LOXL1-AS1 Promotes Cholangiocarcinoma Progression by Regulating the JAK2/STAT3 Signaling Pathway

Academic Background

Cholangiocarcinoma (CCA) is a malignant tumor originating from biliary epithelium, presenting significant medical challenges. Its incidence shows geographical variation, being particularly high in Asia and South America. The occurrence of CCA is associated with various factors such as cholecystitis, cholelithiasis, cirrhosis, and hepatitis virus infection. Due to its insidious early symptoms, many patients are diagnosed at an advanced stage, leading to poor surgical outcomes and prognosis. Currently, in-depth research into the mechanisms of CCA is urgently needed to develop effective targeted therapies, improve patient survival rates, and enhance quality of life. Long non-coding RNAs (lncRNAs) are RNA molecules over 200 nucleotides long, which do not encode proteins. Recent studies have shown that lncRNAs play key regulatory roles in various biological processes, including tumorigenesis and cancer progression.

Source

This study was conducted by a research team from the Second Affiliated Hospital of Harbin Medical University, Heilongjiang Province. The primary authors include Shaobo Yu, Xin Gao, Sidi Liu, among others. The study was published in the 2024 issue of Cancer Gene Therapy (DOI: https://doi.org/10.1038/s41417-024-00726-2).

Research Process

Clinical Samples

Collected 65 pairs of tumor tissues and corresponding non-tumor tissues from cholangiocarcinoma patients. These samples were collected between January 2013 and July 2018 at the Second Affiliated Hospital of Harbin Medical University, immediately frozen after surgery, and stored at -80°C. All patients signed informed consent, and ethical committee approval was obtained.

Cell Culture and Transfection

Cultured and conducted transfection experiments on normal biliary epithelial cell line HIBEC and cholangiocarcinoma cell lines CCLP-1, QBC939, RBE, and HuCCT1. LOXL1-AS1 knockdown and overexpression were achieved through siRNA and plasmid transfection.

qRT-PCR

Total RNA from cell and tissue samples was extracted and reverse transcribed into cDNA, followed by qRT-PCR analysis to detect the expression levels of target genes.

Cell Viability Assay

Cell viability was assessed using CCK-8 and EdU assays, and mitochondrial membrane potential was evaluated using JC-1 assay.

Cell Apoptosis Detection

Cell apoptosis rate was detected by TUNEL assay and flow cytometry.

Cell Migration and Invasion Assays

Cell migration and invasion capabilities were assessed using scratch and Transwell assays.

Western Blot

Western Blot analysis was conducted to determine the expression of proteins related to the JAK2/STAT3 signaling pathway.

Subcellular Localization Assay

Subcellular fractionation and fluorescent in situ hybridization (FISH) were used to determine the localization of LOXL1-AS1 in cells.

RNA Pulldown Assay

RNA pulldown and RIP assays were used to validate the interaction between LOXL1-AS1 and JAK2 protein.

In Vivo Experiments

Subcutaneous tumor and metastasis models were established in nude mice, and evaluations were performed using H&E staining and immunohistochemistry.

Main Results

High Expression of LOXL1-AS1 in CCA

LOXL1-AS1 was found to be significantly overexpressed in cholangiocarcinoma tissues (Figure 1a). This high expression was significantly associated with TNM stage, lymph node metastasis, and patient prognosis (Figures 1b-1e). Evaluated using Kaplan-Meier survival curves and ROC analysis, LOXL1-AS1 was closely linked to DFS and OS in CCA patients (Figure 1f).

Regulation of Tumor Proliferation and Apoptosis by LOXL1-AS1

CCK-8 and EdU assays revealed that LOXL1-AS1 knockdown significantly inhibited the proliferation of cholangiocarcinoma cells (Figures 2c-2d), while LOXL1-AS1 overexpression promoted proliferation. TUNEL and JC-1 assays indicated that LOXL1-AS1 knockdown significantly induced apoptosis in cholangiocarcinoma cells (Figures 2e-2f). In vivo experiments in mice showed that LOXL1-AS1 knockdown significantly inhibited tumor growth (Figures 2g-2h).

Effect on Migration and Invasion by LOXL1-AS1

Scratch and Transwell assays demonstrated that LOXL1-AS1 knockdown significantly inhibited the migration and invasion abilities of cholangiocarcinoma cells (Figures 3a-3c). Analysis of EMT markers revealed that LOXL1-AS1 is closely associated with the initiation of the EMT process (Figure 3d). In vivo experiments indicated that LOXL1-AS1 knockdown significantly reduced liver and lung metastasis (Figures 3e-3f).

Regulation of JAK2/STAT3 Signaling Pathway by LOXL1-AS1

Experiments found that LOXL1-AS1 primarily localizes in the cytoplasm (Figures 4a-4b). Further bioinformatics analysis and experimental results suggested that LOXL1-AS1 influences the phosphorylation state of JAK2, thereby affecting the JAK2/STAT3 signaling pathway (Figures 4c-4d).

Impact on JAK2 Ubiquitination Degradation by LOXL1-AS1

RNA pulldown and RIP assays verified the direct interaction between LOXL1-AS1 and JAK2 (Figures 5a-5b). Following LOXL1-AS1 knockdown, the degradation of JAK2 protein accelerated, while inhibition of the proteasome with MG132 impeded JAK2 degradation (Figures 5d-5e). Further CO-IP experiments showed that LOXL1-AS1 knockdown significantly increased the ubiquitination level of JAK2 (Figure 5f).

Relationship Between YY1 Transcription Factor and LOXL1-AS1

Bioinformatics predictions and experimental validations indicated that YY1 regulates LOXL1-AS1 expression by binding to upstream promoter sites E1 and E2, primarily through the E2 site (Figures 7a-7c). The functional impact of LOXL1-AS1 knockdown on CCA cells could be reversed by YY1 overexpression (Figures 7d-7i).

Discussion and Conclusion

The study indicates that LOXL1-AS1 is highly expressed in cholangiocarcinoma and is significantly associated with clinicopathological features and patient prognosis. LOXL1-AS1 promotes the development of CCA by inhibiting JAK2 ubiquitination degradation and activating the JAK2/STAT3 signaling pathway. The YY1 transcription factor further influences the malignant biological characteristics of CCA by regulating LOXL1-AS1 expression. These findings provide important clues for understanding the pathogenesis of CCA and developing new therapeutic strategies. The study suggests that further exploration of the interactions between LOXL1-AS1 and other molecules, and its role in other signaling pathways, is needed to validate its clinical significance in a larger patient cohort.