RNAi Silence Gene Expression Of TAMs On The Impact Of Invasive Ability Of Laryngeal Hep-2 Cells

Research Purpose and BackgroundSurrounding environment of malignant tumor cells is very important influence on the cell itself,which can change its behavior and properties.As laryngeal cancer with poor prognosis in head and neck cancer,its inflammatory microenvironment plays an important role in promoting growth,infiltration and metastasis of cancer cells.The inflammatory microenvironment around the tumor has become a hotspot in recent studies of tumor,and many inflammatory factors can serve as markers for the prognosis of various malignant tumors.The most common inflammatory cells include macrophages,T cells,mast cells,and natural killer cells.They play an important role in tumor microenvironment and have a double effect on tumor promotion or inhibition.Tumor cells is not isolation growth,but they has the connection between the tumor cells by molecular signal conduct,so the comprehensive understanding of laryngeal cancer invasion biology behaviour need to understand the inflammation cells in the microenvironment.Macrophages play an important role in the inflammatory microenvironment of tumor.Tumor Associated Macrophages(TAMs)promote tumor angiogenesis and inhibit normal anti-tumor immune response.Recent studies have shown that the molecular signaling of cancer cell has achieved the molecular linkage between cells,thus promoting the role of tumor related inflammatory cells and affecting the biological behavior of the tumor.TAMs results in more cytokines secreted by the tumor,which leads to the association with interstitial tissue,which leads to further proliferation of tumor cells.TAMs with tumor tissues and cells,which leading to tumor tissues and cells secrete many proteolytic enzyme,which lead to hydrolysis and damage of the basement membrane around tumor with for implantation of new blood vessels and the growth of tumor cells and metastasis.TAMs are divided into two distinct types:M1 and M2,and M1 has anti-tumor activity caused by microbial product stimulation.The M2 type has the activity of promoting tumor growth,which is produced by IL-4,IL-13,and IL-10 stimulation.Most TAMs have M2 type phenotype,which promotes metabolism,angiogenesis and immunosuppression.Evidence suggests that stromal cells,such as around the tumor infiltrating lymphocytes,TAMs and fiber cells associated with cancer in the microenvironment of cancer are related to the development of cancer,as M2-TAMs play an irreplaceable role in promoting tumor infiltration,lymph node metastases,new blood vessels to form and the immune suppression.Recent studies have found that M2-TAMs can express special markers such as CD 163 and CD204.It was found that TAMs played a role in the tissues of the head and neck squamous cell carcinoma,which may be regulated by IL-10 and PD-L1 regulating T lymphocytes.The high density TAMs expression in the inflammatory microenvironment is closely related to the poor prognosis of patients with rectal cancer.It was found that the peripheral monocytes were correlated with the density of TAMs in tumor microenvironment.Colony Stimulating Factor-1(CSF-1)stimulate TAMs chemotaxis and play an important role in TAMs gathered together in the tumor edge area of tumor and thus involved in the invasion and metastasis of colon cancer cells.Many patients with laryngeal cancer has been diagnosed with late or lymph node metastasis,the not characteristic clinical expression in the early laryngeal cancer performance led to the occurrence of misdiagnosis,biology activity research of laryngeal cancer cell has an important role in early diagnosis of laryngeal cancer,to provide the possibility for master the laryngeal cancer occurrence,development and the molecular mechanisms of invasion and metastasis.From the above inspired,first we studied CD68 antibodies labeled tumor associated macrophage CD68-TAMs in laryngeal squamous carcinoma tissue expression and its relationship with clinical pathological parameters,and discussed the clinical significance of expression of CD68-TAMs.The Hep-2 cells as the research object,using RNAi technology,through cell culture,the technical methods of synthesis and design,have a purpose to TAMs,and synthesis of recombinant plasmid shRNA,through the role of the transfection of Hep 2 cells,using PCR and determined by MTT to determine the purpose gene have substantial change,thus it is concluded that interfere with TAMs gene expression influence on the activity of tumor cells,we need come to the conclusion that with the expected presence of differences,also provide certain theoretical basis for the subsequent laryngeal cancer molecular biology research.PART I EXPRESSION AND CLINICAL SIGNIFICANCE OF CD68-TAMS IN LARYNGEAL SQUAMOUS CARCINOMAObjective To study the expression of CD68-TAMs and the relationship between clinicopathological parameters and the expression of CD68-TAMs,and to investigate the clinical significance of CD68-TAMs expression in the laryngeal caicinoma.Methods To collect 45 cases of laryngeal cancer from August 2010 to October 2016 in the department of pathology of weifang medical college,12 cases of female patients;33 male,aged 45-78,average 64.2 years old,20 cases were taken as control group.Routine pathological examination was established as laryngeal squamous cell carcinoma.According to the 2002 international TNM staging standards promulgated by the cancer alliance(UICC),? period(15 cases),? period(17 cases),? period(9 cases),? period(4 cases).All patients were not treated for