Primary Research On Functions And Mechanisms Of Transcriptional Regulation Of FAM172A

Colorectal cancer (CRC) is one of the most common malignant tumor, with about 1.4 million new cases and at least 700 000 patients died in the world annually. In China, Colorectal cancer has been ranked No.4 to 6 in malignant tumor. Although the current treatments, such as various surgery methods, radiotherapyand chemotherapy programs have improved the early survival rate of patients, most cancer patients accompanied with distant metastases are not suitable for conventional treatment with<10% of the five-year survival rate. Tendency of age of onset to younger seriously affect people’s health level. The drugs of anti-tumor chemotherapy commonly used in clinical treatment possess a certain degrees of side effects, which they can kill tumor cells, but also destruct normal tissue cells at the same time. Meanwhile, the resistance of tumor cells to drugs has increased urgent demand for new anti-cancer drugs at a large extent. However, due to the instability of gene and atypia of tumor cells, research on specific genes or other areas is a daunting task. Therefore, the development of new anticancer drugs, and the altered mode of action of anti-tumor drugs will be an important research topic for scientists. Based on development of immune therapy and molecular biology and continuous progress of cancer research, anticancer protein as a treatment strategies has been recently proposed, which has become a new hot topic in tumor therapy. Anticancer protein characterized by small molecular weight, easy to penetrate tumor cells, and strong stability, can improve the immune response, inhibit tumor angiogenesis, and directly induce apoptosis or cell cycle arrest of tumor to suppress tumor growth and metastasis. Our previous study found a new human gene with unknown-functions, which can inhibit mitosis of LoVo cells of colon cancer cell line, and was named AC-01. Bioinformatics analysis indicated that AC-01, that is FAM172A (family with sequence similarity 172, member A), is located on chromosome 5q15 with its wide range of expression including monocytes, lymphocytes, macrophages, human embryonic kidney cells, urothelial, liver cells, colon epithelial cells and various tumor cells. Further study found that FAM172A could significantly inhibit the proliferation of human colon cancer LoVo, which may inhibit mitosis of human colon cancer cells and thus inhibit its growth. According to recent studies about the anti-cancer proteins, we hypothesized that FAM172A may be an anti-cancer protein, and a typical tumor suppressor gene, which suggests FAM172A protein may become a new anti-cancer protein for colorectal cancer biotherapy.Therefore, this study is to further clarify the central role of FAM172A in the growth of colorectal cancer, and to investigate the minimum promoter and regulatory key transcription factor to explore the regulatory mechanisms of transcription level of FAM172A expression. Our research is to provide experimental evidence for biological treatment of FAM172A in colorectal cancer.Objectives:To further clarify the central role of FAM172A in colorectal cancer, and to explore the mechanisms of transcriptional regulation.Methods:1. Biological information analysisNCBI (http://www.ncbi.nlm.nih.gov/) was used to search mRNA sequence of FAM172A gene online, and BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) with with the human genome sequence was performed to find the best match of the genomic sequence. Then we identified gene transcription start site and positioned the first nucleotide of the transcription start site as+1. Subsequently, We used the UCSC Genome Browse (http://genome.ucsc.edu/) and Ensemble Genome Browse (http://asia.ensembl.org/index.html) to forecast FAM172A gene promoter sequences.2. Genomic DNA preparation and construction of plasmid DNAGenomic DNA was prepared from LoVo cells using a genomic DnA Purification kit (Promega). According to FAM172A mRNA sequence retrieved from NCBI and predicted promoter sequence, the software Vector NTI Advance 11.5.1 was used to design its specific primers to amplify its DNA. Then DNA fragment are cloned into the pcDNA3.1 (-) and pGL4.10 vector respectively and FAM172A gene expression and its promoter plasmid were constructed.3. To clarify clearly whether FAM172A can inhibit proliferation and promote differentiation and apoptosis of colon cancer cell(1)Total RNA was extracted from LoVo cells of colorectal cancer cell line and reverse transcribed