Discovery Of A New TNS4 Mutant And Its Role And Mechanism In Prostate Cancer Cells

Prostate cancer does serious harms to men’s health and the morbidity of prostate cancer is the highest one but the lung cancer.Although prostate cancer has typical geographical difference and racial diifference:the morbidity in Caribbean Sea and Scandinavia and Aframerican is the highest.The morbidity of prostate cancer,in China and Japan,is increasing year by year when people living habits are changing and globalization is speeded up by economic development.Ignorance is denied here.The prostate cancer cells can penetrate the soft tissue of seminal vesicle or encroach on seminal vesicle through ducts ejaculators.The treatment difficult of prostate cancer is no less favorable than any other one.Some related genes are unusual in expression level and existence form as the transfer ability and deterioration degree of tumor boost up.They are promoted or inhibited.Mutation and miss appear also.Studies found that TNS4 is unique high in embryonic tissue and normal prostate tissue and found the contrary fact in others tissues.That is so called tissue specificity.In recent years,some study focuses on the relationship of TNS4(Cten)and prostate cancer.According to reports,TNS4 is extremely low in prostate cancer cells and even cannot be tested in some cell lines.That indicates the intimate connection of TNS4 and prostate cancer development.However,the molecular mechanism of the TNS4 inhibition in prostate cancer is unclear.The effect of high TNS4 expression on prostate cancer cells living was seldom reported.In order to know about what TNS4 will do on cell metastasis,adhersion,invasion and living,we do researches on the expression and existence form of TNS4 in androgen independent prostate cancer cell PC-3,and on the interaction between TNS4 and cell living,apoptosis and cell cycle.Then we can know very well about the action of TNS4 on tumor occurrence and development.To solve the above problems,experiments are divided into several sections as follows:At first,we detect the TNS4 level in two typical prostate cells,BPH1,WPMY-1 and four prostate cancer cells types,LNCaP,CRW22Rvl,DU145 and PC-3.RT=PCR tells the truth that the mRNA level of TNS4 is high in BPH1 and lower in cancer cells,especially in PC-3,and WPMY-1.Then,we sequenced the complete CDS of TNS4 from BPH1,LNCaP,DU145 and PC-3.Only the CDS from BPH1 is exactly same to the sequence from human normal prostate tissue provided by NCBI.The CDS from cancer cells mutate in different degrees.The two single-base-mutations happened in LNCaP and DU 145:426T-C,1454G-A.Besides the two single-base-mutations,there is a base-deficiency at 876th bp site in PC-3.We got the amino acid sequence through Primer Premier 5.0 and compare with each other.The software shows us that the two single-base-mutations in CDS bring about only one mutation in amino acid in LNCaP and DU145:142th Ser amino acid changes to Pro.Because of the special location of the mutation which is in the phosphor rich district,it may affect function seriously.The amino acid sequence in PC-3 changes greatly from 291th,owing to base-deletion.Next,we structure the TNS4 mutant plasmid(TNS4-Mut)using the CDS from DU145 and the TNS4 wildtype plasmid(TNS4-WT)by single-base-mutation technique.And we confirm that the plasmids acomplish expressing by western blot.At last,we found that both TNS4-WT and TNS4-Mut can restrain cell proliferation through MTT assay.At the same time,Flow Cytometry assay was performed to test cell cycle and apoptosis after pTNS4.G2 phase was promoted.On the contrary,apoptosis was not affected seriously by TNS4 overexpression.Then,wound healing assay,adhesion assay,and transwell assay were performed to investigate the alteration of cell migration,adhesion,and invasion ability when TNS4-WT and TNS4-Mut were overexpressed in PC-3.The results show that the migration and invasion of cells are enhanced,at the same time,cell adhesion is inhibited.What’s important,there is obvious distinct between the two types plasmids.The above facts tell us that TNS4 inhibits cancer cells by inhibiting proliferation and cell cycle.And TNS4 can promote cancer development by enhancing cell migration and invasion abilities.And STAT1 is necessary to the process.Besides,we find that STAT1 expression is elevated and be promoted to transfer into nucleus after overexpress TNS4-WT and TNS4-Mut.We put Immuno fluorescence assay into effect and find TNS4 does affect its distribution.The effect of TNS4-Mut is much more obivious than TNS4-WT.Based on the above all,the different mutations of TNS4 in prostate cancer cells are proved.Cell migration and invasion ability are all affected by both wild type-and mutant-TNS4.So do cell living and cycle.The influences degree is significant different between TNS4-WT and TNS4-Mut.Probably,TNS4 promotes tumorigenesis in STAT1 dependent way.Docetaxel is an effective anticancer drug synthesized mainly by some chemical substances which is extracted from yew.It has been widely used in the treatment of breast cancer and non-small cell cancers,and the clinic treatment of prostate cancer also.Studies found that it has a bright prospect fo r docetaxel to cure prostate cancer as it can reduce the survival ability of prostate cancer cells.In this study,PC-3 and LNCaP cells were exposed to docetaxel with different dose and time gradients,then,we test the mRNA and protein level of Smad3 by RT-PCR and Western Blot.This study found that docetaxel can suppress the expression of Smad3 in both mRNA and protein levels.Then total Smad3 and its phospho level were also tested when PC-3 cells were treated with docetaxel and TGF-pl.This study verified that docetaxel can suppress the expression of Smad3 in TGF-pl signaling pathway-independent/dependent ways in PC-3k cell.What’s more,we found the cooperation between Smad3 and STAT1 through co-IP assay.Regarding the facts that STAT1 can bind to the promoter of Smad3 and docetaxel can also reduce the expression of STATl,we do confirm that docetaxel can suppress the expression of Smad3 in TGF-pl signaling pathway-independent way.According to the above all5 docetaxel inhibits cancer cells9 growth by the inhibition of Smad3 in different ways.