Overexpression of circRNA chr7:154954255-154998784+ in cancer-associated pancreatic stellate cells promotes the growth and metastasis of pancreatic cancer by targeting the miR-4459/KIAA0513 axis.

PURPOSE: Circular RNAs (circRNAs) have been reported to act as important regulators in pancreatic cancer. Abnormal expression of circRNAs in pancreatic cancer cells (PCCs) can promote the development of pancreatic cancer; however, the role of circRNAs in cancer-associated pancreatic stellate cells (CaPSCs) remains unclear. PATIENTS AND METHODS: In this study, we isolated CaPSCs from pancreatic cancer tissues from 5 pancreatic cancer patients and NaPSCs from normal pancreatic tissue from 5 patients with benign pancreatic disease. After the PSCs were co-cultured with the pancreatic cancer cell line PANC-1, a CCK-8 assay was used to detect PANC-1 proliferation ability, and CaPSCs1, which had the strongest promoting effect on PANC-1 proliferation, and NaPSCs1, which had the weakest effect, were screened. Then, the circRNA, microRNA (miRNA) and mRNA profiles between CaPSCs1 and NaPSCs1 were compared by RNA-seq. The candidate circRNA/miRNA/target protein axis was selected using bioinformatics analysis. circRNAs were silenced and miRNAs were overexpressed in CaPSCs1, and the expression of circRNAs, miRNAs and target proteins were detected by qRT-PCR and Western blot, respectively. At the same time, CCK8, wound healing, and Transwell assays were used to detect the proliferation, migration and invasion of PANC-1 cells in the different co-culture groups. Moreover, a tumour xenograft model was used to observe the tumorigenic ability of PANC-1 cells in different co-culture groups. Finally, immunohistochemistry was used to detect the expression of target proteins in PDAC tissues, and the clinicopathological features and prognosis were analysed. RESULTS: The expression of the differentially expressed RNAs identified by RNA-seq was verified by qRT-PCR, and the chr7:154954255-154998784+/miR-4459/KIAA0513 axis was selected from the candidate targets. Functional studies of PANC-1 cells after co-culture with chr7:154954255-154998784+-silenced CaPSCs1 showed that the proliferation, invasion and metastasis of PANC-1 cells decreased. Moreover, after chr7:154954255-154998784+ was silenced, the expression of miR-4459 in CaPSCs1 increased, and the expression of KIAA0513 decreased. When PANC-1 cells were co-cultured with CaPSCs1 with miR-4459 overexpression, they showed an increased ability to proliferate, invade and metastasize. Additionally, when miR-4459 was overexpressed in CaPSCs1, the expression of chr7:154954255-154998784+ and KIAA0513 decreased. Animal experiments revealed that silencing chr7:154954255-154998784+ in CaPSCs1 inhibited tumour growth in nude mice inoculated with CaPSCs1+PANC-1 cells. Finally, we performed immunohistochemistry and a prognostic analysis of KIAA0513 expression in paraffin tissue samples from patients with pancreatic cancer and found that high expression of KIAA0513 was associated with more aggressive clinicopathological factors. Furthermore, patients with high expression of KIAA0513 had worse disease-free survival (DFS) and overall survival (OS). CONCLUSION: Chr7:154954255-154998784+ may promote the development of pancreatic cancer through the miR-4459/KIAA0513 axis in CaPSCs and may be an important therapeutic target for patients with pancreatic cancer in the future.

A modified method for isolating human quiescent pancreatic stellate cells.

BACKGROUND: This study explored a simple, high-yield method for isolating quiescent human pancreatic stellate cells (PSCs) to provide sufficient and reliable raw materials for PSC-related studies. MATERIALS AND METHODS: Single-cell suspensions were prepared from normal human pancreatic tissue specimens using the gentleMACS™ tissue processor, which enhanced the yield and viability of the suspensions. Percoll density gradient centrifugation was then performed to isolate quiescent normal PSCs (NPSCs). Cell viability was determined by trypan blue staining, and the states of the NPSCs were determined by autofluorescence and oil red O staining. The purity of human activated PSCs (APSCs) was determined by immunofluorescence assays. RESULTS: The yield of NPSCs was ~(2.75±0.65)×106 cells/g. The maximum cell viability was 92%, whereas the maximum cell purity was 95%. CONCLUSION: The method employed in this study to isolate PSCs is a simple, high-yield and stable method that is worth popularizing.