into cDNA. Then amplified DNA fragment of FAM172A was inserted into pET32a (+) vector and pET32a (+)-FAM172A recombinant plasmid was successfully constructed. The recombinant plasmid was transformed into E. coli BL21 (DE3) followed by IPTG induction of expression and purified polyclonal antibodies were prepared. LoVo cells and SW480 cells of colon cancer cell line were co-cultured with different concentrations (0,0.1,1.0,10 and 100 ng/ml) of extracted FAM172A recombinant protein. After 12,24,36,48h, cells were collected and flow cytometry was used to detect cell cycle and cell number. And XTT assay was used to examine the proliferation of colorectal cancer cells;(2)The colorectal cancer cells were transfected with FAM172A recombinant plasmid and siRNA plasmids. After 48-hour incubation, the cells were collected; Annexin V-7AAD reagents and flow cytometry were used to detect apoptotic cells.4.Clone and preliminary analysis of FAM172A gene promoterGenomic DNA was extracted from human whole blood. According to bioinformatics prediction of the promoter sequence FAM172A gene, we designed its specific primers to amplify DNA, and amplified DNA fragment was inserted into pGL4.10 vector to construct the recombinant plasmid of FAM172A gene promoter. Then the recombinant plasmid of FAM172A gene promoter was transformed into E. coli DH5a, and a large amount of recombinant plasmids were prepared. Transient transfection of FAM172A recombinant plasmids and pGL4.10 vector into LoVo cells was performed and Luciferase reporter assay was used to detect the promoter activity of the recombinant plasmid.5. Identification and analysis of minimum promoter of FAM172A geneThrough continuous truncation of above promoter sequence, specific primers were designed respectively to amplify their DNA, and DNA fragment was cloned into pGL4.10 vector to construct each truncated promoter recombinant plasmid. Transient transfection of each truncated promoter recombinant plasmids and pGL4.10 vector into LoVo cells was performed and Luciferase reporter assay was used to detect the promoter activity of truncated recombinant plasmids. Then their activity was compared and the minimum promoter sequence with the highest activity was identified.6. Impact of the key transcription factor on expression of FAM172A gene.Bioinformatics analysis showed prediction of transcription factors binding to FAM172A gene sequences, and sequences of all relevant putative transcription factors were retrieved from NCBI. Total RNA was extracted from LoVo cells, and reverse transcribed to cDNA. Then specific primers of transcription factors were designed to amplify their cDNA followed by insertion into pcDNA3.1 (-) vector to construct an expression plasmid of transcription factors. Transient co-transfection of the minimum promoter plasmid and transcription factor expression plasmid was performed, and Luciferase reporter assay was carried out to detect the activity of transcription factors. Subsequently EMSA (electrophoretic mobility shift assay) and ChIP (chromatin immunoprecipitation) were conducted to identify whether the key transcription factor could specifically bind to the minimum promoter sequences of FAM172A gene. Transient transfection of the key transcription factor expression and siRNA plasmids into LoVo cells was performed respectively, and Real-time PCR and Western blotting was conducted to observed expression of FAM172A gene from mRNA and protein levels, which illustrated the effect of transcription factor on FAM172A expression.7. Statistical analysisThe SPSS 20.0 software were used to perform all the statistical analysis.And all the results were displayed as means ± SEM. The two-tailed Student’s t-test or one-way analysis of variance (ANOVA) was conducted to compare the differences. p-value<0.05 was set as statistically significant. All relevant experiments were repeated three different times unless stated otherwise.Results:1. To determine the influence of FAM172A on the cell cycle, SW480 colon cancer cells were co-cultured with different concentrations of FAM172A recombinant protein. We found that the percentage of cells in S phase and the total number of colon cancer cells decreased in a dose-dependent manner. Moreover, when at the highest concentration (0.1 μg/ml), the percentage of colon cancer cells in S phase reduced to 3.18%. These results demonstrated that FAM172A could significantly suppress the proliferation of colon cancer cells, especially at a concentration>0.1 μg/ml. using XTT proliferation assay we