Expression And Significance Of ARHGAR10 In Non-small Cell Lung Cancer

Lung cancer is still the second most common cancer in the world,of which non-small cell lung cancer(NSCLC)accounts for about 70%.In the past 20 years,the study of molecular aberration,targeting drugs and immunotherapy for NSCLC,especially in lung adenocarcinoma,has been widely and deeply studied.According to the mutation of the gene,the corresponding targeted drug therapy is used to bring new treatment and curative effect to the patients with lung cancer,but there are still a large number of unknown oncogenes that have not been discovered or unidentified.Up to now,lung cancer is still a heterogeneous,complex and challenging,refractory and malignant disease.The incidence and mortality of lung cancer are still high,and the overall treatment effect is not satisfactory.To study the molecular biological mechanism of the pathogenesis,occurrence,development and metastasis of non-small cell lung cancer,and to explore the early diagnosis,monitoring and treatment of lung cancer related markers and genes and immunotherapy targets are still the focus,hot spot and research direction of the research.ARHGAP10 is homologous to Ras gene and belongs to G protein superfamily.It is positive in many human normal tissues and malignant tumor cell lines.Over expression or deletion of ARHGAP 10 may regulate the invasiveness and migration of multiple malignancies.However,because of the complexity of the ARHGAP 10 structure and its unique expression in different tumor cells,the expression and mechanism of ARHGAP 10 in the lung NSCLC have not been reported.Further study of the expression and regulation function of ARHGAP 10 in NSCLC has important clinical significance for elucidating the regulatory mechanism between ARHGAP 10 and NSCLC.Further in-depth study will hopefully provide a target for clinical treatment of ARHGAP 10 related non-small cell lung cancer,and promote the development of related drugs.Part 1.Clinical study of ARHGAP10 expression in non-small cell lung cancerObjective To study the expression of ARHGAP 10 in benign lung tissue and NSCLC tissues at three stages of early,middle and advanced stages.To analyze the expression of ARHGAP10 in different stages of lung cancer,and the correlation with common immunohistochemical index,circulating blood tumor cell and EGFR gene mutation,in order to provide preliminary research direction and theoretical support for further study of ARHGAP10 in NSCLC.Methods A total of 120 samples were selected,including benign lung tissue,early,middle and late three stages of NSCLC tissue,30 cases each.The expression of Ki67,P53,EGFR,VEGF and ARHGAP10 were detected by immunohistochemistry.Immunomagnetic beads were enriched and probe fluorescence in situ hybridization was used to detect the count of circulating blood tumor cells(CTC)in peripheral blood.The mutation system of scorpion probe amplification block(ARMS)and direct sequencing were used to detect the mutation of EGFR,and the experimental results were classified according to the clinicopathological features of the samples.Results(1)The expression of ARHGAP10 was significantly different between the normal lung tissue and the NSCLC,and the expression of ARHGAP10 protein decreased gradually with the severity of the tumor stage.(2)The positive expression of Ki67,P53,EGFR,VEGF and ARHGAP10 were independent of five indexes.(3)The strong positive expression of VEGF and ARHGAP10 in the four groups showed obvious reverse trend.(4)The positive rates of CTCS between the four groups were statistically different(P<0.01).The NSCLC group with lymph node metastasis(92.3%)was higher than the NSCLC group without lymph node metastasis(61.1%),and the difference was statistically significant(P<0.01).There was no significant correlation between the positive expression of ARHGAP10 and CTCs>1 in the four groups(P<0.01).