G-quadruplex From Proto-oncogene C-myc Promoter On Proliferation Of Colorectal Cancer Cells

Objective:Cancer has become the second largest cause of death for human,Colorectal cancer(CRC) is one of the serious threats to human health, and relevant with oncogene activation and inactivation of tumor suppressor genes lead to gene expression disorders at the level of genetic and molecular biology.With the continuous development of anticancer drugs, gene therapy has become one of the hot topics, large number of netargets of anticancer drugs is gradually discovered and used in clinical trials, in which the study of G-quadruplex for the drug targets caused widespread concern.G-quadruplex is made by tandem repeat rich-guanine nucleotide sequence under certain conditions, it can form telomerase G-quadruplex in gene promoter region,which drugs binding and stable G-quadruplex and inhibit telomerase activity and oncogene expression to inhibit tumor growth.The nucleic acid hypersensitivity element III1(NHE III1) of c-myc gene promoter region, also called Pu27 which is guanine-rich repeats sequences and can form intramolecular G-quadruplex to silence gene expression,effectively inhibit the activity of c-myc gene transcription and protein expression level,become a new drugs target to gene therapy.Oligonucleotides with guanosine-rich sequences can form G-quadruplex structures,some GRO(G-rich oligonucleotide) showed efficient inhibitory effect to the most tumor cells and little toxicity to the normal cells,it gradually become a new class of anti cancer nucleic acid drugs.Pu27 can form G-quadruplex in vitro,and take into the leukemia cells by the way of non-transfected,stable cell c-myc gene promoter region G-quadruplex structure,reduced c-myc gene expression and inhibite cell proliferation, it have the advantage which the traditional macromolecular nticancer drugs such as antisense oligonucleotides, etc are not.To investigate the impact of Pu27 and Mut Pu27 on colon cancer cell apoptosis and proliferation;To investigate the inhibition of c-myc gene expression; Preliminary analysis of possible molecular mechanisms about tumor cell growth inhibition by Pu27.Methods:(1)Read the literature,select and synthesized the sequence of Pu27 and also the specific mutant sequence Mut Pu27;(2)Use human colon cancer cells HCT-116, SW480,SW620 as the experimental subjects, Pu27 and Mut Pu27 treat cells by the non-transfected way.Then detect the Proliferation ability by MTT experiments and detect cell apoptosis by flow cytometry.Set up the experimental groups as HCT-116/Pu27、HCT-116/Mut Pu27、 SW480/Pu27、SW480/ Mut Pu27、SW620/Pu27、SW620/Mut Pu27、FHC/Pu27 and FHC/Mut Pu27;(3)To extract total RNA from cells to detect the level of c-myc m RNA using fluorescence real-time quantitative PCR technology. Then extract total protein from the same group cells as above to detect the level of protein using western blotting;Useing FHIC labeled Pu27 and Mut Pu27 sequences to observe the uptake and stability.Results:(1)Cell proliferation assay MTT results suggested that HCT-116 and SW480 cells’ proliferation ability was decreased with Pu27 and the SW480 /Pu27 group is the lowestinhibition rate in all groups;(2) Flow cytometry results suggested the apoptosis rate were increased in these groups, and the SW480 / Pu27 group is higher than the other Groups. These results demonstrated that Pu27 inhibits cell proliferation and promotes cell Apoptosis;(3)RT-PCR results suggested that the c-myc m RNA and protein expression was decreased,the level of expression on SW480 group was lower than other groups.Conclusion:Pu27 can take into cells by theway of non-transfected.Pu27 sequences can reduce c-myc m RNA and protein expression,inhibite malignant proliferation and induce apoptosis for human colon cancer cell line HCT-116, SW480.SW480/Pu27 group has the highest sensitivity to Pu27 in inhibition of cell proliferation rate and the degree of decrease for the c-myc m RNA and protein expression.This study provides a new ideas and methods which have clinical significance for colon cancer gene therapy.