The Expression Of ALDH1A2in Colonic Cancer And Role Of ALDH1A2in Biological Function Of Colonic Cancer Cell

Colonic cancer is a common and serious digestive tract malignant that threat to the health. The main treatment for colon cancer is surgery combined with systemic or local chemotherapy and radio therapy, but the tumor is easy to recur. The key to reduce mortality and improve the survival rate of colon cancer is early detection, early diagnosis and early treatment. Therefore, in-depth study of gene expression in colorectal is useful to a comprehensive prevention and treatment of colorectal cancer.Aldehyde dehydrogenase family contains17genes translated into different isozymes catalyze different substrates, respectively, and is widely distributed in many tissues, the research results show tumor occurrence and development is closely related to ALDH1.ALDH1A2is key enzyme catalytic retinaldehyde oxidized to retinoic acid, retinoic acid and retinoic acid receptors and retinoid X receptor ligand binding domain combined direct effect on the target genes,regulating the expression of different target genes, regulating normal and tumor cell growth and differentiation.Numerous studies show that ATRA can inhibit tumor cell growth, invasion, metastasis and adhesion. ATRA can decrease athymic mice tumor and agar colony formation ability, decrease gastric cancer cell lines MKN45H adhesion to the basement membrane glue decline, and significantly reduce synthesis of the procollagen Ⅰ, Ⅱ, Ⅲ fibronectin, laminin, Therefore ATRA plays an important role in the inhibition of tumor invasion and metastasis process.Matrix metalloproteinases (MMPs) are a class of proteolytic enzymes that degrade extracellular matrix and basement membrane, MMP2plays an important role in invasion and metastasis of tumor cells, in many physiological and pathological processes of tumor evolution. In many tumor tissues, MMP2is higher MMP2expression, and the expression of the degree of tumor invasiveness.E-cad is adhesion molecules that mediate epithelial cell adhesion, mainly homogeneity mediated cell adhesion, such as tumor cells and the adhesion between the tumor cells, so the reduction in E-cad expression contributes to tumor invasion and metastasis.The present study, The ALDH1A2, MMP2, and the E-cadherin mRNA and protein expression in human colon cancer and adjacent organizations were measured by immunohistochemistry, RT-PCR method and Western blotting. ALDH1A2cDNA sequence was obtained by gene amplification technology.pEGFP-N1-ALDH1A2expression vector is transfected into the human colon cell lines LOVO cells to obtain a stable carrying pEGFP-N1-ALDH1A2LOVO. ALDH1A2mRNA and protein was measured before and after transfection cells, the activity of ALDH1A2is technical determination before and after transfection cells by the use of ultraviolet spectrophotometry. using the MTT assay, colony formation assay, transwell invasion assay, immunofluorescence assay, RT-PCR method, and immune blotting method,we detect proliferation, colony formation ability,the invasive ability and related factors expression in pEGFP-N1-ALDH1A2vector-transfected cell, at the same time, ATRA is used as a positive control. Finally, to explore the ALDH1A2over expression in vivo colon cancer and study over-expression in colon cancer mechanism for further research ALDH1A2,we build xenografts in athymic mice and observed tumor formation time, the average volume of the tumor, the average tumor weight. This research was divided into four parts:Part Ⅰ Expression of Aldehyde Dehydrogenases1A2in Colonic CancerMethods:1. ALDH1A2、E-cadherin and MMP2expression in52Colonic Cancer tissues and para-cacinoma(PC)tissues was detected by immunohistochemistry2. ALDH1A2、E-cadherin and MMP2expression in20Colonic Cancer tissues and para-cacinoma(PC)tissues was detected by RT-PCR3. ALDH1A2、E-cadherin and MMP2expression in52Colonic Cancer tissues and para-cacinoma(PC)tissues was detected by Western blotting.Results:1. Immunohistochemistry、RT-PCR and Western blotting assay showed the expression of ALDH1A2and E-cadherin in Colonic Cancer tissues