MiR-21-5p Modulates Cisplatin-Resistance of CD44+ Gastric Cancer Stem Cells Through Regulating the TGF-β2/SMAD Signaling Pathway

Abstract

Background: Cisplatin (DDP) resistance in gastric cancer (GC) is likely to come from gastric cancer stem cells (GCSC). It is a new idea to study the mechanism of the DDP-resistance in GCSC from miRNA.

Materials and methods: CD44+ GCSCs and CD44- control cells were constructed based on the HGC27 gastric cancer cell line. DDP sensitivities in CD44+ and CD44- cells were detected via CCK-8 assay. The differential expression of miR-21-5p in these cell lines was detected by RT‒qPCR. The expression levels of downstream TGF-β2, SMAD2 and SMAD3 were determined through RT‒PCR and Western blotting. A luciferase assay was used to detect the relationship between miR-21-5p and TGFB2, and the TCGA database, clinical data from our centre, and vivo experiment were used for validation. Finally, we knocked down miR-21-5p to detect changes in cisplatin resistance in GCSCs and to verify changes in the levels of downstream pathways in GCSCs.

Results: CD44+ GCSCs induced cisplatin resistance compared with CD44- cells. miR-21-5p was highly expressed in GCSCs, and the TGF-β2/SMAD pathway was also highly expressed. TGFB2 was proven to be a downstream target gene of miR-21-5p and had a positive relationship with it in phenotype. After knockdown of miR-21-5p, the TGF-β2/SMAD pathway was also inhibited, and the resistance of GCSCs to cisplatin was specifically decreased.

Conclusion: MiR-21-5p promotes cisplatin resistance in gastric cancer stem cells by regulating the TGF-β2/SMAD signalling pathway.

Keywords: GCSCs; TGF-β2 pathway; cisplatin resistance; miR-21-5p.

Figure 1

CD44 is significantly upregulated in GCSCs. (A) The expression of CD44 in HGC27 was analysed by flow cytometry, and about 10% of the upper and lower sides were selected to construct CD44- and CD44+ cell populations by fluorescence-activated cell sorting. (B) CD44+ cells were grown in stemness-culture, and the cells gradually changed from single cells to clusters and finally to spheres of cells over time. (C) Under 200x light microscope, HGC27 CD44- grows adherently, while CD44+ suspends into spheres. (D) The positive rate of CD44 in the CD44+ group by flow cytometry was about 95%, while it was about 40% in CD44- group, and there was a statistical difference between them. The quantitative data were presented as the mean ± SD of triplicate experiments. (****p < 0.0001) (E) Real-time PCR analysis of the CD44 expression level in CD44- and CD44+ cells and it was normalized by GAPDH and presented as the relative ratio. (**p < 0.01) (F) Western blot analysis of CD44 expression in CD44- and CD44+ cells.

Figure 2

Cisplatin resistance in CD44+ and CD44- HGC27 cells in vitro and in vivo. (A): Viabilities of CD44+ and CD44- cells after treatment with different concentration cisplatin as measured by CCK8 assay. Values shown represent averages of half maximal inhibitory concentration; each treatment was carried out in triplicate (**p < 0.01). (B): Tumour growth curves in nude mice inoculated with HGC27 CD44- and HGC27 CD44+ cells that were treated by DDP (*p < 0.05). (C): The mean weight (left) and the size (right) of tumours at the end of the experiment from mice treated with DDP (**p < 0.01).

Figure 3

MicroRNA-21-5p is upregulated in CD44+ cells. (A): miRNA sequencing between HGC27 CD44- and CD44+ cells and the differentially expressed miRNAs were screened in the heatmap. Among them, HGC27a represented CD44+ HGC27 cells, while HGC27b represented CD44- HGC27 cells. (B): The radar chart showed the 15 miRNAs with the largest log ford change (logFC) differences between the two groups and llogFC of miR-21-5p was 1.35 (p < 0.001). (C): Expression levels of miR-21-5p between the two groups of samples with differential expression of CD44 in TCGA database. (**p < 0.01) (D): IHC staining of CD44 under electron microscope, and field of view at 50x (left) and 200x (right) respectively. Here we show the weak positive CD44 (above) as well as the strong positive CD44 (below). (E): Distribution of IHC scores forD44. According to the median CD44 score of 88 samples, they were divided into two groups, which were CD44 low and CD44 high. (F): The miR-21-5p expression levels between two groups of clinical samples by absolute quantitative PCR. (****p < 0.0001) (G): Real-time PCR analysis of the miR-21-5p expression level in HGC27 CD44- and HGC27 CD44+ cells in vitro (left) and in vivo (right). It was normalized by U6 and presented as the relative ratio. (*p < 0.05, **p < 0.01).

Figure 4

TGFB2 is a miR-21-5p target gene. (A): Putative miR-21-5p binding sequences in the 3’-UTR of TGFB2 mRNA. (B): Luciferase activity assay to examine the activity of TGFB2 in negative control or miR-21-5p inhibitor- transfected 293T cells. Data are expressed as a ratio of Firefly luciferase activity to Renilla luciferase activity (** p<0.01). (C): It showed a relationship between miR-21-5p and TGFB2 expression levels in samples in the TCGA database, and there was a positive correlation between the two fittings. (p < 0.0001) (D): The positive relationship between the log copies of miR-21-5p and the IHC staining score of TGF-β2 in clinical samples. (p < 0.001) (E): IHC staining of TGF-β2 under electron microscope, and field of view at 50x (left) and 200x (right) respectively. Here we show the weak positive TGF-β2 (above) as well as the strong positive TGF-β2 (below).

Figure 5

The TGF-β2/SMAD signalling pathway is upregulated in CD44+ cells. (A): Differential mRNAs between HGC27 CD44- and CD44+ cells were identified by sequencing, in which TGFB2 was upregulated in CD44+ cells. (p < 0.05) (B): Expression levels of TGFB2 between the two groups of samples with differential expression of CD44 in TCGA database. (*p < 0.05) (C): IHC staining scores for TGFB2 were significantly higher in CD44 high clinical samples than they were in CD44 low. (****p < 0.0001) (D): Real-time PCR analysis of the TGFB2, SMAD2, and SMAD3 expression level in HGC27 CD44- and CD44+ cells and they were normalized by GAPDH and presented as the relative ratio. (***p < 0.001, ****p < 0.0001) (E): Western blot analysis of TGF-β2, SMAD2/3, phosphorylated SMAD2/3 expression in CD44- and CD44+ cells. The above pathway proteins were activated in CD44+ cells. (****p < 0.0001) (F): The CD44- and CD44+ tumours inoculated in nude mice were stained with HE and IHC and observed at 50x and 200x field respectively.

Figure 6

Inhibition of miR-21-5p decreases TGF-β2/SMAD signalling pathway expression in CD44+ cells. (A): CD44+ HGC27 cells were transfected with miR-21-5p inhibitor and negative control respectively, and there was significant inhibition by PCR detection. (***p < 0.001) (B): Real time PCR showed that the mRNA expression levels of TGFB2, SMAD2, SMAD3 all decreased with the inhibition of miR-21-5p. (*p < 0.05. ***p < 0.001) (C): TGF-β2/SMAD pathway protein levels decreased with miR-21-5p inhibition. (****p < 0.0001) (D): Cisplatin sensitivity in CD44+ HGC27 cells with native control (NC) and with miR-21-5p inhibitor. Viabilities of NC and inhibitor after treatment with different concentration cisplatin as measured by CCK8 assay. Values shown represent averages of half maximal inhibitory concentration; each treatment was carried out in triplicate (**p < 0.01).