Expression And Functional Study Of SLP-2 Gene In Colorectal Cancer

BackgroundColorectal cancer(CRC)is a common malignant tumor in the digestive tract,the incidence and mortality rates of CRC are the third and fourth in the global malignant tumors,respectively;according to the location of the disease,it can be divided into two categories:colon cancer and rectal cancer,but because they have more common pathophysiological features,it is commonly referred to as colorectal cancer(CRC).Since entering into the twenty-first century,the incidence and mortality of CRC have shown a significant upward trend,and there is a trend of getting younger.The clinical features of the young CRC are the relatively silent symptom,lack of specificity,high degree of malignancy,and earlier infiltration and metastasis.Therefore,Colorectal cancer has already had a serious impact on the life and health of our residents,and its prevention and control has been a major social task that we must face.In recent years,especially with the development of colonoscopy,the early diagnosis and treatment of CRC has made some progress,but,the survival rate of patients with advanced CRC has not been substantially improved,the main cause of death is liver and lung metastasis.Therefore,it is of great practical significance to study the pathogenesis and transfer mechanism of colorectal cancer.Since Vogelstein proposed the classic theory is the model of multiple steps and multiple genes,the internal and external factors in the development of CRC are studied by lots of scholars and experts in the world.The study of internal factors is mainly focused on genetic factors,such as the inheritance of susceptibility genes in CRC.According to statistics,the incidence of genetic related colorectal cancer accounts for about 20%of CRC,and the HNPCC and hereditary polyposis coli are the two major categories;external factors are mainly dietary factors such as high fat,high protein,low cellulose diet and the intake of nitrite salts.However,especially gene sequencing technology,tumor research has entered the "genomics era" from"pathological era",the view that play a key role in the development of tumor has been widely accepted by experts and scholars at home and abroad.Proto-oncogenes are genes that are inherent in human or other animal cells,they are the necessary genes to maintain normal life activities,the protein products encoded by them are almost present in various organelles in the body;Tumor suppressor gene,also known as anticancer gene,exists in normal cells and inhibits cell proliferation under activated conditions.Both of them participate in the normal functions of cell growth,differentiation,proliferation and signal transduction,and co-regulate the dynamic balance between cell proliferation and apoptosis.But under certain conditions,over expression of oncogenes or inactivation of tumor suppressor gene,can result in abnormal cell signaling pathways and some changes of cell differentiation?proliferation and invasion,this finally promotes the occurrence and development of the tumor.Therefore,it is important to strengthen the study of molecular etiology of CRC and reveal the mechanism of CRC from the molecular level.SLP-2(stomatin-like protein 2)is the second member of the Stomatin gene superfamily,and is first discovered by the pathology department of Yale university in 2000.SLP-2 gene presents on chromosome 9pl3.1,consisting of 10 exons and 9 introns,It encodes the mRNA of about 1500bp,and directs the synthesis of a polypeptide chain,including 356 amino acid sequences.Studies have shown that SLP-2 protein is a membrane protein,which is an important part of the cytoskeleton,and may be involved in the regulation of cell membrane ion channels.In addition,SLP-2 protein is also a mitochondrial related protein,which exists in the mitochondria of cells.it can regulate the stability of mitochondrial membrane associated proteins and affect the synthesis of ATP.The study found that SLP-2 proteins are expressed in many tissues of the normal human body,especially,there is a high expression in the heart,liver and pancreas.In recent years,reports about the occurrence and development of SLP-2 in malignant tumors have been increasing and it is speculated that it may be a new cancer gene.For example,Zhang Liyong from the Chinese Academy of Medical Sciences applied cDNA A microarray technique to compare esophageal squamous cell carcinoma with the normal epithelium in 54 patients;the results showed that the expression of SLP-2 protein in squamous cell carcinoma of esophagus was increased by more than 6 times;In vitro study,it was found that after the slp-2 silenced,the cell cycle S phase was blocked,the cell growth was inhibited,the cell adhesion and the mitochondrial membrane potential was decreased,suggesting that SLP-2 played an important role in the process of tumorigenesis.Similarly,in breast cancer studies,it was found that the high expression of SLP-2 was significantly associated with axillary lymph node metastasis,systemic distantmetastasis and breast cancer staging;and that the SLP-2 protein was significantly related to the expression of HER2/neu protein,the high expression of SLP-2 was negatively associated with overall survival,especially,in the patients with positive lymph node and HER/neu negative.So the SLP-2 protein could be used as an independent index to evaluate the survival