MiRNAs involved in development of intraepithelial precursor lesions and progression to cholangiocarcinoma

Background and aim:

Biliary intraepithelial neoplasia (BilIN) is a common precursor lesion of distal cholangiocarcinoma (dCCA). However, the sequence leading from non-neoplastic biliary epithelia to intraepithelial precursor lesions and finally to the invasive tumor is poorly understood. Here, we aimed to elucidate key miRNAs involved in this stepwise process and to identify their possible mRNA target genes in distal cholangiocarcinogenesis.

Methods:

As basis of our study, we conducted Laser Capture Microdissection of FFPE tissue sections of 12 patients with distal cholangiocarcinoma. For each patient we isolated three matched sample sets, including non-neoplastic biliary epithelia, high grade BilIN (grade 3), and invasive dCCA, thereby representing different stages of distal cholangiocarcinogenesis. This resulted in a total of 36 samples. Total RNA was extracted and the expression of ˜800 miRNAs was assessed using the Nanostring® technology. Significantly deregulated miRNAs were validated by quantitative RT-PCR. Possible candidates were then transfected in dCCA derived cell lines and functionally tested for affecting cell proliferation and migration. Target genes were identified using the miRWalk2.0 algorithm and validated by qRT-PCR, Western blot and luciferase reporter assay.

Results:

We identified 40 miRNAs with significantly altered expression from non-neoplastic biliary epithelia to dCCA of which 18 showed gradual up- or downregulation. Clustering analyses separated the samples into three groups: non-neoplastic tissue, high-grade BilIN, and invasive dCCA. Next, we selected candidate miRNAs by fold change expression of invasive dCCA versus BilIN grade 3 versus non-neoplastic biliary epithelia to reflect this stepwise tumorigenic process. Thereby, the following top 4 miRNAs were identified: miR-23a-3 p (2.1-fold up), miR-10a-5 p (9.7-fold up), miR-144 – 3 p (6.3-fold down) and miR-451a (10.9-fold down). The expression of all candidate miRNAs was successfully validated by qRT-PCR. Functional cell culture assays indicated a migration inhibitory effect of miR-451a. The online tool miRWalk2.0 predicted the metastasis-related genes ATF2 and ADAM10 to be direct miR-451a targets. In vitro experiments demonstrated that miR-451a leads to a downregulated mRNA expression of ADAM10 as well as mRNA and protein expression of ATF2. Luciferase assay confirmed direct interaction of miR-451a with the predicted binding sites in ATF2 and ADAM10. In addition, ATF2 knockdown mimicked the inhibitory effect on migration of miR-451a in dCCA cell lines.

Conclusion:

Thus, our data support the concept of BilIN as a direct precursor of invasive dCCA. In addition, we identified candidate miRNAs with a possible functional impact on the early stepwise progression to invasive carcinoma and present miR-451a as a putative tumor suppressor. Investigating this mechanism in interaction with other miRNAs of altered expression is subject of our current research.