MicroRNA-320 inhibits invasion and induces apoptosis by targeting CRKL and inhibiting ERK and AKT signaling in gastric cancer cells

Figures & data

Figure 1 Expression pattern of miR-320 in gastric cancer tissue samples.

Notes: (A) Relative expression level of miR-320 in 33 cases of fresh gastric cancer tissues and paired normal tissues. The mean value of miR-320 in normal tissue (2.591±0.615) was higher than that in cancer tissue (1.353±0.761) (mean ± SD, P<0.05). (B) Box plot showed that median/highest/lowest miR-320 levels in fresh gastric cancer tissues were lower than those in corresponding normal tissues. (C) Correlation between miR-320 and CRKL mRNA in gastric cancer tissues using linear correlation.
Abbreviations: SD, standard deviation; miR-320, microRNA-320.

Table 1 Distribution of miR-320 in gastric cancer according to clinicopathological characteristics

Characteristics	Number of patients	miR-320 low expression	miR-320 high expression	P-value
Age, years				0.8812
<60	14	7	7
≥60	19	10	9
Gender				0.2006
Male	24	14	10
Female	9	3	6
Differentiation				0.6193
Poor–moderate	20	11	9
Well	13	6	7
Tumor invasion (T)				0.0488
T1+T2	11	3	8
T3+T4	22	14	8
Lymph node metastasis				0.3930
Absent	14	6	8
Present	19	11	8
TNM stage				0.0380
I	7	1	6
II	9	4	5
III	17	12	5

Abbreviation: tumor-node-metastasis.

Figure 2 Effects of miR-320 on cell proliferation and invasion in gastric cancer.

Notes: (A) Expression level of miR-320 in normal cell line GES-1 and three gastric cancer cell lines (AGS, SGC-7901, and BGC-823). miR-320 mimic upregulated miR-320 level in SGC-7901 cell line and its inhibitor downregulated miR-320 level in BGC-823 cell line. (B) CCK8 assay revealed that miR-320 mimic inhibited growth rate meanwhile miR-320 inhibitor accelerated growth rate. (C) miR-320 mimic decreased colony number of SGC-7901 cells. miR-320 inhibitor increased colony number of BGC-823 cells. (D) Transwell assay revealed that invasive cell number of miR-320 mimic group was lower than that in control. Invasive cell number of miR-320 inhibitor group was higher than that in control (*P<0.05).
Abbreviation: miR-320, microRNA-320.

Figure 3 miR-320 inhibits 5-fluorouracil resistance and downregulates cyclin D1, MMP9, Bcl-2, p-ERK, p-AKT, and CRKL expression.

Notes: (A) miR-320 mimic significantly downregulated SGC-7901 cell viability i at 48 and 72 h of 5-fluorouracil (5 μg/mL) treatment. miR-320 inhibitor showed the opposite effect by upregulating cell viability. (B) Western blot displayed that cyclin D1, MMP9, Bcl-2, p-ERK, and CRKL proteins of SGC-7901 cells were decreased with miR-320 mimic treatment. Cyclin D1, MMP9, Bcl-2, p-ERK, and CRKL proteins of BGC-823 cells were upregulated with miR-320 inhibitor transfection. Real-time RT-PCR displayed that mRNA levels of cyclin D1, MMP9, Bcl-2, and CRKL were decreased with miR-320 mimic treatment. miR-320 inhibitor showed the opposite effects (*P<0.05).
Abbreviation: miR-320, microRNA-320.

Figure 4 CRKL is a direct target of miR-320 in gastric cancer cells.

Notes: (A) Luciferase reporter plasmid with wild-type and mutant binding site was transfected into SGC-7901 cells with miR-320 mimic. When transfected with wild-type reporter, cells showed a lower luciferase activity after transfection with miR-320 mimic. When transfected with mutant site reporter, there was no significant change in activity. (B) CRKL siRNA in SGC-7901 cells decreased protein levels of cyclin D1, MMP9. In CRKL-depleted cells, the inhibiting effect of miR-320 on cyclin D1 and MMP9 was not significant compared with cell without CRKL siRNA. (C) CRKL transfection restored MMP9 and cyclin D1 status in cells transfected with miR-320 mimic. *P<0.05.
Abbreviation: miR-320, microRNA-320.

Figure S1 Effect of miR-320 in AGS cell line and role of CRKL in SGC-7901 cell line.

Notes: (A) Transfection of miR-320 mimic downregulated AGS cell proliferation rate. miR-320 also decreased AGS invading ability. (B) Transfection of pCMV6-CRKL plasmid upregulated proliferation and invasion ability of SGC-7901 cells (*P<0.05).