The Role And Mechanism Of SiRNA^BRAFV600ECarried By Targeted Nanobubbles For Papillary Thyroid Cancer With Cervical Lymph Node Metastasis

Object:BRAFV600E is the most common gene mutation in papillary thyroid carcinoma(PTC),which has been a sufficient and essential diagnostie criteria when the result of Fine-needle Aspiration(FNA)is uncertain.It has been suggested that BRAF E become one of the most powerful predictive factors in terms of cervical lymph node metastasis(LNM),however,the mechanism of which is not illustrated.We found that many research aimed to establish the relationship between LNM and BRAFV600E mutation just by means of qualification,namely“Yes" or "No",while,little attention has been focused on the qualification of this gene mutation.Therefore,in this project,the technology of Ultrasound-targeted Microbubble Destruction(UTMD)combined with RNA interference has been introduced to find out to what extent this mutation is associated with LNM by quantification through inhibition of BRAFV600E in PTC.Methods:(1)Totally,280 PTC patients were selected from our hospital between the year of 2016 and 2017.Then 280 PTCs was grouped into two groups by LNM or not referring to the pathologic result after operation.After comparing the life habit,general characteristics,the ultrasonic feathers of primary tumors,and the status of BRAFV600E,we analyzed the differences of mRNA and protein expression between two groups;the patients were classified from lower level to higher level by the quantitative results mentioned above in order to find out the association with the risks of LNM in PTC patients.(2)We fabricated three kinds of targeted liposomal nanobubbles:Targeted-cationic nanobubbles(FA-CNBs),Cationic nanobubbles(CNBs)and Neutral nanobubbles(NNBs)by co-evaporating method accompanied with carbodiimide method,as well as the connection of nanobubbles to siRNA by electrostatic attraction on their surface.We observed these bubbles' morphology,Particle size and potential difference by Malvern Instruments,as well as their targeted capacity and transfection efficiency by Flow Cytometry when carrying siRNABRAF600E.(3)Research in vitro:The cultured B-CPAP cells was then grouped into four groups:siRNA-FA-CNBs,siRNA-CNBs,siRNA-CNBs and CNBs before being transfected by these nanobubbles carrying siRNABRAFV600E,which were then evaluated in the levels of mRNA and protein expression of BRAFV600E/MEK/ERK?E-Cadherin/Snail among these four groups by PCR and Western blot;CCK8 was then used to test cell viability and toxity,as well as cell cycle and apoptosis by Flow cytometry,and cell invasive and migration ablity were also assayed simultaneously.(4)Resarch in vivo:The nude mouse model of subcutaneous B-CPAP cells xenografts were constructed before being of targeted transfection into the xenografts by the effect of UTMD.The level of mRNA and protein expression about RAFV600E/MEK/ERK?E-Cadherin/Snail was observed by PCR and Western blot,as well as tumor apoptosis and proliferation by the method of TUNEL and EdU,and tumor inhabitation rate(%)was also calculated.Results:(1)There was significant difference of BRAFV600E Ct value and protein intensity between LNM groups and none-LNM groups;by using Logistic regression model,the risks of LNM in PTCs increased with the incremental level of BRAFV600E protein expression.(2)There types of nanobbles were characterized by smooth surface,uniform morphology and distribution,with the particle size of 471.36±23.34nm?469.32±29.18nm and 480.35±22.15nm,respectively(p>0.05)and with the potential of 35.18±3.2mV?32.34±4.18mV and-3.25±0.38mV,respectively(p<0.05).The FA-CNBs presented the highest ability in targeting B-CPAP cells.(3)In vitro,after transfection of siRNABRAFV600E into four groups of B-CPAP cells,the level of BRAF,its downstream MEK/ERK and Snail in FA-CNBs group was most significantly inhibited.Conversely,the E-cadherin of this group was upregulated most significantly(p<0.05),and the cell proliferation and invasive ability also decreased.(4)In vivo,the subcutaneous xenografts was inhibited significantly in FA-CNBs group with the highest tumor inhibition rate of 52.56±5.22%among three groups;the number of apoptotic cells in this group was larger than other two groups,with the lowest extent of its proliferation.Conclusion:There has been a great value of the role of quantitative BRAFV600E in predicting LMN in PTC patients followed by fabricating three kind of nanobubbles successfully as a siRNA vector aimed to be transfected into the PTC cells and xenografs,which was designed to regulate the expression of BRAF quantitatively.As a result,some biological changes have happened,among which the best treatment effects has been observed in group siRNABRAFV600E-FA-CNBs.Hence,it might be another therapeutic method in the PTC patients,especially from the aspect of preventing cervical lymph node metastasis.