ABSTRACT
Objective To construct a recombinant lentiviral plasmid of receptor for advanced glycation end products (RAGE) and establish RAGE stable expressing hepatocellular carcinoma cell models in order to explore the pathogenesis of RAGE in the occurrence and development of hepatocellular carcinoma. Methods RAGE gene fragments were amplified by PCR and cloned into the lentiviral expression vector pCDH-CMV-MCS-EF1-Puro. Then the recombinant lentiviral plasmid and packaging plasmid were used to package lentivirus in HEK293T cells by calcium phosphate transfection. Lentivirus supernatant was collected to infect hepatoma cells HepG2 and Huh7. After that cells were screened by puromycin. Finally, RAGE expression was detected by real-time PCR and Western blot. Results RAGE gene fragment was successfully amplified and inserted into lentiviral expression vector. After packaging the lentivirus-infected liver cancer cells, real-time quantitative PCR and Western blot results showed that the mRNA (P<0.05) and protein expression of RAGE cells stably expressing RAGE were significantly higher than that of the control cells. Conclusions The RAGE overexpressing hepatocarcinoma cell line was successfully constructed by using lentiviral expression vector, which laid a good foundation for further study on the pathogenesis of RAGE.
ABSTRACT
@#Objective: To construct a hsa-microRNA-224(miR-224) lentiviral expression vector and to establish pancreatic mucinous cystadenocarcinoma MCC1 cell line with stable miR-224 over-expression. Methods: Pri-miR-224 gene fragment was designed and amplified by quantitative real-time polymerase chain reaction (qRT-PCR), and then loaded into GV369 lentiviral vectors (GV369-miR224) by gene recombination technology. GV369-miR-224 lentivrial expression vectors were then identified by PCR and DNA sequencing. The GV369-miR-224 vector fluid was then used to infect pancreatic mucinous cystadenocarcinoma MCC1 cell line to establish the MCC1 cell line stably over-expressing miR-224. The transfection efficiency of GV369-NC and GV369-miR-224 was observed under fluorescence microscopy; and the expression levels of miR-224 in MCC1, GV369-miR-224-MCC1 and GV369-NC-MCC1 cell lines were detected by RT-PCR. Results: The GV369-miR-224 lentiviral vectors were successfully constructed. GV369-miR-224-MCC1 and GV369-NC-MCC1 cells all emit green fluorescence under the fluorescence microscope. The expression level of miR-224 in GV369miR-224-MCC1 cell group was significantly higher than that in negative control GV369-miR-224-MCC1 group and blank control MCC1 cell group (23.45±1.94， 1.46±0.1 and 2.11±0.38, P<0.01), however, there was no significant difference between the two control groups (P>0.05). Conclusion: A pancreatic mucinous cystadenocarcinoma MCC1 cell line with stable miR-224 over-expression was successfully established, and this will provide a new cell model for exploring the function and pathogenesis of miR-224 in pancreatic mucinous cystadenocarcinoma.