Ikkα Expression In Gastric Cancer And The Effect On Proliferation And Metastasis Of Gastric Cancer Cells

BackgroundGastric cancer is one of gastrointestinal malignancies，the deadth caused by Gastriccancer is the second leading cause of cancer-related deaths worldwide. A serious threat tothe health of our people and the quality of life of people affected. Incidence of gastriccancer is affected by many factors, the specific mechanism remains to be elucidated indetail. Conventional cytotoxic chemotherapy has limited efficacy for metastatic gastriccancer. Many molecular pathways are currently under investigation as therapeutic targetsin gastric cancer. Advances in molecular diagnostic tools further support the discovery ofnew molecular targets. NF-kB is an important transcriptional factor which controls avariety of biological processes including inflammation, cell survival or death, and cellcycle. It is notable that aberrant NF-kB activation can lead to tumorigenesis. IKK is anessential regulator of the NF-kB pathway, and it is reported to mediate NF-kBdownstream gene expressions by modulating the promoter regions of NF-kB-regulatedgenes in the nucleus.the study suggest that IKK can regulate the expression of genes involved in cell transformation, tumor progression, angiogenesis,it plays a vital role inthe development of tumors.it has been reported that the important role in prostate cancer,breast cancer,but has not been in gastric cancer.Aims1. To detecte the IKK expression in gastric cancer tissues and their correspondingadjacent tissues,and the IKK expression in primary gastric cancer tissues and itslymph node metastasis. To detect the IKK expression at the protein and mRNAlevels of high metastatic potential of gastric cancer cells MKN28-M and lowmetastatic potential of gastric cancer cells MKN28-NM.2. To study the effect of knockdown of IKK on the ability of gastric cancer cell linesmigration and invasion in vitro,and the metastasis ability in vivo.3. To investigate the effect of knockdown of IKK on gastric cancer cell linesproliferation phenotype.Methods1. Immunohistochemical method to detect IKK expression level in gastric cancer andtheir corresponding adjacent tissues,and also to detect IKK expression level in theprimary gastric cancer tissues and the lymph node metastasis.2. IKK interference lentivirus and control lentivirus transfected MKN28-M gastriccancer cell lines, Western blot experiments verify the interference efficiency at theprotein level and quantitative PCR technology verify interference efficiency atmRNA levels.3. Wound healing assay, transwell migration assay, transwell invasion assay to detect theability of migration and invasion of gastric cancer cell with low expression of IKK invitro.4. The methods of intravenous injection in nude mice to detect the ability of metastasis ofgastric cancer cell with low expression of IKK in vivo.5. The methods of MTT assay to describe growth curve of gastric cancer cells with low expression of IKK and the experiment of colony formation assay to observetumorigenicity of the gastric cancer cells.Results1. Immunohistochemistry results showed: IKK mainly localized in the cytoplasm, andno staining of the nucleus. the positive rate of gastric cancer cases was62%.thepositive rate of adjacent non-tumor tissues case was24%. the study of the expressionlevels of IKK in primary gastric cancer tissues and their matched metastatic lymphnode tissues show that the positive rate of primary gastric cancer tissues was32%, thepositive rate of their matched metastatic lymph node tissues was65%.we analyzed therelationship between IKK staining and the clinicopathological parameters of gastriccancer patients.The results showed that neither gender nor age was correlated with theexpression of IKK.However,the IKK expression was statistically correlated withtumor staging,which consisted of tumor size(T,P <0.001),lymph node invasion (N, P<0.001), and distant metastasis (M，P=0.001).2. Western blot and qRT-PCR results showed that: IKK were higher in highly metastaticpotential of gastric cancer cell line MKN28-M than the low metastatic potential ofgastric cancer cell lines MKN28-NM at the protein and mRNA level.3. Western blot and qRT-PCR results showed that: either in the protein level or mRNAlevels, the IKK expression levels is higher in MKN28-M cells line transfected controllentivirus than the MKN28-M cells line transfected IKK interference lentivirus.4. Scratch test results showed that: MKN28-M-shcontrol cells migrate to the opposite at24hours, and the gap between the two sides have basically disappear whileMKN28-M-shIKK cells do not migrate toward to the opposite at24hours, and thegap between the two sides is basically the same as0hour.5. Transwell experiments showed that: the mumber of MKN28-M-shcontrol cell linemigrate to the lower microporous membrane is significantly higher thanMKN28-M-shIKK cells line. 6. Nude mice with tail vein injection experiment showed:the number of the lungmetastases of the group which express the low level of IKK was significantly lesscompared with the control group, and the size and the number of metastases nodulesare less than the control group.7. MTT growth assay and colony formation experiment show that: the capacity of cellproliferation of the experimental group with low IKK expression in gastric cancercell was significantly lower than the control group.the number of colony-forming inthe control group was significantly higher than the experimental group.the colonyformation rate of control group was91.5%while the experimental group was58%.the size of colony formation in the control group is also larger than the experimentalgroup.Conclusions1. The IKK expression levels in gastric cancer was significantly higher than inadjacent normal tissues(47/75vs18/75). the level of expression of IKK in lymph nodemetastasis was significantly higher than in gastric carcinoma primary tumor,(26/40vs13/40), such results suggest that the expression of IKK are close related relationshipwith the metastasis of gastric cancer. we analyzed the relationship between IKK stainingand the clinicopathological parameters of gastric cancer patients The results showed thatneither gender nor age was correlated with the expression of IKK.However,theincreased IKK expression was statistically correlated with tumor staging,whichconsisted of tumor size(T,P <0.001),lymph node invasion (N, P<0.001), and distantmetastasis (M，P=0.001).2. Knockdown of IKK expression in MKN28-M gastric cancer cells significantlyreduce the migration and invasion of the cell in vitro and the capacity of metastasis invivo.3. Knockdown of IKK expression in gastric cancer cells can significantly reduce cellsproliferation of gastric cancer cells. In summary, we believe that IKK may play an important role in the proliferation andmetastasis of gastric cancer process, while provides a the new molecular mechanism forthe diagnosis and treatment of gastric cancer.