| Background Lung cancer(also known as primary bronchogenic carcinoma)is the global first malignant tumor,statistics show that the incidence of lung cancer is up to 20.6~48.2/10 million,about 1.82 million cases of lung cancer were diagnosed and 1.59 million died in 2012,the number of new cases worldwide is increasing by about 3% a year.About 400,000 cases of lung cancer were added in China each year,360,000 cases died,the patient's quality of life is poor,the mortality rate is high,and the incidence rate is increasing year by year,which threatens life safety seriously.The occurrence and development of lung cancer is a complex and diverse biological process involving many factors,the etiology and pathogenesis are complex,environmental pollution,smoking,occupational exposure,previous chronic lung disease,population aging,and family history of cancer are all related to the occurrence of lung cancer.At present,resection of the tumor and metastasis or potential metastatic lesions and lymph nodes are the only possible radical treatment,but the early symptoms of lung cancer patients are not obvious,can not be early diagnosed,delayed the operation time.Chemotherapy is the use of chemical drugs(cisplatin and carboplatin-like chemotherapy drugs,etc.)in the DNA chain and Chain Inner Chain,forming DDP~DNA complex,interfering with DNA replication,after entering the nucleus,binds to nuclear DNA,leads to irreversible DNA damage,cells don't metabolize normally,causes the mitotic tumor cell death to play its anticancer role,and becomes one of the treatment methods of lung cancer.Drug resistance of tumor cells,including primary drug resistance and acquired drug resistance,is one of the mechanisms of self-protection of tumor cells.Because of the mechanism of DNA damage repair in tumor cells,if the repair capability of the DNA repair gene is improved,chemotherapy resistance may be produced,and the effect of chemotherapy drugs will be affected.Primary resistance is the genetic basis for drug resistance,which is a drug-resistant clone caused by mutations,deletions,or gene amplification,translocation,and chromosome rearrangement.Acquired drug resistance is due to cellular dynamics,produced by tumor cells that are not split or dormant.DNA damage caused by chemotherapeutic drugs can be repaired by raising DNA damage repair related factors to repair.Therefore,Study on the main mechanism of drug resistance in lung cancer cells is Helpful to improve the prognosis of patients.PLK1 is a highly conservative member of the serine/threonine kinase PLKs family that regulates mitosis.It is related to the splitting stage,the center body maturation,the cohesin bonding release,the Golgi matrix splitting,the microtubules connecting the silk grains and spindle elongation.It plays an important regulatory role in the process of cell cycle,such as DNA damage test point,center body maturation,chromosome enrichment,chromosome separation,cytoplasmic division,cell proliferation and separation in different periods.It is closely related to DNA damage response,tumor formation,early embryo development and participats in damage repair of genetic information.PLK1 may participate in the development of lung cancer,its expression level and function play an important role in the occurrence,development,invasion and metastasis of lung cancer,and it plays an important role in the mechanism of chemotherapeutic drug resistance in lung cancer cells.Activation of Gst-?,ck2?,p53,P-gp,Ki67 target signaling pathway key factor induces cancer cell expression,then enhances its activity to be able to regulate the cancer cell metastasis and the chemotherapy resistance,may promote the malignant tumor cells proliferation simultaneously suppresses the tumor cell apoptosis,and increases the tumour cell multidrug resistance phenomenon.To sum up,PLK1 expression may play an important role not only in the development of lung cancer,but also in relation to chemotherapy sensitivity and prognosis of patients.This project intends to use case-control study design,through the correlation research method as the clue,using immunohistochemistry and polymerase chain reaction to detect the expression of PLK1 in lung cancer tissues and adjacent tissues,and to explore its relationship with the prognosis of lung cancer patients.At the same time,to analyze the sensitivity of cisplatin-resistant A549 cells to chemotherapeutic drug(cisplatin)after inhibition of PLK1 expression.The completion of this project will help to reveal the main mechanism of chemotherapy sensitivity in patients with lung cancer,and then provide new ideas and plans for finding effective and safe anticancer therapies to solve the drug-resistance problems in lung cancer during chemotherapy.Objective This research intends to compare the expression levels of PLK1 in non-small cell lung cancer tissues and adjacent tissues,which in order to explore the correlation between PLK1 expression and the pathological features of lung cancer and prognosis.This research also intends to analyze the sensitivity of A549 to chemotherapeutic drug(cisplatin)after inhibition of PLK1 expression,which in order to provide