was higher than para-carcinoma tissues (P<0.05), MMP2in Colonic Cancer tissues was higher than para-carcinoma tissues.2. The expression level of ALDH1A2were correlated with MMP2and E-cadherin in Colonic Cancer tissuesPart Ⅱ Construction and expression of pEGFP-N1-ALDH1A2vectorMethods:1. Obtained ALDH1A1cDNAby PCR, Constructed pEGFP-N1-ALDH1A2.2. Using Lipofectamine2000transfected LOVO lines with pEGFP-N1-ALDH1A2, which was selected under G418.3. The expression of ALDH1A2mRNA and protein was detected by RT-PCR and Wb.4. The activity of ALDH1A2was detected with ultraviolet-visible light detector.Results: 1. The restriction analysis showed that the ALDH1A2genes were correctly inserted into pEGFP-N1.2. The vatcor pEGFP-N1-ALDH1A2was successfully transfected into LOVO, which was detected under G418selection.3. The expression of ALDH1A2mRNA and protein in LOVO transfected with pEGFP-N1-ALDH1A2was higher than that in LOVO.4. the assay of ALDH1A2activity showed the ALDH1A2activity of LOVO transfected with pEGFP-N1-ALDH1A2was significantly decreased.Part Ⅲ The impact on the biological function of Colonic Cancer with overexpresion of ALDH1A2Methods:1. The cell cultures were measured for cell proliferation levels after transfection using MTT assay.2. colony forming unit assay was used to evaluate the colony forming of Colonic Cancer cell.3. Transwell test was used to evaluate the invasion of Colonic Cancer cell lines after transfection4.RT-PCR test was used to evaluate the content of ALDH1A2, MMP2and E-cadher in Colonic Cancer cell lines after transfection5. IF test was used to evaluate the content of ALDH1A2, MMP2and E-cadher in Colonic Cancer cell lines after transfection6. Wb test was used to evaluate the content of ALDH1A2, MMP2and E-cadher in Colonic Cancer cell lines after transfectionResults:1. MTT assay indicated that the proliferation level of LOVO cells were obviously decreased after transfection with pEGFP-N1-ALDH1A2as compared with control groups.2. The colony forming assay indicated that the colony forming unit of LOVO cells were obviously decreased after transfection with pEGFP-N1-ALDH1A2as compared with control groups. 3. In the transwell test, LOVO cells transfected with pEGFP-N1-ALDH1A2obviously reduced invasion.4.RT-PCR, IF and wb test indicated that ALDH1A2、E-cadherin mRNA and protein were obviously increased after transfection with pEGFP-N1-ALDH1A2as compared with control groups. MMP2mRNA and protein were obviously rised.Part Ⅳ Influence on athymic mouse tumor transplanted after transfection with pEGFP-N1-ALDH1A2Methods:1. MTT assay indicated that the proliferation level of LOVO cells were obviously decreased after transfection with pEGFP-N1-ALDH1A2as compared with control groups.2. ALDH1A2、E-cadherin and MMP2expression in tumor tissues was detected by immunohistochemistryResults:1. Models of athymic mice pEGFP-N1-ALDH1A2-LOVO transplantation tumor were created sueeessfully. Tumorigenic time was extended in ALDH1A2group athymic mice as compared with control groups, and the average volume and weight were smaller.2. ALDH1A2、E-cadherin protein were obviously increased in ALDH1A2group as compared with control groups (P<0.05). MMP2protein were obviously lower (P<0.05)Conclusions:1. expression of ALDH1A2and E-cadherin in Colonic Cancer tissues was higher than para-carcinoma tissues, MMP2in Colonic Cancer tissues was lower than para-carcinoma tissues.ALDH1A2were correlated with MMP2and E-cadherin in Colonic Cancer tissues.2. The vector pEGFP-N1-ALDH1A2was successfully transfected into LOVO.3. over-expression of ALDH1A2decreased ability of proliferation, the colony forming and invasion. This inhibition may be related with over-expression of ALDH1A2and E-cadherin, low-expression of MMP24. Models of athymic mice pEGFP-N1-ALDH1A2-LOVO transplantation tumor were created successfully. ALDH1A2、E-cadherin protein were obviously increased in ALDH1A2group as compared with control groups. MMP2protein were obviously rised.5. ALDH1A2inhibit colon cancer cell growth, proliferation, invasion and adhesion by adjusting the expression of MMP2and E-cadherin,...