of breast cancer patients.However,in the study of cervical cancer patients,SLP-2 was found to be low expression in cancer tissues,the expression was not related to the clinical stage,lymph node metastasis,depth of tumor invasion and tumor size,but only to the degree of tissue differentiation.Number of studies have showed that SLP-2 is associated with the development of tumor gene,while little is known about the effect of SLP-2 on colorectal cancer,it is not clear that SLP-2 plays a cancer suppression or promotion role in the development of CRC,so it is necessary to carry out further study.We studied the function of SLP-2 in CRC by the following 3 parts:First,the expression levels of SLP-2mRNA and protein in CRC and adjacent normal tissues of 30 cases were detected by real-time quantitative PCR and Western blot;With the help of this immunohistochemistry chemical method,the expression of SLP-2 in 50 cases of CRC patients was detected,then,the clinical data of patients with CRC was analyzed to evaluate the correlation between SLP-2 expression and clinicopathological features,and the effect of SLP-2 expression on the prognosis of CRC patients was also assessed.Second,we examined the SLP-2 expression levels of five common CRC cells by qRT-PCR and Western Blot and chose the cells in which SLP-2 expressed at higher levels for knockdown assay,cells in which SLP-2 expressed at relatively low levels were selected for overexpression experiment.We then performed MTT assay to test the influence of SLP-2 on CRC cell proliferation.Transwell migration assay were used to examine the function of SLP-2 on cell migration;Matrigel assay were used to examine the invasive ability of the cells,to further investigate the biological function of SLP-2 in colorectal cancer.Third,the expression levels of EMT proteins in 50 cases of colorectal cancer tissues were detected by immunohistochemistry;the expressions of EMT related cell markerswere detected by qRT-PCR and Western blot in colorectal cancer cells,which was after overexpression or knockdown of SLP-2;in order to further illuminate the mechanism of SLP-2 in promoting the growth and progression of CRC.Finally,we used the CRC cell line which had been stably transfected with the high expression of SLP-2 to establish a nude mice xenograft and distant metastasis tumor model,through observing the growth and metastasis of tumor animals,to further verify the results of in vitro cell experiments.Part IThe expression and clinical significance of the SLP-2 in colorectal cancerObjectiveTo investigate the expression patterns of SLP-2 in colorectal cancer and its adjacent non-tumorous tissues,and to analyze the SLP-2 and the patients'clinicopathological characteristics,as well as its role in predicting prognosis.Methods30 specimens from January to June in 2015 were collected from dezhou people's Hospital,and all these patients were diagnosed and received radical surgery.After the radical operations,colon cancer tissues and matched adjacent normal tissues were got in time.The mRNA and protein expression levels of SLP-2 were detected through qRT-PCR and Western Blot.In addition,another 50 cases from 2010 to 2011 were randomly selected from dezhou people's Hospital,and all these patients were diagnosed and received radical surgery.The expression of SLP-2 were detected by immunohistochemical analysis.Survival curves are usually plotted using the Kaplan-Meier method and survival rate was analyzed by log-rank test.Multivariate analysis of the prognostic factors was performed with Cox regression model.Results:1.The mRNA level of SLP-2 in tumor tissues is consistent with qRT-PCR results.2.Detection of the expression of SLP-2 proteins in tissues of patients by immunohistochemistry.The expression level of SLP-2 in colorectal cancer tissues is significantly promoted compared with that in cancer-adjacent normal tissues of 50 CRC cases.3.In addition,high SLP-2 expression was correlated with tissue differentiation,lymph node metastasis and TNM stage of CRC patients(P<0.05).Kaplan-Meier analysis showed that CRC patients with high SLP-2 expression had a shorter overall survival(OS)than low SLP-2 expression group(P<0.05).Moreover,multivariate Cox model analysis showed that SLP-2 expression was an independent risk factor for poor OS in CRC patients.ConclusionsThe expression level of SLP-2 and prognosis of patients with colorectal cancer.It is speculated that it plays an important role in the development and progression of CRC,and has clinical value as a potential target for early diagnosis and treatment of colorectal cancer.Part ?Effect of SLP-2 expression on biological behaviours of colorectal cancer cellsObjectiveIn this study,the expression level of SLP-2 in the most common colorectal cancer cell lines with different malignant degrees was detected,then the experimental cell lines were selected;next,the expression of SLP-2 in colorectal cancer cell line was regulated in vitro and RNA interference sequence,in order to determine its effect on cell proliferation and migration in colorectal cancer.Methods1.The expressions of SLP-2 in CRC cell lines(SW620?SW480?LoVo?HCT116?HT29)and normal colonic epithelial cell line FHC were examined by qRT-PCR and Western Blot,in order to select high and low SLP-2 expression cell lines for further research.2.The high expression of pcDNA3.1-SLP-2 plasmid was constructed and SLP-2-siRNA