Effect Of MiR-10a-5p On The Occurrence And Development Of Non-small Cell Lung Cancer And The Intervention Mechanism Of β-Hydroxyisovalerylshikonin

Research backgroundLung cancer is one of the most common malignancies with the highest morbidity and mortality around the world,which is seriously harmful to human health.Lung cancer is generally divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).NSCLC contains of squamous cell carcinoma,adenocarcinoma and large cell carcinoma,which was accounting for approximately 85%of the total number in lung cancers.In the early stage,lung cancer patients usually have little obvious symptoms.Most of them have became in the middle or late stages of lung cancer at the time of diagnosis,and the 5-year survival rate is less than 15%.At present,early detection,diagnosis and treatment are effectively ways to improve the survival rate of lung cancer patients.Therefore,it is significance important to find the high-sensitivity tumor markers and to clarify the molecular mechanism of the occurrence and development of lung cancer.MicroRNA(miRNA,miR)are noncoding small RNAs with a regulatory function,approximately 18-24 nucleotides in length,and are mainly translated by binding to the 3’untranslated region(3’UTR)of mRNA in target gene,which will result in the mRNA translations,inhibition or degradation in order to regulate the expression of target genes.Numerous studies have shown that the abnormal expression of microRNA plays an important role in the formation,growth and metastasis of tumors.It can act as an oncogene,which promotes the occurrence and development of tumors,and can also be used as a tumor suppressor gene to protect the body from canceroccuring.It has been great concerned in the field of biomedical research such as cancer.Therefore,in-depth study of the relationship between miRNAs and lung cancer is expected to provide new solutions and new targets for the diagnosis and treatment of lung cancer.miR-10a-5p is one of the new miRNA molecules discovered in recent years.Studies have shown that miR-10a-5p,as an oncogene,plays a role in the development of various tumors(including lung cancer),and bioinformatics suggests that there is a conserved binding site in a variety of biological species between GATA6 and miR-10a-5p.GATA6(GAT A binding protein6)is an evolutionarily conserved member of the GATA transcription factor family.It is involved in the regulation of cell proliferation and differentiation during embryonic development.There are six molecules in the GATA family:GATA1/2/3 are mainly expressed in hematopoietic cells,and GATA4/5/6 are mainly expressed in mesoderm and endoderm-derived tissues such as lung,heart,digestive tract,and liver.Recent studies have reported that the expression of GATA6 in tumors plays an important regulatory role in the development of tumors.Moreover,GATA6 has a distinctly different expression and biological effects in tumors of different tissues.This phenomenon was interpreted by some scholars as a concept of "organizational source specificity".Therefore,whether miR-10a-5p can affect the occurrence and development of lung cancer by targeting GATA6 gene is worth further study.Shikonin is an active ingredient extracted from the root of comfrey comfrey.Its chemical structure is a naphthoquinone compound,which has an effect of detoxification,anti-inflammatory,antibacterial,and anti-tumor.Shikonin can inhibit the proliferation,invasion and migration of a variety of tumor cells.Since shikonin has strong toxicity and its anti-tumor effect is not specific,it has not been widely used in clinical practice.β-hydroxyisovalerylshikon(β-HIVS)is one of the naturally derivatives of shikonin.Studies have found that P-HIVS can exert its anti-cancer efficacy by inhibiting tumor cell proliferation,invasion and migration,inducing tumor cell apoptosis and interfering with cell cycle,and it has the potential to become a new type of highly effective and low-toxic anti-tumor drug.This research focusons the relationship between miR-10a-5p and the occurrence and development of lung cancer through in vitro and in vivo experiments,and provides novel strategies and theories for the search of highly sensitive and specific tumor markers for lung cancer and targeted therapies for lung cancer.Part I Expression and clinical significance of miR-10a-5p in tissues and serum of patients with non-small cell lung cancerObjective:To detect the expression of miR-10a-5p in tissues and serum of patients with non-small cell lung cancer,and to explore its relationship with clinicopathological features.Methods:(1)Collected 80 serum samples of NSCLC patients and 75 healthy volunteers.Twenty pairsof fresh cancer tissues and adjacent normal tissues were removed from NSCLC patients.(2)The expression level of miR-10a-5p was detected by real-time quantitative PCR.(3)The relationship between the expression levels of miR-10a-5p and the clinicopathological features in both NSCLC tissues and serum were analysed.(4)The receiver operating characteristic curve(ROC)was used to evaluate the diagnostic value of miRNA-10a-5p in NSCLC.Results(1)The expression levels of miRNA-10a-5p in NSCLC tissues were significantly higher than those in adjacent normal tissues.Compared with healthy controls,the miR-10a-5p in serum was overexpressed in NSCLC patients.(2)The higher miR-10a-5p expression levels were positively correlated with advanced tumor stage and positive lymph node metastasis.(3)The area under the curve(AUC)of serum miR-10a-5p in the diagnosis of NSCLC was 0.709.The sensitivity and specificity for identifying patients with NSCLC were 65.98%and 72.71%,respectively.Combinatiing with carcinoembryonic antigen(CEA)can further improve the diagnostic value.The area under the curve(AUC)is 0.801(sensitivity is 76.34%;specificity is 79.26%).Conclusion:Serum miR-10a-5p may be useful biomarkers for clinical diagnosis of NSCLC.Part Ⅱ Effect of miR-10a-5p on Biological Behavior of Lung Cancer CellsObjective:To investigate the effect of miR-10a-5p on the biological behaviors of human lung cancer cells such as proliferation,invasion and migration.Methods:(1)The normal lung epithelial cells(BEAS-2B)were used as a control,and the expression levels of miR-10a-5p in lung cancer cell lines A549,H1650 and H1299 was detected by qRT-PCR.The cell line with relatively high expression was selected as a subject for subsequent experiments.