Novel tumor suppressor role of miRNA-876 in cholangiocarcinoma

Abstract

Cholangiocarcinoma (CCA) is a rare, highly invasive malignancy, and its incidence is increasing globally. MicroRNAs (miRNAs) mediate a wide array of cellular and biological processes and are dysregulated in various tumors. The functional and biological roles of miRNAs in CCA have not been fully elucidated. In this study, we show that miR-876 expression levels and copy number are significantly attenuated in the TCGA cohort of CCA tissue samples. TCGA expression data was consistent with the observed substantial decrease in miR-876 expression in patient samples and CCA cell lines. In-silico algorithm databases revealed BCL-XL as a potential target of miR-876. We observed miR-876 expression to be downregulated, whereas, BCL-XL upregulated in CCA cell lines. BCL-XL was identified as a direct functional target of miR-876 in CCA. miR-876-mediated reduction of BCL-XL regulated cell survival, induced apoptosis and caspase 3/7 expression in CCA. BCL-XL overexpression reversed the miR-876 mediated effect on CCA cell growth and apoptosis. Stable overexpression of miR-876 produced potent tumor suppressor activity and in vivo tumor cell growth reduction. Overexpression of miR-876 in a patient-derived xenograft (PDX) cell line significantly suppressed BCL-XL expression and spheroid formation with a concomitant induction of caspase 3/7 activity and apoptosis. This study demonstrates a novel tumor suppressor role for miR-876 in CCA, identifies BCL-XL as an actionable target, and suggests a potential therapeutic role for miR-876 in CCA.

Fig. 1. miR-876 is suppressed in cholangiocarcinma (CCA).

a Copy number analysis of CCA samples from TCGA database. b Relative miR-876 expression levels of CCA samples from TCGA database. c Relative miR-876 expression levels in a normal human cholangiocyte line (H69) and cholangiocarcinoma cell lines

Fig. 2. BCL-XL is a target of miR-876 action.

a The seed sequence of miR-876 is complementary to the 3′UTR of BCL-XL. b BCL-XL expression levels in CCA samples relative to normal samples. c, d BCL-XL mRNA and protein expression levels in a normal human cholangiocyte line and CCA cell lines. e Luciferase activity after miR-876 overexpression in KMCH and HuCCT1 cell lines. f, g BCL-XL protein expression following miR-876 overexpression in KMCH and HuCCT1 cell lines. *p < 0.05

Fig. 3. miR-876 overexpression regulates cellular proliferation and induces apoptosis.

a miR-876 overexpression in KMCH cells. b Average colony number of KMCH cells following miR-876 overexpression. c miR-876 overexpression induces apoptosis in KMCH cells. d miR-876 increases caspase 3/7 activity in KMCH cells. e Average colony number following miR-876 and pCMV6-BCL-XL co-overexpression in HuCCT1 and KMCH cell lines. f Apoptosis analysis following miR-876 and pCMV6 BCL-XL co-overexpression in KMCH cell line. *p < 0.05

Fig. 4. Stable overexpression of miR-876 suppresses in vivo tumor growth.

a Stable overexpression of miR-876 in the KMCH cell line. b Colony formation ability of KMCH cells stably expressing miR-876. c BCL-XL protein expression levels in KMCH cells expressing miR-876. d Stable expression of miR-876 induces apoptosis. e miR-876 expression suppresses in vivo tumor cell growth. f miR-876 suppresses BCL-XL expression in tumor in vivo. g Western blot analysis showing expression of PARP, caspase 3, and PCNA in miR-876-expressing in vivo tumor samples. *p < 0.05

Fig. 5. miR-876 overexpression regulates proliferation and apoptosis in a patient-derived xenograft cell line.

a miR-876 overexpression in the CHNG6 cell line. b BCL-XL expression levels folllowing miR-876 overexpression in CHNG6 cells. c miR-876 overexpression suppresses cellular spheroid formation. d miR-876 overexpression induces apoptosis in CHNG6 cells. e miR-876 induces caspase 3/7 activity in CHNG6 cells