level of MALAT1 in KGN cells treated with three different siRNA sequences via RT-qPCR (* p < 0.05). (B) Graphs showing the proportion of proliferation cells via Edu assay in granulosa cells treated with the most efficient siRNA sequence in (A). The amount of cells was detected by staining with Hochest (blue), as the proliferated cells were detected by staining with EdU (red). The result was analyzed by fluorescence microscope. (C) Graph showing the percentages of EdU-positive cells (* p < 0.05). (D) Graph showing the viability of KGN cells treated with siRNA-MALAT-1 was measured by using Cell Counting Kit-8 at the indicated time points (* p < 0.05; *** p < 0.01).

• 

  Figure 3. AMH upregulated SMAD signal pathway and MALAT1 expression. (A) KGN cells were treated with a range of AMH dose (5 to 50 ng/ml) for 24 h. The expression level of MALAT1 was determined by RT-qPCR (* p < 0.05; *** p < 0.01). (B) Graph showing the expression level of MALAT was gradually increased in KGN cells treated with 20 ng/ml AMH and was determined by RT-qPCR (* p < 0.05; *** p < 0.01). (C) Representative Western blot images using anti-pSMAD 1/5, anti-SMAD1, and anti-SMAD5 antibodies on total protein lysates extracted from KGN cells treated with AMH (20 ng/ml) for 120 minutes. Quantitative analysis of protein bands was performed by ImageJ. * p < 0.05; ** p < 0.01. (D) Scheme graph of MALAT-induced proliferation decreased in granulosa cells. AMH upregulated the expression of MALAT via activing phosphorylated SMAD1/5, leading to suppress granulosa cell.

• 

  Figure 4. Correlations between MALAT1 expression and clinical parameters. (A) Graph showing correlation between the relative expression of MALAT1 in GCs of control patients and clinical parameters. (B) Graph showing correlation between the relative expression of MALAT1 in GCs of OCOS patients and clinical parameters. (C) ROC curve showing diagnosis value of MALAT in GCs for the positive clinical pregnancy in women without PCOS. (D) ROC curve showing diagnosis value of MALAT in GCs for the positive clinical pregnancy in women with PCOS.