Exosome-Delivered c-Met siRNA Could Reverse Chemoresistance to Cisplatin in Gastric Cancer

Abstract

Background: Drug resistance often occurs in the treatment of gastric cancer, which is the main cause of poor prognosis of chemotherapy. c-Met is overexpressed in a variety of tumors including gastric cancer, often leads to poor prognosis of gastric cancer, therefore regarded as a key target for the treatment of gastric cancer. This study aims to determine whether exosomes with si-c-Met could inhibit the resistance to cisplatin in gastric cancer (GC).

Methods: The protein expression levels of c-Met in tumor tissues and normal tissues of patients were evaluated by Western blot (WB) and immunohistochemistry (IHC), HEK293T cells were transfected with si-c-Met, exosomes were isolated, then co-cultured with gastric cancer cell lines and confirmed that it was incorporated into the cells by transmitted electron microscopy. Functional experiments were performed to examine the inhibitory effect of exo-si-c-Met on gastric cancer cell resistance in vitro, and xenograft models were used to reveal that exo-si-c-Met can enhance the sensitivity of tumors to cisplatin in vivo.

Results: High expression of c-Met is associated with poor prognosis of GC patients. si-c-Met significantly inhibited migration, invasion and promoted apoptosis in vitro, which indicated that si-c-Met sensitizes the response of gastric cancer cells to cisplatin. Exo-si-c-Met sharply reduced c-Met expression in gastric cancer cells and reverse the resistance to cisplatin in vitro and in vivo.

Conclusion: Our results indicate that exo-si-c-Met can inhibit the invasion and migration of gastric cancer cells and promote apoptosis in vitro and inhibit tumor growth in vivo, reversing the resistance to cisplatin in gastric cancer.

Keywords: c-Met; chemoresistance; exosomes; gastric cancer; siRNA.

Figure 1

Exo-si-c-Met increases the sensitivity of gastric cancer tissues to cisplatin in vivo. (A) A flow diagram depicting the in vivo experimental design. (B) the morphology of xenografted tumor tissues (n = 5). (C, D) Quantitative analysis of tumor volume (C) and weight (D). (E, F) Expression levels of c-Met in implanted tumors by qRT-PCR (E) and Western blot analysis (F). (G) Gray analysis of (F). **p < 0.01; ***p < 0.001. All error bars stand for SE.

Figure 2

The expression level of c-Met in GC. (A) The survival curve of GC patients associated with c-Met expression. (p < 0.001). (B) Immunohistochemistry of the paraffin-embedded human gastric cancer tissues and paracancerous normal tissues (n=8, total of 16 samples). (C) Western blotting analysis of c-Met expression in GC tissues and the corresponding adjacent normal tissues (n=8, total of 16 samples). (D) Gray analysis of (C). *p < 0.05, **p < 0.01. All error bars stand for SE.

Figure 3

The role of si-c-Met. (A, B) qRT-PCR analysis was done to exam the inhibition to the expression level of c-Met mRNA. (C, D) Western blotting analysis was done to confirm the inhibiting effect of si-c-Met in gastric cancer cells. (E, F) Gray analysis of (C, D). **p < 0.01, and ***p < 0.001. All error bars stand for SE.

Figure 4

Exo-si-c-met fuses into cells and downregulates the expression c-met. (A) The inhibition rate of two cell lines was observed by CCK-8. (B, C) Cell viability of different drug concentrations in SGC7901 and SGC7901/DDP was tested by CCK-8, and the inhibition ratio was calculated. (D) EdU was stained to confirm the number of newly proliferating cells. (E, F) Quantitative analysis of (D) **, p<0.01; All error bars stand for SE.

Figure 5

Exo-si-c-met fuses into cells and downregulates the expression c-met. (A) Extraction of exosomes from HEK293-T cells. (B) Electron microscope scanning of exosome from HEK293T cells. (C) Exosomes were stained with PKH26 and then fused into gastric cancer cells. (D–F) Expression levels of c-Met in exosomes from medium of SGC7901 and SGC7901/DDP were detected by Western blotting (D) and qRT-PCR (E, F) assays. (G) Gray analysis of (D). **p < 0.01, and ***p < 0.001. All error bars stand for SE.

Figure 6

Exo-si-c-met increases the sensitivity of gastric cancer cells to cisplatin (A) Apoptotic rate was tested by flow cytometry. (B) Transwell assays were completed to examine the cell migration ability. (C) Wound-healing assay demonstrated that exo-si-c-Met increased SGC7901/DDP migration. (D, E) Quantitative analysis of (A). (F, G) Quantitative analysis of (B). ***p < 0.001. All error bars stand for SE.