ITGB6-Knockout Suppresses Cholangiocarcinoma Cell Migration and Invasion with Declining PODXL2 Expression.

Yurie Soejima,Toshio Fukusato,Miho Takeuchi,Motoji Sawabe,Nao Miyamoto

doi:10.3390/ijms22126303

Abstract

Intrahepatic cholangiocarcinoma (iCCA) is a heterogeneous bile duct cancer with a poor prognosis. Integrin αvβ6 (β6) has been shown to be upregulated in iCCA and is associated with its subclassification and clinicopathological features. In the present study, two ITGB6-knockout HuCCT1 CCA cell lines (ITGB6-ko cells) were established using the clustered regulatory interspaced short palindromic repeats (CRISPR), an associated nuclease 9 (Cas9) system, and single-cell cloning. RNA sequencing analysis, real-time polymerase chain reaction (PCR), and immunofluorescent methods were applied to explore possible downstream factors. ITGB6-ko cells showed significantly decreased expression of integrin β6 on flow cytometric analysis. Both cell lines exhibited significant inhibition of cell migration and invasion, decreased wound-healing capability, decreased colony formation ability, and cell cycle dysregulation. RNA sequencing and real-time PCR analysis revealed a remarkable decrease in podocalyxin-like protein 2 (PODXL2) expression in ITGB6-ko cells. Colocalization of PODXL2 and integrin β6 was also observed. S100 calcium-binding protein P and mucin 1, which are associated with CCA subclassification, were downregulated in ITGB6-ko cells. These results describe the successful generation of ITGB6-ko CCA cell clones with decreased migration and invasion and downregulation of PODXL2, suggesting the utility of integrin β6 as a possible therapeutic target or diagnostic marker candidate.

Highlights

Cholangiocarcinoma (CCA) is a heterogeneous bile duct cancer with poor prognosis, usually classified as intrahepatic, perihilar, or distal, based on its anatomical location [1,2,3,4]
Integrin β6 expression was markedly reduced in ITGB6-ko cells such as HuH28 with a low-ITGB6 mRNA level compared to HuCCT1-wt cells, indicating the successful establishment of ITGB6-ko cell lines
Direct sequence analysis after transfection of gRNAs into HuCCT1 cells showed a single-base insertion in the HuCCT1 ITGB6-ko 1 line and a single-base insertion and large deletion in the HuCCT1 ITGB6-ko 2 line. Since these two clones do not have the original sequence of ITGB6, we regarded them as ITGB6-ko cell lines with homozygous mutations and a premature stop codon, the effects of a large deletion in ITGB6-ko 2 line was not evaluated sufficiently

Summary

Introduction

Cholangiocarcinoma (CCA) is a heterogeneous bile duct cancer with poor prognosis, usually classified as intrahepatic, perihilar, or distal, based on its anatomical location [1,2,3,4]. Intrahepatic CCA (iCCA) arises above the second-order bile ducts. Large duct iCCA arises in the large intrahepatic bile ducts near the hepatic hilus and resembles perihilar and extrahepatic distal CCA, exhibiting macroscopic periductal infiltrating patterns and intraductal growth. Small duct iCCA occurs primarily in the hepatic periphery and has a macroscopic mass-forming growth pattern. These subtypes are evidenced in the immune and molecular features of the tumors and are related to patient prognosis [1,2,3,4]

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