new ideas and plans for finding effective and safe anticancer therapies to solve the drug-resistance problems in lung cancer during chemotherapy.Methods Adopt the method of case-control study,baseline data was collected from January 2014 to December 2014,Shenzhen Second People's Hospital pathological diagnosis of 85 patients with non-small cell lung cancer(case group),and in the same period in the same area and gender,age and cases healthy controls matched normal lung tissue specimens of pneumothorax or other benign diseases tissue samples of 100 cases(control group).PLK1 and PLK1 m RNA were detected by IHC and polymerase chain reaction.After using BI2536 to inhibit PLK1 expression and platinum treatment,the cell cycle distribution was analyzed by flow cytometry,the morphological changes of apoptosis were analyzed by staining,changes in expression of PLK1 and expression of E-cadherin and N-cadherin in tumor cells were analyzed by Western Blot method,Transwell method was used to detect the changes of A549 metastasis and invasive ability.Statistical analysis was performed by SPSS20.0 software,and P < 0.05 was considered statistically significant.Results(1)Baseline data indicated that there was no significant difference between the two groups of patients ' basic data indexes,which was meet the objective requirements of the control study.(2)PLK1 positive cells were mainly expressed in cytoplasm.The expression level(score)of PLK1 in NSCLC tissues was 4.89±1.87,1.21±1.46 in adjacent tissues and 0.44±0.86 in normal tissues,and the difference was statistically significant.The positive expression rate of PLK1 in NSCLC tissues was 92.94%(79/85),35.29%(30/85)in adjacent tissue and 14.00%(14/100)in normal tissues,and the difference was statistically significant.(3)The tumor size,TNM stage,lymph node metastasis and PLK1 expression had significant effects on the 3-year survival rate,see in Table 3.Tumor size> 4cm and positive PLK1 expression were independent risk factors for 3-year survival rate.(4)The positive expression rate of GST-?,CK2?,P-gp,Ki67,p53 in NSCLC tissues were 91.76%(78/85),74.12%(63/85),64.71%(55/85)and 57.65(49/85),67.06%(57/85),significantly higher than that of cancer-adjacent tissues and normal tissue groups,and the positive expression rate of GST-?,CK2?,P-gp,Ki67,p53 in cancer-adjacent tissues were significantly higher than those in normal tissues.PLK1 expression was positively correlated with GST-?,CK2?,p53,P-gp and Ki67,and r was 0.999,0.984,0.996,0.984 and 0.993 respectively.(5)The expression of PLK1 m RNA in NSCLC tissues was 0.59 ± 0.15,significantly higher than that in adjacent normal tissues 0.43 ± 0.17,and the difference was statistically significant.(6)PLK1 m RNA expression was not significantly correlated to age and gender.There were significant differences in PLK1 m RNA expression between low stage and middle-high stage,and there were significant differences in PLK1 m RNA expression between low and middle-high differentiation.The expression of PLK1 m RNA in tumor size> 4 cm was 0.65±0.19,significantly higher than that in tumor size? 4 cm,the expression of PLK1 m RNA in the distant transfer and lymph node metastasis was significantly higher than that in the non-metastatic tissue.The expression of PLK1 m RNA in patients with survival rates <3 years was 0.67±0.17,significantly higher than that in patients with survival rates ?3 years.(7)PLK1 expression was significantly inhibited in the cells after co-incubation with PLK1 inhibitor,the relative expression level of PLK1 in the case group was 0.502 ± 0.087,and the relative expression level of PLK1 in the control group was 1.128±0.103,the difference was statistically significant.(8)With the increase of cisplatin concentration,the cell apoptosis rate in the experimental group was significantly higher than that in the control group.In the experimental group,the A549 cells were blocked in the G2/M period,which was significantly different from the control group.(9)After adding inhibitors to the culture fluid,the expression level of E-cadherin was increased from 0.95 + 0.12 to 1.26 + 0.24,while the expression level of N-cadherin was reduced from 0.92 + 0.17 to 0.63 + 0.12,and the difference was statistically significant.(10)Transwell method was used to detect the changes of A549 metastasis and invasion ability.The results showed that the ability of A549 invasion and metastasis decreased significantly after inhibiting PLK1.Conclusions(1)There is a significant correlation between the level of PLK1 and the prognosis of non-small cell lung cancer patients.which can be used as a specific index to evaluate the prognosis of non-small cell lung cancer(2)The biological function of PLK1 in non-small cell lung cancer is mainly related to the regulation of transfer invasion and chemotherapy resistance.There is positive correlation with the expression of GST-?,CK2?,P-gp,Ki67 and p53.(3)Inhibition of PLK1 expression in platinum-resistant A549 cells can improve cisplatin chemotherapy sensitivity and reduce the metastasis and invasion ability of platinum-resistant A549 cells. |
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