| Colorectal cancer(CRC)is one of the most common malignant tumor,with about 1.2 million new cases and at least 600,000 paitents died in the world annually.Metastasis is one of the important factors affecting prognosis of CRC paitents.Most of CRC paitents who accompanied with distant metastasis are not suitable for surgery and their five-year survival rate is 10 percent.The metastatic causes of CRC may be related to genetic mutations whose mutation rate of genes is no more than 50 percent.Therefore,there are other oncogenes or tumor suppressor genes being considered to play a part.Our previous study found a new human gene called FAM172A that was down-regulated in malignant group,which could significantly inhibited the proliferation and promoted apoptosis of LoVo cells,being controlled by transcription factor STAT1 in the transcriptional level.Based on the above research,we hypothesized that FAM172A is a tumor suppressor gene and its further research of the mechanism of transcription regulation is significant.Except the control of transcriptional level,gene expression control at the transcriptional level was a key regulation.In recent years,it has been realized that miRNAs have an important role in transcriptional regulation.miRNAs are relatively new class of regulatory RNAs that are 22 nucleotides long encoded by endogenous genes.The study confirms that abnormal versions of miRNAs may contribute to the metastasis of CRC.The positive expression rate of miR-27a increased significantly in CRC,and miR-27a could inhibit the PDCD4 expression and prevention the invasion of tumor cells.In our previous work,we sifted miR-27a that could probably target to FAM172A mRNA.Therefore,we speculate that miR-27a play important roles at the posttranscriptional level of FAM172A.This study is to further clarify the regulation mechanism of that FAM172A inhibited the invasion and metastasis of CRC cellsproviding supplying scientific and theoretical basis and for targeted gene therapy.Objectives:1、To be clear about the effect of FAM172A on the invasion and metastasis of CRC cells;2、The filter and decision of miRNAs that target regulating FAM172A;3、To be clear about the expression levels of miR-27a in the CRC tissues and cell strains,and to analyze the relationship between the expression levels of miR-27a and clinical parameters;3、To clarify the funtions and mechanism of that miR-27a targets target regulating FAM172A on the invasion and metastasis of CRC cells;Methods:1、To construct the recombinant plasmid of FAM172A and to evaluate the effect of FAM172A on invasion and metastasis of CRC cells by wound healing assay and transwell chambers test;2、Bioinformatics was used to predict the important miRNA of FAM172A:The method of luciferase reporter gene was used to confirm the the combination of FAM172A and miR-27a.3、The expression of miR-27a was evaluated by RT-qPCR in the CRC tissues and cell strains.4、The effect of miR-27a on invasion and metastasis of CRC cells was evaluated by wound healing assay transwell chambers test;The expression change of the important signal molecules of invasion was evaluated by westernblot;The LoVo cell strains stably expressing the miR-27a was established,and the changes were evaluated by above tests after the former was transfected with latter.5.The SPSS 22.0 software were used perform all the statistical analysis.All the results were displayed as means±SEM.Difference of measurement data was compared with the two-tailed Students’ s t-test or one-way analysis of variance.P-value<0.05 was considered as statistical significant.All relevant experiment were repeated three different times unless stated otherwise.Results:1、Over expression or interference of FAM172A in LoVo cells can slow down or quicken the migration speeds and decrease or increase the amount of invasion cells;2、There was a binding site of miR-27a in the FAM172A mRNA;Luciferease assay showed luciferase activity would down-regulate after transfected with miR-27a mimic.3、The expression of miR-27a in CRC tissues and metastatic tissue is higher than the specimens of normal tissue;There was a positive correlation between the increasing expression of miR-27a and TNM staging and lymphatic metastasis and distant metastasis;The expression of miR-27a in LOVO,HT-29,SW480,HCT116,DLD-1,RKO cell strains is higher than the normal colonic epithelium NCM460 cell strain.4、Compared to normal LoVo cells,the invasion and metastasis of LoVo cells with over-expression of miR-27a are increased,which are weaken after transfected with FAM172A recombinant plasmid.Compared to normal LoVo cells,theMMP-2、MMP-9 和 NF-κB expression of LoVo cells with over-expression of miR-27a are increased and the FAM172A expression of that are decreased,which are restorable after transfected with FAM172A recombinant plasmid.Conclusions:1、FAM172A gene has significant effects of anti-migration and anti-invasion on the LoVo cells;2、miR-27a could target binding to FAM172A;3、It suggests that miR-27a is involved with regulating the invasion and metastasis of CRC cells based on the high expression of miR-27a in CRC tissues;The expression level of miR-27a would be extremely valuable for diagnosing and assessing the conditionof CRC patients;4、FAM172A inhibits the invasion and metastasis of LoVo controlled by miR-27a;5、miR-27a targets binding to the FAM172A and promotes the invasion.and metastasis by probably regulating the NF-κB signaling pathway. |
