| Background Colorectal cancer(CRC)including colon cancer and rectal cancer,is one of the most common human gastrointestinal malignant tumor.There are about 1.2 million new patients worldwide each year,and about 600 thousand people died of this disease.Chinese Academy of Medical Sciences/National Cancer Center published China cancer statistics of 2015 in the CA Cancer J Clin magazine showed that in China,incidence and mortality of colorectal cancer ranked the fifth among the whole malignant tumors,including 376 thousand new patients and 191 thousand death patients which became a serious threat to Chinese health.With the development of economy,the improvement of people's living standards and the change of diet,the increase of fat,meat in dietary and the reduce of fiber and excercise resulted the incidence and the mortality of colorectal cancer showing an increasing trend each year,while genetic changes also led to the individual susceptibility to colorectal cancer.The occurrence and development of tumor is a complex pathophysiological process,which involves the changes of multiple genetic.In general,changes of the genome replication,transcription,translation will affect the biological behavior.Therefore,the study of the molecular mechanism of colorectal cancer will be of great significance to the prevention and treatment of colorectal cancer.Our previous studies have found that the expression of SLC5A8 gene in colorectal cancer tissue down or missing suggests that the gene silencing is associated with the occurrence of colorectal cancer.However,the molecular mechanism of SLC5A8 gene silencing in colorectal cancer,the role of substrate,signal pathway,and the interaction with other proteins are not very well understood,and it is needed further research.The solute carrier family fifth family member 8(SLC5A8)was founded by Rodriguez as a new candidate tumor suppressor gene.It is located on human chromosome 12q13 and belong to the sodium / glucose co transporter protein family members.It is a short chain fatty acid(SCFA)as coupling Na + transporter of acetic acid,propionic acid and butyric acid,pyruvic acid and lactic acid.The domestic and foreign scholars have found that the expression of SLC5A8 in human esophageal cancer,cardia cancer,breast cancer,thyroid cancer,lung cancer,pancreatic cancer,glioma,prostate cancer and acute myeloid leukemia down or missing suggest that it may be involved in the occurrence,metastasis and invasion process.It was found that the decrease or loss of SLC5A8 expression in tumor tissues was due to the methylation of GPG islands and histone deacetylation.As one of the substrates of SLC5A8 gene transfer,butyric acid is produced by bacterial fermentation of dietary fiber.In addition to being the substrate of metabolic energy for ?-oxidation and citric acid cycle,butyric acid is also a histone deacetylase(HDAC)inhibitor,especially HDAC1 and HDAC3 isoforms.These endogenous HDAC inhibitors are present in the normal colonic lumen,which is mediated into colonic epithelial cells by SLC5A8,and inhibits HDACs.Because the expression of SLC5A8 gene is reduced or absent,it can not let the butyric acid enter the colonic lumen,which may be one of the causes of the occurrence of colorectal cancer.Objectives Effects of SLC5A8 gene transfection on proliferation and invasion of colorectal cancer cell line SW480.Methods The experiment was divided into three groups.Experimental group: p EX-4 plasmid containing SLC5A8 gene was transfected into colorectal cancer cell line SW480;Blank load transfection group: empty plasmid was transfected into colorectal cancer cell line SW480;Blank control group: Normal colorectal cancer cell line SW480.(1)The expression of SLC5A8 m RNA in the cells was analyzed by q RT-PCR;(2)The inhibition rate of cell proliferation was detected by MTS assay;(3)The changes of invasiveness of SW480 cells transfected with SLC5A8 gene was detected by Transwell cell invasion assay;(4)The effect of transfection of SLC5A8 gene on the migration ability of SW480 cells was studied by cell scratch assay.Statistical method: All the data were analyzed by SPSS19.0 statistical software.The data were measured by the mean standard deviation.The t test and ANOVA analysis were used to compare the differences between the two groups.The test level P < 0.05 was statistically significant.Result(1)Fluorescence microscopy showed:SW480 cells transfected with p EX-4-SLC5A8 and p EX-4 showed high intensity green fluorescence.The dissolution curve of SLC5A8 amplification products showed a single peak.Primer specificity is good,the amplification products and the number of amplification cycles(CT)are available.The results of q RT-PCR showed that the expression of SLC5A8 in the transfected cells was significantly higher than that of m RNA.The results showed that the transfection efficiency was high and the experimental methods and conditions were reliable.(2)After the transfection of SLC5A8 gene,the results of MTS assay showed that when adding butyric acid into the culture mediumthe,the inhibition rate of SW480 in experimental group was higher than that of the control group,and the difference was statistically significant(P =0.013).When there was no butyric acid in the culture medium,there was no significant difference between the control group and the experimental group of the cell proliferation inhibition rate(P=0.957).The inhibition rate of SW480 in the experimental group was higher than that in the culture medium,and the difference was statistically significant(P =0.017).There was no significant difference between the control group in the culture medium and no butyric acid on the inhibition rate of cell SW480(P =0.930).(3)Transwell invasion in vitro showed that the number of cells in the experimental group was lower than that of the empty load control group.The difference was statistically significant(t=14.072,P=0.000).The number of cells in the experimental group was lower than that in the blank control group.The difference was statistically significant(t=17.710,P=0.000).There was no significant difference between the empty load control group and blank control group(t=1.540,P=0.162).In Transwell in vitro invasion assay,the number of transmembrane cells was used as evaluating indicator.These suggest that SLC5A8 gene can inhibit the invasion of colorectal cancer cell line SW480 by the action of butyric acid.(4)Cell scratch test was used to observe the effect of SLC5A8 gene on cell migration.After 24 hours,the migration rate of the experimental group was lower than that of the empty load control group.The difference was statistically significant(t=13.617,P=0.000).The cell migration rate in the experimental group was lower than that in the blank control group.The difference was statistically significant(t=15.997,P=0.000).There was no significant difference between the empty load control group and the blank group(t=1.421,P=0.193).Similarly,after 48 hours,the migration rate of the experimental group was lower than that of the empty load control group.The difference was statistically significant(t=29.526,P=0.000).The cell migration rate in the experimental group was lower than that in the blank control group.The difference was statistically significant(t=48.936,P=0.000).There was no significant difference between the empty load control group and the control group(t=1.988,P=0.082).These results suggest that SLC5A8 gene can inhibit the migration of colorectal cancer cell line SW480 under the action of butyric acid.Conclusion 1.The expression of SLC5A8 gene was down or absent in colorectal cancer cell line SW480.2.In this study,SLC5A8 gene was successfully transfected into colorectal cancer cell line SW480 by gene transfection technique.3.SLC5A8 gene inhibits the proliferation,invasion and migration of colorectal cancer cell line SW480 by butyric acid,and butyric acid is one of its transport substrates.4.This study provides a new method for gene therapy of colorectal cancer. |
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