found that FAM172A significantly inhibited cell growth of SW480 cells compared to control group at each time point (p<0.01).2. The effect of FAM172A on apoptosis was assessed in colon cancer cells by flow cytometry. Up-regulation of FAM172A in SW480 cells induced increase of apoptosis (p<0.01). Moreover, SW480 cells treated with shRNA of FAM172A suppressed apoptosis significantly (p<0.01). FAM172A recombinant protein caused differentiation (magnification ×100 and magnification x400) of SW480 cells compared with control (magnification ×100 and magnification x400).3. In order to identify the FAM172A promoter, we obtained the FAM172A gene mRNA and analyzed and predicted the transcription start sites based on NCBI database and UCSC genome Browser (http://genome.ucsc.edu/). Then the genomic DNA fragment from -740bp to +205bp in the predicted promoter of FAM172A was amplified from LoVo cells. The putative FAM172A promoter region was inserted into a pGL4.10 Basic vector with the restriction sites Xhol I and EcoK V. pGL4.10-FAM172A promoter was named P1 and used for further shortened promoter plasmids as the template. After transfection for 24 h, Luciferase reporter assay was carried out. The results were recorded as Firefly/Renilla ratio. Compared with pGL4.10 Basic, P1 had ～40-fold luciferase activity, which indicated that the putative FAM172A promoter was active in the LoVo cells. Then, we studied the minimal FAM172A promoter. Five recombinants plasmids P2, P3, P4, P5 and P6 were constructed, respectively by sequentially truncating P1 plasmid. Subsequently, luciferase assays were carried out as above. The luciferase activity results showed that P6 had highest promoter activity. The serial truncation analysis suggested that P6,-112bp to+48bp, contained the essential sequences for transcriptional activity.4. We predicted possible transcription factors by searching TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCh. html) and GeneCards (http://www.genecards.org/). Based on our analysis, one major common transcription factor with the highest score, STAT1, was within the minimum promoter. The STAT1 binding site was between -68 and-58 within the mimimal promoter region (-112bp to +48bp).5. In order to investigate whether STAT1 could bind to FAM172A promoter in vivo, chromatin immunoprecipitation (ChIP) was performed followed by the PCR assay to amplify the region between-112bp and +48bp. These results demonstrated that the binding bite of STAT1 was presented on P6 compared with IgG-precipitated controls. This revealed that STAT1 could specifically bind to the FAM172A promoter. Then we carried out electrophoretic mobility shift assays (EMSA) to explore whether STAT1 could bind the FAM172A promoter. The oligonucleotide contained sequences that matched a consensus binding site for STAT1 was chose for EMSA. The nuclear extracts from the LoVo cells revealed strong binding to a labeled probe embracing an STAT1 binding sites. We then conducted competition experiments with unlabeled probes. DNA-protein complexes were specific, as demonstrated by the fact that it could be completely out-competed by a 100-fold excess of unlabeled probe. These results suggested that STAT1 was able to bind to the FAM172A minimum promoter.As shown above, these results revealed that STAT1 could specifically bind to the FAM172A promoter.6. To address whether STAT1 transcription factor could facilitate the expression of FAM172A, we performed western blot analysis together with quantitative real-time PCR to validate the upregulation of FAM172A. The protein samples and total RNA were extracted from cultured LoVo cells transfected with STAT1 gene overexpression plasmid and siRNA. The results demonstrated that the mRNA and protein expression level of FAM172A increased prominently induced by STAT1, which inferred that STAT1 could promote the expression of FAM172A.Conclusions:1. FAM172A can inhibit the proliferation and promote apoptosis of colon cancer cells.2. We have successfully cloned and identified the promoter region (-745bp to +205bp) of FAM172A gene with significant promoter activity. And the minimum promoter region (-112bp/+48bp) was also identified.3. The transcription factors STAT1 could bind specifically to the minimum promoter sequences and enhance the promoter activity of FAM172A.4. The transcription factor STAT1 could promote the expression of FAM172A gene.5. These findings offer a comprehensive understanding of the basic functions FAM172A gene and the mechanisms of its transcriptional regulation.