| BackgroudColorectal cancer (CRC) is the one of common cancers in the world. The incidence rate of CRC in China is increasing fast during the past decades. The occurrence and development of CRC have the following characteristics:inapparent pristine symptoms, middle or advanced stage when final diagnosis in most of patients, half of patients finally develops metastases. Accordingly, screening and identification of special and important genes during the occurrence and development of CRC, illumination of the mechanism of CRC occurrence and metastasis and finding tumor markers for early diagnosis and anticancer medical targets are the key points for increasing the survival rate and cure rate for CRC patients, meanwhile, are the urgent missions for cancer study in present age.In previous studies, we prepared Affymetrix mRNA microarray to screen CRC-associated genes in human CRC tissues and normal colon tissues and obtained 1950 candidate genes closely related to occurrence and development of CRC contain 1180 upexpressed genes and 770 downexpresed genes.12 genes of which were selected as important genes of colorectal cancer by literature mining such as MMP28, PINK1, CEACAM1 and CK2a. Significately, we found an interesting gene, Protein kinase CK2a, one of the 12 candidate CRC genes, which was validated in the protein level by Western Blot.Protein kinase CK2 has traditionally been classified as a ubiquitous and highly conserved messenger-independent protein serine/threonine kinase that is typically found in tetrameric complexes consisting of two catalytic (αand/orα) subunits and two regulatoryβsubunits. Human CK2a subunit can use both ATP and GTP as phosphorylating agents. The construction features and highly conservatism of CK2a subunit powerfully illustrate that CK2αgene may has an important physiological function. Present study proved that CK2αparticipates in the regulation of cell proliferation, differentiation, metabolism, motion, oncogene and antioncogene.In 1995 Seldin et al. studied the function of CK2 in lymphomagenesis and firstly proved the important function of CK2 in this cancer and infered that CK2αis a pro-oncogene. After then, it is proved that CK2a can promote occurrence and metastasis in many tumors, such as Lung cancer, prostate cancer and breast cancer. CK2αfacilitates p53 deficiency and misexpression to the development of thymic lymphomas in mice. CK2 activity has been linked to Her-2/neu oncogene. Increased CK2αactivity is a key factor in the promotion of the Her-2/neu survival pathways leading to elevated growth rates and the development of tumorigenesis. Upexpressed CK2 subunit resulted in increased IKK-i/IKKεmRNA and protein expression. It is inferred that CK2αactivated the NF-кB signal pathway by induced activity of IKK-i/IKKεand NF-кB. All the results manifested that CK2a is a pro-oncogene of many tumors-which participates in the occurrence and metastasis of cancer via regulation of many cell signaling pathways. So far, the study of CK2a in CRC development is still unreported and the function and regulation mechanism in CRC occurrence and development still remain to study. ObjectivesInvestigate the function of CK2αin CRC occurrence and development to identify the relationship between CK2αand CRC occurrence and development. Illuminate the regulation mechanism of CK2a in CRC occurrence and development to provide a new theory of CRC occurrence and development.MethodsWestern Blot and Immunohistochemistry were used to examine the expressions of CK2αin colorectal carcinoma cell lines, colorectal cancer tissues, colorectal adenomas and the normal colon tissues. And then we investigated the association between CK2αexpression and clinicopathological characteristics of CRC cases to initially definited the correlation between CK2αexpression and CRC occurrence and development. After that, RNAi (RNA interfere) was performed to explore CK2αfunctions in proliferation, cell cycle, senescence, motion and invasion of colorectal carcinoma cells. At last, we used immunofluorescence staining and western blot to explore the regulation mechanism of CK2αin CRC occurrence and development.ResultsPARTⅠ:The expressions of CK2αin colorectal adenomas, CRC tissues and cell lines and the association between CK2αexpression and clinicopathological characteristics of CRC casesWestern Blot and immunohistochemistry were used to examine the expression of CK2αin colorectal carcinoma cell lines and colorectal cancer tissues, colorectal adenomas and the normal colon tissues. CK2αprotein expression was analyzed in 104 cases with CRC,40 with colorectal adenomas and 86 with normal colon tissues. The results of immunohistochemistry showed that nuclear staining for CK2αwas extremely weak in only 11 samples of normal colon epithelium (11 of 86,12.8%), positive in 17 of 40 (42.5%) samples of colorectal adenomas, and