Promotion of miR-221-5p on the Sensitivity of Gastric Cancer Cells to Cisplatin and Its Effects on Cell Proliferation and Apoptosis by Regulating DDR1

Abstract

Purpose: This research aimed to explore the role of miR-221-5p on the sensitivity of gastric cancer cells to cisplatin, and the proliferation and invasion of gastric cancer cells by regulating DDR1.

Patients and methods: Altogether 69 patients who treated with radical gastrectomy from January 2014 to January 2016 were collected. With the agree of the patients, 69 gastric carcinoma and 69 adjacent tissues were taken, respectively, during the operation, and gastric carcinoma and human gastric mucosa cells were purchased. RT-PCR was used for detection of the expression level of miR-221-5p and DDR1. Wound healing assay and CCK-8 assay were used for exploration of the cell migration and viability. Western blot and double luciferase reporter gene were performed to determine the target gene of miR-221-5p.

Results: It was showed that miR-221-5p expression was decreased in GC tissues and cell lines. The high expression of miR-221-5p reduced the resistance of GC cells to cisplatin and inhibited the proliferation and migration of gastric cancer cells. The high expression of miR-221-5p promoted the proliferation, invasion and migration of GC cells. In addition, we found that DDR1 was a direct target gene of miR-221-5p in GC cells. We found that DDR1 expression increased in gastric carcinoma. Moreover, there was a negative correlation of DDR1 with the expression level of miR-221-5p. The increase of miR-221-5p increased the chemosensitivity of GC cells to cisplatin, and inhibited the proliferation, invasion, migration and EMT of GC cells by targeting DDR1.

Conclusion: The above research indicated that miR-221-5p may be a target for enhancing cisplatin chemotherapy sensitivity in gastric cancer patients.

Keywords: DDR1; apoptosis; cisplatin; gastric cancer cells; miR-221-5p; proliferation; sensitivity.

Figure 1

Expression and clinical significance of miR-221-5p and DDR1 in gastric cancer tissues. (A) expression of miR-221-5p in gastric cancer tissue; (B) expression of DDR1 in gastric cancer tissue; (C) miR-221-5p and DDR1 were negatively correlated in gastric cancer tissues; (D) the overall survival rate of patients with miR-221-5p high expression group was significantly higher than that of patients with miR-221-5p low expression group. ** indicates that P<0.05.

Figure 2

Effect of miR-221-5p on cell proliferation, invasion, migration and apoptosis. (A) miR-221-5p low expression in gastric cancer cells; (B) expression of miR-221-5p after transfection; (C and D) effects of up-regulating miR-221-5p and down-regulating miR-221-5p expression on proliferation of gastric cancer cells; (E) effect of up-regulating miR-221-5p and down-regulating miR-221-5p expression on invasion of gastric cancer cells; (F) effect of up-regulating miR-221-5p and down-regulating miR-221-5p expression on gastric cancer cell migration; (G) effect of up-regulating miR-221-5p and down-regulating miR-221-5p expression on apoptosis of gastric cancer cells; (H) Flow cytometry of apoptosis; (I) effects of up-regulating miR-221-5p and down-regulating miR-221-5p expression on EMT-related proteins N-cadherin, vimentin and E-Cadherin in gastric cancer cells; (J) protein image; (K) effect of up-regulating miR-221-5p and down-regulating miR-221-5p expression on apoptosis-related proteins Bax, Bcl-2 and Caspase-3 in gastric cancer cells; (L) protein image. ** indicates that P<0.05.

Figure 3

Over-expression of miR-221-5p on cisplatin sensitivity of cells. (A and B) effect of up-regulation and down-regulation of miR-221-5p on survival rate of gastric cancer cells under cisplatin intervention; (C) effect of up-regulation and down-regulation of miR-221-5p on apoptosis rate of gastric cancer cells under cisplatin intervention; (D) Flow cytometry of apoptosis; (E) effect of up-regulation and down-regulation of miR-221-5p on apoptosis-related proteins in gastric cancer cells under cisplatin intervention; (F) protein image. ** indicates that P<0.05.

Figure 4

Effect of DDR1 on cell proliferation, invasion, migration and apoptosis. (A) high expression of DDR1 in gastric cancer cells; (B) expression of DDR1 after transfection; (C and D) effects of up-regulating DDR1 and down-regulating DDR1 expression on proliferation of gastric cancer cells; (E) effect of up-regulating DDR1 and down-regulating DDR1 expression on invasion of gastric cancer cells; (F) effect of up-regulating DDR1 and down-regulating DDR1 expression on gastric cancer cell migration; (G) effect of up-regulating DDR1 and down-regulating DDR1 expression on apoptosis of gastric cancer cells; (H) Flow cytometry of apoptosis; (I) effect of up-regulating DDR1 and down-regulating DDR1 expression on EMT-related proteins N-cadherin, vimentin and E-Cadherin in gastric cancer cells; (J) protein image; (K) effect of up-regulating DDR1 and down-regulating DDR1 expression on apoptosis-related proteins Bax, Bcl-2 and Caspase-3 in gastric cancer cells; (L) protein image. ** indicates that P<0.05.

Figure 5

Inhibition of DDR1 expression on cisplatin sensitivity of cells. (A and B) effect of DDR1 up-regulation and down-regulation on survival rate of gastric cancer cells under cisplatin intervention; (C) effect of DDR1 up-regulation and down-regulation on apoptosis rate of gastric cancer cells under cisplatin intervention; (D) Flow cytometry of apoptosis; (E) effect of up-regulation and down-regulation of DDR1 on apoptosis-related proteins in gastric cancer cells under cisplatin intervention; (F) protein image. ** indicates that P<0.05.

Figure 6

Identification of miR-221-5p target gene. (A) Double luciferin reporter enzyme; (B) effect of up-regulation and down-regulation of miR-221-5p expression on DDR1 protein; (C) protein image. ** indicates that P<0.05.