The Molecular Mechanism Of MiRNA-302d Inducing The Lung Adenocarcinoma And Lung Saquanious Ceil Carcinomas Proliferation By Inhibitoring The Expression Of DACH1Gene

The Drosophila Dachshund (Dac) gene, cloned as a dominant inhibitor of thehyperactive growth factor mutant ellipse, encodes a keycomponent of the retinaldetermination gene network that governs cell fate. The human DACH1gene encodes aprotein with a predicted helix-turn-helix family structure.DACH1protein as antranscription factor could inhibitor breast tumor and endometrial cancer growth. Thisresearch confirmed DACH1protein expression lower in lung adenocarcinoma andsquamous carcinoma tissue by using Western blotting methods. This researchcooperation unit of fudan university, ministry of education key laboratory of modernanthropology found that miRNA302d abnormal expression in lung adenocarcinoma andlung squamous carcinoma and DACH1is its target genes by using Micrornas chipanalysis and Database prediction. This study using rt-pcr technology to detectmiRNA302d expression level between lung adenocarcinoma/lung squamous carcinomaand its corresponding adjacent tissues. We found that miRNA302d expression higher inlung adenocarcinoma/lung squamous carcinoma. So, We predict that MiRNA302d mayaffect the biological behavior of lung cancer by regulating the expression of DACH1.Further research, we carry out the cytological experiments with the result thatmiRNA302d promoting proliferation of lung tumor in lung adenocarcinoma/lungsquamous cell carcinomas and enhance miRNA302d inhibits DACH1protein expressionin lung adenocarcinoma and squamous cancer cells. According to the above researchresults, We study the mechanism further more by using the luciferase report experimentand RNAi technology. We found that MiRNA302d can directly affect the3’UTR regionof DACH1gene and inhibiting DACH1gene expression. We also found that Directblocking DACH1expression can inhibitor cancer cell proliferation and reverse theinhibition effect on cancer cell proliferation affected by MiRNA302d. Comprehensive theabove results, We believe that in human lung adenocarcinoma/lung squamous cellcarcinomas, miRNA302d can promote tumor proliferation and the mechanism mainlythrough direct suppressing the DACH1expression. Part I DACH1protein expression abnormal in lung adenocarcinomaand lung squamous cell carcinomasPurpose: Detecting DACH1protein expression in lung adenocarcinoma and lungsquamous cell carcinomas by Western-blotting method and RT-PCR method to ClearDACH1expression level.Method:(1)1. Using pyrolysis liquid protein extraction the lung tissue proteins；2.Using Bradford method determination of protein concentration;3. Prepared sds-pageelectrophoresis liquid and carry out the Sds-page electrophoresis;4. According to theconditions, using PVDF membrane to transfer membrane;5. Carry out immune responseby joining the antibody;6. According to the different light intensity adjustment exposureconditions and carry out chemiluminescence reaction;7. Gel image analysis.(2) TotalRNA of40pairs of lung cancer samples (tumor tissues and their corresponding normaltissues) was extracted and reversely translated into cDNA. Using real-time quantitativepolymerase chain reaction, the relative level of DACH1mRNA was detected.Results:(1)DACH1expression in tumor samples were below than the normal tissues.Statistical results indicate that the specimens of lung cancer about DACH1expressionwas lower than normal tissue has a statistically significant.(2) DACH1expression level isassociated with clinical stageConclusion: Lung adenocarcinoma and lungsquamous carcinoma tissues lowerexpress the DACH1protein,which means the lower expression of DACH1protein maybe related to the occurrence and development of lung cancer.PartII miRNA302d expression abnormal in lung adenocarcinoma andlung squamous carcinomasPurpose: Detecting miRNA302d expression in lung adenocarcinoma and lungsquamous carcinomas by RT-PCR method to Clear miRNA302d expression level.Methord:1.Using tissue grinding machine under the condition of the liquid nitrogengrinding organization adequately;2. Accordance with the operation manual requirementsand methods, extract the RNA by using RNA extraction kit;3. According to theexperiment requiremens to prepare the RNA transcription reaction solution,;4.amplificateMiRNA302d by RT–PCR;5. The experimental data were collected for statisticalanalysis. Results: miRNA302d expression in tumor samples were higher than the normaltissues. Statistical results indicate that the specimens of lung cancer about miRNA302dexpression was higher than normal tissue has a statistically significant.Conclusion: Lung adenocarcinoma and lung squamous carcinoma tissues higherexpress the miRNA302d,which means the higher expression of miRNA302d may berelated to the occurrence and development of lung cancer.PartIII The mechanism of MiRNA302d affecting lung cancerbiology behavior by controlling the DACH1expressionPurpose:1.Confirm the influence on the biological behavior of lungcancer by highexpressing the miRNA302d level.Methord:1.Transfected miRNA302d-mimics or suppress the Lung adenocarcinomaand lung squamous carcinoma cells by using liop2000;2. Flow cytometry instrument todetect the cell cycle change;3. Determined by MTT method to observe changes in cellproliferation activity;4. Observe cell ChengKeLong ablitity through Cell ChengKeLongexperiment;5. Test cell DACH1protein expression by using Western-blotting;6.Building the luciferase report gene carrier and carrying out the Luciferase report geneexperiments to confirm MiRNA302d combining DACH1directly;7. Direct blockingDACH1gene expression in the cell to observe the Cell ability changes by using RNAitechnology.Results:1. The high expression of MiRNA302d promoted the lung cancer cellproliferation and MiRNA302d play the role of promoting the proliferation in Lungadenocarcinoma and lung squamous carcinoma cells;2. MiRNA302d combine DACH1directly in3’UTR region, Direct blocking DACH1gene expression can reverse thenegative effects which the MiRNA302d affect on Lung adenocarcinoma and lungsquamous carcinoma cells.Conclusion: MiRNA302d can promote proliferation of the tumor and mechanism ismainly implemented by directly inhibiting DACH1expression.