| The incidence of malignant neoplasms,deaths related to malignant neoplasms are increasing rapidly in the Worldwide.At the same time,breast cancer is a kind of complex disease,which is mainly characterized by large heterogeneity and many types between and within tumors.Hypoxic tumor microenvironment and long non-coding RNAs play an important role in tumor metabolism,proliferation and invasion.Triple negative breast cancer,due to its unique molecular phenotypic characteristics,has limited treatment methods compared with other types of breast cancer,and is prone to recurrence and metastasis,which also has a poor prognosis.Therefore,exploring the mechanism of action of long non-coding RNA on triple negative breast cancer under hypoxia and the relationship between the expression level of long non-coding RNA and related factors has important clinical theoretical and application value for better accurate diagnosis and treatment of this type of breast cancer in the future.Part I LncRNA GHET1 Promotes Hypoxia-Induced Glycolysis,Proliferation and Invasion in Triple-Negative Breast Cancer Through the Hippo/YAP Signaling PathwayObjectiveBreast cancer is one of the most important cancers threatening women’s health all over the world.Triple negative breast cancer is a type of breast cancer in which all 3 genes are negative.Meanwhile,hypoxia microenvironment and lncRNA play an important role in the occurrence and development of breast cancer.Therefore,this study aimed at screening specific lncRNA in triple negative breast cancer under hypoxic induction and exploring the relevant mechanism of this specific lncRNA on triple negative breast cancer patients,which is in order to find new targets and provide effective theoretical basis for triple negative breast cancer treatment and diagnosis.Experimental materials and methods●Tumor tissue samples(TNBC group)and adjacent tissues with normal histology(normal group)from patients with triple negative breast cancer treated in our hospital from January 2017 to June 2019 were enrolled.All tumor tissue samples from breast cancer patients were negative for progesterone receptor(PR),estrogen receptor(ER)and human epidermal growth factor receptor 2(HER-2)by immunohistochemistry after surgery;●4 kinds of human normal breast cell lines(MCF10A),triple negative breast cancer cell lines MDA-MB-231,MDA-MB-468,HCC1937 and BT-549 were used in this study,and the cell lines were resuscitated,subcultured and cryo-preserved according to laboratory procedures;●TRizol method was used to extract total RNA from clinical samples of TNBC group and Normal group and from each cell and tissue treatment group in this study,and reverse transcription and amplification were performed according to PCR method;●The cell lines that were treated by different ways and were inoculated into 24-well plates,stained and sealed according to the INSTRUCTIONS of The EdU staining kit,and the number of positive cells in each field was recorded by fluorescence microscope at random to calculate the EdU labeling rate;●The cell lines that were treated by different ways and were digested and beaten evenly,the cells were re-suspended,inoculated in 6-well plates,and then fixed,rinsed and stained with 0.1%crystal violet solution for observation;●Cells were inoculated into Transwell cells,cultured,fixed,rinsed and stained,and observed for statistics;●Cell lines cultured under Normoxia or Hypoxia conditions were tested for glucose and lactic acid levels in the medium according to kit instructions;●Proteins extrication,protein concentrations measurement and Western blot assay procedures were performed according to standard experimental procedures;●12 BALB/C nude male mice aged 4 to 6 weeks were randomly divided into 4 groups.The cell lines were subcutaneously injected into the right axilla of nude mice,and tumor size was measured and tumor weight was weighed after death;●SPSS 25.0 was used for statistical analysis of one-way ANOVA and independent sample t-test。