The microRNA cluster miR-30b/-30d prevents tumor cell switch from an epithelial to a mesenchymal-like phenotype in GBC

Abstract

As a malignancy of the gastrointestinal tract, gallbladder cancer (GBC) continues to exhibit notable rates of mortality. The current study aimed at investigating the effects associated with miR-30b and miR-30d (miR-30b/-30d) patterns in tumor cells undergoing epithelial-to-mesenchymal transition (EMT) in GBC. It identified that miR-30b and miR-30d, composed as a miRNA cluster, exhibited lower levels in the cancerous tissues from 50 patients with GBC relative to the gallbladder tissues from 35 patients with chronic cholecystitis. As expected, elevated expression of miR-30b/-30d was found to inhibit the EMT process, as evidenced by enhanced E-cadherin and reduced N-cadherin and vimentin in human GBC cells treated with miR-30b mimic, miR-30d mimic, and miR-30b/-30d mimic. Semaphorin-6B (SEMA6B) was identified as a target gene of miR-30b/-30d. Silencing of SEMA6B by its specific small interfering RNA (siRNA) mimicked the effect of miR-30b/-30d upregulation on the GBC cell EMT. Consistently, SEMA6B overexpression promoted this phenotypic switch even in the presence of miR-30b/-30d mimic. The tumorigenicity assay data obtained from nude mice also further supported the notion that miR-30b/-30d inhibited EMT of GBC cells. Thus, based on the key findings of the current study, we concluded that the miR-30b/-30d cluster may provide a potential avenue for targeting mesenchymal-like, invasive tumor cells in GBC.

Keywords: SEMA6B; epithelial-to-mesenchymal transition; gallbladder cancer; microRNA cluster; microRNA-30b; microRNA-30d.

Graphical abstract

Figure 1

The miR-30b/-30d miRNA cluster is downregulated in GBC (A) Heatmap presenting the top 50 miRNAs with the most significant expression difference between GBC (n = 40) and normal gallbladder (n = 8) tissue samples in the GEO: GSE104165 dataset. Color scale indicates the degree of upregulation (red) or downregulation (green). (B) Expression of miR-30b/-30d in cancerous tissues (n = 30) and normal gallbladder tissues (n = 18), normalized to that of U6. (C) Epithelial nature of HGBECs (left map: original magnification, ×40; right map: original magnification, ×200). (D) Expression of miR-30b/-30d in cultured GBC cells (GBC-SD, Mz-ChA-1, NOZ, and SGC-996) and HGBECs. (E) The expression of E-cadherin and vimentin in cultured GBC cells (GBC-SD, Mz-ChA-1, NOZ, and SGC-996) and HGBECs. ∗p < 0.05 compared with the corresponding control (independent sample t test for statistical comparisons between two groups and one-way ANOVA followed by Tukey’s test among multiple groups).

Figure 2

The miR-30b/-30d miRNA cluster represses the EMT process in GBC cells The miR-30b mimic, miR-30d mimic, and miR-30b/-30d mimic were introduced into GBC-SD cells to elevate the expression of miR-30b, miR-30d, and miR-30b/-30d. (A) Quantification of E-cadherin, N-cadherin, and vimentin proteins in GBC-SD cells. (B) Statistics of GBC-SD cells invading from Matrigel-coated upper Transwell chambers into lower ones. (C) Statistics of GBC-SD cell migration. ∗p < 0.05 compared with GBC-SD cells treated with miRNA-mimic NC by independent sample t test.

