Abstract:

Objective To discuss the effect of long non-coding RNA （lncRNA） urothelial carcinoembryonic antigen 1 （UCA1） on the anoikis of human gastrointestinal stromal tumor （GIST） cells， and to clarify its mechanism of action. Methods The human GIST cell line GIST-T1 was cultured under adherent and anoikis conditions， and the GIST-T1 cells resistant to anoikis were induced. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression level of lncRNA UCA1 in the two kinds of cells； Western blotting method was used to detect the expression levels of autophagy markers microtubule-associated protein light chain 3 （LC3）-Ⅰ， LC3-Ⅱ， and p62 proteins in the cells， and the LC3-Ⅱ/LC3-Ⅰ ratio was calculated. The GIST-T1 cells were transfected with lncRNA UCA1 knockdown or negative control lentivirus and treated with the autophagy activator rapamycin （RAPA）. The GIST-T1 cells were divided into control group， sh-NC group， sh-UCA1 group， sh-NC+RAPA group， and sh-UCA1+RAPA group， and the cells were subjected to anoikis induction. Flow cytometry was used to detect the anoikis rates of cells in various groups； Western blotting method was used to detect the expression levels of LC3-Ⅰ， LC3-Ⅱ， and p62 proteins in the cells in various groups； cell counting kit-8 （CCK-8） method was used to detect the proliferation activities of the cells in various groups； cell scratch assay was used to detect the scratch healing rates of the cells in various groups； Transwell chamber assay was used to detect the number of invasive cells in various groups. The GIST-T1 cells from sh-NC group and sh-UCA1 group were collected and injected via the tail vein to establish two groups of nude mouse xenograft models， respectively. After 4 weeks， a fluorescence in vivo imaging system was used to detect the tumor growth and liver metastasis in the nude mice in two groups， and the tumor tissue mass was measured. TUNEL staining was used to observe cell apoptosis in the tumor tissue of the nude mice in two groups； immunohistochemical staining was used to detect the expression levels of LC3B and p62 proteins in the tumor tissue of the nude mice in two groups. Results Compared with adherently cultured GIST-T1 cells， the expression level of lncRNA UCA1 in anoikis-resistant GIST-T1 cells was significantly increased （P<0.05）， the LC3-Ⅱ/LC3-Ⅰ ratio was significantly increased （P<0.05）， and the expression level of p62 protein was significantly decreased （P<0.05）. Compared with sh-NC group， the anoikis rate of GIST-T1 cells in sh-UCA1 group was increased （P<0.05）， the LC3-Ⅱ/LC3-Ⅰ ratio was significantly decreased （P<0.05）， the expression level of p62 protein was significantly increased （P<0.05）， the cell proliferation activity and scratch healing rate were significantly decreased （P<0.05）， and the number of invasive cells was significantly decreased （P<0.05）； compared with sh-UCA1 group， the anoikis rate of GIST-T1 cells in sh-UCA1+RAPA group was significantly decreased （P<0.05）， the LC3-Ⅱ/LC3-Ⅰ ratio was significantly increased （P<0.05）， the expression level of p62 protein was significantly decreased （P<0.05）， the cell proliferation activity and scratch healing rate were significantly increased （P<0.05）， and the number of invasive cells was significantly increased （P<0.05）. In the nude mouse xenograft experiment， compared with sh-NC group， the fluorescence intensity in liver tissue of the nude mice in sh-UCA1 group was weakened， the tumor mass was significantly decreased （P<0.05）， the cell apoptotic rate in tumor tissue was significantly increased （P<0.05）， the expression level of LC3B protein was significantly decreased （P<0.05）， and the expression level of p62 protein was significantly increased （P<0.05）. Conclusion LncRNA UCA1 is highly expressed in anoikis-resistant GIST-T1 cells， and downregulating its expression can promote anoikis of GIST-T1 cells by reducing the autophagy level， thereby inhibiting cell growth， migration， and invasion.

Key words: Gastrointestinal stromal tumor, Long non-coding RNA, Urothelial carcinoembryonic antigen 1, Anoikis, Autophagy