Results:●lncRNA GHET1 expression is upregulated in triple negative breast cancer tissues,and hypoxia can promote the expression of lncRNA GHET1 in breast cancer cell lines,and the expression level of lncRNA GHET1 increases with prolonged hypoxia intervention;●Down-regulated lncRNA GHET1 expression inhibits triple negative breast cancer cell proliferation,replication and invasion,as well as cell glycolysis levels in hypoxia;●Interference with lncRNA GHET1 expression can inhibit Hippo/YAP pathway activation and YAP nuclear translocation in triple negative breast cancer cells under hypoxia;●High lncRNA GHET1 expression promotes the growth of weight and size of triple negative breast cancer tumors in nude mice;●High expression of lncRNA GHET1 in vivo can promote Hippo/YAP pathway in triple negative breast cancer and promote nuclear shift of YAP.ConclusionlncRNA GHET1 expression can be induced,leading to overactivation of Hippo/YAP signaling pathway under hypoxia,which can improve TNBC cell proliferation,cell viability and invasion ability,and increase glucose consumption and lactic acid production levels.At the same time,it may promote the growth rate of TNBC tumor tissue.Part Ⅱ lncRNA GHET1 expression and related influencing factorsObjectivePreliminary basic research results of this project showed that the expression level of lncRNA GHET1 in TNBC tissues or cells was significantly increased.Similar studies have shown that the significantly increased expression of lncRNA GHET1 in lung cancer specimens is related to tumor size,tumor cell differentiation and lymph node metastasis in clinical specimens.High expression of lncRNA GHET1 in patients with lung cancer and cervical cancer suggests poor prognosis in the meanwhile.Therefore,the expression of lncRNA GHET1 in TNBC patients was grouped and followed up in the preliminary basic study to explore the influencing factors of lncRNA GHET1 expression and the impact of lncRNA GHET1 expression on the prognosis of patients in this study.Besides,this study would provide scientific theoretical basis for personalized diagnosis and follow-up of TNBC.Materials and Methods●Patients with triple-negative breast cancer admitted to the hospital between January 2017 and June 2019 were collected for this study,and tumor tissue samples were collected from all enrolled patients;●Demographic,clinical history,and biochemical parameters of study subjects were collected;●Collect surgical and pathologic indicators of study subjects;●According to the expression level of lncRNA GHET1 in tumor tissues of patients with triple negative breast cancer,the mean expression level of lncRNA GHET1 was used as the discriminating value;Patients with below-average GHET1 expression were included in the low-expression group and patients with higher-than-average GHET1 expression were included in the high-expression group,which were divided into 2 groups.●Patients were followed up from the beginning of the operation to 2 years after the operation,and the follow-up methods were outpatient visits,telephone and Wechat follow-up;●SPSS 25.0 was used for statistical analysis of independent sample t-test and Chi-square test(when theoretical value and actual value<5,then the Fisher’s exact probability method was used),and P<0.05 was used as the judgment standard of statistically significant difference.Results●The level of lncRNA GHET1 expression in tumor tissues of patients with triple negative breast cancer was not affected by age,BMI,disease history and blood lipids.●The level of lncRNA GHET1 expression in tumor tissues of patients with triple-negative breast cancer is not affected by the surgical method,the presence of tumor thrombi or not,and postoperative radiotherapy and chemotherapy,but is affected by metastasis and invasion factors such as distal metastasis,lymphatic metastasis,infiltration into surrounding tissues,and tumor stage.●The level of lncRNA GHET1 expression in tumor tissues of triple negative breast cancer patients was not affected by the number of peripheral blood T cells and the proportion of CD8+T cells,but was affected by the proportion of peripheral blood CD4+T cells and the ratio of CD4+/CD8+;●High lncRNA GHET1 expression could shorten patients’ survival time,reduces disease-free survival,and increases the rate of recurrence.ConclusionHigh expression of lncRNA GHET1 is closely related to tumor stage,distal metastasis,lymphatic metastasis and invasion in TNBC patients,and patients with high expression of lncRNA GHET1 have shorter survival time,lower disease-free survival rate and higher recurrence rate.It may be through direct or indirect regulation of CD4+T cells and CD4+/CD8+ratio,and then affect the immune function of TNBC patients,thus affecting the prognosis of patients. |
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