MicroRNA signature characterizes primary tumors that metastasize in an esophageal adenocarcinoma rat model

Ali H Zaidi, Lindsey T Saldin, Lori A Kelly, Linda Bergal, Ricardo Londono, Juliann E Kosovec, Yoshihiro Komatsu, Pashtoon M Kasi, Amit A Shetty, Timothy J Keane, Shyam J Thakkar, Luai Huleihel, Rodney J Landreneau, Stephen F Badylak, Blair A Jobe, Ali H Zaidi, Lindsey T Saldin, Lori A Kelly, Linda Bergal, Ricardo Londono, Juliann E Kosovec, Yoshihiro Komatsu, Pashtoon M Kasi, Amit A Shetty, Timothy J Keane, Shyam J Thakkar, Luai Huleihel, Rodney J Landreneau, Stephen F Badylak, Blair A Jobe

Abstract

Objective: To establish a miRNA signature for metastasis in an animal model of esophageal adenocarcinoma (EAC).

Background: The incidence of esophageal adenocarcinoma (EAC) has dramatically increased and esophageal cancer is now the sixth leading cause of cancer deaths worldwide. Mortality rates remain high among patients with advanced stage disease and esophagectomy is associated with high complication rates. Hence, early identification of potentially metastatic disease would better guide treatment strategies.

Methods: The modified Levrat's surgery was performed to induce EAC in Sprague-Dawley rats. Primary EAC and distant metastatic sites were confirmed via histology and immunofluorescence. miRNA profiling was performed on primary tumors with or without metastasis. A unique subset of miRNAs expressed in primary tumors and metastases was identified with Ingenuity Pathway Analysis (IPA) along with upstream and downstream targets. miRNA-linked gene expression analysis was performed on a secondary cohort of metastasis positive (n=5) and metastasis negative (n=28) primary tumors.

Results: The epithelial origin of distant metastasis was established by IF using villin (VIL1) and mucin 5AC (MUC5AC) antibodies. miRNome analysis identified four down-regulated miRNAs in metastasis positive primary tumors compared to metastasis negative tumors: miR-92a-3p (p=0.0001), miR-141-3p (p=0.0022), miR-451-1a (p=0.0181) and miR133a-3p (p=0.0304). Six target genes identified in the top scoring networks by IPA were validated as significantly, differentially expressed in metastasis positive primary tumors: Ago2, Akt1, Kras, Bcl2L11, CDKN1B and Zeb2.

Conclusion: In vivo metastasis was confirmed in the modified Levrat's model. Analysis of the primary tumor identified a distinctive miRNA signature for primary tumors that metastasized.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Study schema outlining the major…
Fig 1. Study schema outlining the major steps in the experimental design and miRNA analysis.
Fig 2. Immunofluorescence staining of rat tissue…
Fig 2. Immunofluorescence staining of rat tissue with MUC5AC and villin.
Panel A and B show MUC5AC immunofluorescence staining for primary adenocarcinoma in Levrat esophagus and normal esophagus, respectively. Panel C and D show villin immunofluorescence staining in Levrat esophagus and normal esophagus, respectively. Positive MUC5AC and villin staining were detected in primary tumor with the Alexa Fluor 488 secondary antibody, conjugated to a green fluorophore and the Alexa 594 secondary antibody, conjugated to a red fluorophore, respectively. Normal esophagus shows the absence of MUC5AC and villin staining.
Fig 3. Immunofluorescence staining of rat tissue…
Fig 3. Immunofluorescence staining of rat tissue with MUC5AC.
Panel A to F show immunofluorescence staining for metastatic lung, metastatic liver, metastatic stomach, non-metastatic lung, non-metastatic liver and non-metastatic stomach, respectively for representative cases. Positive MUC5AC cytoplasmic staining was detected in all metastatic samples with the Alexa Fluor 488 secondary antibody, conjugated to a green fluorophore. Metastasis negative liver and lung show the absence of MUC5AC staining. However, metastasis negative stomach stains positive for MUC5AC as gastric mucin M1 antigen is found in mucus cells of gastric epithelium.
Fig 4. Immunofluorescence staining of rat tissue…
Fig 4. Immunofluorescence staining of rat tissue with villin.
Representative case of (A)metastatic lung, (B) metastatic liver, (C) metastatic stomach, (D)non-metastatic lung, (E) non-metastatic liver and (F) non-metastatic stomach. Positive villin cytoplasmic staining was detected in all metastatic samples with the Alexa Fluor 594 secondary antibody, conjugated to a red fluorophore. Metastasis negative liver, lung and stomach show the absence of villin staining.
Fig 5. Cellular interactions between miRNAs and…
Fig 5. Cellular interactions between miRNAs and target genes.
Green represents downregulation and red represents upregulation of gene expression. A two-tailed, two-sample equal variance T-test was performed to obtain the p-values.
Fig 6. Relative gene expression levels in…
Fig 6. Relative gene expression levels in a secondary cohort of metastasis negative and metastasis positive primary EAC in the Levrat Model.
Quantitative reverse transcription-PCR (QRT-PCR) was performed on seven gene targets identified by the metastatic miRNA signature in IPA. Six of seven target genes were significantly upregulated in primary tumors with metastasis.

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