SPINT1-AS1 promotes oxidative damage and apoptosis of gastric cancer cells via the miR-656-3p/PLCXD3 axis

Abstract

Background and objectives: In the study of gastric cancer (GC), long non-coding RNAs (lncRNAs) have been identified and their functions have been partly characterized; however, the specific function of SPINT1 antisense RNA 1 (SPINT1-AS1) in GC remains unclear. This study aimed to investigate the role of SPINT1-AS1 in GC cells and elucidate its downstream molecular mechanisms.

Methods: SPINT1-AS1, microRNA-656-3p (miR-656-3p), and phospholipase C, X domain containing 3 (PLCXD3) levels were modulated in GC cells through transfection experiments. Quantitative reverse transcription polymerase chain reaction and Western blot analyses were performed to assess SPINT1-AS1, miR-656-3p, and PLCXD3 levels. The proliferative capacity, apoptosis, invasion, migration, and oxidative stress levels of GC cells were evaluated using 5-ethynyl-2'-deoxyuridine assay, colony formation assay, flow cytometry, Transwell assays, and commercial kits, respectively. Dual-luciferase reporter assay and RNA immunoprecipitation assay were conducted to assess the targeting relationships among SPINT1-AS1, miR-656-3p, and PLCXD3. The impact of SPINT1-AS1 on GC tumor growth was examined in xenograft tumor models.

Results: SPINT1-AS1 and PLCXD3 were found to be downregulated in GC, whereas miR-656-3p was upregulated. SPINT1-AS1 elevation inhibited GC cell proliferation, invasion, migration, and oxidative stress, and promoted apoptosis. SPINT1-AS1 knockdown had opposite effects. The pro-tumor effects induced by SPINT1-AS1 knockdown were reversed by concomitant knockdown of miR-656-3p. Similarly, the inhibitory effects of SPINT1-AS1 elevation on GC malignancy were abrogated by PLCXD3 knockdown. SPINT1-AS1 knockdown suppressed GC tumor growth in mice. SPINT1-AS1 competitively bound to miR-656-3p to mediate PLCXD3 expression.

Conclusions: SPINT1-AS1 suppresses GC malignancy through the regulation of the miR-656-3p/PLCXD3 axis. These findings provide robust data supporting the biological functions of lncRNAs in GC and offer potential targets for therapeutic intervention.

Keywords: Gastric cancer; Oxidative damage; PLCXD3; SPINT1-AS1; miR-656-3p.

Fig. 1

Abnormally low expression of SPINT1-AS1 in GC. A RT-qPCR measured SPINT1-AS1 in 5 GC cell lines and normal gastric mucosa cell line. B FISH assay analyzed the subcellular localization of SPINT1-AS1. C The website (http://lncatlas.crg.eu/) queried the subcellular location of SPINT1-AS1. D Analysis of differential expression of SPINT1-AS1 in GC tissue samples (375 cases) and normal tissue samples (32 cases) based on TCGA database. E Correlation between SPINT1-AS1 and TNM stage of GC patients based on TCGA database; F TCGA database analyzed the relationship between SPINT1-AS1 and survival rate of GC patients. G Expression patterns of five potential RBPs interacting with SPINT1-AS1 in GC analyzed by TCGA database. Data were expressed as mean ± SD (n = 3). *P < 0.05

Fig. 2

SPINT1-AS1 inhibits GC proliferation, invasion and migration, and oxidative stress and promotes apoptosis. MKN-28 and HGC-27 cells were transfected with pcDNA 3.1 overexpression vector targeting SPINT1-AS1. A RT-qPCR measured SPINT1-AS1. B, C EdU assay and colony formation assay analyzed cell proliferation. D Flow cytometry evaluated cell apoptosis rate. E Transwell detected cell invasion and migration. F DCFH-DA combined with flow cytometry assessed ROS levels in cells. G Commercial kits or ELISA measured MDA, SOD, CAT and GSH-Px. Data were expressed as mean ± SD (n = 3). *P < 0.05

Fig. 3

Competitive adsorption of miR-656-3p by SPINT1-AS1. A RT-qPCR measured five potential miRNAs with binding sites to SPINT1-AS1 in MKN-28 and HGC-27 cells. B RIP test examined the binding between SPINT1-AS1 and potential miRNAs. C starbase predicted the binding sites of SPINT1-AS1 and miR-656-3p. D Dual luciferase reporting assay verified the targeting relationship between SPINT1-AS1 and miR-656-3p. E RT-qPCR measured miR-656-3p in 5 GC cell lines and normal gastric mucosa cell line. F RT-qPCR studied the effect of SPINT1-AS1 overexpression on miR-656-3p expression. Data were expressed as mean ± SD (n = 3). *P < 0.05

