ALKBH5 Inhibits TGF-?-induced Epithelial Mesenchymal Transition In Non-small-cell Lung Cancer

Background and Objectives:Lung cancer is one of the most malignant cancers that threaten human health and life.It has high morbidity and mortality.About 80%to 90%of all lung cancer patients are non-small cell lung cancer(NSCLC)patients.Up to now,the main treatments for lung cancer patients have been a variety of combined treatments mainly based on surgery.With the development of medical technology,molecular targeted medicine has become the first choice for treating lung cancer,which is an early treatment for NSCLC patients.However,the prognosis of most lung cancer patients is still very poor.According to statistics,the 5-year survival rate is only about 15%.And more than 90%of lung cancer patients die quickly due to the continuous metastasis of lung cancer cells.During the metastasis of lung cancer cells,EMT plays an important role,The occurrence of EMT is related to a variety of cytokines,transcription factors,signaling pathways and so on,among which transforming growth factor-? plays an important role in the process of NSCLC metastasis and invasion.In recent years,in the field of epigenetic research,it has been found that the RNA level N6-methyladenine(m6A)demethylase alk B homolog 5 is involved in the modification of many types of cancer development.However,its role in lung cancer and the study of its internal molecular mechanisms have been seldom described.especially in non-small cell lung cancer.Therefore,in this thesis,we start with the TGF-?/Smad-mediated EMT signaling pathway to explore the role played by ALKBH5 and the molecular mechanism by which this effect occurs in NSCLC cell linesMethods:(1)Real-time PCR was used to detect the expression levels of ALKBH5,SMAD3,SMAD7 and TGF?R2 mRNA in 97 pairs of NSCLC clinical samples,and to further analyze the correlation between ALKBH5 and SMAD3,SMAD7 and TGF?R2 mRNA expression levels.(2)After A549 and SPC-A1 cells were treated with TGF-?1 for a predetermined time,the expression levels of ALKBH5 mRNA and protein were detected by Real-time PCR and Western Blotting.(3)Construct and screen A549 stable transfected cell lines stably overexpressing ALKBH5,Real-time PCR and western blot were used to detect the overexpression efficiency of ALKBH5.TGF-?1 treatment induced EMT in A549 stable transfected cell lines for 24h.The effect of overexpression of ALKBH5 on the expression level of EMT marker protein under TGF-?1 treatment was further examined.(4)siRNAs(si-ALKBH5-1 and si-ALKBH5-2)targeting ALKBH5 were transiently transfected in A549 cells and SPC-A1 cells.Real-time PCR and Western Blotting were used to detect the knockdown effect of ALKBH5.TGF-?1 treatment induced EMT in NSCLC cells for 24h,and further examined the effect of interfering with ALKBH5 on the expression level of EMT marker protein under TGF-?1 treatment.(5)Transwell migration and invasion experiments were used to examine the effect of overexpression or knockdown of ALKBH5 on the migration and invasion ability of NSCLC cells induced by TGF-?1.(6)Establish nude mice lung cancer metastasis model and detect the effect of ALKBH5 overexpression on the transfer ability of A549 cells in vivo.(7)Real-time PCR and Western Blotting were used to analyze the effect of overexpression of ALKBH5 on the mRNA and protein expression levels of key factors of TGF-?/Smad signaling pathway(such as TGFBR1,TGFBR2 and Smads).(8)The RNA Binding Protein Immunoprecipitation technique was used to detect the effect of overexpression of ALKBH5 on the level of m6A modification of key genes in TGF-?/Smad signaling pathway(such as TGFBR1,TGFBR2 and Smads).(9)siRNAs were used to knock down the YTHDF1-3 family of m6A recognition proteins,and Real-time PCR was used to detect the knockdown effect of YTHDF1-3.Further test whether knocking down YTHDF1-3 affects the regulation of ALKBH5 on the mRNA level of key genes in TGF-(3/Smad signaling pathwayResults:(1)The detection of 97 clinical samples of NSCLC found that the expression level of ALKBH5 mRNA in cancer tissues was significantly down-regulated(p<0.001)compared with adjacent tissues;compared with non-metastatic group(no lymph node metastasis and distant metastasis),metastatic ALKBH5,SMAD3 and TGF?R2 mRNA(T/N)levels were significantly up-regulated(p<0.05),while SMAD7 mRNA(T/N)levels were significantly down-regulated(p<0.001).Further analysis revealed that ALKBH5 was positively correlated with SMAD3 and TGF?R2 mRNA levels(p<0.05),but negatively correlated with SMAD7 mRNA expression levels(p<0.05)(2)TGF-? inhibited the expression of ALKKB5 mRNA and protein in NSCLC cells,and it gradually decreased with the increase of TGF-?treatment time(0,2,4,8,12,24h).(3)The NSCLC over-expressing NSCLC stable cell line was successfully constructed and screened.TGF-?1 stimulation for 24 h can induce EMT in A549 and SPC-A1 stable transfected cell lines.Overexpression of ALKBH5 significantly promotes the expression of TGF-?1 stimulated epithelial marker molecules and significantly inhibits the expression of TGF-?1 stimulated interstitial marker molecules.(4)si-ALKBH5-1/2 can effectively knock down the expression of ALKBH5.TGF-?1 stimulation for 24h can induce EMT in A549 and SPC-A1 transient knockdown cell lines,knocking down ALKBH5 significantly inhibits the expression of epithelial marker molecules under TGF-?1 stimulation,and significantly promotes the expression of interstitial marker molecules under TGF-?1 stimulation.(5)Overexpression of ALKBH5 significantly inhibited the migration and invasion ability of NSCLC cells induced by TGF-?1,while knocking down ALKBH5 significantly promoted the migration and invasion ability of NSCLC cells induced by TGF-?1.(6)In the nude mice lung metastasis model,overexpression of ALKBH5 significantly inhibited the ability of A549 cells to metastasize in vivo.(7)Overexpression of ALKBH5 inhibits the mRNA and protein levels of the positive regulators of TGF?/Smad pathway SMAD3 and TGFOR2,and promotes the mRNA and protein levels of the negative regulators of TGF?/Smad pathway SMAD7.(8)Overexpression of ALKBH5 broadly inhibits m6A levels of key genes SMAD3,SMAD7 and TGF?R2 in the TGF-?/Smad signaling pathway.(9)m6A recognition proteins YTHDF1 and 3 play a role in the negative regulation of ALKBH5 expression of SMAD3 and TGF?R2,while YTHDF2 plays a key role in ALKBH5 promoting SMAD7 expression.Conclusion:This study initially confirmed that ALKBH5 inhibits TGF-?1-induced EMT and metastasis of NSCLC.The possible mechanism is that ALKBH5 accurately inhibits the m6A levels of TGF?R2,SMAD3,and SMAD7,which the key factors of TGF-?/Smad signaling pathway,and then through the m6A recognition enzymes YTHDF1 and YTHDF3 negatively regulate the expression of SMAD3 and TGF?R2 and YTHDF2 positively regulates the expression of SMAD7,which play a synergistic role in cancer suppression.This study provides a new theoretical basis for inhibiting the transfer of NSCLC through the regulation of ALKBH5-mediated m6A modification to regulate TGF-?/Smad signaling pathway.