LncRNA SNHG4 promotes malignant biological behaviors and immune escape of colorectal cancer cells by regulating the miR-144-3p/MET axis

Abstract

Objective: This study aimed to explore the underlying mechanism of long noncoding RNA (lncRNA) SNHG4 regulating MET to participate in the malignant biologic behaviors and immune escape of colorectal cancer (CRC) by sponging miR-144-3p.

Methods: CRC tissues were collected and the expression levels of lncRNA SNHG4, miR-144-3p, and MET were detected by quantitative real-time PCR (qRT-PCR). Then, the localization of lncRNA SNHG4 was studied by fluorescence in situ hybridization (FISH), and the regulatory relationship among lncRNA SNHG4, miR-144-3p, and MET was verified by dual-luciferase reporter assay. Next, cell counting kit-8 (CCK-8), Clone formation assay, and Transwell migration assay were carried out to evaluate cell proliferation, colony formation, and invasion, respectively. Flow cytometry was performed to evaluate cell apoptosis. Western blotting was applied to semi-quantify the expression levels of MET and PD-L1 in cells.

Results: LncRNA SNHG4 expression was upregulated in CRC tissues. Knockdown of lncRNA SNHG4 suppressed the proliferation, colony formation and invasion of CRC cells (all P<0.05). LncRNA SNHG4 directly regulated miR-144-3p, by which either lncRNA SNHG4 knockdown or miR-144-3p overexpression can inhibit CD4+ T cell apoptosis (both P<0.05) to suppress immune escape. Either overexpression of lncRNA SNHG4 or knockdown of miR-144-3p activated PD-1/PD-L1 and induced CD4+ T cell apoptosis (both P<0.05). LncRNA SNHG4 targeted and regulated MET through the regulation of miR-144-3p, while overexpression of MET can partially reverse the effect of lncRNA SNHG4 knockdown on CD4+ T cells.

Conclusion: LncRNA SNHG4 sponges miR-144-3p and upregulates MET to promote the proliferation, colony formation, invasion, and immune escape of CRC cells, leading to the progression of CRC.

Keywords: LncRNA SNHG4; MET; colorectal cancer; immune escape; miR-144-3p.

Figure 1

LncRNA SNHG4 may be correlated to the progression of CRC. A: Volcano map to show 434 upregulated lncRNAs and 617 downregulated lncRNAs in CRC; B: The top 100 differentially expressed lncRNAs to draw a heat map; C: GEPIA database to show the significantly upregulated expression of lncRNA SNHG4 in COAD; D: qRT-PCR to detect the expression of lncRNA SNHG4 in CRC tissues; E: qRT-PCR to detect the expression of lncRNA SNHG4 in CRC cells; F: ROC curve to show the sensitivity of lncRNA SNHG4 in the diagnosis of CRC. Compared with normal tissues and cells, *P<0.05. CRC: colorectal cancer; lncRNAs: long non-coding RNAs; GEPIA: gene expression profiling interactive analysis; COAD: colon adenocarcinoma; qRT-PCR: quantitative real-time PCR; ROC: receiver operator characteristic.

Figure 2

Knockdown of lncRNA SNHG4 can inhibit cell growth and invasion. A: Transfection efficiency of silencing lncRNA SNHG4; B: CCK8 to detect the cell proliferation; C: Clonogenic assay to detect cell colony formation; D: Transwell assay to detect cell invasion (×200). Compared with the si-NC group, *P<0.05.

Figure 3

Verification of a targeting relationship between lncRNA SNHG4 and miR-144-3p. A: FISH experiment to detect the localization and expression of lncRNA SNHG4 (×200); B: Overlapped target miRNAs of lncRNA SNHG4; C: Specific binding site of lncRNA SNHG4 and miR-144-3p; D: Dual-luciferase reporter assay to verify the targeting relationship; E: qRT-PCR to detect the expression of miR-144-3p in CRC tissue; F: qRT-PCR to detect the expression of miR-144-3p in CRC cells; G: LncRNA SNHG4 and miR-144-3p correlation analysis; H: Effect of knockdown of lncRNA SNHG4 on the expression of miR-144-3p. Compared with the miR-144-3p NC group, *P<0.05; compared with normal tissues and cells, &P<0.05; compared with the si-NC group, %P<0.05. FISH: fluorescence in situ hybridization; CRC: colorectal cancer; qRT-PCR: quantitative real-time PCR.

Figure 4

LncRNA SNHG4/miR-144-3p induces an interaction between CACO2 cells and CD4+ T cells and CD4+ T cell apoptosis through the PD-1/PD-L1 immune checkpoint. A: Flow cytometry to detect the percentage of CD4+ T cells; B: Flow cytometry to detect the apoptotic rate of CD4+ T cells; C: Flow cytometry to detect the effect of PD-1/PD-L1 antibody on the percentage CD4+ T cells; D: Flow cytometry to detect the effect of PD-1/PD-L1 antibody on the apoptotic rate of CD4+ T cells. Compared with the si-NC group, *P<0.05; compared with the NC mimic group, &P<0.05; compared with the pcDNA group, %P<0.05; compared with the SNHG4 group, ^P<0.05; compared with the NC inhibitor group, #P<0.05; compared with the miR-144-3p inhibitor group, @P<0.05.

Figure 5

LncRNA SNHG4/miR-144-3p targets and regulates MET. A: Venn diagram to show the number of overlapped target genes of miR-144-3p; B: KEGG analysis of overlapped target genes; C: Analysis of the interaction between overlapped target genes and CRC risk genes (red representing CRC risk genes); D: The dual-luciferase report assay to verify the targeting relationship between miR-144-3p and MET; E: qRT-PCR to detect the expression of MET in CRC tissue; F: qRT-PCR to detect the expression of MET in CRC cells; G: Correlation analysis of MET and lncRNA SNHG4 and miR-144-3p; H: Immune analysis of MET using the Timer online website; I: The effect of lncRNA SNHG4 and miR-144-3p on the expression of MET/PD-L1. Compared with the miR-144-3p NC group, *P<0.05; compared with normal tissues and cells, ^P<0.05; compared with the si-NC group, #P<0.05; compared with the si-SNHG4+NC inhibitor group, &P<0.05. CRC: colorectal cancer; qRT-PCR: quantitative real-time PCR.

Figure 6

LncRNA SNHG4 inhibits CD4+ T cell activity through MET and promotes the progression of CACO2 cells. A: The effect of lncRNA SNHG4 on the expression of MET and PD-L1; B: Flow cytometry to analyze of CD4+ T cell activity and apoptosis; C: CCK8 to detect cell proliferation; D: Clonogenic assay to detect cell colony formation; E: Transwell migration assay to detect the cell invasion (×200). Compared with the si-NC group, *P<0.05; compared with the si-SNHG4+pcDNA group, ^P<0.05. PD-L1: programmed death-ligand 1.

Figure 7

LncRNA SNHG4 regulates the miR-144-3p/MET axis to promote the immune escape and progression of colorectal cancer.