| OBJECTIVE:To explore the expression profile of CD73 in lung adenocarcinoma and paracancerous tissues and to analyze the association of its expression differences with clinical individual characteristics and prognosis of patients;In vitro cell experiments were conducted to explore the effects of CD73 on the proliferation,migration,and invasion abilities of H1975 lung adenocarcinoma cells and the underlying mechanisms,with a view to providing an experimental basis and theoretical basis for mechanistic studies and potential therapeutic approaches for lung adenocarcinoma development.METHODS:Clinical study:the expression of CD73 in lung adenocarcinoma tissues vs.para carcinoma tissues was detected by immunohistochemical techniques and the relationship between its expression and the clinical individual characteristics and prognosis of patients was analyzed.Cell culture:human lung adenocarcinoma H1975 cell line was cultured in vitro,the expression level of CD73 in H1975 lung adenocarcinoma cells was knocked down by CD73 sh RNA lentivirus transfection,and the green fluorescence protein expression after lentivirus transfection of H1975 cells was observed under ultraviolet excitation fluorescence microscopy to determine the success of CD73knockdown transfection,The expression of CD73 protein of H1975 cells after lentivirus transfection was verified by Western blot experiment again and the target sequence with the best interference efficiency was determined.After successful lentiviral transfection of H1975-sh CD73 cell line and H1975-NC blank transfection control cell line,CCK-8 cell proliferation assay,cell scratch assay and Transwell invasion assay were performed to explore the effect of silencing CD73 on the proliferation,migration and invasive ability of H1975 lung adenocarcinoma cells,Western blot was used to verify that H1975 lung adenocarcinoma cell line interfered with the epithelial marker protein E-cadherin before and after CD73 expression Differences in the expression of vimentin,a mesenchymal marker protein,as well as proteins involved in the PI3K/Akt signaling pathway were observed to explore the specific mechanism underlying the effect of CD73 in H1975 cells.RESULTS:1.Clinical study:(1)62 cases(73.8%)of 84 lung adenocarcinoma tissues were positive for CD73 protein expression,of which 21 cases(25.0%)were high expression and 22 cases(26.2%)were negative expression;CD73 protein expression was positive in 2(2.4%)of the 84 adjacent noncancerous tissues,and 82(97.6%)of these samples showed negative expression without high expression sections.(2)CD73expression was differentially and statistically significant between lung adenocarcinoma tissues and adjacent normal tissues(X~2=58.06452,P<0.05),and the expression level of CD73 was associated with lymph node metastasis and TNM stage of patients(P<0.05),but not with age,gender,smoking history and differentiation grade of patients(P<0.05).(3)The 5-year survival rate was 87.1%and the 5-year progression free survival rate was69.8%in the CD73 low expression group;The 5-year survival rate was 76.1%and 5-year progression free survival rate was 57.1%in the CD73 high expression group,and both the five-year survival rate and progression free survival rate were lower in the CD73 high expression group than in the low expression group,but the differences were not statistically significant(P>0.05).2.Cell culture:(1)the results of CCK-8 proliferation assay showed that the OD values(450 nm)of H1975-sh group were lower than normal H1975-NC control group at 24h,48h and 72h,and the differences were statistically significant(P<0.05).(2)Cell scratch healing assay of lung adenocarcinoma cells in H1975-NC or H1975-sh group the results showed that the cell scratch area of H1975-NC group was 17.99%±2.90%,28.62%±3.10%after 24 h and 48 h incubation in incubator,respectively;The scratch area healing rates of cells in H1975-sh group cultured for 24 h and 48 h were 11.39%±2.43%,17.13%±1.86%,respectively,which were lower than that of H1975-NC group(P<0.05).(3)Transwell invasion assay was performed for lung adenocarcinoma cells in H1975-NC group and H1975-sh group,and the results showed that the number of cells that penetrated through the Matrigel and chamber microwells of mtrigel respectively after H1975-NC group vs H1975-sh group cultured under the same culture conditions for the same time was 254±22 vs 146±15,respectively,and the H1975-sh group had significantly fewer invasive cells than the H1975-NC group,with significant differences(P<0.05).(4)When silencing the expression of CD73 protein,compared with the H1975,H1975-NC group cells,the expression of E-cadherin protein in H1975-sh cells increased,vimentin protein expression decreased,all of which were statistically significant(P<0.05),and the ratios of phosphorylated protein in H1975-NC cells,p-pi3k/PI3K and p-Akt/Akt,were significantly decreased,all of which were statistically significant(P<0.05).CONCLUSIONS:CD73 expression is elevated in lung adenocarcinoma tissues and its high expression correlates with lymph node metastasis and TNM stage in patients;CD73promotes the proliferation,migration and invasion of H1975 lung adenocarcinoma cells,and the mechanism may be related to the activation of PI3K/Akt signaling pathway to promote EMT,which provides a theoretical basis and ideas for the development of new treatments for lung adenocarcinoma. |
PDF Full Text Request