Abstract: Objective  To investigate the biological behavior of transmembrane 6 superfamily member 1 (TM6SF1) in intrahepatic cholangiocarcinoma (iCCA) and related mechanisms. Methods Tumor tissues and corresponding adjacent normal tissues of 26 iCCA patients, who were admitted to Oriental Hepatobiliary Surgery Hospital (Shanghai, China) from August 2008 to December 2012, were collected. The expression of TM6SF1 mRNA was analyzed by RT⁃qPCR. The clinical data and tumor tissues mRNA sequencing data of iCCA patients from the FU⁃iCCA cohort were downloaded to analyze the correlation between TM6SF1 and the clinical features of iCCA patients as well as molecules related to the KRAS⁃MEK⁃ERK signaling pathway, and the pathway enrichment analysis was performed. The relationship between the expression level of TM6SF1 and the prognosis of the iCCA patients was analyzed by using Log⁃rank test base on the Cancer Genome Atlas Program (TCGA) database. Transient transfection technology was used to construct hepatocellular⁃cholangiocarcinoma cell lines with transient knockdown or overexpress TM6SF1 in human hepatocellular⁃cholangiocarcinoma cell lines RBE and HCCC⁃9810. CCK⁃8, plate clone formation assays, and Transwell assays were used to evaluate the effects of TM6SF1 on the proliferation, migration, and invasion abilities of RBE and HCCC⁃9810 cells. Flowcytometry assays were used to evaluate the effects of TM6SF1 on cell cycle and apoptosis of RBE and HCCC⁃9810. Ras activity detection assays were used to detect GTP⁃RAS activity. Western blot was used to detect the expression of signaling pathway node molecules. Results Compared with corresponding adjacent normal tissues, TM6SF1 mRNA expression level was significantly down⁃regulated in iCCA cancer tissues (P<0.001), and iCCA patients with TM6SF1 low expression had a higher tumor malignancy and lower overall survival and disease⁃free survival rates (all P<0.05). Knockdown of TM6SF1 promoted the proliferation, migration and invasion ability of RBE and HCCC⁃9810 cells and inhibited apoptosis (all P<0.01), while overexpression of TM6SF1 inhibited the proliferation, migration and invasion ability of RBE and HCCC⁃9810 cells and promoted apoptosis (all P<0.05), though neither affected cell cycle. Pathway enrichment analysis based on FU⁃iCCA cohort mRNA sequencing data showed that the low expression of TM6SF1 was related to the activation of KRAS signaling pathway. Knockdown of TM6SF1  enhanced GTP⁃RAS activity and up⁃regulated the protein expression of p⁃MEK and p⁃ERK in RBE and HCCC⁃9810 cells (all P<0.001), while overexpression of TM6SF1 inhibited GTP⁃RAS activity and down⁃regulated the protein expression of p⁃MEK and p⁃ERK (all P<0.01). The expression levels of GTP⁃RAS, p⁃MEK, p⁃ERK protein were increased after treatment with the KRAS agonist KRA⁃533 in cell lines transiently overexpressing TM6SF1 (allP<0.05). Conclusions TM6SF1 may inhibit the proliferation, migration and invasion ability of iCCA cells and promote apoptosis by inhibiting the KRAS⁃MEK⁃ERK signaling pathway, and the low expression of TM6SF1 is associated with the poor prognosis of iCCA.

Key words: Intrahepatic cholangiocarcinoma, TM6SF1, Proliferation, Migration, Invasion, Apoptosis, KRAS