MicroRNA-1225-5p behaves as a tumor suppressor in human glioblastoma via targeting of IRS1

Figures & data

Table 1 Clinicopathological features of 44 patients with glio-blastoma

Features	WHO I	WHO II	WHO III	WHO IV
No of patients	10	6	13	15
Mean age (years)	45.3	49.5	48.6	51.2
Gender
Male	6	3	7	9
Female	4	3	6	6
Surgery
Gross total resection	10	6	7	6
Partial resection	0	0	4	6
Biopsy	0	0	2	3
Adjuvant treatment
Radiotherapy	0	0	9	9
Chemotherapy	0	0	0	2
Radiotherapy and chemotherapy	0	0	4	4

Figure 1 miR-1225-5p is downregulated in glioblastoma and correlated with patient survival.

Notes: (A) The expression of miR-1225-5p in glioblastoma cell lines (A172, SW1783, U87, LN-229, SW1088, and T98G) and normal human astrocytes was measured by reverse transcription PCR (RT-PCR) and normalized against U6. (B) The expression of miR-1225-5p in five normal brain tissues and 44 glioblastoma samples was measured by RT-PCR and normalized against U6. (C) The expression of miR-1225-5p in stage I–II and III–IV glioblastoma tumors was measured by RT-PCR and normalized against U6. (D) The correlation between the miR-1225-5p level (low or high) with overall patient survival was assessed with Kaplan–Meier analysis. **P<0.01 vs astrocytes; **P<0.01 or ***P<0.001 vs indicated group.

Table 2 Correlation between miR-1225-5p expression level and clinicopathological characteristics in 44 patients with glioblastoma

Factor	Low-level miR-1225-5p expression (n=25)	High-level miR-1225-5p expression (n=18)	P-value
Gender			0.63
Male	13	12
Female	8	11
Age (median), in years			0.55
>48	10	11
<48	12	11
Histopathologic grade			0.023
Low grade (I+II)	5	11
High grade (III+IV)	20	8

Figure 2 Overexpression of miR-1225-5p suppresses the proliferation of glioblastoma cells in vitro and in vivo.

Notes: (A) The expression of miR-1225-5p in U87 and U87-1225 (stably expressing miR-1225-5p) cells was measured by reverse transcription PCR (RT-PCR) and normalized against U6. (B) U87 and U87-1225 cells were subjected to an MTT assay to assess cell proliferation at each time point. (C) Representative image of crystal violet-stained cell colonies analyzed by colony-formation assay at 14 days. (D) Quantification plots of (C). (E) Tumor-growth curves demonstrated that miR-1225-5p inhibited xenograft tumor growth in nude mice. (F) Relative tumor weight at 22 days after inoculation. **P<0.01 or ***P<0.001 vs U87 group.

Figure 3 Overexpression of miR-1225-5p suppresses migration and invasion of glioblastoma cells.

Notes: (A) Representative picture of U87 and U87-1225 cell migration was measured by scratch wound assay. (B) The wound-healing percentage is plotted in (A). (C) Representative image of U87 and U87-1225 cell invasion using Matrigel-based invasion assay. (D) The average number of invading cells of five randomly selected fields from each sample was calculated. **P<0.01 or ***P<0.001 vs U87 group.

Figure 4 Insulin receptor substrate 1 (IRS1) is a direct target of miR-1225-5p in glioblastoma.

Notes: (A) Protein expression in U87 and U87-1225 cells was measured by Western blotting. (B) The expression of IRS1 in U87 and U87-1225 (stably expressing miR-1225-5p) cells was measured by reverse transcription PCR (RT-PCR) and normalized against U6. (C) Top, wild-type (wt) miR-1225-5p target sequences of IRS1 3′-untranslated region (UTR) and mutant (mut) miR-1225-5p target sequences of IRS1 3′-UTR. Bottom, relative luciferase activity of wt or mut pGL3-IRS1-3′-UTR in U87 and U87-1225 cells. (D) The mRNA (top) and protein (bottom) expression level of IRS1 in glioblastoma cell lines (A172, SW1783, U87, LN-229, SW1088, and T98G) and normal human astrocytes was measured by Western blotting or RT-PCR. (E) Scatter plot of the RNA Sequence data (TCGA) of 63 samples from glioblastoma patients. The red line indicates an inverse correlation of expression between the samples for miR-1225-5p and IRS1 genes (R=−0.5132; P=2.248e-05). **P<0.01.

Figure S1 (A) The correlation between the miR-1225-5p level (low or high) with overall patient survival was assessed with Kaplan–Meier analysis in patients with grade I/II tumors. (B) The correlation between the miR-1225-5p level (low or high) with overall patient survival was assessed with Kaplan–Meier analysis in patients with grade III/IV tumors.

Figure S2 (A) The expression of miR-1225-5p in SW1088 and SW1088-1225 (stably expressing miR-1225-5p) cells was measured by reverse transcription PCR (RT-PCR) and normalized against U6. (B) SW1088 and SW1088-1225 cells were subjected to an MTT assay to assess cell proliferation at each time point. (C) Representative image of crystal violet-stained cell colonies analyzed by colony-formation assay at 14 days. (D) Quantification plots of (C). **P<0.01; ***P<0.001.

Figure S3 (A) Representative picture of SW1088 and SW1088-1225 cell migration was measured by scratch wound assay. (B) The wound healing percentage was plotted in (A). (C) Representative image of SW1088 and SW1088-1225 cell invasion using Matrigel-based invasion assay. (D) The average number of invading cells of five randomly selected fields from each sample was calculated. **P<0.01 vs SW1088 group.

Figure S4 (A) Western blotting of IRS1 in SW1088 cell transfected with si IRS1. (B) SW1088 si con and SW1088 si IRS1 cells were subjected to an MTT assay to assess cell proliferation at each time point. (C) Representative picture of SW1088 si con and SW1088 si IRS1 cell migration was measured by scratch wound assay. (D) The wound-healing percentage was plotted in (C). (E) Representative image of SW1088 si con and SW1088 si IRS1 cell invasion using Matrigel-based invasion assay. (F) The average number of invading cells of five randomly selected fields from each sample was calculated. **P<0.01 vs SW1088 si con group.