positive in 61 of 104 (58.7%) samples of CRC. The results of Chi-square criterion showed that CK2a immunoexpression was much stronger in CRC than that in adenoma, while its expression was greater in adenoma than in normal colon epithelium, and the difference was significant (x2=42.035, P=0.000). Western Blot was used to examine the expression of CK2a in 5 colorectal carcinoma cell lines and 8 normal-CRC tissue pairs. The results of Western Blot showed that CK2a expression was markedly higher in colon tumor tissues than normal colon tissues. In addition, CK2a was expressed in all five CRC cell lines.We investigated the association between CK2a expression and clinicopathological characteristics of CRC cases and found that CK2a overexpression was significantly associated with T classification (x2=9.534, P=0.002). Expression of CK2a protein in CRC of T3-4 stage (72.7%) was significantly higher than those in CRC of T1-2 stage (57.1%). However, no significant correlation was found between CK2a expression and gender, age, degree of differentiation, N classification, distant metastasis, or location (P>0.05).In this part of study, we found CK2a was overexpressed in CRC tissues and cell lines, while it was low or negative expressed in normal colon tissues. Immunohistochemistry results showed that CK2a expression was much stronger in CRC than that in adenoma, while its expression was greater in adenoma than that in normal colon epithelium. CK2a overexpression is correlated with T classification in colorectal cancer. Accordingly, it is infered that CK2a gene is an important promoter of CRC occurrence and development which may has an important biological function.PARTⅡ:RNAi of CK2a in colorectal cancer cells, investigating the influence of CK2αexpression in cell biology The specific RNA interference of CK2a was produced and transfected into CRC cell lines. The changes of biological behaviors in CRC cell lines were investigated by several in vitro assays. Western Blot was used to detect the transfection efficiency after transfected the CK2a siRNA.70% of the transfection efficiency was detected by Western Blot. The MTT assay showed that knockdown of CK2a significantly decreased CRC cell proliferation compared with control (nonspecific siRNA) (F=208.051, P<0.001). The colony formation assay showed that inhibition of CK2a expression significantly decreased the number of CRC colonies in LoVo cell (t=20.252, P=0.001). Knockdown of CK2a significantly promoted CRC cell senescence (t=-13.890, P=0.001) and the percent of G0/G1 phase cells increased (t =-9.577, P=0.003) and the percent of S phase cells decreased (t=8.749, P=0.004), indicating that CK2a knockdown induced G0/G1 phase arrest. The migration and matrigel transwell invasion assays were performed to examine the effect of CK2a on tumor cell migration and invasion, respectively. Knockdown of CK2a greatly inhibited wound closure (F=160.886, P<0.001) and invasion (t=5.955, P=0.025).All the results showed that CK2a knockdown significantly inhibited cell proliferation and migration and invasion ability, induced G0/G1 phase arrest, promoted cell senescence which infered that CK2a is closely for proliferation and invasion of CRC and it is a promoter of CRC cell proliferation and invasion.PART III:The mechanism of CK2a in CRC occurrence and development by immunofluorescence staining and western blotImmunofluorescence staining was used to detect the location and expressions of EMT related genes such as E-cadherin, P-catenin and vimentin after knockdown of CK2a. The results showed that knockdown of CK2a reversed cytoplasmic-to-nuclear transfer of P-catenin, an epithelial marker, resulted by EGF stimuli and increased the E-cadherin expression, another epithelial marker, decreased the vimentin expression, a mesenchymal marker.Western blot was used to detect the protein expressions of E-cadherin,β-catenin, vimentin and the proliferation related genes such as p53, p21, C-myc. Cells transfected with CK2a siRNA dramatically increased the level of E-cadherin, had no effect on the expression level of (3-catenin, and decreased the level of vimentin. The results of Western blot were well consistent with the results of immunofluorescence staining. Moreover, CK2a knockdown increased endogenous p53 and p21 expressions and decreased endogenous C-myc expression.All the results showed CK2a regulates the EMT-related genes expressions such as E-cadherin,β-catenin, vimentin and proliferation-related genes such as p53, p21, C-myc, promotes epithelial-mesenchymal transition, modulates the biologic behavior of CRC and promotes CRC occurrence and development.ConclusionsCK2a is a promoter of CRC occurrence and development and has the important biological function. CK2a may be an effective target of molecule targeted therapy. |
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