| Breast cancer is the most common malignant tumor in women,which is a serious threat to women's health.It is reported that the incidence of breast cancer in China and the number of new cases is increasing year by year.Although with the development of early screening and the improvement of comprehensive treatment,the mortality rate of breast cancer has decreased gradually,but the mortality rate of breast cancer in China is still on the rise,and the situation is not optimistic.Lymph node metastasis is the main cause of poor prognosis in breast cancer patients,more than 30% of patients died from recurrence and metastasis.Therefore,in-depth study of how breast cancer recurrence and metastasis,search for the cause of recurrence and metastasis of breast cancer biomarkers and therapeutic targets is currently considered the most effective means of controlling and monitoring,preventing the recurrence of breast cancer,it has important clinical significance to improve the overall level of treatment of breast cancer.Long non-coding RNAs(lncRNAs)is a diverse class of RNA transcripts>200 nucleotides in length with limited protein coding potential,due to the lack of significant open reading frame.Many previous studies have found that lncRNA can through epigenetic modification,transcription and post transcriptional regulation and translation level regulating process of tumor invasion and metastasis regulates multiple pathways,vascular proliferation,effect of tumor formation,cell adhesion and other biological process,play an important role in the development of malignant tumor.In the past,the primary and normal tissues were used to analyze the differences of lncRNA.But compared with the primary tumor,the content of lncRNA in the metastatic lesions often changed significantly,and the change of expression level is closely related to tumor invasion and metastasis and prognosis of patients.Therefore,in order to find the molecular markers and target targets for the recurrence and metastasis of breast cancer,this study mainly selected lncRNA gene chip screening in primary and metastatic breast cancer.In order to analyze the differential expression of lncRNA in breast cancer and its metastatic lymph nodes,this study focus on the breast cancer IncRNA expression profiles.In order to investigate the expression of lncRNA in breast cancer cells and its molecular biological mechanism and the mechanism of tumor regulation,the research includes two parts.The first part: Part one: Expression and analysis of lncRNAs between breast cancer and metastatic lymph node tissues;the second part: in vitro and in vivo studies on the inhibitory effect of lncRNA00499 expression on the biological function of breast cancer.Part one: Expression and analysis of lncRNAs between breast cancer and metastatic lymph node tissues;Objective:To investigate the pathogenesis of breast cancer,we observed the expression profile of lncRNA in breast cancer by microarray technique,analyed differential expression of lncRNA in breast cancer and its metastatic lymph node tissues and verified the results of microarray analysis in the cell and tissue level,finally selected out the relationship between lncRNA and clinicopathological parameters and prognosis.Methods:1.Fifty-nine pairs of breast cancer tissues and its metastatic lymph node tissues was used and the clinical data of 183 cases of breast cancer were analyzed.2.LncRNA chip was used to detect expression of lncRNA in 5 cases of breast cancer tissues and its metastatic lymph node tissues and analysis the differential expression of lncRNA3.The qRT–PCR method was used to detect expressions of lncRNA00499 in fifty-four cases of breast cancer tissues and its metastatic lymph node tissues and the correlation between the expression level of lncRNA00499 and clinicopathological data of breast cancer was analyzed.4.The qRT–PCR method was used to detect expressions of lncRNA00499 in 183 cases of breast cancer tissues and the correlation between the expression level of lncRNA00499 and prognosis of breast cancer patients was analyzed.Results:1.Compared with breast cancer tissues,metastatic lymph node tissues exhibited 198 differentially expressed lnRNAs,lnRNAs,including 45 lncRNAs upregulated and 153 lncRNAs downregulated.LncRNA00499 is upregulated in metastatic lymph node tissues(P<0.05).2.qRT-PCR detection showed that compared to breast cancer tissues,expression level of lncRNA00499 has a significant upregulation in metastatic lymph node tissues(P<0.05).The results of lnRNA chip and qRT-PCR are consistent.3.LncRNA00499 expression level in breast cancer tissues was associated with TNM stage(P<0.05).There was no statistically correlation between lncRNA00499 