Thymoquinone,a Natural Small Molecular Compound,inhibits The Growth,migration And Invasion Of Triple Negative Breast Cancer Cells By Up-regulating The Expression Of HSPA6

Objective:Since the 21st century,the number of cancer patients in the world is increasing at an alarming rate.Millions of people are diagnosed with cancer every year,and breast cancer accounts for a large share of all tumors.Breast cancer is a malignant tumor that occurs in the breast epithelium or ductal epithelium.Its etiology is not clear.It may be related to family history,carrying breast cancer related genes,reproductive factors,sex hormones,nutrition,diet and environmental factors.The incidence rate of breast cancer is the highest in the world,and the mortality rate is second.In China,the incidence rate of breast cancer is increasing year by year,which seriously threatens women s health.The treatment of breast cancer is mainly based on the principle of precision and comprehensive treatments,including radiotherapy,chemotherapy,endocrine therapy,biological targeted therapy and traditional Chinese medicine adjuvant therapy.Although there are many means of diagnosis and treatment,the treatments are more complex,the treatment cycles are longer,the curative effects are not ideal.So we need to further find new treatment options,and patients can benefit more.Black seed is a traditional plant active medicinal material,which has a wide range of pharmacological effects,such as anti-tumor,anti-hypertension,anti-diabetes,anti-bacterial,anti-inflammatory,anti-oxidant,immune regulation and so on.Thymoquinone（TQ）is the main active ingredient in black grass seed.Its chemical formula is C₁₀H₁₂O₂（2-isopropyl-5-methyl-1,4-benzoquinone）.It has anti-cancer effect on a variety of tumors,including triple negative breast cancer（TNBC）.In order to further explore the anti-tumor pathway of TQ,we will analyze and discover the differentially expressed genes in tumor cells without and with TQ treatments following transcriptome sequencing（RNA-seq）,and find the new target gene and molecular mechanism of TQ inhibiting the growth,migration and invasion of breast cancer cells.Methods:1.The human triple negative breast cancer cell line BT-549with high invasion and high migration characteristics was selected as the research material.2.The control group and TQ treatment group were set up,and the two groups of cells were analyzed and compared by RNA-seq to find out the differentially expressed genes.3.Through quality control,sequence alignment,expression analysis,classification and annotation of Gene Ontology（GO）database and analysis of Kyoto Encyclopedia of genes and genomes（KEGG）database,valuable differential genes were screened.4.Furthermore,Reverse transcription polymerase chain reaction（RT-PCR）,polymerase chain reaction（PCR）and Western blot（WB）were used to verify whether the expression changes of the differentially expressed genes were consistent with the sequencing results.That is to say,there was a difference in expression before and after TQ treatmens.5.Through up-regulation or down-regulation of HSPA6 expression to study and analyze its biological function characteristics.6.Objective to study the effect of HSPA6 on the survival rate of breast cancer patients and its regulatory mechanism by bioinformatics analysis and online database retrieval.Results:1.After detailed analysis of RNA-seq data,we screened out 169differentially expressed genes,including 141 down-regulated differentially expressed genes and 28 up-regulated differentially expressed genes,and obtained 18 differentially expressed genes that may be closely related to tumors.2.Further analysis showed that the expression of HSPA6 gene was significantly up-regulated after TQ treatment,and showed the effect of inhibiting the growth,migration and invasion of TNBC cells.3.The high expression of HSPA6 is positively correlated with the long-term overall survival（OS）of breast cancer patients,which also suggests that HSPA6 has tumor inhibitory effect.4.DNA methylation of HSPA6 may not be the regulatory mechanism of up-regulation of HSPA6 m RNA in breast cancer tissues,although the expression level of HSPA6 m RNA in breast cancer tissues is significantly higher than that in normal tissues.5.When overexpressing HSPA6,TQ further enhanced its inhibitory effect on tumor cell migration and invasion;when knocking down HSPA6,TQ further weakened the ability of tumor cell migration and invasion,showing a partially HSPA6-dependent way of action of TQ.Conclusion:We successfully identified a new TQ targeting gene HSPA6.Both experimental and clinical data showed that TQ could inhibit the growth,migration and invasion of TNBC cells through HSPA6 gene.However,the mechanism of HSPA6 is still unclear,which is of great significance to further explore.The discovery of HSPA6 not only reveals a new regulatory mechanism of TQ,but also provides a new candidate gene target for clinical treatment of breast cancer,especially TNBC.