| Objectives:Bone metastasis is treated as one of the most frequent complications of advanced lung cancer.At present,the pathogenesis of lung cancer bone metastasis is unclear.Studies have shown that miRNAs play an important role in distant metastasis of tumors.Through high-throughput sequencing,the research group found 28 miRNAs differentially expressed in blood samples from patients with lung adenocarcinoma bone metastasis(BM)and non bone metastasis(NBM)patients.Among them,miR-106a was significantly highly expressed in BM samples.Real-time Quantitative PCR(RT-qPCR)was used to verify that the expression level of miR106a was consistent with the results of miRNA high-throughput sequencing.It suggests that miR-106a may closely related to bone metastasis of lung adenocarcinoma.Subsequently,the miRNA-target prediction databases results shown that miR-106a has a targeted binding relationship with Tumor Protein 53-induced Nuclear Protein 1(TP53INP1)in the p53 signaling pathway.This project intends to investigate the expression of miR-106a in patients with lung adenocarcinoma bone metastasis and non-bone metastasis and its clinical prognostic significance,and to explain the phenotypic function of miR-106a in promoting the proliferation and metastasis of lung adenocarcinoma cells through in vitro and in vivo experiments.Then,we further verified whether TP53INP1 is the direct target of miR-106a and analyzed the interaction between them,in order to explore the molecular mechanism of miR-106a regulating lung adenocarcinoma invasion and metastasis by targeting TP53INP1.Through the research project,the new mechanism of miR-106a regulating bone metastasis of lung adenocarcinoma will be clarified,and it will provide a theoretical basis for the further development of new drugs targeting lung adenocarcinoma bone metastasis in the clinic.MethodsPart 1:In Situ Hybridization(ISH)and RT-qPCR were used to detect the expression of miR-106a in paraffin sections of lung tissueswhich were from 80 patients with BM and NBM of lung adenocarcinoma.The Kaplan-Meier method was used to draw the survival curve,and the Cox regression hazard function model was established for multivariate analysis of patients’ survival and prognosis.Part 2:CCK-8 assays were used to detect the effect of mimic and inhibitor transfected with miR-106a on the proliferation of SPC-A1 and A549 cells.Wound Healing assay and Transwell migration assay were used to observe the effects of mimic and inhibitor transfection with miR-106a on the migration and invasion of SPC-A1 and A549 cells.The nude mice model of lung adenocarcinoma bone metastasis were X-ray,micro-CT,HE and other methods were used to compare the effects of miR-106a agomir/antagomir transfection on tumour cell growth in bone and tumour-induced bone production.The differentially expressed genes in the most important biochemical metabolic pathways and signal transduction pathways were analyzed through transcriptome RNA-seq KEGG.After transfection of miR-106a mimic and miR-106a inhibitor in SPC-A1 and A549 cells,and the changes in p53 expression levels were detected by RT-qPCR and Western blot.Subsequently,the GFP-P53 plasmid was transfected to detect the localization of p53 between cells.Bioinformatics analysis was used to screen target genes directly acting downstream of miR-106a,and correlation analysis with miR-106a expression in the tissue samples of two groups via immunohistochemical analysis(IHC)was also performed.Finally,the results were verified by dual luciferase reporter gene detection and retro-assays.Part 3:(1)At the cellular level,the effect of miR-106a/TP53INP1 on the migration and invasion abilities of SPC-A1 and A549 cells were detected.At the animal level,the effects of miR-106a/TP53INP1 on the formation and growth of bone metastases of lung adenocarcinoma in nude mice were compared and analyzed.(2)Western blot was used to analyze the expression of EMT-related proteins after miR106a mimic transfection.(3)SPC-A1 cells treated with miR-106a mimic and TP53INP1 were subjected to Fluorescence activated Cell Sorting(FACS)to analyze the proportion of apoptotic cells in different treatment groups.The expression of p21,Bax and Pig3 were detected by Western blot.(4)The GFP-LC3 indicator system was constructed and the effect of miR-106a/TP53INP1 on autophagy activity in SPC-A1 and A549 cells was evaluated by fluorescence microscope.Western blot was used to detect the effect of miR-106a/TP53INP1 on the expression of autophagy markers LC3II and P62.The cell clone formation assay was used to detect the effect of the clonal formation rate of SPC-A1 cells which was regulated by autophagy-related protein Atg5 on miR-106a/TP53INP1.After adding the autophagy inhibitor 3methyladenine(3-MA)and knocking out Atg5,the changes in miR-106a regulating the migration and invasion of SPC-A1 and A549 cells were detected.The Atg5 gene was further used to silence the cell line and the changes in EMT were analyzed after transfection of TP53INP1 for 48 hours.Finally,IHC experiments were used to determine the correlation between the expression of key autophagy-related proteins and bone metastasis