Research of the mechanism on miRNA193 in exosomes promotes cisplatin resistance in esophageal cancer cells

Abstract

Purpose: Chemotherapy resistance of esophageal cancer is a key factor affecting the postoperative treatment of esophageal cancer. Among the media that transmit signals between cells, the exosomes secreted by tumor cells mediate information transmission between tumor cells, which can make sensitive cells obtain resistance. Although some cellular exosomes play an important role in tumor's acquired drug resistance, the related action mechanism is still not explored specifically.

Methods: To elucidate this process, we constructed a cisplatin-resistant esophageal cancer cell line, and proved that exosomes conferring cellular resistance in esophageal cancer can promote cisplatin resistance in sensitive cells. Through high-throughput sequencing analysis of the exosome and of cells after stimulation by exosomes, we determined that the miRNA193 in exosomes conferring cellular resistance played a key role in sensitive cells acquiring resistance to cisplatin. In vitro experiments showed that miRNA193 can regulate the cell cycle of esophageal cancer cells and inhibit apoptosis, so that sensitive cells can acquire resistance to cisplatin. An in vivo experiment proved that miRNA193 can promote tumor proliferation through the exosomes, and provide sensitive cells with slight resistance to cisplatin.

Results: Small RNA sequencing of exosomes showed that exosomes in drug-resistant cells have 189 up-regulated and 304 down-regulated miRNAs; transcriptome results showed that drug-sensitive cells treated with drug-resistant cellular exosomes have 3446 high-expression and 1709 low-expression genes; correlation analysis showed that drug-resistant cellular exosomes mainly affect the drug resistance of sensitive cells through paths such as cytokine-cytokine receptor interaction, and the VEGF and Jak-STAT signaling pathways; miRNA193, one of the high-expression miRNAs in drug-resistant cellular exosomes, can promote drug resistance by removing cisplatin's inhibition of the cell cycle of sensitive cells.

Conclusion: Sensitive cells can become resistant to cisplatin through acquired drug-resistant cellular exosomes, and miRNA193 can make tumor cells acquire cisplatin resistance by regulating the cell cycle.

Fig 1. Identification of drug-resistant cell lines and exosomes.

A: Detection of cell viability of original cell line (TE-1) and drug-resistant cell line (TE1/DDP) under 2.5 μM DDP conditions. B: Apoptosis detection in original cell line (TE-1) and drug-resistant cell line (TE1/DDP) under 2.5 μM DDP. C: Scanning electron microscopy to identify exosomes. D: Western blot analysis of good exosome membrane protein marker CD63. E: CCK-8 detects cell viability showing that drug-resistant exosomes (TE1/DDP/exo) promote DDP resistance in TE1 cells.

Fig 2. Analysis and identification of next-generation sequencing results.

A: miRNA length distribution in different exosomes samples. B: Difference in miRNA expression levels in exogenous samples from different sources. C: Sample correlation analysis between sample sequencing data sets. D: Analysis of difference in gene expression between cells after drug-resistant exosome stimulation. E: Differential gene Go analysis. F, G: RT-qPCR validation of the significantly different expression of miRNAs and genes. H: Wayne diagram analysis shows the intersection of miRNA target genes and differential genes.

Fig 3. Verification that TFAP2C is the target gene of miRNA193.

A: qPCR detection of differential expression of miRNA193 and TFAP2C in different cells. B: TargentScan 7.2 predicts the targeting relationship between TAFAP2C and miRNA193, and its dual luciferase experimental mutation site. C: Dual luciferase assay for targeted binding of miRNA193 to TFAP2C. D: Changes in resistance to cisplatin in esophageal cancer cells after addition of TE1/DDP-Exo, transfection of miRNA193 minic or TFAP2C siRNA.

Fig 4. In vitro experiments demonstrate that both drug-resistant exosomes and miRNA193 increase the sensitivity of sensitive cells to cisplatin.

A, B: nude mice and postoperative tumor photos. C: Comparison of tumor size in different groups of nude mice. D: Tumor growth curves in different groups and different locations. The group situation is shown in the upper left corner of Fig A. For Group A and Group D, TE-1/DDP was injected into the left underarm of nude mice (L, the corresponding tumor in Fig B was the tumor on the top of each group), and TE-1 was inoculated in the right underarm (R, the corresponding tumor in Fig B was the tumor on the bottom of each group). For Group B and Group E, TE-1/miR193 mimic was inoculated into the left underarm of nude mice, and TE-1 was inoculated in the right underarm. For Group C and Group F, TE-1 was inoculated in both underarms of nude mice. In the Fig C D, the mark “A-L” means the tumor in the left underarm of nude mice in group A, which was injected TE-1/DDP.

Fig 5. TFAP2C and mIRNA193 affect cell cycle and apoptosis.

A: Effect of DDP, transfection of miRNA193 mimic or TFAP2C siRNA on TE1 cell cycle. B: qPCR detection of mRNA expression differences of cell cycle-related genes in differently treated cells. C: Western blot analysis of protein expression differences in cell cycle-related genes in differently treated cells. D: Apoptosis assay showing that overexpression of TFAP2C promotes apoptosis, and overexpression of miRNA193 inhibits cisplatin-induced apoptosis of esophageal cancer cells. E: qPCR detection of mRNA expression differences of apoptosis-related genes in differently treated cells. F: Western blot analysis of protein expression differences in apoptosis-related genes in differently treated cells.