Study On The Mechanism Of Compound Genome - Wide APA In The Treatment Of Compound Chinese Medicine Jin

Eukaryotic mRNA (except histone mRNA), as well as some ncRNA, miRNA’s maturation undergoes a selective polyadenylation (Alternative Polyadenylation, APA) is formed poly A tail. By generating different length of 3’UTR (Untranslated Region), APA can affect a series of biological characteristics, including stability, translation activity and intracellular localization of transcripts, and so on. Recent research shows that in the case of none genetic mutations, APA could produce shorter 3’UTR in oncogenes which could lead loss of miRNA binding. Escaping the negative regulation by miRNA, oncogenes promote malignant transformation of cells. Conversely, when 3 ’UTR of oncogene mRNA is shorter which is mediated by APA, the inhibition of miRNA is absent either, leading to the increased expression of apoptotic genes, so as to achieve tumor suppressor role. From this perspective, this study discuss the tumor suppress mechanism of compound Chinese medicine Jinfukang (JFK).JFK is an anti-cancer oral medicine, officially approved by China Food and Drug Administration for the treatment of lung cancer in clinical application. As a kind of Astragalus-based Chinese herbal compound, JFK has been reported to improve the effect of chemotherapy in patients with non-small cell lung cancer. In vivo experiments in mice shows JFK can inhibit the growth of tumor cells. However, its specific mechanism is not very clear, pending further study.We used JFK ethanol extract, acting on the human non-small cell lung cancer cell line A549. Through a special method-3T-seq (TT-mRNA Terminal Sequencing) technology developed by our lab previously, we could locate PAS (Poly A Site) sites precisely, thereby establishing APA tagged libraries. By high-throughput sequencing technology, we built genome-wide PAS spectrum, and analyzed the transcriptional regulation by JFK in human non-small cell lung cancer cell line A549.We found, a few of tumor suppressor genes’ 3’UTR were significantly truncated through the process of APA after adding JFK in A549 cells. At the meantime, the expression levels of these genes are increased. Functional analysis of these genes, such as MANF, UBE2Z, SGK223, CKAP4, reveals that they are apoptosis-related genes. Real time PCR validation shows that these four genes’ expression levels were up-regulated after JFK treatment in A549 cell; APA sites were verified by 3’RACE, which can prove the accuracy and authenticity of the APA tag database made by 3T-seq. The results suggest that JFK may affect APA sites of the apoptosis related genes, and regulate the expression of these genes, thus inhibit the growth of tumor cells. Our studies not only provide important information in the regulation of gene expression in lung cancer cells, but also offer theoretical basis molecular mechanism of JFK’s tumor suppression. In short, our research opens up a way to study the molecular mechanism of anti-tumor effect of Chinese medicine.