Promotion of Cell Proliferation through Inhibition of Cell Autophagy Signalling Pathway by Rab3IP is Restrained by MicroRNA-532-3p in Gastric Cancer

Abstract

Background: RAB3A-interacting protein (Rab3IP) is known to be involved in cancer; however, its function during the proliferation of gastric cancer (GC) cells remains unknown. Therefore, this study aimed to explore the potential function of Rab3IP in GC. Methods: The expression of Rab3IP and its clinical pathology value were determined by quantitative real-time PCR and immunohistochemistry. Rab3IP (knockdown and overexpression) and light chain 3 (LC3) lentiviruses were transfected into GC cells, and cell proliferation was measured using cell counting kit-8, plate clone formation, flow cytometry, and tumorigenesis assays. Cell autophagy was measured using a confocal laser scanning microscope and by western blotting. Luciferase reporter assay was performed to analyse the regulation of Rab3IP by microRNA-532-3p (miR-532-3p). Results: Overexpression of Rab3IP in GC samples enhanced the cell proliferation ability, but decreased the number of autophagosomes and expression of LC3-II and sequestosome-1 (SQSTM1 or p62) markers. Furthermore, we found that miR-532-3p can bind to the 3'UTR region of RAB3IP and inhibit the proliferation ability of GC cells. Further, the expression of miR-532-3p negatively correlated with that of Rab3IP. Conclusions: Our study elucidates the central role of Rab3IP in inducing proliferation of GC cells through its involvement in autophagy. miR-532-3p directly targets Rab3IP and represses its function, thereby demonstrating a novel regulatory link in GC.

Keywords: Rab3IP; cell autophagy; cell proliferation; gastric cancer; miR-532-3p.

Figure 1

Rab3IP is overexpressed in gastric cancer (GC). A. Expression of Rab3IP transcripts in paired GC tissues and peritumoral normal tissues. B. Western blot analysis of Rab3IP in paired GC tissues and peritumoral normal tissues. C. Western blot analysis of Rab3IP in GC cell lines. D. Tissue microarray (TMA) analysis of Rab3IP in primary human GC tissues. ** means P<0.001. Scar bar, 100 nm.

Figure 2

Depletion of Rab3IP suppresses cell proliferation in gastric cancer. A. Quantitative real-time-PCR analysis to confirm knock-down efficiency of Rab3IP transcripts in AGS cells. B. CCK-8 assay using AGS-Rab3IP (-) and AGS- Rab3IP (-)-NC cells. C. Clone formation assay using AGS-Rab3IP (-) and AGS- Rab3IP (-)-NC cells. D. Apoptosis rates of AGS-Rab3IP (-) and AGS- Rab3IP (-)-NC cells using Annexin V-FITC/PI kit. E. Tumour xenograft model established by subcutaneous injection in nude mice (n = 3). Data points are presented as mean tumour weight (gram) and tumour volume (mm³). *means P<0.05, ** means P<0.01, *** means P<0.001.

Figure 3

Overexpression of Rab3IP promotes cell proliferation in gastric cancer. A. Quantitative real-time-PCR analysis to show overexpression of Rab3IP transcripts in MKN45 cells. B. CCK-8 assay using MKN45-Rab3IP (+) and MKN45- Rab3IP (+)-NC cells. C. Clone formation assay using MKN45-Rab3IP (+) and MKN45- Rab3IP (+)-NC cells. D. Apoptosis rates of MKN45-Rab3IP (+) and MKN45- Rab3IP (+)-NC cells using Annexin V-FITC/PI kit. E. Tumour xenograft model established by subcutaneous injection in nude mice (n = 3). Data points are presented as mean tumour weight (gram) and tumour volume (mm³). *means P<0.05, ** means P<0.01, *** means P<0.001.

Figure 4

Rab3IP promotes cell autophagy in gastric cancer. A. Effect of downregulation of Rab3IP and addition of 3-methyladenine (3-MA) on cell proliferation using CCK-8 assay. B Effect of overexpression of Rab3IP and addition of RAPA on cell proliferation using CCK-8 assay. C LC3 fluorescence in AGS cells after downregulation of Rab3IP and addition of 3-MA. D LC-3 fluorescence in MKN45 cells after overexpression of Rab3IP and addition of RAPA. E Western blot analysis of Rab3IP, LC3-I, LC3-II, p62, and GAPDH in AGS and MKN45 cells. *** means P<0.001. Scale bar, 50 nm.

Figure 5

Rab3IP is a direct target of miR-532-3p in gastric cancer (GC). A. Western blot analysis of Rab3IP in the indicated cells treated with different mimics. B. Expression analysis of Rab3IP in the indicated cells treated with different mimics. C. Luciferase assay revealed that miR-532-3p directly binds to the 3ʹUTR region of Rab3IP. D. Correlation between miR-532-3p and Rab3IP at mRNA level in 40 paired human gastric cancer tissues and adjacent normal mucosa tissues. E. Upregulation of miR-532-3p was found in (31/40) 77.5% of GC cases. Data represents the log2 ratio of miR-532-3p level in GC tissues compared to the corresponding non-tumour tissues. F. The expression of miR-532-3p in GC tumour (T) versus non-tumour (NT) tissues, as detected by qRT-PCR. *means P<0.05.

Figure 6

Enhanced cell proliferation induced by Rab3IP is restrained by miR-532-3p in gastric cancer. A. Effect of overexpression of miR-532-3p and Rab3IP on cell proliferation using CCK-8 assay. B. Effect of overexpression of Rab3IP and addition of miR-532-3p on cell apoptosis using Annexin V-FITC/PI assay. C. Tumour xenograft model established by injecting Rab3IP-overexpressed MKN45 cells, while miR-532-3p agomir, a small molecule inhibitor, was injected into the tumour (n = 3). Data points are presented as mean tumour weight (gram) and tumour volume (mm³). D. LC-3 fluorescence in MKN45 after introduction of Rab3IP and addition of miR-532-3p. E. Western blot analysis of Rab3IP, LC3-I, LC3-II, p62, and GAPDH in MKN45 cells after introduction of Rab3IP and addition of miR-532-3p. *means P<0.05, ** means P<0.01, *** means P<0.001. Scar bar, 50 nm.