Long noncoding RNA FAM225A promotes the malignant progression of gastric cancer through the miR-326/PADI2 axis

Abstract

Gastric cancer (GC) is a global health problem and further studies of its molecular mechanisms are needed to identify effective therapeutic targets. Although some long noncoding RNAs (lncRNAs) have been found to be involved in the progression of GC, the molecular mechanisms of many GC-related lncRNAs remain unclear. In this study, a series of in vivo and in vitro assays were performed to study the relationship between FAM225A and GC, which showed that FAM225A levels were correlated with poor prognosis in GC. Higher FAM225A expression tended to be correlated with a more profound lymphatic metastasis rate, larger tumor size, and more advanced tumor stage. FAM225A also promoted gastric cell proliferation, invasion, and migration. Further mechanistic investigation showed that FAM225A acted as a miR-326 sponge to upregulate its direct target PADI2 in GC. Overall, our findings indicated that FAM225A promoted GC development and progression via a competitive endogenous RNA network of FAM225A/miR-326/PADI2 in GC, providing insight into possible therapeutic targets and prognosis of GC.

Fig. 1. FAM225A is upregulated in GC and predicts a poor prognosis.

A, B Analyzing the expression of FAM225A in unpaired and paired GC tissues by using TCGA database. C Relative mRNA expression of FAM225A in GC tissues according to GEO database. D, E The expression of FAM225A was detected in our own 68 paired GC tissues, GC cells and GES-1. F Survival analysis of the association between FAM225A level and the overall survival (OS) rate of GC patients. p values were obtained by Student’s t test (A–C, E) or Wilcoxon test (D). FC fold change, **p < 0.01, ***p < 0.001.

Fig. 2. FAM225A promotes GC cell proliferation, migration, and invasion and induces cell apoptosis.

A–D Measuring MKN45 or AGS cell proliferation ability when knockdown or overexpression of FAM225A through colony formation assays and the Edu assays (scale bar: 100 μm for Edu assay). E, F Flow cytometric analysis of MKN45 or AGS cells by downregulating or upregulating FAM225A expression to detect the cell apoptosis. G–J Wound healing assay and Transwell assay revealed that silencing FAM225A could dramatically attenuate MKN45 cells migration ability, while overexpression of FAM225A has the opposite effects in AGS cells (scale bar: 200 μm for transwell assay, 100 μm for wound healing assay). K, L Western blot analysis of the EMT-related molecules in GC cells when knockdown or overexpression of FAM225A. p values were obtained by Student’s t test. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. Knockdown of FAM225A inhibits GC tumor growth and metastasis in vivo.

A Tumor tissues harvested from shFAM225A and shNC groups of female nude mice. B Tumor volumes in both groups were measured every 5 days. C Tumor weight in different groups. D, E HE staining of specimen to show lung metastasis in shFAM225A and shNC groups (scale bar: 50 μm). p values were obtained by Student’s t test (C, E) or ANOVA (B). Data are presented as means ± SD. **p < 0.01, ***p < 0.001.

Fig. 4. FAM225A functions as a ceRNA and competitively absorbs miR-326 in GC.

A, B FISH and qRT-PCR analysis of the subcellular localization of FAM225A in gastric cancer cells (scale bar: 50 μm for FISH assay). C, D A dual-luciferase reporter assay suggested that the relative luciferase activity of the wild-type FAM225A was reduced by miR-326. E, F Relative expression of miR-326 in GC tissues according to TCGA database and our own 68 GC tissues. G The correlation between the expression of FAM225A and miR-326 was analyzed in 68 GC tissues. H Detecting miR-326 expression when knockdown or overexpression of FAM225A in MKN45 or AGS cells by using qRT-PCR. p values were obtained by Student’s t test. Data are presented as means ± SD. FC fold change, ***p < 0.001.

Fig. 5. The functional effects of FAM225A can be regulated by miR-326 in GC.

A, B Colony formation assays was used to detect the cell proliferation ability after transfecting MAN45 cells with negative control, shFAM225A, miR-326 inhibitor or shFAM225A + miR-326 inhibitor, and transfecting AGS cells with negative control, FAM225A, miR-326 mimics or FAM225A + miR-326 mimics. C–F The wound healing assays and transwell assays were used to detect the cell migration and invasion ability after transfecting GC cells (scale bar: 100 μm for wound healing assay, 200 μm for transwell assay). p values were obtained by Student’s t test. Data are presented as means ± SD. *p < 0.05, **p < 0.01.

Fig. 6. FAM225A modulates PADI2 expression by competitively binding miR-326.

A, B Relative expression of PADI2 in GC tissues according to TCGA database and our own 68 GC tissues. C–F Detecting PADI2 mRNA and protein expression when knockdown or overexpression of miR-326 in MKN45 or AGS cells by using qRT-PCR and western blot. G Predicting binding sites between PADI2 and miR-326. H, I The relative luciferase activity of the wild-type PADI2 was reduced by upregulating miR-326. J–M Detecting PADI2 mRNA and protein expression when MKN45 cells transfected with siNC + Anti-NC, Anti-NC + siFAM225A, siFAM225A + miR-326 inhibitor, or when AGS cells transfected with Vector + miR-NC, miR-NC + FAM225A, FAM225A + miR-326 mimics by using qRT-PCR and western blot. N, O The correlation between the expression of PADI2 and the expression of miR-326 or FAM225A was analyzed in 68 GC tissues. p values were obtained by Student’s t test. Data are presented as means ± SD. FC fold change, **p < 0.01, ***p < 0.001.

Fig. 7. The effect of FAM225A on GC cell proliferation, migration, and invasion could be reversed by PADI2.

A–D Detecting PADI2 mRNA and protein expression when MKN45 cells transfected with siNC + Vector, siFAM225A + Vector, siFAM225A + PADI2, or when AGS cells transfected with Vector + siNC, FAM225A + siNC, FAM225A + siPADI2 by using qRT-PCR and western blot. E–H Colony formation assay and edu assay demonstrated that knockdown of FAM225A mediated inhibition of MKN45 cell proliferation were partially rescued by overexpression of PADI2, and silencing PADI2 could partially counteract overexpression of FAM225A mediated promotion of AGS cell proliferation (scale bar: 100 μm for Edu). I–L Detecting the cell migration and invasion ability after transfecting GC cells through wound healing assays and transwell assays (scale bar: 200 μm for transwell assay, 100 μm for wound healing assay). M, N Western blot analysis of the EMT-related molecules in different treatment groups. p values were obtained by Student’s t test. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.