| ObjectiveGlobally,lung cancer has become the most commonly diagnosed cancer in the past few decades and is one of the leading causes of death for men and women worldwide.Althoμgh smoking is the main risk factor for lung cancer,accounting for approximately 75%to 90%of all lung cancer diagnoses,many other risk factors have been found to be related to the incidence of lung cancer.In recent years,new chemotherapeutic drμgs and treatment measures have continued to appear,and the treatment of lung cancer has also been continuously improved.However,surgical treatment is still the main treatment for non-small cell lung cancer.It is encouraging that with the clarification and in-depth analysis of lung cancer genome,more and more targeted treatment methods have emerged,but at the same time,these methods have little effect on lung squamous cell carcinoma.Therefore,it is imperative to find an effective targeted therapy for lung squamous cell carcinoma.Existing studies have found that the overexpression of LASP1 is closely related to tumor invasiveness,and it plays an important role in the occurrence,development and metastasis of a variety of tumors.Small RNA(miRNA)is an important gene regμlatory factor,and its abnormal expression is closely associated with the appearance,development and metastases of tumours.Current studies have found that miRNA-20a exhibits different up-regμlation or down-regμlation in a variety of tumors,and it plays a role in suppressing or promoting cancer.The purpose of this study is to clarify the expression of LASP1 and miRNA-20a in lung squamous cell carcinoma,analyze and prove that LASP1 is the direct target gene of miRNA-20a,and study the regμlatory mechanism of LASP1 and miRNA-20a expression in lung squamous cell carcinoma.To explore the role of miRNA20a/LASP1 regμlatory pathway in the invasion and metastasis of lung squamous cell carcinoma,and provide new ideas for clarifying the invasion and metastasis mechanism of lung squamous cell carcinoma and discovering possible targeted therapy targets.Method1.Using immunohistochemical method to detect the expression of LASP1 protein in 120 cases of lung squamous cell carcinoma paraffin tissue specimens,and analyze the relationship between the expression level of LASP1 and the clinicopathological characteristics of lung squamous cell carcinoma.RT-PCR and westernblot methods were used to analyze the expression of LASP1 mRNA and protein in fresh cancer tissues and adjacent tissues of 30 patients with lung squamous cell carcinoma.2.In order to detect the expression of miRNA-20a in fresh cancer tissues and adjacent tissues of 30 patients with lung squamous cell carcinoma,we used RT-PCR to detect the expression level and analyze the relationship between its expression level and clinicopathological factors.Analysis of the correlation between the expression of miRNA-20a and LASP1 in lung squamous cell carcinoma.3.In order to verify whether miRNA-20a uses LASP1 as a direct regulatory target,we used mirdb.org,TargetScan and other websites to analyze the possible interaction sites between miRNA-20a and LASP1 gene,and verify that LASP1 is a direct target of miRNA-20a throμgh luciferase report analysis.4.Transfect miRNA-20a inhibitor,miRNA-20a inhibitor NC,miRNA-20a mimics,miRNA-20a mimics NC into lung squamous cell carcinoma H520 cells,and analyze the effect of miRNA-20a on the expression of LASP1 mRNA and protein by RT-PCR and western blot methods.5.Transfect LASP1 plasmid,LASP1 plasmid NC,LASP1 shRNA plasmid,and LASP1 shRNA plasmid NC into lung squamous cell carcinoma H520 cells,combined with MTT experiment,scratch experiment and transwell experiment to analyze the effect of LASP1 on the proliferation,migration and invasion of H520 cells.6.Transfection of miRNA-20a inhibitor,miRNA-20a inhibitor NC,miRNA-20a mimics,miRNA-20a mimics NC into lung squamous cell carcinoma H520 cells,combined with MTT experiment,scratch experiment,and transwell experiment to analyze the effect of miRNA-20a on the proliferation,migration and invasion of H520 cells.Results1.The expression of LASP1 in lung squamous cell carcinoma tissues is elevated,and the expression level of LASP1 is correlated with the degree of tumor differentiation,T stage,lymph node metastasis and TNM stage in patients with lung squamous cell carcinoma;Patients with lung squamous cell carcinoma with high LASP1 expression have shorter disease-free survival.2.The expression of miRNA-20a in lung squamous cell carcinoma tissues is reduced.And the miRNA-20 expression level is correlated with the degree of differentiation of pulmonary squamous cell carcinoma,T stage,lymph node metastasis and TNM stage.3.miRNA-20a can directly bind to the mRNA of LASP1.The increase of miRNA-20a can significantly decrease the relative expression level of LASP1 mRNA and protein;the decrease of miRNA-20a can significantly increase the relative expression level of LASP1 mRNA and protein.4.After up-regμlating LASP1,the proliferation,migration,and invasion ability of H520 cells was significantly enhanced,and at the same time,the invasion,migration and invasion ability of H520 cells was significantly weakened after LASP1 was downregμlated;the proliferation,migration,and invasion ability of H520 cells was significantly weakened after miRNA-20a was up-regμlated,At the same time,the proliferation,migration,and invasion ability of H520 cells was significantly enhanced after miRNA-20a was down-regμlated.Conclusions1.LASP1 can promote tumor proliferation,invasion and metastasis.2.miRNA-20a has the potential to inhibit tumour proliferation,invasion and metastasis.3.miRNA-20a can directly target and regμlate LASP-1 to affect the proliferation,migration and invasion of lung squamous cell carcinoma cell H520. |
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