Regulation Of NF-κB Activation By LINC00365-SCGB2A1 Signaling Pathway On Proliferation And Apoptosis Of Breast Cancer Cells

Breast cancer is the first female malignant tumor in the world.The focus of biological research on breast cancer is to find effective prognostic indicators and therapeutic targets at the molecular level.At present,the clinical limitation of molecular marker application is mainly due to the complexity and intersection of biomolecules,so the new biomolecules and their complex mechanisms in cancer cells are discussed.It is helpful to develop new effective prognostic markers and promote the individualized strategy of comprehensive therapy for breast cancer.With the development of gene sequencing and bioinformatics,the relationship between the regulation of non-coding RNA sequences in cancer genome and the complex biological characteristics of tumors has become a hot topic.Similar to other common tumors,numerous studies have reported that many long non-coding RNAs(LncRNAs)are abnormally expressed in breast cancer,which is related to biological characteristics or prognosis of tumors.LncRNA is a non-coding RNA,with a length of more than 200 nucleotides,because of its complex spatial structure,the mechanisms involved in expression regulation are more diverse and complex.Characterization of the functional mechanisms of LncRNA in tumors not only contributes to the application of biomarker clinical markers,but also promotes the development of new cancer therapeutic targets.In previous bioinformatics analysis,it was found that the expression of long non-coding RNA LINC00365 on chromosome 13q12.3 was significantly different between cancer,paracancerous and normal tissues of patients with gastric cancer,and SCGB2A1(mammaglobin B)was positively correlated with the expression of LINC00365 by expression correlation analysis.Biological experiments show that LINC00365 can affect the biological characteristics of gastric cancer cells by regulating SCGB2A1.NF-κB has been discovered since 1986 as a transcription factor induced by cell rapid reaction,because of its extensive involvement in the regulation of immunity,inflammation,cancer and other processes,it has always been a hotspot of pharmacological targeted research.The activation of nuclear transcription factor NF-κB is involved in the transcriptional regulation of various genes,apoptosis associated proteins,chemokine and transforming growth factor(TGF)promote the proliferation and escape from apoptosis of tumor cells.Several studies have proved that NF-kappa B is overactivated in breast cancer and can be used as an important molecular target for breast cancer therapy.Based on the literature review and previous studies,it is speculated that LINC00365-SCGB2A1 pathway may affect the proliferation and apoptosis of breast cancer cells by affecting NF-κB transcriptional activity.This study is based on clinical specimens of breast cancer patients and breast cancer cell lines.Firstly,the correlation between LINC00365 and SCGB2A1 expression and the regulatory relationship between them were verified.Then the biological function of LINC00365 in breast cancer cells was characterized based on cell experiments.To explore the role of LINC00365-SCGB2A1 signaling pathway in the development of breast cancer and its specific mechanism.Methods:1.Real-time quantitative PCR was used to detect the transcription levels of LINC00365 and SCGB2A1 in tissue samples of 30 breast cancer patients(matched with cancer tissues and adjacent tissues),normal breast MCF-10A cells,breast cancer MCF-7 and T47D cells.2.The overexpression vector of LINC00365 was constructed and transfected.After overexpression of LINC00365 in MCF-7,T47D cells,the changes of transcription and protein level expression of SCGB2A1 were detected by qPCR and Western Blot.3.The overexpression vector of SCGB2A1 was constructed and transfected.After overexpression of SCGB2A1 in MCF-7,T47D cells,the transcription level of LINC00365 was detected by qPCR.4.The experiment is divided into six groups:(1)MCF-7_NC group:transfection of empty vector into MCF-7 cell.(2)MCF-7_LINC00365 group:transfection of LINC00365 overexpression vector into MCF-7 cell.(3)MCF-7_SCGB2A1 group:transfection of SCGB2A1 overexpression vector into MCF-7 cell.(4)T47D_NC group:transfection of empty vector into T47D cell.(5)T47D_LINC00365 group:transfection of LINC00365 overexpression vector into T47D cell.(6)T47D_SCGB2A1 group:transfection of SCGB2A1 overexpression vector into T47D cell.5.Cell proliferation and apoptosis detection:RTCA real-time was monitoring cell survival status,EdU cell proliferation and clone formation to detect cell proliferation ability.Flow cytometry was used to detect the apoptosis rate.Western Blot was used to detect the expression of Bc1-2 family protein and cleaved-Caspase3 protein expression.6.Detection of NF-κB activity:double luciferase reporter gene was used to detect the transcription activity of NF-κB,Western Blot was used to detect the expression of p-IκBα and IκBα protein,and indirect immunofluorescence was used to detect the expression of p50 protein in nucleus.The transcription levels of downstream target genes CXCL-8,TGF-β and c-IAP1 of NF-κB was detected by qPCR.Results:1.Compared with the adjacent tissues,the transcription expression of LINC00365 and SCGB2A2 was lower in breast cancer tissues,Pearson analysis showed that LINC00365 was positively correlated with the expression of SCGB2A1 in tissues(r = 0.682).Compared with normal MCF-10A cells,LINC00365 and SCGB2A1 transcription level showed low expression in breast cancer MCF-7 and T47D cells.2.After transfection with LINC00365 overexpression vector in MCF-7 and T47D cells,the LINC00365 transcription level,SCGB2A1 transcription level and protein expression were increased.3.After transfection with SCGB2A1 overexpression vector in MCF-7 and T47D cells,the transcription level and protein expression of SCGB2A1 was increased,while the transcription level of LINC00365 did not change significantly.4.Compared with MCF-7_NC group,the survival rate and proliferation ability of MCF-7_LINC00365 group and MCF-7_SCGB2A1 group were decreased,compared with T47D_NC group,the survival rate and proliferation ability of T47D_LINC00365 group and T47D_SCGB2A1 group were decreased.5.Compared with the MCF-7_NC group,the apoptosis rate of MCF-7_LINC00365 group and MCF-7_SCGB2A1 group were increased,the expression of cleaved-Caspase3 protein expression was increased,and the ratio of Bax/Bcl-2 was increased.Compared with T47D_NC group,the apoptosis rate of T47D_LINC00365 group and T47D_SCGB2A1 group were increased,the expression of cleaved-Caspase3 protein was increased,and the ratio of Bax/Bcl-2 was increased.6.Compared with MCF-7_NC group,NF-κB transcriptional activity was decreased in MCF-7_LINC00365 group and MCF-7_SCGB2A1 group,p-IκBαprotein expression was decreased,IκBα protein expression was increased,and p50 protein expression in nuclear was decreased,the transcription level of CXCL-8,TGF-β and c-IAP1 was decreased.Compared with T47D_NC group,NF-κB transcriptional activity was decreased in T47D_LINC00365 group and T47D_SCGB2A1 group,p-IκBα protein expression was decreased,IκBα protein expression was increased,and p50 protein expression in nuclear was decreased,CXCL-8,TGF-p and c-IAP1 transcription level was reduced.Conclusions:1.Compared with adjacent tissues and normal breast cells,LINC00365 and SCGB2A1 in breast cancer tissues and breast cancer cells showed low expression in transcription level and LINC00365 could positively regulate SCGBWA1 transcription and protein expression2.The LINC00365-SCGB2A1 signaling pathway inhibits breast cancer cell proliferation and promotes apoptosis by regulating NF-κB activation.