| BackgroundLaryngeal cancer is the common type of malignant tumors of head and neck,ranks second in the respiratory system tumor incidence,accounts for about 1% ～ 5% of systemic malignant tumors,and primary laryngeal squamous cell carcinoma（LSCCs）is the predominant type.There are a variety of clinical manifestations for voice hoarse,dyspnea,cough,difficulty swallowing and ear pain and other symptoms,which seriously affect the patient’s quality of life.Laryngoscopy and imaging techniques are often used to examine laryngeal conditions,precancerous lesions and early laryngeal cancer lesions,and total laryngectomy or concurrent radiotherapy and chemotherapy are often used to treat laryngeal lesions and achieve favorable results.The pathophysiological mechanism of laryngeal cancer is still unclear,and its etiology may be related to viral infection,alcohol consumption,smoking and sex hormones.The incidence of laryngeal cancer was significantly higher in males than in females.At present,the role of inflammatory response and/or systemic immune state in the occurrence,development and metastasis of malignant tumors is a hot research topic.In order to find the ideal biomarkers to evaluate the induction and prognosis of laryngeal cancer,we conducted blood cytology examination and anticoagulant function evaluation of patients with laryngeal cancer before surgery,and analyzed the parameters related to platelets.The effects of platelets on the proliferation,apoptosis,invasion and secretion of inflammatory cytokines of laryngeal carcinoma cells were studied by CCK8 assay,flow cytometry,real-time fluorescence quantitative PCR（q RT-PCR）and Transwell invasion assay to explore the molecular mechanism of platelet influence on the progression and metastasis of laryngeal cancer,and provide theoretical basis for early clinical diagnosis and treatment of laryngeal cancer.ObjectiveThis study aimed to observe the changes of blood platelets and related indicators in patients with laryngeal cancer and to find platelet-related biomarkers for early prediction or diagnosis of laryngeal cancer.And to study the effects of platelets on the proliferation,apoptosis,invasion and expression of inflammatory cytokines in laryngeal carcinoma Hep-2 cells,and in order to further explore the molecular mechanism of platelets promoting the progression and metastasis of laryngeal cancer.Methods1.Clinical subjectsA total of 38 patients with laryngeal carcinoma in the First Affiliated Hospital of Xinxiang Medical College were selected as the experimental group,including 37 males and 1 female,aged 52～79 years old.All patients were confirmed by laryngeal CT,MRI and electronic laryngoscopy before surgery.During the same period,49 healthy people were randomly selected from the outpatient department,including 42 males and 7 females,aged from 44 to 88 years.All the subjects had informed consent,and there was no significant difference in age and gender.2.Evaluation of laboratory test indicators（1）Detection of platelet-related indicators: The blood routine indexes were detected in all patients before surgery and normal group by automatic blood cell analysis instrument,and Focusing on indicators including the blood platelet count（PC）,platelet distribution width（PDW）,mean platelet volume（MPV）,large ratio of platelet（P-LCR）and platelet volume（PCT）.Based on blood routine results,platelet to lymphocyte ratio（PLR）,platelet to MPV ratio（PC/MPV）,platelet to PDW ratio（PC/PDW）were calculated.（2）Assessment of coagulation and fibrinolytic function: Coagulation and fibrinolytic indexes in some patients with laryngeal cancer before surgical treatment:（1）Coagulation indexes: prothrombin time（PT）,PT international standardized ratio（PT-INR）,activated partial thrombin time（APTT）,fibrinogen（Fbg）and thrombin time（TT）.（2）Fibrinolytic index: D-dimer（D-D）and fibrinogen degradation products（FDPs）.（3）Comparison of laboratory results: SPSS 22.0 software was used for statistical analysis of experimental data.Independent samples t-test was used to process experimental results between the patients with laryngeal cancer before treatment and the control,and the clinical diagnostic efficacy of platelet-related indexes was evaluated by ROC curve.P<0.05 was considered as statistically significant.2.Effects of platelets on the biological function of Hep-2 cells（1）The effect of platelets on the proliferation of Hep-2 cells: Instead of fetal bovine serum,Hep-2 cells were cultured with platelet-rich plasma（PRP）at different concentrations from the same individual,and the effect of platelets on the proliferation degree of Hep-2 cells was observed by CCK8 assay.（2）The effect of platelets on the apoptosis of Hep-2 cells: Hep-2 cells were cultured with PRP at different concentrations from the same individual,and the effect of platelets on the apoptosis of Hep-2 cells was observed by flow cytometry.（3）The effect of platelets on the invasion of Hep-2 cells: Hep-2 cells were cultured with PRP and platelet-poor plasma（PPP）,respectively,and the effect of platelets on the invasion ability of Hep-2 cells was observed by Transwell invasion assay.（4）The effect of platelets on the expression of cytokines in Hep-2 cells: Hep-2 cells were cultured in PRP and PPP medium,respectively,and the expression of m RNA of cytokines（IL-1,IL-6,IL-10 and TNF-α）in Hep-2 cells were evaluated by q RT-PCR assay.Results1.Clinical significance of platelet-related indexes in patients with laryngeal carcinomaCompared with normal controls,platelet to lymphocyte ratio（PLR）was abnormally elevated in patients with laryngeal cancer before treatment（P<0.01）,and there was relatively good sensitivity and specificity in PLR（AUC=0.817）by ROC curve,which could assist early clinical prediction or diagnosis of laryngeal cancer.2.Evaluation of the function of coagulation and fibrinolysis in patients with laryngeal cancer before treatmentBefore treatment,coagulation and fibrinolytic function indexes of patients with laryngeal cancer were basically within the normal reference range without abnormal changes.3.Platelets affect the biological function of Hep-2 cellsIt was found that platelets could enhance the proliferation and invasion ability of Hep-2cells and inhibit their apoptosis when laryngeal carcinoma Hep-2 cells were cultured with platelets of different concentrations instead of fetal bovine serum.4.Platelets affect the expression of cytokines in Hep-2 cellsPlatelets in PRP could enhance the m RNA expression of some inflammatory cytokines,such as IL-1,IL-6,TNF-α and inhibit the IL-10 m RNA of anti-inflammatory cytokine in Hep-2 cells.Conclusions1.The platelet-related PLR can be used as an inflammatory indicator to early prediction or auxiliary diagnosis for laryngeal cancer.2.Platelets can affect the biological function of Hep-2 cells and enhance the expression of some inflammatory cytokines. |
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