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

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.


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with the manufacturer's operating instructions, the CCk-8 kit was used to test the cell vitality.

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Small RNA high-throughput sequencing analysis 119 TRIzol was used to extract total RNA from the sample, and AGE (agarose gel 120 electrophoresis) was used to cut and select 18-30 nucleotide (nt) segments. The 3' joint and 121 5' joint were connected, respectively; then, reverse transcription and PCR amplification were 122 conducted on the small RNAs with joints at both ends; finally, AGE was used to recover and 123 purify a ladder of around 140 base pairs (bp), and library construction was completed. Agilent 124 2100 (Agilent, Santa Clara, CA, USA) and qPCR were used for quality control of the constructed 125 library, and computer-controlled sequencing was conducted.

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For the small RNA sequencing data, first of all, we used a dynamic programming algorithm 127 to filter out low-quality reads that included polyA, and sequences with reads smaller than 16 nt 128 or larger than 35 nt were all eliminated. After filtering, the quality and length distribution of the 129 sequencing data were summarized. Then, standard bioinformatics analysis of the small RNA  The total miRNA reads were standardized to select the miRNAs with differential 141 expressions between the two libraries. Per million (TPM) was used to standardize the miRNA 142 counts. Old known and newly known miRNA expression levels between the two libraries (TE-1/exo was used as control group) were compared to find the differential expressions of the 144 miRNA. Here, the selection method was based on the method of Audic and Claverie. 21 The

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IDEG6 software was used to analyze differential expressions of miRNA. 22

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The exosomes in TE-1/DDP medium were collected by ultracentrifugation. represented as n = 1 to complete for three times, and the U6sniRNA and β-actin genes were 183 respectively used as internal controls. The Ct value is presented as the average value of three 184 independent repetitions; the relative expression level was calculated with the Ct method.

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The standard error of the mean for the repetitions was also calculated. Test of cell cycle 205 The PBS solution was used to process around 1 × 10 6 cells to single-cell suspension, and Biosciences, San Jose, CA, USA) was used to analyze the apoptosis rate.

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The PBS solution was used to process around 1 × 10 6 cells to single-cell suspension twice, which was fixed with 70°C ice alcohol for 24 h. Then, after washing and centrifugation with 211 PBS, the propidium iodide solution (containing 100 mg/l RNaseA) was added, and it was 212 incubated at room temperature away from the light. Then, the FACScan system (BD into the body of the mice by intraperitoneal injection every day, and this lasted for seven days.

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On the seventh, tenth, fourteenth, sixteenth, eighteenth and twenty-first days, the tumor size

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was measured with calipers. The tumor volume was estimated according to the formula of (L ×

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S 2 )/2 (where L is the longest diameter and S is the shortest diameter). The mice were killed, than that of the TE-1 cell line (p＜0.001) (Fig. 1A).

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The flow cytometer test results showed that 24 h after being processed with 2.5 μM  1C). Western blotting was used to detect the exosome's marker protein molecule CD63.

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As a result, the CD63 protein was detected in the TE-1 cell and the TE-1/DDP cell, as well as 257 in the extracted exosomes of the TE-1/DDP cell and the TE-1 cell (Fig. 1D).

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The CCK-8 kit was used to test the cell vitality, and we identified the influence of TE-1/DDP 259 exosome on the tolerance of TE-1 cells for cisplatin. The results showed that under the IC 50 TE- of expression level (Fig. 2B).

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After sequencing, the adapter sequences, fuzzy reads and low-quality reads were 285 eliminated, and around 55 × 10 5 to 110 × 10 5 clean reads were generated for each sample.

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Compared to the reference sequence of genome complex GRCh37/hg19, 92% of all reads

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In total, 5155 differential expression genes were determined as being related to the effects 293 of TE-1/DDP/exo. Of these, 3446 genes had up-regulated expression, and 1709 genes had 294 down-regulated expression (Fig. 2D). In order to further analyze the function of these differential 295 genes, we conducted GO analysis based on the GO terminologies (Fig. 2E). We identified 16 296 overrepresented GO terminologies related to the biological process (p < 0.05). These 297 terminologies included genes that participated in cell proliferation, cell adhesion, angiogenesis 298 regulation and metabolic processes. Eight overrepresented GO terminologies were related to 299 the molecular function (p < 0.05). These genes belonged to the genes with nucleic acid binding 300 transcription factor activity, protein binding transcription factor activity, signal transduction 301 activity and structural analysis activity. Ten GO terminologies with significant difference (p < 302 0.05) were related to the components of cells, and they mainly belonged to the extracellular 303 components and organelles (Fig. 2E).

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In order to verify the expression level of miRNAs with differential expression (TOP24) in 305 the sequencing data of TE-1/exo and TE-1/DDP/exo, we used miRNA fluorescent quantitative difference in the transcriptome (Fig. 2G). The results showed that the expression levels of some 309 of the miRNAs and mRNA with significant differences screened by us in the separate exosomes 310 or cells were consistent with the expression results of the sequencing data.

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(http://www.targetscan.org) was used to predict that miRNA-193 and TFAP2C had a targeting 334 sequence relation (Fig. 3B). The test results of the dual luciferase reporter showed that miR193 335 was only combined with correct TFAP2C gene 3'-UTR area, indicating that TFAP2C was the 336 target gene of miR193 (Fig. 3C).

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We added TE-1/DDP/exo to the esophageal cancer cell TE-1 and breast cancer cell ECA-

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We wished to prove the hypothesis that exosomes or miRNA19 could cause drug

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We further adopted the RT-qPCR technology and western blot to test the influence of

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Flow cytometry results showed that compared to TE-1/OE-control, the apoptosis rate of

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The activator 2 (TFAP2) family consists of five homologous developmental regulation 438 transcription factors: TFAP2A-E, and each transcription factor is coded by independent genes.

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In structure, the TFAP2 protein contains a highly conserved C-end helix-span-helix sequence