(2)Lentivirus infected human lung cancer A549,and then the miR-10a-5p expression was down-regulated.(3)The proliferationand migration,cell cycle,and apoptosis of A549 cells were detected by CCK-8,flow cytometry,and Transwell chamber method.(4)The effect of miR-10a-5p on tumor growth was examined in nude mice xenografts as well.The tumor volume and quality were measured.The tumor growth curve was plotted and the tumor inhibition rate was calculated.Results:(1)The miR-10a-5p expression is relatively high in A549.(2)The expression of miR-10a-5p in A549 cells infected with lentivirus was significantly decreased(P<0.05).(3)In vitro cell experiments:Down-regulation of miR-10a-5p expression in A549 cell could inhibite its proliferation,induce apoptosis,block G1 to S phase progression,and inhibit cell invasion and migration.(4)In vivo experiments:Down-regulate miR-10a-5p inhibited the growth of lung cancer xenografts in nude mice,which could significantly reduce the tumor volume.Conclusion:The results in both vivo and in vitro are consistent to prove miR-10a-5p maybe an oncogene.Down-regulation its expression could inhibit the proliferation,invasion,migration of A549 cells..Part Ⅲ miR-10a-5p target gene and its mechanism of actionObjective:To study the target genes and signaling pathways of miR-10a-5p in NSCLC.Methods:(1)Bioinformatics methods were used to predict the target gene of miR-10a-5p.(2)The dual luciferase reporter gene assay verified whether miR-10a-5p has a direct targeted regulatory effect on the target gene.(3)The expression of target gene in lung cancer tissues and adjacent normal tissues was analyzed by immunohistochemistry.(4)Western blotting and qRT-PCR technique were used to detect the effect of miR-10a-5p on the protein and mRNA expression of target genes and the downstream genes.Results:(1)Biological information suggests that the 3’UTR region of GATA6 has a conserved binding site with miR-10a-5p in a variety of biological cells including human being.(2)The dual luciferase reporter assay showed miR-10a-5p is able to directly target the regulation of GATA6 Gene.(3)The immunohistochemistry showed that the expression of GATA6 in lung cancer tissues was lower than that in adjacent normal tissues.Comparing to blank control group and the negative control group,the GATA6 expression in transplanted tumors of nude mice would be higher after the down-regulation of the miR-10a-5p.(4)After down-regulating the expression of miR-10a-5p in A549 cells,the proteins and mRNA expression levels of GATA6 was increased,while the protein and mRNA expression levels of EGFR,CTNNB1 were decreased.Conclusion:GATA6 may be a direct target gene of miR-10a-5p,and the pathways are associated with EGFR and Wnt signals pathway.Part Ⅳ The Inhibitory Effect of p-Hydroxyisovalerylshikonin on Lung Adenocarcinoma A549 Cells and Its MechanismObjective:To study the effect of β-Hydroxyisovalerylshikonin(β-HIVS)on the biological function of human lung cancer A549 cells and to explore its mechanism.Methods:(1)In vitro cell experiments:① A549 Cells treated with different concentrations of β-HIVS.② The effects of β-HIVS on cell proliferation,cell cycle,apoptosis,invasion and migration were detected by CCK-8 assay,flow cytometry,and Transwell assay.③ The expression of miR-10a-5p in A549 cells were detected by real-time fluorescence quantitative PCR and Western blotting after β-HIVS treatment,and the mRNA and protein expression of GATA6,EGFR,CTNNB1,P53,and Bcl-2 were detected as well.(2)In vivo experiments:① A human lung cancer xenograft model of nude mice was established and randomly divided into a control group,low-,medium-,high-dose of p-HIVS(10,20,40 mg/kg),and paclitaxel(30 mg/kg)groups② Each group was given intraperitoneal injections,each 0.2 mL with 2 times per week,and continuous medication for 4 weeks.③ The body mass,tumor volume and tumor quality of the nude mice were measured.The tumor growth curve was plotted,and the tumor inhibition rate was calculated.The TUNEL method was used to detect the apoptosis of transplanted tumors in each group after β-HIVS treatment.④ Immunohistochemistry,Western blotting and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of miR-10a-5p,GATA6,EGFR,CTNNB1,P53,Bcl-2 in all groups of transplanted tumors after P-HIVS treated.Results:(1)In vitro cell experiments:① β-HIVS inhibited the proliferation of lung adenocarcinoma A549 cells;and induced apoptosis of A549 cells;blocked the development of G phase to S phase;inhibited cell invasion and migration.② β-HIVS can up-regulate the protein and mRNA expression of p53 and down-regulate the protein and mRNA expression of Bcl-2 in a concentration-dependent manner.③ The miR-10a-5p and GATA6 expression did not change significantly(P>0.05)after β-HIVS treated.(2)In vivo experiments:①β-HIVS can inhibit the growth of lung cancer xenografts in nude mice,and can significantly reduce the tumor volume in a dose-dependent manner.② TUNEL assay showed that the apoptosis of tumor cells after β-HIVS treatment was significantly increased compared with the control group.③ The results of immunohistochemistry,Western blotting and qRT-PCR showed that the protein and mRNA expression of p53 increased after treatment with β-HIVS,while the expression of Bcl-2 protein and mRNA decreased.Conclusion:The in vivo and in vitro results consistently demonstrate that β-HIVS can significantly inhibit the proliferation,invasion,and migration of A549 cells and induce apoptosis.The mechanism may be related to up-regulation of p53 expression and down-regulation of Bcl-2 expression.