Cancer Res Treat.  2021 Jan;53(1):184-198. 10.4143/crt.2020.192.

Long Non-coding RNA CASC15 Promotes Intrahepatic Cholangiocarcinoma Possibly through Inducing PRDX2/PI3K/AKT Axis

• Zhang Y
• Zhang L
• Lu S
• Xiang Y
• Zeng C
• He T
• Ding Y
• Wang W

• KMID: 2510660
• DOI: http://doi.org/10.4143/crt.2020.192

Abstract

Purpose
Intrahepatic cholangiocarcinoma (ICC) is one of the most common liver primary tumors but its treatments are limited. Bioinformatics showed that the expression level of long non-coding RNA cancer-associated susceptibility 15 gene (CASC15) is correlated with ICC progression, but its functional mechanism remains unclear.
Materials and Methods
Tissues from ICC patients, tumor and adjacent tissue, were used for detection of the expression of CASC15. Clinical data were also collected for clinicopathologic and survival analysis. Short interfering RNA and lentiviral short hairpin RNA were used to knock down CASC15 and PRDX2 expression in ICC cell lines, for the analysis of changes of cell function and xenografts. RNA-pulldown and RNA immunoprecipitation assays were used to detect RNA-binding protein, PRDX2. Male nude mice were used for ICC xenografts, and livers were collected after 4 weeks for immunohistochemistry.
Results
CASC15 is highly expressed in ICC tissues and is related to higher TNM stage. Knockdown of CASC15 in ICC cells reduced cell proliferation, migration, invasiveness and increased apoptosis, and G1/S block. PRDX2 bound to CASC15. Knockdown of CASC15 decreased PRDX2 expression which was rescued by the inhibition of proteasome formation. Downregulation of PRDX2 resulted in G1/S block, reduced ICC cell invasion. Downregulation of CASC15 inhibited phosphoinositide 3-kinase (PI3K)/AKT/c-Myc pathway through downregulating of PRDX2 and overexpressed PRDX2 rescued the block. CASC15 knockout in ICC xenografts suppressed tumor development in vivo, decreased the expression of PRDX2 and Ki67 and inhibited PI3K/AKT pathway.
Conclusion
CASC15 promotes ICC possibly by targeting PRDX2 via the PI3K/AKT pathway, indicating poor prognosis and high degree of malignancy of ICC.

Keyword

Long non-coding RNA; CASC15; Intrahepatic cholangiocarcinoma; PRDX2

Figure

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  Fig. 1 Cancer-associated susceptibility 15 (CASC15) affected the prognosis of patients with intrahepatic cholangiocarcinoma (ICC). (A) Relative expression of long non-coding RNA (lncRNA)-CASC15 in tissues of 95 patients. (B) Survival curve analysis of the relationship between lncRNA-CANSC15 expression differences and survival time of patients. (C) Relative expression of ICC cell lines (CCLP, 9810, HuCCT1, and RBE). (D) The efficiency of knockdown of CASC15 in HuCCT1 and RBE cells. **p<0.01, ***p<0.001.

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  Fig. 2 The knockdown of cancer-associated susceptibility 15 (CASC15) expression inhibited cell invasion and proliferation. (A, B) Transwell invasion experiment and quantitative results. (C–E) EdU experiment showed that cell proliferation capacity was decreased in the siCASC15 group. Scale bars=100 μm. (F, G) Cell Counting Kit-8 (CCK-8) assay showed that the cell proliferation capacity decreased in the siCASC15 group. NC, negative control. **p<0.01, ***p<0.001.

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  Fig. 3 The knockdown of cancer-associated susceptibility 15 (CASC15) expression promoted cell apoptosis and inhibited G1/S phase transformation. (A, B) Flow cytometry diagram of cell apoptosis and quantitative results. (C, D) Flow cytometry diagram of cell cycle and quantitative results. (E) Western blotting revealed that the expression of apoptosis and cell cycle-related proteins were changed in the siCASC15 group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, negative control; PARP, poly(ADP-ribose) polymerase. *p<0.05, **p<0.01, ***p<0.001.

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  Fig. 4 Cancer-associated susceptibility 15 (CASC15) was associated with peroxiredoxin 2 (PRDX2) and regulated its expression. (A) Location of CASC15 in intrahepatic cholangiocarcinoma (ICC) cells. (B) RNA-protein complexes were resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis and visualized by silver staining, showing bands at 25–170 kDa. (C) RNA immunoprecipitation (IP)–quantitative polymerase chain reaction showed PRDX2 bound to CASC15. (D) Relationship of PRDX2 and disease-free survival of patients with ICC in The Cancer Genome Atlas database database. (E) Western blotting revealed that PRDX2 expression was decreased in the siCASC15 group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, negative control. Scale bars=200 μm. **p<0.01, ***p<0.001.

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  Fig. 5 The knockdown of peroxiredoxin 2 (PRDX2) expression inhibited the invasion and G1/S phase transformation of intrahepatic cholangiocarcinoma (ICC) cells. (A) Efficiency of PRDX2 knockdown in RBE cells. (B) Flow cytometry diagram of cell cycle and quantitative results. (C) Transwell invasion experiment and quantitative results of RBE cells. Scale bars=100 μm. (D) Western blotting showed that the expression levels of cell cycle and endothelial mesenchymal transition (EMT)–related proteins were altered in the siPRDX2 group of RBE cells. (E) Western blotting showed that the phosphoinositide 3-kinase (PI3K)/AKT/c-Myc pathway was inhibited in the siPRDX2 group of RBE cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, negative control. *p<0.05 and **p<0.01.

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  Fig. 6 Cancer-associated susceptibility 15 (CASC15) affected phosphoinositide 3-kinase (PI3K)/AKT/c-Myc pathway through peroxiredoxin 2 (PRDX2) and CASC15 regulated PRDX2 by inhibiting ubiquitination. (A) Western blotting showed that the PI3K/AKT/c-Myc pathway was inhibited in the siCASC15 group. (B) Western blotting showed that the PI3K/AKT/c-Myc pathway was enhanced when PRDX2 was overexpressed in the siCASC15 group. (C, D) Expression of PRDX2 was increased and the PI3K/AKT pathway was activated when proteasome formation was inhibited (left panel). Relative expression of PRDX2 changed little in the siCASC15 group (right panel). (E, F) Flow cytometry diagram of cell apoptosis and quantitative results. 5-FU, 5-fluorouracil; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, negative control. **p<0.01.

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  Fig. 7 The knockdown of cancer-associated susceptibility 15 (CASC15) in xenograft model inhibited the development of intrahepatic cholangiocarcinoma (ICC). (A–D) Knockdown of CASC15 in xenograft model decreased tumor size and weight. (E) Immunohistochemistry assay showed peroxiredoxin 2 (PRDX2), phosphoinositide 3-kinase (PI3K), AKT, pAKT, and Ki67 expression levels were decreased in the ShCASC15 group compared to in the control group. NC, negative control. Scale bar=100 μm. **p<0.01.

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  Fig. 8 Knockdown of cancer-associated susceptibility 15 (CASC-15) inhibits the phosphoinositide 3-kinase (PI3K)/AKT/c-Myc signal pathway possibly by binding to peroxiredoxin 2 (PRDX2) and promoting its degradation, resulting in the decrease of intrahepatic cholangiocarcinoma (ICC) development.

Reference

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