The Change And Function Of Related Molecules: Occludin, PHF14and KIF4A In Colorectal Cancer

Objective: Colorectal cancer is one of the most common malignant tumors so far. Thebetter early intervention has become a direct factor of reducing the incidence of colorectalcancer and effectively reduced the mortality of colorectal cancer patients. To furtherinvestigate the molecular mechanism of carcinogenesis and development of colorectalcancer, we have done preliminary researches on the tight junction protein-Occludin,Homo Plant Homodomain (PHD) finger protein14-PHF14and kinesin superfamilyprotein KIF4A, Through proteomics, molecular biology and cell biology method, wehave analyzed the possible relation between these three molecules and colorectal cancer.Methods: Real-time PCR was used to analyze the different mRNA expression ofOccludin, PHF14and KIF4A in colorectal cancer samples, and statistical methods wereutilized to find the correlation between the different mRNA expression level and theclinical information; Besides Western Blot as well as two-dimensional electrophoresiswere performed to identify the different protein expression of Occludin in colorectal cancersamples. Since then, we also cultured colorectal cancer cell-lines for the observationwhether change of Occludin would affect their morphology.Results: Compared with para-cancer normal tissues, Occludin mRNA expression wasgenerally down-regulated significantly in colorectal cancer tissues, reaching about63%of normal tissues.And Occludin low expression was correlated with the existence ofbloody diarrhea (P=0.011*);Besides Occludin expression was also correlated with APCexpression (P <0.01**); on protein level, we verified Occludin low expression and foundthat a specific modification or isoform was generally lost in colorectal cancer tissues. Thatlost did not exist in the colorectal neoplastic polyps. However, direct evidence affectingcolorectal cancer cell lines was not observed through cellular experiments. In addition,Real-time PCR results of55colorectal cancer samples showed that PHF14mRNAexpression was normal and was not correlated with clinical factors.however,KIF4A washighly expressed in colorectal cancer tissues, It is about2.8times of that in normal tissues.and it is more significant(P=0.046*, P=0.044*). in female patients and in patients whosetumor diameter were less than or equal to5cmConclusion: Occludin has a potential function as tumor suppressor gene. Not only itsexpression was downregulated but also a lost of special isoform in colorectal cancer samples. It could not be determined whether Occlduin affect the colorectal cancer cell linesin their morphology or other biological functions. Different with occludin, PHF14mRNAexpression was not generally changed in colorectal cancer tissues; However, KIF4A wassignificantly upregulated in colorectal cancer tissues indicating its characteristics of apotential oncogene. Objective: Patients’ tissue samples are extremely valuable resources in clinical research.The amount of clinical tissue samples is very limited, commonly in a milligram (mg) level.The genetic information and pathophysiological changes of patients are implied in thevariation of DNA, RNA and protein. At present, there hasn’t been an mature technique tosimultaneously extract DNA, RNA and protein from micro-tissues due to differentextraction principles.Methods: To extract high-quality total RNA from micro-tissues and to extract genomicDNA and total protein at the same time, We modified the traditional TRIzol reagentmethod here. Mouse liver total DNA, RNA and protein were extracted by modified TRIzolreagent method, which was compared with RNA extracted by traditional TRIzol reagentmethod and genomic DNA extracted by commercial kit, besides protein was also comparedwith that of extraction by traditional RIPA lysis buffer.Results: the average yield of RNA extracted from mouse liver tissue by modified TRIzolreagent was3.64μg/mg, while the tradition method was1.95μg/mg. The average OD260／OD280value was2.1and2.01respectively. The modified TRIzol method can acquiremore intact RNA than traditional one, besides the ratio of28s/18s is more approximate to2:1which is seen from the picture of agarose gel. Although the yield of DNA extraction bymodified TRIzol reagent was obviously lower than that by commercial kit, which is0.49ug/mg,1.78ug/mg separately, purity is kept good. The average protein yield ofextraction using TRIzol reagent is50-60%of that of RIPA lysis method. After thevalidation of SDS-PAGE electrophoresis and Coomassie blue staining, no significantprotein loss was found.Conclusion: In conclusion, Total RNA,DNA as well as protein were able to be extractedby modified TRIzol reagent method simultaneously，which was more efficient andqualified in total RNA extraction than traditional TRIzol method; more convenient andeconomical in genomic DNA extraction than commercial kit, better in preserving smallprotein in total protein extraction than RIPA buffer lysis method. Our method minimized the usage of tissue sample to the best, and it is a rational choice when the quantity ofsample is relatively small.