Study On The Role And Mechanism Of Netrin-1 In Cisplatin Resistance Of Lung Cancer

Objective:Lung cancer is the leading cause of cancer morbidity and mortality in the world.Chemotherapy is the most important treatment for advanced lung cancer,in which platinum-based standard chemotherapy is widely used in clinic.The resistance of lung cancer to chemotherapeutic drugs is often an important reason for the failure of chemotherapy,in which the decrease of the activity of apoptosis pathway is one of the main factors of cisplatin resistance in lung cancer.Hypoxia is an important microenvironment for tumor tissue growth,and hypoxia is closely related to tumor drug resistance.Some studies have shown that Netrin-1 promotes the invasion and metastasis of lung cancer under hypoxia,but as an axon guiding factor,the role of Netrin-1 in drug resistance of lung cancer is not clear.The purpose of this study was to observe the expression level of Netrin-1 in tissue and serum of patients with lung cancer before and after chemotherapy,and to analyze the relationship between it and clinical data.At the same time,the effect of Netrin-1 on cisplatin-induced apoptosis of non-small cell lung cancer cells under hypoxia was studied in order to provide a new idea for the diagnosis and treatment of chemotherapy resistance of lung cancer.Methods:1.Thirty pairs of cancer tissues and corresponding paracancerous tissues from patients with lung cancer in our hospital were collected.There were 13 males and 17 females.The average age was 61.53±8.26 years old.All the clinical stages were I-II stage.All cancerous tissues were confirmed as lung adenocarcinoma by pathological examination.In addition,30 pairs of serum samples of inpatients with lung cancer in oncology department were collected before the use of chemotherapeutic drugs and after 4 cycles of platinum-based chemotherapeutic drugs.There were 17 pairs of adenocarcinoma,5 pairs of squamous cell carcinoma and 8 pairs of small cell lung cancer.There were 18 males and 12 females.The average age was 56.60±10.73 years old.The serum samples of 22 patients with normal physical examination in outpatient clinic were healthy control group,including 13males and 9 females,with an average age of 46.64±8.03 years.2.The expression of Netrin-1 in tissue and serum of patients with lung cancer was detected by immunohistochemical method and ELISA method,and the relationship between Netrin-1 and clinical parameters was analyzed.3.ELISA method was used to detect the expression level of serum Netrin-1 in patients with lung cancer before the application of chemotherapeutic drugs and 4 cycles after the application of platinum-based chemotherapeutic drugs.The relationship between Netrin-1 and clinical data was analyzed.4.The hypoxia model was established by Cocl₂,and the sensitivity of lung cancer A549 cells to cisplatin under normoxic and hypoxic conditions was observed by MTT method.5.The low expression cell line of Netrin-1 was constructed by Si RNA,and the expression level of Netrin-1 was verified by Western blot.Apoptosis experiment was used to detect the effect of Netrin-1 on apoptosis of lung cancer cells under normoxic and hypoxic conditions.The effect of Netrin-1 on cisplatin-induced apoptosis of non-small cell lung cancer cells was detected by simulating tumor hypoxia microenvironment.Results:1.The positive expression rate of Netrin-1 protein in patients with lung adenocarcinoma was significantly higher than that in paracancerous tissues,and there was significant difference between the two groups（P<0.01）.2.The expression of Netrin-1 protein in serum of patients with lung cancer was significantly higher than that of healthy controls,and there was significant difference between the two groups（P<0.01）.There was significant difference between Netrin-1 expression level and pathological types（P<0.05）,including lung adenocarcinoma and small cell lung cancer（P<0.05）,but there was no significant difference between lung squamous cell carcinoma and lung adenocarcinoma and small cell lung cancer（P>0.05）.There was no significant correlation between the expression of Netrin-1 and age,sex,smoking history,lymph node metastasis and clinical stage（P>0.05）.3.The expression of serum Netrin-1 protein in patients with lung cancer before the use of chemotherapeutic drugs was higher than that after 4cycles of platinum-based chemotherapeutic drugs,and there was significant difference between the two groups（P<0.05）.To consult the clinical data of22 patients with lung cancer,the serum Netrin-1 index decreased after 4cycles of chemotherapy,and the curative effect was evaluated as SD or PR（stable disease or partial response）,and continued the original chemotherapy regimen.After 4 cycles of chemotherapy,the Netrin-1 index of 2 patients with lung cancer was higher than that before,and the curative effect was evaluated as PD（progressive disease）,which was treated with modified chemotherapeutic drugs.4.The sensitivity of lung cancer A549 cells to cisplatin under hypoxia was significantly lower than that under normoxia.5.After knocking down the expression of Netrin-1 in lung cancer A549 cells under normoxic condition,the apoptosis rate of lung cancer A549cells was higher than that of the blank control group.There was significant difference in the apoptosis rate between the experimental group and the blank control group（P<0.05）,but there was no significant difference in the apoptosis rate between the negative control group and the blank control group（P>0.05）.After knocking down the expression of Netrin-1 in lung cancer A549 cells under hypoxia,the apoptosis rate of lung cancer A549cells was higher than that of the blank control group,and the apoptosis rate of lung cancer A549 cells induced by cisplatin was higher than that of non-knockdown group.Conclusions:In this study,we found that the expression of Netrin-1 in serum and tissues of patients with lung cancer increased.After 4 cycles of platinum-based chemotherapeutic drugs,the expression of Netrin-1 in clinically stable and response patients decreased significantly,which is expected to become a potential marker for the diagnosis and evaluation of chemosensitivity.Netrin-1 may participate in the process of cisplatin resistance by reducing the activity of cisplatin-induced apoptosis pathway,which provides a new idea for the intervention of cisplatin resistance in lung cancer.