Study On Radiosensitization Effects Of Natrual Product CAPE Based On LncRNA SNHG8 Regulation

Radiotherapy is one of the common methods for the treatment of malignant tumors.However,ionizing radiation(IR)could cause radiation resistance of tumor cells and damage normal tissues and cells,thus failing to achieve the expected therapeutic effects.How to improve the radiosensitivity of tumor cells and reduce radiation damage to normal tissues and cells has become an important problem to be solved urgently in the field of radiotherapy.Caffeic acid phenethyl ester(CAPE),a natural product,is the main active component in propolis.It has many biological effects,such as antioxidant and antitumor activities.Some studies have shown that the expression of long non-coding RNAs(lncRNAs)is related to the radiosensitivityof tumors.However,the radiosensitization effects and mechanisms of CAPE regulated by lncRNAs are still unclear.In this study,CAPE was selected as the research material to reveal the radiosensitization effects of CAPE based on lncRNA levels in vitro and explore possible regulatory mechanisms.The main research contents are as follows:1.The radiosensitization effects of CAPE in hepatoma H22 cellsThe radiosensitization effects of CAPE were studied by detecting cell viability,intracellular reactive oxygen species(ROS)levels and cell apoptosis in a 60Coγ-radiation(60Coγ-IR)damaged H22 cells model.The results of cell viability showed that CAPE combined with IR could effectively inhibit H22 cell proliferation(P<0.001)compared with the 60Coy-IR(4 Gy)alone.In addition,the ROS levels of H22 cells were significantly increased(P<0.001)after IR treatment,while CAPE combined with IR significantly enhanced the ROS levels(P<0.001).Correspondingly,the apoptosis rate of H22 cells was significantly increased after CAPE combined with IR treatment.These results indicated that CAPE has a significant radiosensitization effect in hepatoma H22 cells.2.Screening of CAPE-induced DElncRNAs and identification of radiosensitization effects related DElncRNAsHigh-throughput sequencing was used to screen differentially expressed lncRNAs(DElncRNAs)induced by CAPE.The targeting miRNAs of candidate DElncRNAs were predicted using miRnada software.The quantitative realtime PCR(qRT-PCR)technology was used to screen key DElncRNA involved in the radiosensitization effects of CAPE.The results showed that a total of 153 DElncRNAs in CAPE-treated group were identified,of which 65 DElncRNAs were up-regulated and 88 DElncRNAs were down-regulated.And the results of qRT-PCR were consistent with the those of high-throughput sequencing.KEGG pathways analysis showed that the mRNAs co-expressed by DElncRNAs were mainly involved in tumor-related pathways,DNA damage repair,cell cycle,apoptosis and p53 signaling pathways.The miRnada software prediction results showed that candidate DElncRNAs had targeted binding sites with miR-200c-3p,miR-138-5p,miR-29b-3p,miR-101a-3p,miR-9-3p and miR-101a-5p.And the qRT-PCR results showed that CAPE-induced small nucleolar RNA host gene 8(SNHG8)could respond significantly to IR treatment(P<0.01),suggesting that SNHG8 may be a potential radiosensitization gene regulated by CAPE.3.The role of SNHG8 in the radiosensitization process of CAPE and its functional conservatism analysisFirstly,the hepatoma H22 cell line with the lower/over expression of SNHG8 was constructed,and the role of SNHG8 in the radiosensitization process of CAPE was verified by qRT-PCR and Cell Counting Kit-8(CCK-8)methods.Then,the BLASTn software was used to compare the SNHG8 sequences between Homo sapiens and Mus musculus genomes.Finally,the conservative functions of SNHG8 in human hepatoma HepG2 cells were verified.The results showed that CAPE could regulate the expression of SNHG8 and correspondingly enhance the radiosensitivity of H22 cells,suggesting that SNHG8 could participate in the radiosensitization process of CAPE.Furthermore,BLASTn alignment results showed that SNHG8 sequences had short and highly conserved sequences between Homo sapiens and Mus musculus,suggesting that SNHG8 may have similar functions in both Homo sapiens and Mus musculus genomes.Finally,the results of cell viability showed that SNHG8 was also involved in the radiosensitization process of CAPE in human hepatoma HepG2 cells,which was consistent with the trend of H22 cell viability.It suggested that the functions of SNHG8 were conserved in both Homo sapiens and Mus musculus.4.Exploring the radiosensitization mechanisms of CAPE based on SNHG8 regulationFirstly,the dual luciferase reporter assay was used to verify the binding relationship between SNHG8 and the predicted target miR-29b-3p.Then,the TargetScan software was used to predict the downstream regulated target gene of miR-29b-3p.And the prediction result was verified by the dual luciferase reporter assay.Finally,the H22 cell lines with the lower/over expression of SNHG8 and lower/over expression of miR-29b-3p were constructed respectively by cell transfection experiments.And the radiosensitization mechanisms of CAPE based on SNHG8 regulation were revealed by CCK-8,qRT-PCR and Western Blot methods.The results showed that CAPE-induced SNHG8 could negatively regulate the expression of miR-29b-3p,weaken the regulation of miR-29b-3p on the expression of Bcl-2,and then affect the expression of apoptosis-related proteins including Caspase 9,Caspase 3 and Bak,thus exerting its radiosensitization role.In conclusion,CAPE has a significant radiosensitization effect against hepatoma H22 cells.Its mechanisms may be related to its regulation of SNHG8/miR-29b-3p/Bcl-2 axis and its influence on the expression of related apoptotic proteins Caspase 9,Caspase 3 and Bak.This research can provide a theoretical basis for the development and application of natural products as radiosensitizers.