Upregulated FFAR4 correlates with the epithelial-mesenchymal transition and an unfavorable prognosis in human cholangiocarcinoma.

BACKGROUND: Free fatty acid receptor 4 (FFAR4) is associated with the epithelial mesenchymal transition (EMT) and is involved in the progression of several types of cancer. However, the role of FFAR4 in cholangiocarcinoma (CCA) remains unclear. OBJECTIVE: The present study evaluated the diagnosis and prognosis of CCA using FFAR4 as a biomarker. METHODS: Immunohistochemistry was employed to detect expression of FFAR4 in 98 samples of CCA tissues and adjacent tissues. In addition, expression of E-cadherin, vimentin, Snail-1, CK7 and CK19 in the 98 samples of CCA tissues was detected, and relationships with FFAR4 were analyzed. Correlation between FFAR4 and clinical pathological factors and prognosis was also analyzed. RESULTS: FFAR4 was highly expressed in 72.4% (71/98) of CCA tissues and 29.6% (29/98) of adjacent tissues, with a statistically significant difference between the two tissue types (P< 0.05). A negative correlation between high expression FFAR4 and E-cadherin expression in CCA tissues was also observed (r=-0.445, P< 0.001), and high expression of FFAR4 was positively correlated with vimentin (r= 0.354, P< 0.001), Snail-1(r= 0.496, P< 0.001), CK7(r= 0.494, P< 0.001) and CK19 (r= 0.532, P< 0.001). Moreover, the degree of FFAR4 expression was associated with aggressive clinicopathological characteristics, such as histological grade, perineural invasion (PNI), lymph node metastasis (LNM), advanced TNM stage and preoperative serum CA19-9 level (P< 0.05). In terms of prognosis, CCA patients with high FFAR4 expression showed shorter disease-free survival (DFS) (P< 0.05) and overall survival (OS) (P< 0.05) than did patients with low FFAR4 expression. CONCLUSIONS: FFAR4 overexpression may mediate the process of CCA EMT. In addition, FFAR4 is promising as a new diagnostic molecule and therapeutic target for CCA.

Role of palliative radiotherapy in unresectable intrahepatic cholangiocarcinoma: population-based analysis with propensity score matching.

BACKGROUND: This population-based study evaluated the overall (OS) and cancer-specific survival (CSS) benefit from palliative radiotherapy (RT) in patients with unresectable intrahepatic cholangiocarcinoma (ICC). METHODS: We queried The Surveillance, Epidemiology, and End Results (SEER) database for the patients with unresectable ICC diagnosed from 1973 to 2013. Propensity score-matched analysis was performed to reduce the impact of the selection bias between the palliative RT group and the nonpalliative RT group. Kaplan-Meier survival curves were used to estimate the survival outcome before and after propensity score matching. OS and CSS were compared between patients with and without palliative RT using univariate and multivariate Cox proportional hazards regression analyses. RESULTS: A total of 4,027 of 15,803 patients diagnosed with ICC were included in this study. Of those, 847 (21%) patients underwent palliative RT, whereas 3,180 (79%) did not. In the unmatched population, patients treated with palliative RT had improved OS and CSS relative to those treated without palliative RT (adjusted HR =0.9065, 95% CI =0.8360-0.982, P=0.01735) and CSS (adjusted HR =0.8874, 95% CI =0.8160-0.9652, P=0.00532). After propensity score matching, palliative RT was associated with a significantly improved OS (adjusted HR =0.8544, 95% CI =0.7722-0.9453, P=0.00228) and CSS (adjusted HR =0.8563, 95% CI =0.7711-0.9509, P=0.0037). CONCLUSION: Palliative RT seems to improve the prognosis of patients with unresectable ICC.