| Background and purpose:Colorectal cancer is one of the most common malignant tumors of the digestive system, and the incidence and mortality are among the upper third in the Western countries. With the development of economy and the changes of lifestyle especially changes in diet, the incidence and the mortality rate of colorectal cancer has increased.The development of colorectal cancer is a complex process that multi-factors, multi-steps and multi-genes have involved in it,90% of the patients can be cured after surgery in the initial stages of the disease, however, clinically approximately 40% -50% of the patients with colorectal cancer have been foud micrometastases at the process of initial diagnosis, and the prognosis could be very poor.Local transfer of colorectal cancer occous much earlier than distant metastasis of colorectal cancer, approximately 60% -80% of the patients could be found local transfer 2 years after surgery, metastatic is an important cause of death in patients. Colorectal cancer has seriously effects on patien’s quality of life and physical health, but also brings heavy financial burdens to patients and society.Recently the research on the molecular mechanisms associated with colorectal cancer metastasis attracte people’s attention. At present,although we have a lot of research and investigate on it, but so far, the molecular basis and mechanisms of formation and metastasis of colorectal cancer is not fully understood.The occurrence of colorectal cancer is a complex process:variety of tumorigenic effect of factors, a variety of oncogenic factors acting on the body, intestinal epithelial cells at the genetic level to its loss of the normal regulation of growth, leading to cell cycle disorders, intestinal epithelial cells were immortalized, eventually leading to cancer. Metastasis of colorectal cancer is a complex process involved multiple steps, multiple stages and multiple genes, including cancer cells in the primary tumor site escape into the surrounding matrix, and thence into the circulatory system or lymphatic system, adhering tumor endothelial cell wall cells into extravascular migration, then the cells invasion in distant, tumor angiogenesis, eventually forming new metastases.The process is accompanied by a plurality of mutant genes, which comprises activating a number of oncogenes. There are already a lot of research on metastasis of colorectal cancer-related molecules, and hundreds of genes have been reported to participate in metastasis of colorectal cancer, and the number continues to increase.In view of this, in-depth study of the molecular mechanisms of colorectal cancer, and to seek to develop effective, sensitive and specific molecular markers of colorectal cancer in a large number of factors, and thus early warning of colorectal cancer, diagnosis, intervention, and outcomes judgment have important clinical value and social benefits.LIM kinase 1 (LIM Kinase 1, LIMK-1) gene is one of the upregulated genes identified by our pre-screened in microarray of colorectal cancer that occured lymphatic metastasis. LIMK-1 gene is located on human chromosome 7q 11.23, containing 39,499 bases, with 16 exons of the gene produces messenger mRNA, that may encode full-length LIMK-1 protein. LIMK-1 is recognized the role of phosphorylate of the first three serine (Ser3) of ADF/cofilin and make it inactivate, which in turn inhibits F-actin formation of G-acting, thus inhibits actin depolymerization caused by cofilin.LIMK-1 is involved in actin cytoskeleton reorganization, because the polymerization of actin can cause the formation of pseudopodia structure, thereby regulating the movement and migration of cells. Recent studies have found that LIMK-1 protein is not only plays an important role in the regulation of cell cytoskeleton reorganization, but also capable of mediating membrane structure in dynamic actin assembly and disassembly, such as the formation of lamellipodia and filopodia, so LIMK-1 is considered to be the basis of cell motility, cell migration and invasion. Recent studies have reported LIMK-1 is highly expressed in breast and prostate cancer and other cancers, and is closely