Kinesin family member C1 (KIFC1, also known as HSET) is a minus end-directed motor protein, which is critical in centrosome clustering. The present study investigated the expression of KIFC1 in paired hepatocellular carcinoma (HCC) tissues and adjacent non-cancerous tissues from 91 patients by immunohistochemical analysis; clinical data were concomitantly collected. KIFC1 was expressed at high levels in HCC tissues, compared with that in peritumoral tissues (54.9 vs. 14.3%; P<0.01), and its expression correlated with tumor emboli, metastasis, recurrence and time of recurrence. Kaplan-Meier analysis showed that the expression of KIFC1 was significantly associated with tumor-free survival rates. In addition, multivariate analyses revealed that the overexpression of KIFC1was an independent predictive marker in patients with HCC. Consistently, data derived from GEPIA was in agreement with the results. In vitro, KIFC1 knockdown effectively decreased HCC cell viability, and induced apoptosis and cell death. KIFC1 knockdown also significantly suppressed tumor cell migration and invasion in vitro. Mechanistically, the apoptosis-related protein, B-cell lymphoma-2 (Bcl-2), was downregulated in KIFC1 small interfering RNA-treated groups, whereas thee levels of Bcl-2-associated X protein and p53 were upregulated. In addition, the expression levels of phosphorylated phosphoinositide 3-kinase and phosphorylated AKT were decreased significantly when KIFC1 was silenced. The epithelial-mesenchymal transition-related proteins, N-cadherin, matrix metalloproteinase-2 (MMP-2), β-catenin, Slug, and Zinc finger E-box-binding homeobox 1, were downregulated, whereas the expression of E-cadherin was upregulated. The overexpression of KIFC1 was correlated closely with the progression of HCC and poor prognosis, and suggested that the expression levels of KIFC1 are a potential prognostic biomarker and therapeutic target in HCC.

N6-methyladenosine (m6A) has been reported as an important mechanism of post-transcriptional regulation. Programmed death ligand 1 (PD-L1) is a primary immune inhibitory molecule expressed on tumor cells that promotes immune evasion. In addition, seven in absentia homolog 2 (Siah2), a RING E3 ubiquitin ligase, has been involved in tumorigenesis and cancer progression. However, the role of m6A-METTL14-Siah2-PD-L1 axis in immunotherapy remains to be elucidated. In this study, we showed that METTL14, a component of the m⁶A methyltransferase complex, induced Siah2 expression in cholangiocarcinoma (CCA). METTL14 was shown to enrich m⁶A modifications in the 3’UTR region of the Siah2 mRNA, thereby promoting its degradation in an YTHDF2-dependent manner. Furthermore, co-immunoprecipitation experiments demonstrated that Siah2 interacted with PD-L1 by promoting its K63-linked ubiquitination. We also observed that in vitro and in vivo Siah2 knockdown inhibited T cells expansion and cytotoxicity by sustaining tumor cell PD-L1 expression. The METTL14-Siah2-PD-L1–regulating axis was further confirmed in human CCA specimens. Analysis of specimens from patients receiving anti-PD1 immunotherapy suggested that tumors with low Siah2 levels were more sensitive to anti-PD1 immunotherapy. Taken together, our results evidenced a new regulatory mechanism of Siah2 by METTL14-induced mRNA epigenetic modification and the potential role of Siah2 in cancer immunotherapy.

SLC25A22, a member of mitochondrial carrier system (MCS) family encoding a mitochondrial glutamate transporter, has been reported to have vital roles in promoting proliferation and migration in cancer. Gallbladder cancer (GBC) is the most common biliary tract malignancy and has a poor prognosis. We aimed to determine the expression and function of SLC25A22 in GBC. Immunohistochemistry (IHC) staining analysis and quantitative real-time PCR (qRT-PCR) were conducted to determine the expression of SLC25A22 in GBC tissues. Human NOZ and GBC-SD cells were used to perform the experiments. The protein expression was detected by western-blot analysis. Cell viability was evaluated via CCK-8 assay and colony formation assay. Cell migration and invasion in vitro were investigated by wound healing and transwell assay. Annexin V/PI staining assay for apoptosis were measured by flow cytometry. The effect of SLC25A22 in vivo was conducted with subcutaneous xenograft. We indicated that the expression of SLC25A22 was significantly upregulated in GBC tumor tissues as well as cell lines. Downregulation of SLC25A22 inhibited GBC cell growth and proliferation in vitro and in vivo and also had an effect on metastasis of GBC cells through the EMT processes. In addition, inhibition of SLC25A22 promoted mitochondrial apoptosis via downregulating BCL-2 and upregulating cleaved PARP, Cytochrome-c, and BAX mediated by MAPK/ERK pathway. Our study identified that SLC25A22 promoted development of GBC activating MAPK/ERK pathway. SLC25A22 has a potential to be used as a target for cancer diagnosis of GBC and related therapies.