| DNA methylation is a process where the methyl group of S-adenosylmethionine(SAM) is transferred to a specific base with the catalysis of DNA methyltransferase(DNMT). Two forms of DNMT function distinctively. One is maintenance methyltransferase DNMT 1, mainly involved in methylation of newly synthesized strand during DNA replication. The other is de novo methyltransferase, including DNMT 3A and DNMT 3B, which catalyzes the methylation of Cp G sites, modulating cellular growth and differentiation epigenetically. DNA methylation plays essential roles in genomic imprinting, X chromosome inactivation and inhibition of repetitive elements, which are key processes during normal development. In decades, the disturbed DNA methylation is thought to promote tumorigenesis by activating oncogenes and repressing tumor suppressor genes.Colorectal cancer is one of the common gastrointestinal malignancies, and arises as the consequence of genetic mutations and epigenetic alterations. And epigenetic alterations are recognized as more common events in colorectal cancer and affect hundreds of tumor-related genes. Aberrant DNA methylation is the major form of epigenetic alterations, including global DNA hypomethylation and specific DNA hypermethylation. The former leads to oncogenes activation, and the latter mainly occurs in Cp G of promoter region, resulting in tumor suppressor genes silencing, both of which are important driving force in colorectal carcinogenesis.Recently a novel class of non-coding RNAs, called piwi-interacting RNAs(pi RNAs), was discovered. They are named by exclusive association with piwi subfamily proteins of Argonaute family, but not Ago subfamily proteins. Initially, pi RNAs are thought to present only in germline stem cells, mediating transposon silencing via DNA methylation to maintain the integrity of genome, which indicates that pi RNAs carry out epigenetic regulation functions in germline stem cells. In decades, pi RNAs have been identified a variety of cancers, including gastric cancer, cervical cancer, breast cancer and bladder cancer, etc. And an increasing body of evidence suggests that cancer cells show a similarity of epigenetic signature with stem cells. These data imply that pi RNAs may play a similar epigenetic regulation role in cancer context as well.pi RNA-823, a member of pi RNAs, has a diversity of expression and roles in different cancer types. It has been demonstrated to be down-regulated in gastric cancer, but over-expressed in multiple myeloma, and promote de novo methylation. However, the role of pi RNA-823 in colorectal cancer is not fully understood.Our study aims to investigate the expression of pi RNA-823 in colorectal cancer and the effect on proliferation, apoptosis and colony formation of human colon cancer cell line HCT 116. To further explore the role of pi RNA-823 in epigenetic regulation of colorectal cancer, and provide a theoretical foundation for the development of pi RNA-targeted therapies for colorectal cancer.Objectives: To investigate the expression of pi RNA-823 in colorectal cancer and the effect on biological behavior of colon cancer cell line HCT 116, and to further explore its role in the expression of DNA methyltransferase.Methods: Fresh surgically resected specimens, including colorectal cancer tissues and corresponding normal adjacent tissues from 14 patients, were obtained from the Second Hospital of Hebei Medical University. Real time RT-PCR was used to detect the expression difference of pi RNA-823 in colorectal cancer tissues and normal adjacent tissues, and subsequently its functions was studied in colon cancer cell line HCT 116. The inhibitor of pi RNA-823, pi RNA-823 antagomir, was transfected into HCT 116 cells, and efficiency of transfection was evaluated by real time RT-PCR. Cell proliferation was assessed by CCK-8 assay, and cell cycle was analyzed by flow cytometry. Annexin V/PI double staining and TUNEL technology was used to examine cell apoptosis, and the activity of apoptosis related proteins, such as caspase-8, caspase-9 and caspase-3, was further examined by western blot. Finally, the role of pi RNA-823 in expression of DNMT 1, DNMT 3A and DNMT 3B was used to evaluate the relationship between pi RNA-823 and DNA methylation by western blot.Results: ①Real time RT-PCR indicated that the expression level of pi RNA-823 in colorectal cancer tissues was significantly higher than that in paired adjacent tissues(P<0.05), and a similar result was observed in colon cancer cell line HCT 116(P<0.001). ②In CCK-8 assay, it was showed that the proliferation of HCT 116 was significantly inhibited by three different concentrations of pi RNA-823 antagomir for 10 nmol/L, 30 nmol/L and 100 nmol/L in a dose-dependent manner(P<0.05). ③Real time RT-PCR showed that pi RNA-823 was significantly down-regulated by 100 nmol/L pi RNA-823 antagomir in 48 h after transfection(P<0.05). ④pi RNA-823 inhibition led to an increase in G1 phase population(P<0.001), and a corresponding reduction in S phase and G2/M phase population(P<0.01) by flow cytometry, which indicated that pi RNA-823 antagomir induced G1 phase arrest and suppressed G1/S phase transition in HCT 116 cells. ⑤pi RNA-823 antagomir led to a significantly increased percentage of apoptosis cells by Annexin V/PI double staining(P<0.001), a remarkable increase of TUNEL positive cells by TUNEL technology(P<0.001) and an elevated activity of caspase-8, caspase-9 and caspase-3 by western blot(P<0.01). These data indicated that pi RNA-823 inhibition promoted HCT 116 cells apoptosis. ⑥ Both plate colony formation assay and soft agar assay showed that the colony formation ability of HCT 116 was significantly reduced when pi RNA-823 was inhibited(P<0.01). ⑦ Western blot suggested that pi RNA-823 inhibition led to a marked reduction of DNMT 3A and DNMT 3B at protein level(P<0.05), but DNMT 1 showed no significant difference(P=0.88). This indicated that pi RNA-823 might contribute to colorectal carcinogenesis by regulating de novo methylation.Conclusions: pi RNA-823 contributed to colorectal carcinogenesis by regulating the expression of de novo methyltransferase DNMT 3A and DNMT 3B to promote cell apoptosis and suppress cell proliferation. pi RNA-823 could be a new therapeutic target for colorectal cancer. |
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