related to tumorigenesis, cell invasion and tumour metastasis.Therefore, we believe LIMK-1 may also play an important role in colorectal cancer metastasis. However, there is no studies that have been reported about the role and mechanism of LIMK-1 in the development of colorectal cancer. The role and molecular mechanism of LIMK-1 in the development of colorectal cancer is poorly understood. Our paper intends to detect the expression of LIMK-1 in colorectal cancer, and to analyze the relationship between the expression and clinicopathological parameters; to understand the role and molecular mechanisms associated with LIMK-1 in the occurrence, development and metastasis of colorectal cancerMethods:1.Tissue microarray analysis found that LIMK-1 is a differentially expressed genes between colorectal cancer that occured lymphatic metastasis and did not occured lymphatic metastasis;2.Immunohistochemistry(IHC), immunoblotting(Western blot) and immunofluo-rescence were used to detect LIMK-1 expression in paraffin-embedded and fresh CRC samples;the relationship between LIMK-1 expression and clinical pathology parameters, including tumor stage, metastasis and clinical prognosis, was analysed;3. We introduce nuclear localization, nuclear export signal, and constructed vectors that express LIMK-1 in different subcellular localization, and synthesised SiRNA that can interference the expression of LIMK-1 by transient transfection of colorectal cancer cell lines.Cell proliferation and migration were measured by CCK8, Transwell,scratch experiments in vitro after LIMK-1 transient overexpression and interference;4. Establish colorectal cancer cell lines that overexpress LIMK-1 stablely in cytoplasm and nucleus subcellular localization. Cell proliferation and migration were measured by CCK8, Transwell assay in vitro; tumor cell proliferation and the ability to transfer were measured by subcutaneous tumor experiment and tail vein injection of nude mice in vivo;5. Western blot was used to detect the activation of signaling pathways and the expression of EMT markers in CRC cells with LIMK-1 overexpression;6. Agroase with HA antibody was used to screen protein that interact with LIMK-1, mass spectrometry screening was used to identify the protein,and co-immunoprecipitation was used to verify the candidate proteins that interact with LIMK-1.Result:1. The expression of LIMK-1 in CRC tissue and CRC cell lines1) Tissue microarray analysis found that LIMK-1 is a differentially expressed gene between colorectal cancer that occured lymphatic metastasis and did not occured lymphatic metastasisWe use eight samples of colorectal cancer that occured lymphatic metastasis and did not occured lymphatic metastasis, eight gene chip were made. A careful analysis of the results from the chip showed that LIMK-1 is a 2-fold differential gene between the colorectal cancer that occured lymphatic metastasis and did not occured lymphatic metastasis (p<0.05). So far, LIMK-1 has not yet been reported in colorectal cancer.2)The expression of LIMK-1 in colorectal cancer tissuesIHC experiments shows that LIMK-1 was mainly localized in the cytoplasm and nucleus of normal colorectal tissue. In normal colorectal tissues the level of LIMK-1 protein is less than the paired tumor tissue, the overexpression rate of LIMK-1 protein expressed in cytoplasm in colorectal cancer tissue is 17.7%(27/152) wich is higher than the overexpression rate of LIMK-1 protein expressed in cytoplasm in normal colorectal tissue 0%(0/78) (p= 0.000); the overexpression rate of LIMK-1 protein expressed in nucleus in colorectal cancer tissue is 57.9%(88/152) wich is higher than the overexpression rate of LIMK-1 protein expressed in cytoplasm in normal colorectal tissue 17.9%(14/78) (p= 0.000); Western blot test results also show that in normal colorectal tissues the level of LIMK-1 protein is less than the paired tumor tissue; immunofluorescence showed a low expression levels of LIMK-1 in normal tissues, LIMK-1 is mainly expressed in Cytoplasm; the expression of LIMK-1 in cancer tissues is high, mainly expressed in the cytoplasm and nucleus.3) The expression of LIMK-1 in colorectal cancer