(5)The positive expression of ARHGAP10 was not correlated with the clinicopathological features of NSCLC.There was no significant correlation between the positive expression of EGFR and the mutation of EGFR gene.The positive expression of ARHGAP10 was not correlated with the mutation of EGFR gene and the type of mutation.Conclusion It is suggested that there is a certain relationship between the expression of ARHGAP10 and the occurrence and development of NSCLC,but there is no correlation with the clinicopathological features of NSCLC.There may be a certain correlation between strong positive expression of VEGF and strong positive expression of ARHGAP10 in different clinical stages of NSCLC,and the possibility of negative correlation is larger.It is worth further studying the relationship between the two in the subsequent experiments.Positive detection of CTCS has some reference value for lymph node metastasis in patients with NSCLC.EGFR positive results of immunohistochemistry did not predict that there was a EGFR gene mutation in NSCLC.There was no significant correlation between ARHGAP10 expression and EGFR gene mutation.It is suggested that ARHGAP10 may not be associated with the EGFR tyrosine kinase pathway signal pathway,and the value of further in-depth study of the signal pathway needs to be carefully considered.Part 2.The basic research on the function of ARHGAP10 in NSCLCObjective Base on the content of part 1 about the level of ARHGAP 10-expression on different phases of lung cancer.To explore the correlation between ARHGAP10 and lung cancer furtherly,in this study the series experiments were proved the ARHGAP 10 impact on proliferation,migration and invasion in lung cancer.Methods Firstly,the four kinds of lung cancer cell lines(A549,NCI-1975,NCI-H1299 and NCI-H460)were selected and detected to compare the level of protein and mRNA in cell lines respectively.The low level of ARHGAP 10-expression cell lines was selected and transfected the lenti viral-ARHGAP 10 plasmid for detection of ARHGAP10-expression at gene level and protein level by RT-PCR and WB respectively.Meanwhile the physiological function of cell lines was observed,proliferation,migration and invasion,after the lentiviral-ARHGAP10 expression plasmid was transfected into cell lines.To get the mechanism of ARHGAP10 impacting on physiological function,the GSEA was used for analyzing and predicting the signal pathway and some key factors were proved in next experiments.In addition,this study tumor-bearing model of nude mice were established by tail vein injecting of A549 cells,the pre or post transfected lentiviral-ARHGAPIO expression plasmid.Finally,the neoplastic foci of lung tissue was observed and evaluated.Results The data showed the level of ARHGAP 10 in NCI-H460 and A549 cell lines were lower than in NCI-1975 and NCI-H1299 cell lines.Following the constructed lentiviral-ARHGAP10 expression plasmid was transfected into NCI-H460 and A549 cell lines.The cells with lentiviral-ARHGAP10 expression plasmid produced more protein and gene of ARHGAP 10.The data of growth rate showed cells(NCI-H460 and A549)with lentiviral-ARHGAP10 expression plasmid get more proliferation than wild type and cells with vector at three different time point,24h,48h and 72h.Meanwhile the cell with lentiviral-ARHGAP10 expression plasmid had a lower number than without effective constructed plasmid.To get further clearness the mechanism of ARHGAP10 in lung cancer,the GSEA was used to predict the signal pathway and the key factors in Wnt signal pathway.Of course,the key factors of metastasis(MMP-2,MMP-9 and VEGF)and Wnt signal pathway(?-catenin c-myc and p21)was related to high level of ARHGAP10.The level of factors(MMP-2,MMP-9,p-catenin and c-myc)was lower in A549 and NCI-H460 cell lines with lentiviral-ARHGAP10 plasmid than without,and the factor p21 was opposite in this treatment.The level of ?-catenin was rised in lentiviral-ARHGAP10 plasmid transfected A549 cell line with lOmM Lithium chloride stimulating.In nude mouse tumor-bearing model,the tumor node was visible to the naked eyes in lung tissue in vein injection by A549 cells.The cell nucleus was bigger and hyperchromatic and it has tumor typical feature.However,the lung tissue of nude mouse had no visible tumor node with A549 transfected lentiviral-ARHGAPIO plasmid in vein injection in naked eyes and microscope.Conclusion This study established the correlation between ARHGAP10 and lung cancer by GSEA.The data of enrichment analysis showed the level of ARHGAP10 was negative correlated with metastasis and Wnt signal pathway.Our experiment showed high level of ARHGAP10 impacted on metastasis key factors(MMP-2,MMP-9 and VEGF)and key factors of Wnt signal pathway(?-catenin,c-myc and p21).These data suggested that high level of ARHGAP10 can inhibit proliferation,metastasis and invasion significantly..In short,the experiment results showed that ARHGAP10 maybe as a biomarker for lung cancer diagnosis and ARHGAP10 be as a target for clinical treatment furtherly.