TGFβ1 Promotes Epithelial-mesenchymal Transition And Stem Cell Characteristics By Down-regulating MiR-138-5p In Lung Cancer Cells And Its Mechanism

BackgroundLung cancer is the world’s highest morbidity and mortality,causing approximately 1.6 million deaths worldwide each year.The symptoms of early stage lung cancer are dormant and characterless,which often leads to the loss of surgical opportunities when patients are newly diagnosed,making chemotherapy or chemotherapy combined with radiotherapy the main treatment for patients with advanced lung cancer.Despite the continuous innovation in the therapy of lung cancer in recent years,the 5-year survival rate of lung cancer patients is still as low as 15%.Insufficient understanding of the pathophysiological mechanisms of lung cancer incidence and progression may be one of the reasons for the development of lung cancer treatment into the bottleneck period.In recent years,the theory of cancer stem cells has been proposed and confirmed,introducing new ideas to the concept and treatment of tumors.The study found that tumor tissue is not composed of a single type of tumor cells,but its constituent cells are highly heterogeneous.Cancer stem cell subsets account for only a small proportion of tumor tissue,but have strong ability to form spheroid,self-renew,migrate and initiate tumor.The origin of tumor stem cell subsets is unclear,and studies have suggested that its origin associate with reprogramming during tumor cell epithelial-mesenchymal transition.Although cancer stem cells play a key role in tumor progression and treatment,the regulation mechanism of its intrinsic stem cell characteristics at the molecular level is unclear.As a member of the TGF-β superfamily,transforming growth factor β1 is a pleiotropic cellular negative regulator associated with epithelial tumor growth,which induces epithelial-mesenchymal transition in tumor cells,thereby conferring stem cell-like properties to transformed cells.A large number of studies have shown that TGFβ1 plays its regulatory role mainly through protein-protein interactions,and there is no literature on the evidence that miRNAs participate in this process.MiRNAs are 18-22 nt non-coding RNAs and are involved in the regulation of a variety of cellular biological processes.MiRNAs are associated with multiple human diseases,including malignant tumors.Unlike mRNA,miRNAs are relatively stable,short in length,and can significantly regulate target gene expression,making miRNA a new biomarker and a potential therapeutic target.At present,the role of miRNAs in TGFβ1-regulated lung cancer cell EMT and CSC maintenance is still unclear.Therefore,in order to find miRNAs that may be involved in TGFβ1-induced EMT and CSC maintenance,this study intends to screen key miRNAs involved in the regulation of TGFβ1-induced lung cancer stem cell subpopulation enrichment using next-generation sequencing technology.Furthermore,in combination with literature research,miR-138-5p,which has significant differences in expression profiles,was selected for further study.PartⅠ: Screening of key miRNAs Involved in the regulation of TGFβ1-mediated Lung Cancer Stem Cell Subpopulation Enrichment ObjectivesTo screen key miRNAs involved in the regulation of TGFβ1-mediated lung cancer stem cell subpopulation enrichment.Methods1.The morphology of primary lung cancer cells LC006 and LC021 was observed to detect the epithelial-mesenchymal transition by induced by TGFβ1.2.Real-time quantitative PCR was used to evaluate the expression of epithelial-mesenchymal transition-related genes in primary lung cancer cells LC006 and LC021 induced by TGFβ1.3.Flow cytometry was performed to analyze the changes of cancer stem cells ratio in primary lung cancer cells LC006 and LC021 induced by TGFβ1.4.The CSC and non-CSC subpopulations in TGFβ1-induced LC021 cell lines were sorted by flow cytometry,and the miRNA differential expression profile in lung CSC subsets enriched by TGFβ1 were scanned and analyzed by next-generation sequencing.Results1.TGFβ1 treatment can induce epithelial to mesenchymal transition in primary lung cancer cells in a time-dependent manner.2.TGFβ1 can enrich cancer stem cell subsets in primary lung cancer cells in a time-dependent manner.3.There are 65 significantly different known miRNAs and 8 significantly different novel miRNAs in lung cancer stem cell subpopulation enriched by TGFβ1.ConclusionsThere are 65 significantly different known miRNAs and 8 significantly different novel miRNAs in the TGFβ1-induced enriched lung cancer stem cell subpopulation.PartⅡ: TGFβ1 Promotes Epithelial-mesenchymal Transition and Stem Cell Characteristics by Down-regulating miR-138-5p in Lung Cancer Cells and its Mechanism ObjectivesTo explore the regulation mechanism of TGFβ1 promoting epithelial-mesenchymal transition and stem cell characteristics by down-regulating miR-138-5p in lung cancer cells.Methods1.Real-time quantitative PCR was used to detect the expression of miR-138-5p in primary lung cancer cells LC006 and LC021 and lung cancer cell line A549 induced by TGFβ1.2.The lung cancer cell lines stably overexpressing and underexpressing miR-138-5p were established by constructing the recombinant lentiviral vector overexpressing and knocking-down miR-138-5p.3.Real-time quantitative PCR was used to detect the expression level of epithelial-mesenchymal transition-related genes in lung cancer cells with different miR-138-5p expression levels.4.The cell migration ability of the lung cancer cells with different miR-138-5p expression levels was compared by cell scratch assay.5.Through the monoclonal formation experiment,the difference in single cell clone formation ability of lung cancer cells with different miR-138-5p expression levels was observed.6.Using plate colony formation assay,the difference in plate colony forming ability of lung cancer cells with different miR-138-5p expression levels was analyzed.7.MTS assay was performed to detect the difference in sensitivity of lung cancer cells with different miR-138-5p expression levels to chemotherapeutic drugs cisplatin and paclitaxel.Results1.The expression level of miR-138-5p in lung cancer cells was down-regulated in a time-dependent manner after TGFβ1 treatment.2.MiR-138-5p can reverse the occurrence of epithelial-mesenchymal transition in lung cancer cells.3.MiR-138-5p can attenuate the migration ability of lung cancer cells.4.MiR-138-5p can reduce the single cell clone formation ability of lung cancer cells.5.MiR-138-5p can inhibit the colony forming ability of lung cancer cells.6.MiR-138-5p can increase the sensitivity of lung cancer cells to chemotherapy.ConclusionsTGFβ1 promotes epithelial-mesenchymal transition and its stem cell characteristics by down-regulating miR-138-5p in lung cancer cells,thereby reducing the sensitivity of lung cancer cells to chemotherapy drugs.Conclusions1.There are 65 significantly different known miRNAs and 8 significantly different novel miRNAs in the TGFβ1-induced enriched lung cancer stem cell subpopulation.2.TGFβ1 promotes epithelial-mesenchymal transition and its stem cell characteristics by down-regulating miR-138-5p in lung cancer cells,thereby reducing the sensitivity of lung cancer cells to chemotherapy drugs.