Fig. 4

MiR-656-3p participates in the SPINT1-AS1-regulated GC progression. si-SPINT1-AS1 and miR-656-3p-inhibitor were co-transfected into MKN-28 and HGC-27 cells. A RT-qPCR measured miR-656-3p. B, C EdU assay and colony formation assay analyzed cell proliferation. D Flow cytometry evaluated cell apoptosis rate. E Transwell detected cell invasion and migration. F DCFH-DA combined with flow cytometry assessed ROS levels in cells. G Commercial kits or ELISA measured MDA, SOD, CAT and GSH-Px. Data were expressed as mean ± SD (n = 3). *P < 0.05

Fig. 5

PLCXD3 is regulated by miR-656-3p. A RT-qPCR measured expression levels of five potential mRNAs with miR-656-3p binding sites in MKN-28 and HGC-27 cells. B TCGA database analyzed PLCXD3 in gastric adenocarcinoma. C RIP test checked the binding relationship between miR-656-3p and PLCXD3. D starbase predicted the binding sites of miR-656-3p to PLCXD3. E Dual luciferase reporting assay verified the targeting relationship between miR-656-3p and PLCXD3. F Western blot detected PLCXD3 in 5 GC cell lines and normal gastric mucosa cell line. G Analysis of PLCXD3 expression in GC tissue samples (375 cases) and normal tissue samples (32 cases) based on the TCGA database. H Correlation between PLCXD3 and TNM staging in GC patients. I GEPIA database analyzed the relationship between PLCXD3 and survival rate of patients with gastric adenocarcinoma. J Western blot detected the effect of overexpression or knockdown of miR-656-3p on PLCXD3. Data were expressed as mean ± SD (n = 3). *P < 0.05

Fig. 6

SPINT1-AS1 regulates GC malignant behaviors by regulating the miR-656-3p/PLCXD3 axis. pcDNA 3.1-SPINT1-AS1 and si-PLCXD3 were co-transfected into MKN-28 and HGC-27 cells. A RT-qPCR measured SPINT1-AS1 and miR-656-3p. B Western blot detected PLCXD3. C, D EdU assay and colony formation assay analyzed cell proliferation. E Flow cytometry evaluated cell apoptosis rate. F Transwell detected cell invasion and migration. G DCFH-DA combined with flow cytometry assessed ROS levels in cells. H Commercial kits or ELISA measured MDA, SOD, CAT and GSH-Px. Data were expressed as mean ± SD. *P < 0.05

Fig. 7

SPINT1-AS inhibits Wnt/β-catenin pathway activation in GC by regulating miR-656-3p/PLCXD3 axis. A, B Western blot for protein expression of active-β-catenin, p-GSK-3β and GSK-3β in MKN-28 cells. C, D Western blot for protein expression of Nrf2 and HO-1 in MKN-28 cells. Data were expressed as mean ± SD. *P < 0.05

Fig. 8

PLCXD3 inhibits Wnt/β-catenin pathway activation by promoting MALAT1 degradation. A starbase website analyzed the potential binding sites of PLCXD3 and MALAT1. B RT-qPCR detected the expression pattern of MALAT1 in normal cell lines and five GC cell lines. C TCGA and GTEx data analyzed the expression pattern of MALAT1 in GC tissues and normal tissues. D RNase assay detected the effect of overexpression of PLCXD3 on MALAT1 RNA stability. E RIP assay detected the binding relationship between PLCXD3 and MALAT1. F RT-qPCR detected the effect of knockdown or overexpression of PLCXD3 on MALAT1 RNA expression. G Western blot detected the effect of overexpression of PLCXD3 while overexpression of MALAT1 on the expression of active-β-catenin, p-GSK-3β and GSK-3β protein expression. Data were expressed as mean ± SD (n = 3). *P < 0.05

Fig. 9

SPINT1-AS1 inhibits GC tumor growth. A Representative pictures of tumors. B Tumor volume. C Tumor weight. D IHC staining analyzed Ki-67 and PLCXD3 in tumor tissues. The experiment was repeated three times independently. Data were expressed as mean ± SD (5 mice/group). *P < 0.05