expression and either age,body mass index,menstrual status,family history,tumor size,differentiation,lymph node metastasis,vascular invasion,hormone receptor status,HER2 gene expression and Ki67 expression(P>0.05).4.The expression of lncRNA00499 is closely related with the prognosis of breast cancer patients.Patients with high expression of lncRNA00499 in the 5 year overall survival rate had a tendency to be higher than patients with low expression(p=0.057),especially in luminal B subtype breast cancer patients(P<0.05).Conclusion:The lncRNA expression profile between reast cancer tissues and its metastatic lymph node tissues was significantly different.LncRNA00499 was potential non-coding oncogenes in breast cancer and is an independent prognostic factor of breast cancer.But their detailed functional mechanisms need further exploration.Part two: In vitro and in vivo studies on the inhibitory effect of lnc RNA00499 expression on the biological function of breast cancer.Objective: RNA interference technique was used to inhibit the expression of lnc RNA00499 in breast cancer cells MDA-MB-231.The effects of lnc RNA00499 on the proliferation and migration of MDA-MB-231 in breast cancer cells were analyzed in vitro and in vitro,and two generation sequencing analysis of the relevant signal pathways were used to provide experimental basis for the treatment of breast cancer with lnc RNA00499 as the target.Methods: 1.Construct lentiviral vector with lnc RNA00499 sh RNA and non-sense sh RNA,which were transfected into MDA-MB-231 cells,screen a stable infected cell line that downregulation the expression level of lnc RNA00499.2.The cells are divided into two groups: control group(recombined lnc RNA00499 sh RNA lentivirus infected cells),negative control(Ctrl)group(lnc RNA00499 non-sense sh RNA lentivirus infected cells).3.Using Incu Cyte system and plate clone formation experiment to detect proliferation and growth ability of cells in each group.4.Using transwell chambers experiment and scratch to test invasion and metastasis ability of cells in each group.5.Using Tumor xenograft model to detect the influence of downregulating lnc RNA00499 expression expression in MDA-MB-231 cells on BALB/c nude mice.6.Two generation sequencing analysis effect of lnc RNA00499 silencing on expression of lnc RNA/m RNA in breast cancer cells.Results: 1.Lentiviruses with lnc RNA00499 sh RNA(Sh)and Ctrl were successfully constructed.Compared with Ctrl groups,the expression of lnc RNA00499 was significantly decreased in Sh-2 and Sh-3 group(P < 0.05).2.Incu Cyte system results showed that compared to Ctrl groups,OD450 value of Sh group has a significant reduce in MDA-MB-231 cells,which was moremore obvious with time passing.passing.The difference has statistically(P < 0.05).Plate clone formation experiment results that compared to Ctrl groups,the colony formation number of Sh group has a significant reduce in MDA-MB-231 cells.The difference has statistically significant(P<0.05).3.Transwell invasion experiment results showed that compared to Ctrl groups,the cell number went through membrane of Sh group has a significant reduce.The difference has statistically significant(P<0.05).Scratch experiments results showed that compared to Ctrl groups,after recombination of lentivirus infected cells,scratch healing speed after 48 h of Sh group was significantly lower in MDA-MB-231 cells.The difference was statistically significant(P<0.05).4.To confirm the growth inhibitory effect of Sh-group in vivo,tumor xenograft transplant experiment was performed.Tumor size was significantly reduced in the Sh mice group as compared to control mice at the fourth week(P<0.05).5.The two generation sequencing results showed that compared to the control group,the silence of lnc RNA00499 expression in breast cancer cells were screened and 604 transcripts were up-regulated and 363 down regulated the expression of transcription,Pathway analysis showed that the signal pathway of lnc RNA00499 silencing expression in breast cancer cells was significantly changed.In addition to tumor related signaling pathways,there are also other signal pathways involved in tumor metastasis,such as Regulation of actin cytoskeleton;involved in tumor proliferation,such as TGF-beta signaling pathway,Hippo signaling pathway,Hedgehog signaling pathway?Conclusion: Lnc RNA00499 is involved in the proliferation and migration of breast cancer cells.Two generation sequencing showed that lnc RNA00499 is closely related to the proliferation and metastasis of breast cancer cells.The silencing lnc RNA00499 gene is expected to be used in the targeted therapy and prognosis of breast cancer. |
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