cell linesWestern blot results are displayed in colorectal cancer cell lines,LIMK-1 protein is highly expressed in LOVO, SW620 which are cell lines with lymph node metastasis and subline M5 which is cell lines with liver metastasis, and the expression is relatively lower in HCT116, LS174T, SW480 and HT29 wich are cell lines with poor metastatic potential.Immunofluorescence results show that HCT116, LS174T, SW480 and HT29 cell lines wich have poor metastatic potential express relatively low level of LIMK-1, and mainly located in the cytoplasm, but the expression of LIMK-1 is relatively higher, in LOVO, SW620 cell lines which occurred lymph node metastasis mainly located in the cytoplasm and nucleus.4)The relationship between the expression of LIMK-1 and the clinicopathological parametersThere were no significant differences of LIMK-1 expression in different age groups, different genders and different differentiation (P>0.05); but there were significant differences of LIMK-1 expression in different TNM stage.in which although there were no significant differences of LIMK-1 expression in the different depth of invasion (T)(P> 0.05),but with the number of lymph node metastasis (N) increases, the expression of LIMK-1 increases. The overexpression rate of LIMK-1 in cytoplasm of lymph node metastasis (N1+N2) stage was higher than those without lymph node metastasis(N0)(P=0.007), The overexpression rate of LIMK-1 in nucleu of lymph node metastasis (N1+N2) stage was higher than those without lymph node metastasis(N0)(P=0.000).And with distant metastases(M) increases, the overexpression rate of LIMK-1 in cytoplasm of distant metastasis (Ml) group was higher than those without without distant metastasis (MO) group(p= 0.029), the overexpression rate of LIMK-1 in nucleu of distant metastasis (M1) group was higher than those without without distant metastasis (MO) group(P<0.05);The overexpression rate of LIMK-1 in cytoplasm of the recurrence group was higher than those without recurrence (P= 0.049), The overexpression rate of LIMK-1 in nucleu of the recurrence group was higher than those without recurrence (P= 0.000);5) correlation analysis of LIMK-1 expression levels and clinical outcomes in patientsKaplan-Meier Survival analysis revealed that 5-year overall survival rate of CRC patients with high LIMK-1 expression in cytoplasm and nucleus was significantly lower than that with low LIMK-1 expression.(Log-Rank, P<0.05). 5-year overall survival rate of CRC patients in T3+T4 and NO stage with high LIMK-1 expression in cytoplasm and nucleus was significantly lower than that with low LIMK-1 expression.(Log-Rank, P<0.05)6) single factor affecting the prognosis of patients with CRC multivariate analysisUnivariate analysis showed cytoplasmic and nuclear LIMK-1 overexpression have relationship with colorectal cancer prognosis, However, with the concerning of present study results, LIMK-1 overexpression in the cytoplasm and the nucleus can not be used as a predictor of patient independent indicator of poor prognosis (p> 0.05).2.The impact of different subcellular localization of LIMK-1 on the biological characteristics of colorectal cancer1) The effects of LIMK-1 transient overexpression on proliferation and invasion in colorectal cancer cell① The verify of LIMK-1 transient overexpressionWe introduced the localization, nuclear export signal synthesis constructed LIMK-1 cytoplasm and nucleus subcellular localization expression vector (HA-NLS-LIMK1, HA-NES-LIMK-1, HA-LIMK1) and vector control group (HA), Western blot detection was used after transiently transfected with those vectors in SW480 cell, the results show that cells transfected with HA-LIMK-1, HA-NES-LIMK-1, HA-NLS-LIMK-1 can express HA-LIMK1 fusion protein.② The effects of LIMK-1 transient overexpression on proliferation in colorectal cancer cellCCK8 shows that,, the growth of SW480 and HCT116 cells with LIMK-1 overexpression (HA-LIMK1, HA-NLS-LIMK1, HA-NES-LIMK1) increased markedly compared with the control group (HA) (SW480, F=33.025, p <0.001;HCT116,F=55.276, p<0.001). The results show that transient overexpression of LIMK-1 in vitro can promote the ability of proliferation of colorectal cancer cells.