NOV/CCN3 Promotes Cell Migration and Invasion in Intrahepatic Cholangiocarcinoma via miR-92a-3p

Abstract

Intrahepatic cholangiocarcinoma (ICC) is a common type of human cancer with a poor prognosis, and investigating the potential molecular mechanisms that can contribute to gene diagnosis and therapy. Herein, based on the recently concerned vertebrate-specific Cyr61/CTGF/NOV (CCN) gene family because of its important roles in diverse diseases, we obtained NOV/CCN3 to query for its potential roles in tumorigenesis via bioinformatics analysis. Experimental validations confirmed that both NOV mRNA and protein are up-regulated in two ICC cell lines, suggesting that it may promote cell migration and invasion by promoting EMT. To elucidate the detailed regulatory mechanism, miR-92a-3p is screened and identified as a negative regulatory small RNA targeting NOV, and further experimental validation demonstrates that miR-92a-3p contributes to NOV-mediated migration and invasion of ICC via the Notch signaling pathway. Our study reveals that NOV may be a potential target for diagnosing and treating ICC, which will provide experimental data and molecular theoretical foundation for cancer treatment, particularly for future precision medicine.

Keywords: NOV/CCN3; cell migration; intrahepatic cholangiocarcinoma; invasion; miR-92a-3p.

Figure 1

Pan-cancer analysis of expression pattern of CCN gene family. (A) Expression distributions of homologous genes in the CCN gene family. * indicates significantly deregulated genes (|log₂FC| > 1.5, padj < 0.05). (B) Expression patterns of five genes in normal and tumor samples in CHOL. (C) Paired analysis of deregulated NOV and WISP3 across diverse cancer types. The abnormally expressed genes are presented (|log₂FC| > 1.2, padj < 0.05 for paired analysis).

Figure 2

Expression analysis and experimental validation. (A) Pan-cancer analysis of screened NOV across cancer types based on tumor and normal samples. The red circled part means that there are significant difference on the expression of NOV in BRCA and CHOL (Tumor vs. Normal) (B) NOV mRNA expression based on the q-PCR method. *** indicates p < 0.001. (C) NOV protein expression based on Western blot. *** indicates p < 0.001.

Figure 3

Overexpression and silencing of NOV in HCCC-9810 and RBE. (A). Expression efficiency of NOV mRNA (left) and protein (right) in HCCC-9810. (B) Expression efficiency of NOV mRNA (left) and protein (right) in RBE. (C) Gene silencing of NOV mRNA (left) and protein (right) in HCCC-9810. (D). Gene silencing of NOV mRNA (left) and protein (right) in RBE. All experiments are repeated three times independently, and the data are analyzed by t-test, ** indicates p < 0.01, *** indicates p < 0.001.

Figure 4

NOV regulates the proliferation of ICC cells. (A) The effect of NOV overexpression on cell proliferation via CCK-8 method in HCCC-9810. (B). The effect of NOV overexpression on cell proliferation via the CCK-8 method in RBE. (C) The effect of NOV silencing on cell proliferation via CCK-8 method in HCCC-9810. (D) The effect of NOV silencing on cell proliferation via the CCK-8 method in RBE. (E) The effect of NOV overexpression and silencing on cell proliferation via colony-forming assay in HCCC-9810. (F) The effect of NOV overexpression and silencing on cell proliferation via colony-forming assay in RBE.

Figure 5

NOV promotes the migration of ICC cells. (A) The effect of NOV overexpression on cell migration via wound healing assay. (B) The effect of NOV silencing on cell migration via wound-healing assay. (C) The effect of NOV overexpression on cell migration via transwell migration (100×). (D) The effect of NOV silencing on cell migration via transwell migration (100×). ** indicates p < 0.01 and *** indicates p < 0.001.

Figure 6

NOV promotes the invasion of ICC cells. (A) The effect of NOV overexpression on invasion ability (100×). (B) The effect of NOV silencing on invasion ability (100×). (C) The effect of NOV overexpression on EMT proteins confirmed by Western blot. (D) The effect of NOV silencing on EMT proteins confirmed by Western blot. All experiments are repeated three times independently, and the data are analyzed by t-test. * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001.

Figure 7

The effect of NOV on the Notch1 pathway. (A) The effect of NOV overexpression on Notch1 protein. (B) The effect of NOV silencing on Notch1 protein. ** indicates p < 0.01 and *** indicates p < 0.001.

Figure 8

NOV is the direct target of miR-92a-3p. (A) Related miRNAs of NOV via different prediction methods. (B). Expression levels of miR-92a-3p, miR-30e-5p and miR-455-5p in HCCC-9810 (left) and RBE (right) cells. (C) Relative expression level of miR-92a-3p in tumor and adjacent normal samples. (D) Expression correlation of miR-92a-3p and NOV. (E) The effect of miR-92a-3p on NOV mRNA in HCCC-9810 (left) and RBE (right) cells. (F) The effect of miR-92a-3p on NOV protein in HCCC-9810 (up) and RBE (down) cells. (G). The binding site of miR-92a-3 and NOV (3′UTR WT and 3′UTR MUT). (H) Luciferase activity measurements of WT and MUT after cotransfection of NOV 3′UTR WT or NOV 3′UTR MUT and miR-92a-3p mimic or miR-NC. *** indicates p < 0.001.

Figure 9

miR-92a-3p contributes to cell transformation and migration mediated by NOV. (A). Transwell analysis of migratory and invasive ability after transfection of miR-92a-3p mimics and NOV overexpression plasmid in HCCC-9810 cells. (B–C) The number of migratory (B) and invasive ability (C) after transfection of miR-92a-3p mimics and NOV overexpression plasmid in HCCC-9810 cells. (D–E) Expression levels of E-cadherin, N-cadherin, MMP9, vimentin and Snail after transfecting miR-92a-3p mimics and NOV overexpression plasmid in HCCC-9810 cells based on Western blot. (F) Schematic figure indicating the miR-92a-3p /NOV/Notch1 regulatory pathways and resulting biological effects in ICC. pcDNA3.0 + miR-NC vs. NOV + miR-NC: ** indicates p < 0.01, *** indicates p < 0.001; NOV + miR-NC vs. NOV + miR-92a-3p mimic: ^# indicates p < 0.05, ^## indicates p < 0.01, ^### indicates p < 0.001.