The Effect Of Nrf3 On The Growth Of Human Colon Cancer Lo Vo Cells In Vitro

Colorectal cancer (CRC) is one of the most frequent cancers in human beings. The incidence has an increasing tendency worldwide in recent years. With the development of molecular biology, tumor has been identified as a kind of genic disease. The intrinsical properties of tumor are activation of oncogene and inactivation of antioncogene. With the post genomic era arriving, there have been many researches on colorectal genes by microarray technique with the character of high-throughput, high-flux, miniaturization and automation. As a result, colorectal genic data-bank is becoming more and more abundant. However, there are no systemic researches based on the strategy "data mining of gene profiles → screening candidated genes →? investigating function of these genes".This study is the extension of Hong-Min Xu s research, "Gene expression profiling of human colorectal cancer with different differentiation grades". Hong-Min Xu et al firstly used Affymetrix GenecChip HG-U133 to monitor gene expression of about 19308 known genes and 12956 ESTs (altogether 32264) on 9 single samples of CRC with different differentiation grades (3 well differentiated, 3 moderately and 3 poorly ) paired normal mucosa tissue (9 samples in one pools), and 17 of most important CRC-related genes & ESTs were obtained. Based on this result, we applied literature profiling to analyze above-mentioned 17 genes & ESTs, and identified that Nrf3 was importantly worthy of investigation.In order to understand the function of Nrf3 in CRC, the methods were applied as following. ① Full length of Nrf3 s cDNA was obtained from CRC tissues, and its expression was detected on 3 CRC samples and paired normal mucosa tissues by RT-PCR. ② With the strategy of subcloning, Nrf3 was cloned into eukaryotic expression vector pEGFP-N1 by Sal Ⅰ / BamH Ⅰ directional insertion. As a result, we constructed the eukaryotic expression vector pEGFP-Nl-Nrf3 for Nrf3 gene fused with EGFP. ③ The human colon cancer LoVo cells were infected with this fusing protein in vitro by means of liposome mediation, apoptosis and cell cycle of whichwere detected by FCM. 0 Subcellular localization of Nrf3 was observed by fluorescent microscope. (D In order to verify whether Nrf3 influences the growth of CRC or not, we transfected it into the human liver cancer SMMC7721 cells, and studied with the same methods.The results were as follows:1. The interested gene Nrf3 was identified by literature profiling analysis.2. Nrf3 were expressed in all of 3 CRC tissues, but in 1 paired normal mucosa tissue at lower level and not in others, which were similar with the result of gene chip;High level of Nrf3 was detected in the human colon cancer LoVo cells.3. Nrf3-EGFP fusing protein was distributed in the cell nucleus by fluorescent microscope observation.4. The influence on cell cycle of LoVo cells infected with Nrf3 in vitro were analyzed by FCM. The results were as follows: number of LoVo cells in phase G2/M + S significantly decreasing compared to control group. On the contrary, obviously increasing in phase G0/G1. As a result, Nrf3 was capable to inhibit the growth of LoVo cells in vitro, which can arrest LoVo cells at phase G0/G1 by suppressing the DNA synthesis and mitotic division.5. The influence on cell cycle of SMMC7721 cells infected with Nrf3 in vitro was similar with the influence on LoVo cells. It suggested that Nrf3 could inhibit the growth of SMMC7721 cells by arresting SMMC772 cells at phase G0/G1.6. The influence on apoptosis of LoVo and SMMC7721 cells infected with Nrf3 in vitro were analyzed by FCM. There was no evidence of Sub-Gl peak (or apoptotic peak) of those cells. So it suggested that Nrf3 could not induct apoptosis of LoVo and SMMC7721 cells.The new contributions of our study may be as follows:? The expression of Nrf3 was firstly detected in CRC.? Anew tumor-suppressing gene NrO was affirmed in vitro, which had the role of inhibiting proliferation of CRC and liver cancer.(D Nrf3' s role of infulencing apoptosis of CRC and liver cancer was not observed.