The Effects Of Knocking Down Reg Gene On Proliferation,Cell Cycle And Apoptosis In Thyroid Carcinoma

PART ONEEXPRESSION AND SIGNIFICANCE OF REG,P21,P27AND PCNA IN THYROID CARCINOMA AND NORMOLTISSUESObjective:To study the expression and significance of REG,p21,p27and PCNA in human thyroid carcinoma and normal tissues.Methods:immunohistochemistry,Western blotting and Dot blot wereapplied to detect the expression of REG,p21,p27and PCNA in thyroidcarcinoma and normal tissues.Results:①expression of REG: REG levels of the thyroid carcinomatissue were obviously higher than those of normal tissues；REG levels ofthe well-differentiated thyroid carcinoma tissues with lymph nodemetastatic were obviously higher than those of well-differentiated thyroidcarcinoma tissues without lymph node metastatic。②expression of p21:p21levels of the thyroid carcinoma tissue were obviously lower than those ofnormal tissues；p21levels of the poorly differentiated thyroid carcinomatissues were obviously lower than those of well-differentiated thyroid carcinoma。p21levels of the well-differentiated thyroid carcinoma tissueswith lymph node metastatic were obviously lower than those ofwell-differentiated thyroid carcinoma tissues without lymph nodemetastatic③expression of p27: p27levels of the thyroid carcinoma tissuewere obviously lower than those of normal tissues；p27levels of the poorlydifferentiated thyroid carcinoma tissues were obviously lower than those ofwell-differentiated thyroid carcinoma。p27levels of the well-differentiatedthyroid carcinoma tissues with lymph node metastatic were obviouslylower than those of well-differentiated thyroid carcinoma tissues withoutlymph node metastatic④expression of PCNA: PCNA levels of the thyroidcarcinoma tissue were obviously higher than those of normal tissues；PCNA levels of the poorly differentiated thyroid carcinoma tissues wereobviously higher than those of well-differentiated thyroid carcinoma。PCNA levels of the well-differentiated thyroid carcinoma tissues withlymph node metastatic were obviously higher than those ofwell-differentiated thyroid carcinoma tissues without lymph nodemetastatic.Conclusion: Abnormally higher expression of REG and PCNA arerelated with tumor development, lymph node metastatic and malignancydegree in thyroid carcinoma tissues. Abnormally lower expression of p21and p27are related with tumor development, lymph node metastatic andmalignancy degree in thyroid carcinoma tissues. PART TWOCONSTRUCTION AND IDENDIFICATION OFRECOMBINED shRNA-REG PLASMID INTHYROID CARCINOMA CELLSObjective: To construct and idendify the recombined small hairpinRNAs (shRNA) plasmid targeted to REG gene, which can knock down theREG gene of thyroid carcinoma cells.Methods: Small hairpin RNAs (shRNA) targeted to REG codinggene were designed and synthesized according to the principle mentionedby Elbashir and Reynolds. It was cloned into pGenesil-1which worked as atranscription vector to construct recombined plasmid named pshRNA1-REG, pshRNA2-REG and pshRNA-HK (negative control plasmid). Therecombined plasmid was extracted in middle quantity and transferred intothyroid carcinoma cells by LipofectamineTM2000. RT-PCT, western blotand Dot blot were used to detect REG mRNA and protein respectively.Results: Three recombined plasmids: pshRNA1-REG, pshRNA2-REG and pshRNA-HK, were constructed successfully. The transfectionrate is70%by LipofetamineTM2000. The transfection effect ofpshRNA2-REG is better than that of pshRNA1-REG.The expression ofREG was obviously lower in thyroid carcinoma cells transfected bypshRNA-REG than those by pshRNA-HK.Conclusions: REG protein and mRNA in thyroid carcinoma cells canbe knocked down effectively by recombined plasmid pshRNA-REG. PART THREETHE EFFECTS OF pshRNA-REGγ ON EXPRESSION OFP21,P27AND PCNA IN THYROID CARCINOMA CELLSObjective: To study the effects on expression of p21,p27and PCNAin thyroid carcinoma cell, while the phsRNA-REGγ inhibit the expressionof REGγ gene.Methods: Recombined plasmids pshRNA-REG and pshRNA-HKwere transfected into thyroid carcinoma cells, then these cells werescreened by G418. Western blotting,Dot blot and RT-PCR were applied todetect the expression of p21,p27and PCNA in thyroid carcinoma cellstransfected by pshRNA-REGγ, contrasting to those transfected bypshRNA-HK.Results:①Expression of p21: The expression of p21protein inthyroid carcinoma cells transfected by pshRNA-REGγ are obviously higherthan those transfected by pshRNA-HK. The expression of p27mRNA inthyroid carcinoma cells transfected by pshRNA-REGγ are not obviouslydifferent in contrast to those transfected by pshRNA-HK.②Expression of p27: The expression of p27protein and mRNA inthyroid carcinoma cells transfected by pshRNA-REGγ are not obviouslydifferent in contrast to those transfected by pshRNA-HK.③Expression of PCNA:The expression of PCNA protein and mRNAin thyroid carcinoma cells transfected by pshRNA-REGγ are obviouslyhigher than those transfected by pshRNA-HK.Conclusions: That recombined plasmid pshRNA-REG knock downthe REG gene can lead to increasing of p21protein and depressing ofPCNA protein in thyroid carcinoma cells. PART FOURTHE EFFECTS OF phsRNA-REG ON PROLIFERATIONAND CELL CYCLE IN THYROID CARCINOMA CELLSObjective: To study the effects on expression of on proliferation andcell cycle in thyroid carcinoma cell, while the phsRNA-REGγ inhibit theexpression of REGγ gene.Methods: Recombined plasmids pshRNA-REG and pshRNA-HKwere transfected into thyroid carcinoma cells, then these cells werescreened by G418. MTT and flow cytometry were applied to detect theproliferation and cell cycle in thyroid carcinoma cells influenced byphs-REGγ, contrasting to those not influenced by SiRNA-REGγ.Results: The growth velocity of thyroid carcinoma cells transfected bypshRNA-REGγ are obviously slower than those transfected bypshRNA-HK.Conclusions: That recombined plasmid pshRNA-REG knock downthe REG gene can promote thyroid carcinoma cells proliferation and leadto thyroid carcinoma cell cycle arrest. PART FIVETHE EFFECTS OF phsRNA-REG ON APOPTOSIS INTHYROID CARCINOMA CELLObjective: To study the effects on expression ofapoptosis in thyroidcarcinoma cell, while the phsRNA-REGγ inhibit the expression of REGγgene.Methods: Recombined plasmids pshRNA-REG and pshRNA-HKwere transfected into thyroid carcinoma cells, then these cells werescreened by G418., TUNEL and Western blot were applied to detect theapoptotic index and the expression of caspases-3in thyroid carcinoma cellsinfluenced by phsRNA-REGγ, contrasting to those not influenced byphsRNA-REGγ.Results: The apoptotic index and the expression of caspases-3proteinin thyroid carcinoma cells transfected by pshRNA-REGγ are obviouslyhigher than those transfected by pshRNA-HK.Conclusions: That recombined plasmid pshRNA-REG knock downthe REG gene can promote apoptotic of thyroid carcinoma cells.