| Breast cancer is the most common malignant tumor of women worldwide,and one of the most important diseases threatening women’s health.The incidence of breast cancer is increasing year by year and showing a younger trend.The occurrence and development of breast cancer is a long-term,multi-stage,multi-gene change cumulative process.However,the exact molecular mechanism of its occurrence and development is still unclear.With the rapid development of molecular biology and gene diagnosis technology,in addition to many genes related to breast cancer,some genes closely related to human breast cancer have been found in recent years.The role of these genes in breast cancer is attracting more and more attention.Long-stranded noncoding RNAs(1ncRNAs)are a class of RNA molecules with transcriptional transcripts exceeding 200 nt.They do not encode proteins,but regulate gene expression at various levels(epigenetic regulation,transcriptional regulation,post-transcriptional regulation,etc.)in the form of RNA.They interact with other biological molecules and participate in many physiological and pathological regulation.HOTTIP is derived from long-chain non-coding RNA of Hox gene.HOTTIP plays an important role in the progression of various tumors.HOTTIP can promote the invasion and metastasis of a variety of cancer cells,play a potential carcinogenic role in tumors,and is a predictor of poor prognosis of patients.Studies have shown that high expression of lncRNA HOTTIP can be used as a poor prognostic indicator of breast cancer.However,the effect of lncRNA HOTTIP on invasion and metastasis of breast cancer cells and its related molecular mechanisms remain unclear,which needs further study.Objective: To investigate the expression of HOTTIP in breast cancer and its relationship with clinicopathological parameters.To investigate the effects of HOTTIP on the migration and invasion of breast cancer cells,the effects of HOTTIP on EMT of breast cancer cells,and the effects of HOTTIP on the activation of Wnt/beta-catenin pathway.To determine the effect of HOTTIP interference on metastasis of breast cancer cells in vivo.Methods: The expression of HOTTIP in breast cancer tissues and matched adjacent tissues was detected by qRT-PCR,and the relationship between the expression of HOTTIP and clinicopathological parameters was analyzed.HOTTIP interference plasmid was constructed and a breast cancer cell line stably silenced by HOTTIP was established by screening with G418.Cell migration and invasion were measured by scratch test and Transwell test.HOTTIP was down-regulated and the expression of EMT key protein was detected by Western blot.HOTTIP was down-regulated and the expression level of key molecules in Wnt/beta-catenin pathway was detected by Western blot.HOTTIP interferes with the transfection of plasmid beta-catenin-S33 Y,reversely activates Wnt/beta-catenin signaling pathway,detects the change of cell migration ability by scratch test,detects the change of cell invasion ability by Transwell test,and detects the change of expression level of key molecules in Wnt/beta-catenin pathway by Western blot.Two groups(NC group and HOTTIP interference group)of 2.5 x 105 cell suspensions in logarithmic phase were injected into nude mice aged 6-8 weeks by tail vein at 100 ml PBS.After 6 weeks of cell inoculation,all mice were killed to take lung tissue and take photos.The morphology of lung tissue in each group was detected by HE staining,and the number of pulmonary metastatic nodules in each group was analyzed.Results: The results showed that breast cancer tissues had higher HOTTIP expression than adjacent tissues.Patients with TNM stage III-IV and lymph node metastasis had higher HOTTIP expression.HOTTIP knockdown inhibited the migration and invasion of breast cancer cells.HOTTIP knockdown inhibits epithelial-mesenchymal transition(EMT).HOTTIP down-regulation inhibits cell migration,invasion and EMT by blocking Wnt/beta-catenin protein pathway.The number of pulmonary metastatic nodules in HOTTIP silenced mice(average 7.67)and in control group(average 12.67).Conclusion: HOTTIP is highly expressed in breast cancer.Compared with patients with low expression of HOTTIP,patients with high expression of HOTTIP had higher clinical stage and more prone to lymph node metastasis.HOTTIP affects the migration,invasion and EMT of breast cancer cells by regulating Wnt/beta-catenin pathway.Compared with control group,HOTTIP silenced mice had significantly fewer pulmonary metastatic nodules,suggesting that HOTTIP knockdown can inhibit tumor metastasis in vivo. |
