Preliminary Study On Autophagy Of Cells To Maintain The Stemness Of CNE2 Stem Cell-like Cells In Nasopharyngeal Carcinoma

Objective To study the function of autophagy in maintaining the biological characteristics of Nasopharyngeal carcinoma CNE2 stem-like stem cells,and to provide a new experimental basis for further elucidation of the role of autophagy in the regulation of Nasopharyngeal carcinoma.Methods1 Serum-free suspension culture method induced Nasopharyngeal carcinoma CNE2stem-like cells(CNE2-SC)from Nasopharyngeal carcinoma CNE2 cell line:1)The differentiation ability of CNE2 cells and CNE2-SC in Nasopharyngeal carcinoma was detected by cell differentiation assay.The clones formation ability of CNE2 cells and CNE2-SC was detected by soft agar colony formation assay.2)Flow cytometry to detect the ratio of CD133 positive labeled cells in CNE2 cells and CNE2-SC.3)Real-time quantitative PCR(RT-q PCR)and Western blot(WB)were used to detect the expression levels of stem genes and proteins(Bmi-1,Twist1)in Nasopharyngeal carcinoma CNE2 cells and CNE2-SC.4)Transmission electron microscopy observe the formation of autophagosomes in Nasopharyngeal carcinoma CNE2 cells and CNE2-SC.5)Real-time quantitative PCR and Western blot analyse and detection autophagy-related genes and proteins(Beclin1,LC3B)expression in CNE2 cells and CNE2-SC.2 Construction and identification of Beclin1-sh RNA lentiviral vector:1)Three target sequences and a control sequence targeting the si RNA target of Human Beclin1 gene were designed.After synthesizing the single-stranded DNA oligo fragment,the sense strand and antisense strand of single-stranded DNA oligo were prepared into double-stranded DNA oligo.2)The lentiviral plasmid p HBLV-U6-MCS-CMV-Zs Green-PGK-PURO vector was double-digested with Bam H I and Eco R I restriction enzymes.The double-stranded DNA oligo is ligated to form a recombinant plasmid,transforming competent cells,and sequencing verification of the positive clones.3)The 293 T cells were co-transfected with p SPAX2,p MD2 G and shuttle plasmid three plasmid systems.The cell supernatants were collected for concentration to determine virus titers.4)The Nasopharyngeal carcinoma CNE2-SC was infected with the constructed Beclin1-sh RNA lentivirus,and the endogenous target sieve was performed by RT-q PCR.3 The function of autophagy in Nasopharyngeal carcinoma stem-like cells:1)The effect of silencing Beclin1 expression on the biological characteristics of Nasopharyngeal carcinoma stem-like cell clone formation by soft agar colony formation assay.2)The expression difference of stem cell genes and proteins(Bmi-1,Twist1)after silencing Beclin1 was detected by RT-q PCR and Western blot.3)Flow cytometry was used to analyze the expression of stem cell marker CD133.Results1 The Nasopharyngeal carcinoma CNE2 stem-like cells can be enriched by serum-free suspension culture.After 15 days,they can be stably passaged.1)The CNE2-SC was reset in 10% serum medium and differentiated into adherent cells with the same characteristics as CNE2 cells.The results of soft agar colony formation showed that the colony formation rate of CNE2-SC compared with CNE2 was 25±4% vs 8±3%.The difference was statistically significant(P<0.05).2)The CNE2-SC(9.6%)CD133 positive marker cell ratio was higher than Nasopharyngeal carcinoma CNE2 cell(0.3 %)(P<0.01).3)Compared with the nasopharyngeal carcinoma CNE2 cells,the CNE2-SC highly expressed stem related genes and proteins(Bmi-1,Twist1)(P<0.01).4)Transmission electron microscopic observe that the autophagosomes of the Nasopharyngeal carcinoma CNE2 stem-like cells increased significantly compared with CNE2 cells of the Nasopharyngeal carcinoma.5)Compared with the Nasopharyngeal carcinoma CNE2 cells,the Nasopharyngeal carcinoma CNE2-SC highly expressed autophagy-related genes and proteins(Beclin1,LC3B).(P < 0.01).2 The construction and identification of the Beclin1-RNAi lentiviral vector:1)The recombinant Beclin1-RNAi lentiviral plasmid designed and constructed was verified by PCR and sequencing,and the designed sh Beclin1 sequence was successfully inserted into the vector.2)The results of virus titer determination of target sequence 1-3 and control sequence were respectively 3.5 × 10~8TU/ml,3 × 10~8TU/ml,2 × 10~9TU/ml and 2 × 10~9TU/ml.3)Real-time PCR endogenous screening target confirmed that LV-Beclin1-sh RNA1 lentivirus had the highest silencing inhibition rate of Beclin1 gene expression in the Nasopharyngeal carcinoma CNE2-SC.3 The function of autophagy in Nasopharyngeal carcinoma CNE2-SC:1)The constructed sh Beclin1 lentiviral vector can effectively inhibit the expression level of the autophagy key gene Beclin1.2)The sh Beclin1 can inhibit the colony formation of the Nasopharyngeal carcinoma CNE2-SC.3)The sh Beclin1 can down-regulate the expression levels of stem cell corrlative genes and proteins such as Bmi-1 and Twist1.4)The sh Beclin1 can decrease the expression of stem-like cell marker CD133;Conclusion1 Serum-free suspension culture method can enrich nasopharyngeal carcinoma stem-like cells;2 Nasopharyngeal carcinoma CNE2 stem-like cells have stem cell characteristics and high levels of autophagy expression;3 Cell autophagy is continuously activated in the stem cell-like cells of nasopharyngeal carcinoma to maintain the stemness of stem-like cells of nasopharyngeal carcinoma.