The Role And Mechanism Of LINC00665 In Breast Cancer Progression

BackgroudBreast cancer is one of the most common cancers in the world and the main leading cause of female-related mortality.Although some progress has been made in early diagnosis and treatment and systemic treatment,recurrence,metastasis,and drug resistance are still the keys to successful breast cancer treatment.Therefore,genes related to breast cancer cell proliferation,invasion,and metastasis were discovered,and new breast cancer potentials were sought Targets and targeted drugs are imminent.Long non-coding RNA(lnc RNA)is a type of nc RNAs with a length of more than 200 nucleotides and no protein coding function.lnc RNAs regulates the expression of genes through transcription,post-transcription,epigenetics,etc.It plays an important role in the biological process and has gradually attracted the attention of researchers.In recent years,with the deepening of understanding of lnc RNA,more and more evidence shows that long non-coding RNAs(lnc RNAs)play a key role in the occurrence and development of breast cancer.But the role of most lnc RNA in breast cancer remains unclear.A lot of evidence shows that many lnc RNAs can play the role of endogenous miRNA sponges by competitively binding ordinary miRNAs.The study found that the abnormal expression of LINC00665 exists in various malignant tumors,including liver cancer,gastric cancer,and lung cancer.However,whether LINC00665 affects breast cancer progression and its mechanism of action has not been reported.Research purposes:This study aims to reveal the role of LINC00665 in the development and progression of breast cancer and the molecular mechanisms involved,to provide potential therapeutic targets for the treatment of breast cancer,and provide new diagnostic indicators for the prognosis of breast cancer.Methods1.RT-qPCR was used to detect the difference in expression of LINC00665 in commonly used breast cancer cell lines.Cell lines with lower expression levels were selected for overexpression,and cell lines with higher expression levels were down-expressed.MTT,clone formation,and Ed U experiments were used to determine the effect of LINC00665 on the proliferation of breast cancer cells.Transwell and cell wound healing experiments were used to evaluate the changes in cell migration ability after LINC00665 regulation.2.After overexpression of LINC00665 in breast cancer cells,the expression changes of EMT markers were detected using Western Blotting,RT-qPCR and immunofluorescence experiments.3.The online database predicts that LINC00665 may interact with its competing endogenous RNA and its interacting miRNA.By predicting the binding site of both LINC00665 and ce RNA and their cooperating miRNAs,the corresponding binding site mutation vector and tool vector are constructed,and the luciferase reporter gene experiment and RNA co-precipitation(RIP)experiment are used to jointly verify LINC00665 Direct binding to downstream miRNAs and specific binding sites.4.After overexpression or interference with LINC00665,the RT-qPCR experimental method was used to verify the change in the expression level of the candidate miRNA,thereby further verifying the key downstream miRNA molecules of LINC00665.5.The Dual Reporter Gene Assay was passed to verify the interaction of LIN28 B with the target of miR-379-5p,and the expression level of LIN28 B mRNA in cells overexpressing or interfering with miR-379-5p was verified by RT-qPCR.The protein expression level of LIN28 B in cells interfered by miR-379-5p and cells overexpressing miR-379-5p was detected by Western Blotting.6.After interfering with LINC00665 in MDA-MB-231 cell line or overexpressing LINC00665 in T47 D cell line,RT-qPCR and Western Blotting experiments were used to detect the expression changes of LIN28 B and the expression changes of EMT indicators.A "Rescue" experiment was carried out to co-transfect the si RNA of LINC00665 and LIN28 B,and the expression difference of LIN28 B and EMT markers was again detected by Western Blotting experiment.And through cloning formation experiment,MTT to observe the proliferation ability of breast cancer cells,and Transwell to observe whether it reduced the migration ability of breast cancer cells.Results1.RT-qPCR results show that LINC00665 has different expression levels in different breast cancer cell lines.The expression level of LINC00665 is higher in MDA-MB-231 and BT549 cell lines,and lower in T47 D cell line.MTT,clone formation,and Ed U experiments showed that after interfering with LINC00665 in the MDA-MB-231 and BT549 cell lines,the cell proliferation ability decreased;after overexpression of LINC00665 in the T47 D cell line,the cell proliferation ability was enhanced.Transwell and cell wound healing experiments showed that after interfering with LINC00665 in MDA-MB-231 and BT549 cell lines,the cell migration ability decreased;after overexpression of LINC00665 in T47 D cell line,the cell migration ability increased.2.Western Blotting results showed that the mesenchymal markers Vimentin and N-cadherin in T47 D cells overexpressing LINC00665 were significantly up-regulated,and the expression of E-cadherin was significantly down-regulated.3.According to the star Base database prediction,the downstream target genes that may be regulated by LINC00665 include LIN28 B,miR-379-5p is selected for miRNA,and RTq-PCR experiment is used to verify the sponge adsorption of LINC00665 to miR-379-5p.Next,the interaction between LINC00665 and downstream miR-379-5p was further confirmed by RIP and luciferase reporters4.Dual reporter gene assay,RT-qPCR,Western Blotting experiment results have confirmed that miR-379-5p can target the regulation of LIN28 B expression.5.Western Blotting experiments showed that after overexpression of LINC00665,the expression of LIN28 B increased and E-cadherin was significantly down-regulated,but the mesenchymal markers Vimentin and N-cadherin were significantly up-regulated.the “rescue” experimental results,the expression of LIN28 B was down-regulated,the expression of mesenchymal markers N-cadherin and Vimentin decreased,and E-cadherin was significantly up-regulated.Moreover,after co-transfection of LINC00665 and LIN28 B si RNA,the proliferation and migration ability of the cells were reduced.Conclusion1.LINC00665 promotes breast cancer proliferation migration and tumor growth in vivo;2.LINC00665 produces an EMT-like phenotype in breast cancer;3.LINC00665 function as a sponge for miR-379-5p to regulate the LIN28 B expression;...