Figure 3

The miR-30b/-30d miRNA cluster targets and negatively regulates the expression of SEMA6B (A) Target genes of miR-30b and miR-30d obtained from four public databases, including DIANA (the top 400 scores, http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index); miRDB (>80 scores, http://mirdb.org/miRDB/index.html); mirDIP (the top 400 scores, http://ophid.utoronto.ca/mirDIP/index.jsp#r); and TargetScan (the top 400 scores, http://www.targetscan.org/vert_71/). (B) Overlapping target genes of miR-30b/-30d from the GEO: GSE74048 dataset and four public databases by Venn diagrams (http://jvenn.toulouse.inra.fr/app/example.html). (C) Interactions between 10 recognized GBC-associated genes (the MalaCards database, https://www.malacards.org/) and 13 miR-30b/-30d-binding downstream genes predicted using the STRING database (https://string-db.org/). (D) Differential analysis of SEMA6B expression between human GBC (n = 3) and matched paracancerous (n = 3) tissues in the GEO: GSE74048 dataset. (E) The predicted seed sequence of miR-30b/-30d and SEMA6B in the miRNA target detection program (http://www.mirdb.org/). (F) Luciferase activity of SEMA6B-WT and SEMA6B-MUT in HEK293T cells transfected with miR-30b mimic, miR-30d mimic, or miR-30b/-30d mimic. (G) mRNA expression of SEMA6B in GBC-SD cells transfected with miR-30b mimic, miR-30d mimic, or miR-30b/-30d mimic. (H) Protein expression of SEMA6B in GBC-SD cells transfected with miR-30b mimic, miR-30d mimic, or miR-30b/-30d mimic. ∗p < 0.05 compared with the corresponding control (independent sample t test for statistical comparisons between two groups and one-way ANOVA followed by Tukey’s test among multiple groups).

Figure 4

Silencing of SEMA6B inhibits the EMT process in GBC cells (A) mRNA expression of SEMA6B in GBC tissues relative to normal gallbladder tissues. (B) Protein expression of SEMA6B in GBC tissues relative to normal gallbladder tissues. (C) mRNA expression of SEMA6B in GBC cells relative to HGBECs. (D) Protein expression of SEMA6B in GBC cells relative to HGBECs. In the following experiments, siRNA targeting SEMA6B and scramble siRNA were delivered into GBC-SD cells to specifically blunt the expression of SEMA6B. (E) Immunoblots and quantification of E-cadherin, N-cadherin, and vimentin proteins in GBC-SD cells. (F) Representative view and statistics of GBC-SD cells invading from Matrigel-coated upper Transwell chambers into lower ones. (G) Representative view and statistics of GBC-SD cell migration. ∗p < 0.05 compared with GBC-SD cells treated with scramble siRNA by an independent sample t test.

Figure 5

The miR-30b/-30d cluster prevents the EMT process by repressing SEMA6B expression GBC-SD cells treated with miR-30b/-30d mimic (miR-mimic NC as negative control) were transfected with an expression vector containing the SEMA6B gene (empty vector as negative control). (A) mRNA expression of SEMA6B in GBC-SD cells. (B) Protein expression of SEMA6B in GBC-SD cells. (C) Quantification of E-cadherin, N-cadherin, and vimentin proteins in GBC-SD cells. (D) Statistics of GBC-SD cells invading from Matrigel-coated upper Transwell chambers into lower ones. (E) Statistics of GBC-SD cell migration. ∗p < 0.05 compared with the corresponding control by one-way ANOVA followed by Tukey’s test.

Figure 6

miR-30b/-30d cluster inhibits tumorigenesis and EMT of human GBC cells in vivo by downregulating SEMA6B (A) Volume of mouse xenotransplanted tumors of GBC-SD cells at indicated time points; a repeated-measures ANOVA with a Bonferroni test was used for statistical comparison. (B) Weight of mouse xenotransplanted tumors of GBC-SD cells. (C) Immunoblots and quantification of SEMA6B, E-cadherin, N-cadherin, and vimentin proteins in mouse tumor tissues. (D) Immunohistochemical staining for SEMA6B, E-cadherin, N-cadherin, and vimentin proteins in mouse tumor tissues and extent of staining. ∗p < 0.05 compared with the corresponding control by one-way ANOVA followed by Tukey’s test.

Figure 7

A diagram showing the mechanism by which miR-30b/-30d exerts synergistic effects on GBC progression Overexpression of miR-30b/-30d inhibits GBC cell migration, invasion, and EMT process in vitro as well as weakening tumorigenesis of GBC cells in vivo through target inhibition of SEMA6B.