was purchased,Then we transfected the pcDNA-SLP-2 to CRC cell SW480,which had low endogenous expression of SLP-2 and transfected SLP-2-siRNA to the CRC cell HCT116,which had high endogenous expression of SLP-2.After the transfection of 48h,the efficiency was detected by qRT-PCR.3.With the stable transfected cell lines,cell proliferation was detected by MTT assayand cell cloning assay,transwell chamber and Matrigel invasion assay were used to measure the effects of SLP-2 on cell migration and invasion in CRC cells respectively.Results1.SLP-2 mRNA and protein levels were much higher in CRC cells than those in FHC;while SLP-2 expression levels are different in various CRC cell lines,indicating that SLP-2 is closely related to the invasive property of CRC cells preliminarily?2.The SLP-2 stable overexpression and the silencing cell lines were successfully established;the qRT-PCR results showed that the expression of SLP-2 was downregulated in siRNA-group and was increased in pcDNA-group,compared with control-group.3.MTT assay and cell cloning assay demonstrated that knockdown of the SLP-2 decreased the cell proliferation;in contrast,overexpression of SLP-2 increased the cell proliferation;Transwell chamber experiment and Matrigel invasion assay suggested that downregulation of SLP-2 inhibited the cell migration and invasion ability,whereas,over expression of SLP-2 promoted the migration and invasion ability of CRC cells significantly.ConclusionsThe expression of SLP-2 is different in colorectal cancer cell lines with different malignant degree,which can significantly promote the proliferation,migration and invasion activities in colorectal cancer cells?Part IIIResearch on molecular mechanisms of SLP-2 inducing EMT,invasion and metastasis in CRCObjectivesTo investigate the relationship between SLP-2 and EMT in colorectal cancer and its signal pathways.Methods1.The high expression and low expression cells of transfected SLP-2 were observed under light microscope,in order to determine whether there was a change in the morphology of EMT cells or not.2.The expression of EMT epithelial marker E-cadherin and the interstitial cell marker vimentin in colorectal cancer tissues and adjacent tissues of 50 cases were detected by imnunohistochemistry,then the correlation between the two marker protein and the SLP-2 gene was preliminarily analyzed.3.EMT epithelial markers(E-cadherin,CK),interstitial cell markers(vimentin and fibronectin),transcription factor markers(snail,slug)and downstream target genes of the Wnt signaling pathway(p-catenin,c-myc,cyclinDl)were detected in colorectal cancer cells after overexpression or knockdown of SLP-2,in order to investigate the mechanisms and signaling pathways of SLP-2 in promoting proliferation and invasion of colorectal cancer.4.To study whether the up-regulated SLP-2 can promote the invasion and metastasis ability of colorectal cancer cells,we established xenograft models and distant metastasis model of nude mouse with the application of the constructed over-expressing and control cell lines,the proliferation of the transplanted tumor and the expression of SLP-2,E-cadherin and Vimentin in the metastatic tumor were detected through the qRT-PCR and immunohistochemistry,to further verify the function of SLP-2 which had been confirmed in vitro experiments.Results:1.Under the light microscope,it was observed that the cell morphology of the SLP-2 high expression changed from round to spindle-shaped mesenchymal cell,which was a typical EMT change trend.2.The results of immunohistochemistry showed that in the 50 cases of CRC patients,Correlation analysis showed that SLP-2 was positively related to the expression of vimentin,but negatively correlated with the expression of E-cadherin,it was suggested that the expression of SLP-2 in colorectal cancer was related to EMT phenomenon and the metastasis of colorectal cancer cells might be promoted through EMT.3.Real time quantitative RT-PCR and westem-blot test results showed that in the SLP-2 overexpressing cell line SW480,mesenchymal cell markers(vimentin,fibronectin),while the epithelial cell marker(E-cadherin,CK)was significantly reduced;at the same time,the expressions of 3 target genes(?-catenin,c-myc,cyclinDl)of Wnt signaling pathway were also up-regulated;however,in the SLP-2 interferenced cell line HCT116,the detection results were on the contrary.This further suggested that SLP-2 can promote invasion and metastasis by inducing the EMT of colorectal cancer,and EMT may also be promoted by activating Wnt signaling pathway.4.The xenograft tumor and distant metastasis tumor model of nude mice were successfully established;In the xenograft tumor model,tumors of the SLP-2 overexpressing cell group were found to be much higher in weight and growth rate than the control group,and the expression of tumor proliferation related marker Ki67 in tumor tissue was also significantly higher than that in the control group;in the metastatic tumor model:the expression of SLP-2 in the tumor tissues of the overexpressing cells was significantly higher than that in the control group,the expression of EMT epithelial marker E-cadherin in tumor tissues was significantly decreased,while the expression of mesenchymal marker vimentin was obviously up-regulated.Conclusions1.SLP-2 is associated with colorectal cancer EMT;2.SLP-2 may promote the colorectal cancer EMT by Wnt pathway.