| ObjectiveUsing High-throughput sequencing technology and bioinformatics analysis technology,the study explore the molecular network mechanism of non-coding RNA-mediated to acupuncture improves the hot flushes rats induced by tamoxifen in hypothalamus.It will provide a scientific basis for acupuncture to improve the role of tamoxifen in the endocrine therapy of breast cancer.MethodThirty-six female SD rats with basal body temperature were randomly divided into control group(12 nude)and drug group(24 nude),establish hot flash rat model by intragastric injection administration of tamoxifen.After creating a successful model,the drug group is subdivided into a model group and acupuncture group,12 rats per group.Intervening“SP6+LR3+CV4”via acupuncture treatment for every other day in total of 10 times.1.Observing the efficacy of acupuncture on hot flashes rats induced by tamoxifen.The tail temperature of rats were measured by continuous monitoring of tail temperature with thermociuple,the serum estrogen levels of rats were measured ELESA.2.To reveal the mechanism of acupuncture improves the hot flushes rats induced by tamoxifen in hypothalamus:Through high-throughput sequencing,create the lnc RNA expression profile on Hot flushes model which caused by tamoxifen in hypothalamus.Looking for the differential expression lnc RNA and perform bioinformatics analysis.3.Revealing the mechanism of circ RNA-mi RNA-m RNA-mediated acupuncture to improve the hot flushes with tamoxifen in rats :(1)Screening for difference expression of circ RNA on hot flushes model which caused by tamoxifen in hypothalamus by hiseq X high throughput sequencing platform,and High-throughput sequencing results were verified by RT-PCR;(2)GO and KEGG database bioinformatics analysis methods were used to screen GO biological processes and signaling pathways that were significantly enriched with differentially expressed circ RNA-related target genes;The mi Randa software is used to predict mi RNAs that target circRNAs for differential expression;(3)The Target Scan database is used to predict the target gene of miRNA,then the key target gene are found by compared with differentially expressed mRNA and create circ RNA-miRNA-mRNA ce RNA network for revealing the mechanism of tamoxifen-induced hot flash in rats by ceRNA network;(4)miRNAs were detected by RT-PCR to verify whether the mi RNAs expression trends were consistent with the predicted results.The miRNAs quantitative detection in order to confirm the reliability of the circ RNA-miRNA-mRNA ceRNA network.Result1.The effect of acupuncture on improving the hot flushes induce tamoxifen in rats :After 28 days of tamoxifen administration,the tail temperature of the drug group was significantly higher than the control group.After acupuncture treatment,the tail temperature of the acupuncture group was significantly lower than the model group,and the two differences were statistically significant.Serum estrogen levels in the model group were lower than those in the control group,the acupuncture group were higher than the model group,which was close to the control group.2.lncRNA-mediated the mechanism of acupuncture in rats with tamoxifen-induced:(1)With p≤0.05 as a screening criterion,comparison model group and control group: total of 21 differentially expressed circ RNA,including 10 up regulated and 11 down regulated,comparison acupuncture group and model group:total of 16 different expressed circ RNA,including 5 up regulated and 11 down regulated and total of 440 different expressed m RNA,including 281 up regulated and 159 down regulated.(2)There are 2 target genes were screened in compassion acupuncture group and model group,which had the same downward trend with differentially expressed mRNA in acupuncture group/model group,namely LOC102553099 and Sra1 of LNC＿002164.Biological pathway and Pathyway enrichment results show 85 biological pathways,mainly purineribonucleoside triphosphate biosynthetic process,energy coupled proton transport,down electrochemical gradient,ATP synthesis coupled coupled proton transport,etc,and 9 signaling pathways mainly Oxidative phosphorylation,Parkinson’s disease,Huntington’s disease,Alzheimer’s disease,etc.3.CircRNA-miRNA-mRNA network mediated regulation of Acupuncture in rats with tamoxifen-induced:(1)The control group was compared with the acupuncture group with the screening results of circ RNA different expression in hypothalamus(p≤0.05):total of 74 differentially expressed circ RNAs,including 25 up regulated and 49 down regulated;compassion acupuncture group and model group:total of 53 differentially expressed circ RNA,including 28 up regulated and 25 down regulated.There were 5 differential circ RNA co-expressed between the model group/control group and the acupuncture group/model group.(2)Compassion acupuncture group and model group:biological pathways and single pathyways enrichment results suggest that 293 biological pathways were significantly enriched,mainly including insulin receptor signaling pathway via phosphatidylinositol 3-kinasecas-cade,synaptic transmission,intracellular transport,etc.KEGG enrichment analysis showed that Glutamatergic synapse,Calcium signaling pathway,Gastric acid secretion,Cholinergic synapse were significantly enriched.circRNA/ miRNA binding site analysis revealed that novel＿circ＿0004124 can be combined with rnomi R-6320,rno-miR-132-3p,rno-miR-212-3p,rno-miR-6316,rno-miR-6319(the top five).(3)Target Scan database predicted that there were 412 target genes of mir-212-3p,5 of which had the same downward trend with differentially expressed m RNAs in acupuncture group/model group,namely Hnrnpu,Pam,N4bp212,Atp6v1 h,Hnrnpr.(4)The results of RTPCR shows that:the expression of mi R-212-3p in the acupuncture group was significantly higher than that in the model group,and the difference was statistically significant(p≤0.05).Conclusions1.Tamoxifen can induce hot flushes in rats.Acupuncture can obviously improve hot flushes rats induced by tamoxifen,and it can increase the level of estrogen in hot flushes rats.2.The differentially expressed genes of LNC＿002164 in the hypothalamus may be involved in the process of acupuncture to improve the hot flushes induced by tamoxifen.3.The involvement of circRNA in acupuncture regulation of hot flushes is a multi-target,multi-biological approach and multi-signal pathway.The novel＿circ＿0004124 is the most significant circ RNA,synaptic transmission may be important biological approach,glutamatergic synapses and Calcium signaling pathway may be important involved signal pathway,involved in the hypothalamus of hot flushes rats treated with acupuncture for tamoxifen.The novel＿circ＿0004124-mi R132/212-3p-Pam may be a key regulatory link for circulator-mediated acupuncture to improve hot flushes induced by tamoxifen in hypothalamus. |
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