in lung and bone tissue samples of patients with lung adenocarcinoma.ResultsPart 1:ISH detection found that the expression of miR-106a in lung tissue with bone metastasis was significantly higher than that in lung tissue without bone metastasis(P<0.01).The RT-qPCR test results also verified that the expression of miR-106a in the BM group of lung adenocarcinoma was significantly up-regulated(P<0.001).Correlation analysis found that bone metastasis and the expression of miR106a are factors influencing the overall survival of patients(both P<0.05),and patients with high miR-106a expression had a longer overall survival time than those with low expression.BM and high expression of miR-106a were independent risk factors for poor prognosis in patients with lung adenocarcinoma.Part 2:Cell function test results showed that compared with miR-NCtransfected cells,the miR-106a mimic significantly enhanced the proliferation,migration and invasion capacities of SPC-A1 and A549 cells(P<0.05).The results of X-ray imaging and micro-CT results both confirmed that miR-106a agomir transfection promoted the metastasis of cancer cells to bones and other tissues:The area of tibia bone destruction in model nude mice was significantly increased(P<0.05),while the transfection of miR-106a antagomir had the opposite results.The bone HE stained sections of nude mice injected with miR-106a agomir lung adenocarcinoma cells showed that the bone marrow cavity of the tibia was completely occupied by dark purple tumor cells,and the proportion of bone destruction area increased.The differentially expressed genes between miR-NC and miR-106a-transfected cells are significantly enriched in the p53 signaling pathway.Bioinformatics showed that TP53INP1,SFN,IGF1 and ZMAT3 may be the target genes of miR-106a.The expression levels of TP53INP1,Stratifin(SFN),Insulin-like Growth Factor(IGF1)and Zinc Finger Matrin-Type 3(ZMAT3)detected by qRT-PCR were consistent with the RNA sequencing results.Correlation analysis verified that the expression levels of miR-106a and TP53INP1 were in the lung glands.There was a significant negative correlation in cancer bone metastases(P=0.04,Rz=0.101).The dual luciferase reporter gene assay and the retro-assays verified the targeted interaction of miR-106a and TP53INP1.Part 3:(1)The overexpression of TP53INP1 significantly reversed the promotion of miR-106a on the migration and invasion of lung adenocarcinoma cells(P<0.05).TP53INP1 partially antagonized the promotion of miR-106a on bone metastasis of lung adenocarcinoma in nude mice(P<0.05).(2)miR-106a reduced the level of E-cadherin and increased the levels of vimentin and Snail,and miR-106a enhanced the phosphorylation level of Smad2/3.But this effect was partially blocked by the up-regulation of TP53INP1.(3)The overexpression of TP53INP1 significantly increased the late apoptosis of SPC-A1 cells and the expression levels of the apoptosis-related proteins p21,Bax and Pig3,but miR-106a partially reversed this promotion.(4)Transfection of miR-106a mimic did not affect autophagy in A549 and SPC-A1 cells,but miR-106a mimic could significantly antagonize the increase of intracellular GFP-LC3 fluorescent spots induced by overexpression of TP53INP1.Compared with the miR-106a mimic group,the expression of LC3II protein in the miR-NC+TP53INP1 group increased,and the expression of P62 protein decreased.Simultaneously,the miR-106a mimic+TP53INP1 group could partially reverse this regulatory effect.Cell colony formation assays confirmed that TP53INP1 partially reversed miR-106a’s promotion of lung adenocarcinoma clone formation rate.At the same time,silencing Atg5 significantly reduced miR-106a’s promotion of colony number.Wound healing and Transwell migration assay found that adding 3-MA or silencing Atg5 can significantly reduce the cell migration and invasion induced by miR-106a.Western blot results for LC3II and P62 confirmed that autophagy was inhibited in Atg5 KD cells.Compared with normal cells,the expression level of Ecadherin in autophagic inhibiting cells was significantly upregulated,and the expression level of Snail and Vimentin was significantly reduced.Moreover,the inhibiting effect of TP53INP1 on EMT was antagonized by autophagy inhibition.IHC detection found that p53 and TP53INP1 protein in BM group was lower than that of NBM,and the expression levels of LC3 and mTOR in metastatic bone tissue were higher than those in primary lung adenocarcinoma tissue.Conclusions1.Compared with the NBM group,the expression of miR-106a was significantly higher in the BM group,and was related to the poor clinical prognosis of patients with lung adenocarcinoma.2.In vivo and in vitro experiments had verified that miR-106a has the function of promoting bone metastasis of lung adenocarcinoma.3.The direct target of miR-106a was TP53INP1,and TP53INP1 was negatively correlated with bone metastasis of lung cancer.4.TP53INP1 could partially reverse the promoting effect of miR-106a on bone metastasis.5.The regulation of miR-106a/TP53INP1 on lung adenocarcinoma bone metastasis partly depended on EMT and autophagy-dependent cell death. |
PDF Full Text Request