③ The effects of LIMK-1 transient overexpression on invasion in colorectal cancer cellTranswell assay shows that, the number of invaded cells of SW480 and HCT116 cells with LIMK-1 overexpression (HA-LIMK1, HA-NLS-LIMK1, HA-NES-LIMK1) increased markedly compared with the control group (HA) (P=0.000). Scratches experimental shows that the mobility of SW480 and HCT116 cells with LIMK-1 overexpression (HA-LIMK1, HA-NLS-LIMK1, HA-NES-LIMK 1) increased markedly compared with the control group (HA) (P=0.000).The results show that transient overexpression of LIMK-1 in vitro can promote the ability of invasion of colorectal cancer cells.2) The effects of LIMK-1 transient silencing on proliferation and invasion in colorectal cancer cell① The verify of LIMK-1 transient silencingThe negative control substance labeled with a fluorescent marker with fluorescence, it can be used for transfection efficiency evaluation, the results shows that:siRNA oligo transfection efficiency is up to 60% -70%, which is a high transfection efficiency that can be easily for us to achieve a better gene silencing effect.We use the siRNA-377, siRNA-391, siRNA-1782 fragment transfected SW620 cells, Western blot results showed that:the expression levels of LIMK-1 protein drop after transfect two interfering cell fragments (siRNA-377, siRNA-391), it means that the design and synthesis of siRNA-377, siRNA-391 can interfere significantly knock down of LIMK-1.② The effects of LIMK-1 transient silencing on proliferation in colorectal cancer cellCCK8 shows that,, the growth of SW620 cells with LIMK-1 knokdown (SiRNA-377, SiRNA-391) decreased markedly compared with the control group (NC) (F=22.827, p<0.001). The results show that transient silencing of LIMK-1 in vitro can suppress the ability of proliferation of colorectal cancer cells.③ The effects of LIMK-1 transient silencing on invasion in colorectal cancer cellTranswell assay shows that,the number of invaded cells of SW620 with LIMK-1 knokdown ((SiRNA-377,SiRNA-391)) decreased markedly compared with the control group (NC) (P=0.001). The results show that transient silencing of LIMK-1 in vitro can suppress the ability of invasion of colorectal cancer cells.3) Impact on cell proliferation and invasion of colorectal cancer after LIMK-1 stably overexpressing① The construction of LIMK-1 stable overexpression cell linesRecombinant expression vector(HA, HA-LIMK1, HA-NLS-LIMK1, HA-NES-LIMK1) was transfected into SW480 cells, G418 resistant monoclonal pressurized screening were picked, expanded the culture Western blot test results indicating the successful construction of LIMK-1 stably overexpressing cells and the cells were named as SW480/HA, SW480/HA-LIMK1, SW480/HA-NLS-LIMK1, SW480/HA-NES-LIMK1; immunofluorescence showed that SW480/HA-LIMK1, exogenous LIMK-1 localized in the cytoplasm and SW480/HA-NLS-LIMK1, exogenous LIMK1 localized in the nucleus;SW480/HA-NES-LIMK1,exogenous LIMK-1 localized in the cytoplasm, immunofluorescence results indicating the successfully construction of LIMK-1 stably overexpressing cells, NLS nuclear localization signal can make LIMK-1 localized in nucleus,NES nuclear export signal can make LIMK-1 localized in the cytoplasm.② Experiments of LIMK-1 stably overexpressing in vitroCCK8 shows that,, the growth of cell line with LIMK-1 stable overexpression (SW480/HA-LIMK1, SW480/HA-NLS-LIMK1, SW480/HA-NES-LIMK1) increased markedly compared with the control group (SW480/HA) (F= 47.904, P <0.001). The results show that cytoplasm and nucleus subcellular localization stable overexpression of LIMK-1 can promote the ability of proliferation of colorectal cancer cells in vitro;Transwell cell migration assay results showed that the number of invaded cells cell line with LIMK-1 stable overexpression (SW480/HA-LIMK1, SW480/HA-NLS-LIMK1, SW480/HA-NES-LIMK1) increased markedly compared with the control group (SW480/HA) was significantly increased compared to migrating cells (P=0.000), The results show that cytoplasm and nucleus subcellular localization stable overexpression of LIMK-1 can promote the ability of metastasis of colorectal cancer cells in vitro.