Exo-hsa_circRNA₀056616: The Potential Biomarker Research Of Lymph Node Metastasis In Lungadenocarcinoma

Objective:To investigate the relationship between CXCR4 expression and lymph node metastasis of lung adenocarcinoma,to screen the CXCR4-related circ RNA and find the biological markers of lymph node metastasis of lung adenocarcinoma.Methods:1.The expression of CXCR4 protein in lung adenocarcinoma tissue was detected by western blot.The relationship between CXCR4 protein expression and lymph node metastasis was analyzed.2.The PC-9 cell line with stable CXCR4 knock-down was constructed by lentivirus-mediated RNA interference,the effects of CXCR4 expression on the proliferation and metastasis of lung adenocarcinoma PC-9 cells were detected by soft agar clone formation assay,flow cytometry,CCK8,transwell assay and cell scratches.3.CXCR4-related circ RNAs were found on databases such as circbase,circ RNABase,Circ2 Traits.The expression of CXCR4-related circ RNAs were detected by real-time fluorescence quantitative PCR in PC-9 cells and exosomes before and after silencing CXCR4 expression.4.The expression of plasma exo-hsa_circ RNA₀056616 in lung adenocarcinoma was detected by real-time fluorescence quantitative PCR.The relationship between the expression of plasma exo-hsa_circ RNA₀056616 and the expression of CXCR4 protein in lung adenocarcinoma was analyzed.5.Clinical and pathological data of lung adenocarcinoma patients were collected,the relationship between plasma exo-hsa_circ RNA₀056616 expression and lymph node metastasis in lung adenocarcinoma patients was analyzed.Results:1.The expression level of CXCR4 protein in patients with lymph node metastasis of lung adenocarcinoma was higher than that in patients without lymph node metastasis oflung adenocarcinoma,the difference was statistically significant?P = 0.001?.2.The results of the soft agar clone formation assay showed that the number of PC-9 /CXCR4-sh RNA cells were significantly decreased compared from the negative control PC-9/sh Ctrl group and blank control PC-9 cells group?P<0.001?.PC-9 /CXCR4-sh RNA cells were significantly different from those of negative control PC-9/sh Ctrl group and blank control PC9 cells group from the the outcome of the experiment CCK8?P<0.01?.Transwellassay was also performed to verify whether PC-9 / CXCR4-sh RNA cells would be decreased compared from the negative control PC-9 / sh Ctrl group and blank control PC-9 cells group,the experiment results are in accordance with the expected?P<0.001?.Cell scratch test also showed that cell migration rate was significantly reduced in PC-9/CXCR4-sh RNA cells compared with negative control group PC-9/sh Ctrl and blank control PC-9 cells group?P<0.001?.3.The circ RNAs associated with CXCR4 were hsa_circ RNA₀056615,hsa_circ RNA₀056616,hsa_circ RNA₀117403 which were searched on databases such as circbase,circ RNABase,Circ2 Traits.4.The expression of circ RNA hsa_circ RNA₀056616 associated with CXCR4 in PC-9 / CXCR4-sh RNA cells and exosomes was significantly higher than that in PC-9 / sh Ctrl group,blank control PC-9 cells group after targeting the expression of CXCR4 in silenced PC-9 cells,the difference was statistically significant?P<0.001?.5.After logarithmic conversion,pearson's correlation coefficient of the proteinexpression of CXCR4 in lung adenocarcinoma and expression of exo-hsa_circ RNA₀056616 in plasma was-0.979?P<0.05?.6.The expression of exo-hsa_circ RNA₀056616 in plasma of patients with stage T1 / T2 was significantly higher than that with stage T3 / T4 by Wilcoxon rank test.Other groups?stage M0 versus stage M1,I-II grade versus III-IV grade?comparisons were similar in plasma,the P values were 0.002,0.004,0.001,respectively.And the above difference of expression were not found among age,sex,tumor size and histological type of lung adenocarcinoma,the P values were 0.869,0.765,0.122,and 0.205,respectively.The Spearman correlation coefficients of plasma exo-hsa_circ RNA₀056616 and T stage,M stage,TNM grade,tumor size and histological type of lung adenocarcinomawere-0.318,-0.308,-0.466,-0.232,0.154,respectively,the P values were 0.003,0.003,0.001,0.060 and 0.207,respectively.7.exo-hsa_circ RNA₀056616 expression in patients with lymph node metastasis was significantly downregulated than that without lymph node metastasis?P<0.05?based on an analysis of 90 cases of lung adenocarcinoma plasma samples and the area under the ROC curve?AUC?was 0.63,and the cut off value,maximum Youden index,sensitivity,and specificity were 0.394,0.602,0.792,0.810,respectively.Conclusions:1.CXCR4 expression level was related with lung adenocarcinoma lymph node metastasis and CXCR4 expression with lymph node metastasis in patients was higher than those without lymph node metastasis.2.There was a negative correlation between the expression level of ex-hsa_circ RNA₀056616 in plasma and the expression of CXCR4 in lung adenocarcinoma.A clear positive correlation was also found between lymph node metastasis and CXCR4 expression.The higher the expression level of CXCR4 in lung adenocarcinoma tissue,the lower the expression level of ex-hsa_circ RNA₀056616 in plasma.3.The exo-hsa_circ RNA₀056616 may be considered as a potential biomarker associated with poor survival in patients with lung adenocarcinoma.When the cut off value is 0.394,the sensitivity is 0.792,the specificity is 0.810,the exo-hsa_circ RNA₀056616 in plasma is probably related to lymph node metastasis of lung adenocarcinoma.