operation and chemotherapy.Using immunohistochemical SP method and positive staining decision criteria,positive staining in tumor cell cytoplasm and periphery nucleus for brown and yellow,with positive dying cells number exceeds more than 10%as the positive response.Data analysis use SPSS 16.0,t test,Spearman rank correlation processing data and ?2 as the general statistical methods.The significance of p<0.05 was statistically significant.Results The positive expression rate of CD68 was 83.3%(37/45),15%(3/20)in tumor tissue and tumor of the tissue adjacent to tumor,there is significant difference(P<0.05)between two comparisons groups.The positive expression rate of CD68-TAMs was 70.6%(12/17),89.2%(25/28)in the glottis area and the supraglottic area,there was not significant difference(?2=2.54,P>2.54)between two comparisons groups.The positive expression rate of CD-TAMs was 100.0%and 57.9%in lymph node metastasis group and without lymph node metastasis group respectively,there is significantly differences between two groups p<0.05.The positive expression rate of CD68 in the high,middle and low pathologic differentiation,there was not significant difference(p>0.05)between two groups.Conclusion CD68-TAMs in a high expression in laryngeal squamous carcinoma cells,suggests CD68-TAMs involved in the process of laryngeal cancer biology behavior,there were positive correlation with lymph node metastasis and clinical staging of tumor.In the middle-late laryngeal squamous cell carcinoma tissues will produce more inflammatory microenvironment,which form more easily in the positive expression of CD68-TAMs.CD68-TAMs play a role in promoting the metastasis and formation of new blood vessels of laryngeal squamous carcinoma tissues,it can be used as one of the important markers of invasion and metastasiof laryngeal cancer tissues.PART ? RNAi SILENCE GENE EXPRESSION OF TAMS ON THE IMPACT OF INVASIVE ABILITY OF LARYNGEAL HEP-2 CELLSObjective Using RNA interference technology silent tumor associated macrophages(TAMs)gene expression of laryngeal Hep-2 cells,and discusses influence of silencing TAMs gene expression on invasion ability of laryngeal Hep 2 cells,provides a new and effective theory basis for laryngeal cancer gene therapy.Methods Extraction and culture of macrophage;Construction of shRNA of eukaryotic cell expression vector;Oligos was designed for the shRNA target design,and oligos was synthesized according to the TAM gene sequence reference Genbank database.Dilution and annealing of oligos;Connect the expression vectors.Thermally excited transformation E.col celli;Identification of positive clones.Plasmid extraction and sequencing;Laryngeal cancer Hep-2 cell culture,counting,generation,freezing.Cell transfection and transfection conditions optimization,observed transfection rate under inverted microscope,according to the SiRNA requirements,TAMs genes can be divided into the experimental group,blank control group,negative control group,the fluorescence quantitative PCR detection.According to the TAM gene sequence of gene database,the Primer5 PCR amplification and primers were designed by using molecular genetic software in the conservative area,and the primers were synthesized and designed by the ?-act in.Extraction of reverse transcription and total RNA;In vitro proliferation experiment(MTT)was used to detect the number and growth of cells.All statistics and data using SPSS 16.0 statistical software for data statistics and processing,the mean and standard deviation x ± S statistical measurement data and information,between the two groups compared by t test and x 2 test,using analysis of variance as difference analysis between more groups.Results Laryngeal cancer hep-2 cells appear adherent growth in culture bottle cells liquids,single-layer,refraction,cell growth is rapid,over time,the cells rapidly growing in dense arrangement,fine granular material within the cell cytoplasm,lead to cell refraction is abate,cause cells to disappear;The sequence of eukaryotic cytoplasmic granule was determined by enzyme digestion,the sequence of three groups were correct.Transfection results under the microscope showed that cell transfection appear strong expression of the fluorescent state after 24 hours,and found that about 45%of transfection rate in NC(negative control),AC(blank control group)transfection rate at about 35%,about 42%of the transfection rate for shRNA-1,transfection rate of shRNA-2 was about 47%,transfection rate of shRNA-3 was about 55%.The results of TAM transfection in the experimental interference group were lower than that of NC and AC.NC,AC two groups have close to Hep-2 cell growth curve,and lead to weak abilityof Hep-2 after transfection silent of siRNA TAMs,its cell growth curve interference(experiment group)is relatively less by comparison with NC,AC,there was significant difference between two groups(P<0.05).Conclusion The expression of TAMs gene in the hep-2 cells in vitro culture can be effectively silenced after RNAi interference technology transfected with Hep-2 cells.After application specific shRNA successfully cut TAMs gene expression of Hep 2 cells,it results show that the Hep-2 cells in vitro proliferation ability weakened obviously by MTT test:The high TAMs gene expression in laryngeal cancer cells may closely relate to occur,recurrence,and metastasis of laryngeal cancer cell;The abnormal expression of TAMs may be closely related to the biological behavior of hep-2 cells,and may be an important tumor marker in the metastasis mechanism of laryngeal squamous carcinoma cell.