③ Experiments of LIMK-1 stably overexpressing in vivoSubcutaneous tumor Experimental results show that cell lines with LIMK-1 stable overexpression (SW480/HA-LIMK1, SW480/HA-NLS-LIMK1, SW480/ HA-NES-LIMK1) and control group (SW480/HA), tumor growth increased, tumor weight increased (P=0.005), tumor volume increased (P=0.017). The positive rate of Ki67, SW480/HA-LIMK1 (80.97%), SW480/HA-NLS-LIMK1 (74.08%), SW480/HA-NES-LIMK1(80.61%) was significantly higher than SW480/HA (39.54%) (p= 0.000). Subcutaneous tumor experiments showed cytoplasm and nucleus subcellular localization stable overexpression of LIMK-1 can promote the ability of growth and proliferation of colorectal cancer cell in vivo. Mice intravenously injected experimental results show that pulmonary metastasis of tumor cells and the number of lung metastatic nodules of cell line with LIMK-1 stable overexpression (SW480/HA-LIMK1, SW480/HA-NLS-LIMK1, SW480 /HA-NES-LIMK1) increased markedly compared with the control group (SW480/HA) (p=0.009), intravenous injection experiments show that the cytoplasm and nucleus subcellular localization stable overexpression of LIMK-1 can promote the ability of metastasis of colorectal cancer cells in vivo.3.The molecular mechanisms of LIMK-1 regulating the proliferation and metastasis of colorectal cancer1) Impact of AKT signaling pathway protein p-PTEN, p-AKT (473), p-AKT (308) and AKT expression after LIMK-1 overexpressionWestern blotting was used to detect the expression of AKT signaling pathway protein p-PTEN, p-AKT (473), p-AKT (308) and AKT, after transiently transfected four plasmids (HA, HA-LIMK1, HA-NLS-LIMK1and HA-NES-LIMK1)in SW480, and HCT116 cells, Quantity One software was used to strip gradation analysis. The results show:in SW480 and HCT116 cells, the expression of p-PTEN is low in HA-LIMK1, HA-NLS-LIMK1, HA-NES-LIMK1 three treatment groups compared to the control group HA, and the expression of p-AKT (473), p-AKT (308) in HA-LIMK1, HA-NLS-LIMK1, HA-NES-LIMK1 three treated groups are higher than the control group(HA), AKT total protein was no significant difference among the four kinds of processing cells, suggesting that cytoplasmic and the nucleus LIMK-1 can regulate the activity of AKT signaling pathway.2) The impact of EMT-associated molecular marker expression after LIMK-1 overexpression on CRC cellWestern blotting was used to detect the expression of EMT-associated molecular marker after transiently transfected four plasmids (HA, HA-LIMK1, HA-NLS-LIMKland HA-NES-LIMK1)in SW480, and HCT116 cells, Quantity One software was used to strip gradation analysis. The results show:in SW480 and HCT116 cells, the expression of epithelial markers (E-cadherin,β-catenin) is low in HA-LIMK1, HA-NLS-LIMK1, HA-NES-LIMK1 three treatment groups compared to the control group HA, and the expression of mesenchymal markers (N-cadherin, Fibronectin) is higher in HA-LIMK1, HA-NLS-LIMK1, HA-NES-LIMK1 three treated groups than in the control group(HA), suggesting that cytoplasmic and the nucleus LIMK-1 can can induce EMT.3) Screening of LIMK-1 interacting proteinAgarose tagged HA antibody was use to capture HA-LIMK-1 fusion protein, Binding of total protein was isolated by SDS-PAGE electrophoresis. There are three experimental groups SW480/HA-NES-LIMK1, SW480/HA-NLS-LIMK1, SW480/HA-LIMK1, the control group was SW480/HA. After SDS-PAGE electrophoresis separation, mass spectrometry compatible silver staining, clearly visible protein can be seen.Preliminary screening identified as MYH9 (myosin-9, myosin heavy chain 9), ACTN4 (alpha-actinin4, cell auxiliary power protein), DBN1 (Drebrin).4) Direct binding assays of LIMK-1 and MYH9, ACTN4The use of Co-IP to detect the direct binding between LIMK-1 and MYH9、 ACTN4,the results showed:LIMK-1 and MYH9, ACTN4 have a direct binding.conclusion:Cytoplasm and nucleus LIMK-1 are significantly up-regulated in colorectal cancer.Cytoplasm and nucleus LIMK-1 promote cell proliferation and metastasis in colorectal cancer. The over-expression of cytoplasm and nucleus LIMK-1 have a positive correlation between the clinical and pathological parameters. |
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