The Role And Underlying Mechanisms Of Wnt/β-catenin Pathway In Regulating Cancer Stem Cells Of Non-small Cell Lung Cancer Under Hypoxic Microenvironment

Lung cancer is the most common malignant tumor all over the world,and it has the highest incidence and cause of death in malignant tumors in China.According to pathological types,lung cancer can be divided into small cell lung cancer and non-small cell lung cancer(NSCLC).Non-small cell lung cancer is the most common pathological type,accounting for about 85%of the total number of lung cancer.With the deterioration of air pollution and the continuous growth of smoking population in China in recent years,the incidence of lung cancer is still increasing year by year.Although the cure rate and survival time of some lung cancer subtypes have increased in recent years,the overall therapeutic effect is still not satisfactory.We urgently need to explore related factors and participation mechanisms that affect the development of non-small cell lung cancer,and provide a theoretical basis for the improvement of treatment strategies.Cancer stem cells(CSCs)are the root cause of infinite proliferation and recurrence of malignant tumors,and have been a research hotspot in the field of cancer in recent years.CSCs are closely related to many malignant phenotypes such as tumor cell proliferation,invasion and metastasis,and therapeutic resistance.Studies have shown that CSCs play a key role in the development,development and drug resistance of non-small cell lung cancer.The treatment strategy for cancer stem cells has become one of the important development directions of cancer treatment such as lung cancer.At present,most of the lung cancer CSCs markers used in research include ALDH,CD 133 and ABCG2.However,the specific mechanism for regulating the proportion and function of CSCs in non-small cell lung cancer is still not clear.Wnt/β-catenin signaling pathway is one of the important signaling mechanisms in cells.It is involved in the regulation of various physiological processes in the body.Its abnormal expression and regulation are involved in the occurrence and development of various diseases,especially tumors.This pathway is named for its promoter protein WNT.WNT is a cysteine-rich glycoprotein.When it binds to the Frizzled family of transmembrane protein receptors on the cell membrane surface,it activates the classical WNT/β-catenin signaling pathway,allowing β-catenin to be stably present in the cytoplasm.Part of β-catenin enters the nucleus and interacts with the TCF/LEF transcription factor family and promotes the expression of specific target genes.Previous studies have confirmed that the Wnt/β-catenin pathway plays an important role in tumorigenesis,tumor angiogenesis and tumor stem cell self-renewal.However,in the tumor microenvironment,the Wnt/β-catenin pathway has little research on the regulation of CSCs and the factors involved in different pathological types of non-small cell lung cancer.Hypoxia is one of the important features of the solid tumor microenvironment.It not only affects the biological behavior of tumor cells themselves,but also may significantly affect the various characteristics of CSCs,including stem cell ratio,invasion and metastasis ability and sensitivity to drugs.Studying the regulatory mechanisms and participating molecules of CSCs in hypoxic tumor microenvironment can better reflect the regulatory pathways and mechanisms involved in the local CSCs.At present,there are few related reports,and it can provide a more accurate theoretical basis for the improvement of malignant phenotype and treatment strategy for non-small cell lung cancer.This study investigated the effects of Wnt/β-catenin signaling pathway on the proportion of tumor stem cells and invasion ability in non-small cell lung cancer(including different types of lung cancer cells:lung adenocarcinoma A549 cells and lung squamous cell carcinoma H520 cells)in hypoxic microenvironment.This study examined the expression of Wnt/β-catenin pathway and HIF-1α,a key regulator of hypoxia in clinicopathological specimens of patients with non-small cell lung cancer,and analyzed the correlation between them and the survival prognosis of patients.Further validation was provided for the results of the in vitro studies.Basic fibroblast growth factor(bFGF)is widely expressed in various malignant tumors including lung cancer.It not only plays an important role in the self-renewal of normal stem cells such as bone marrow mesenchymal stem cells and embryonic stem cells,but also participates in the process of dry maintenance and invasion of CSCs and treatment of drug resistance.However,the relationship between bFGF expression and Wnt/β-catenin signaling pathway is still unclear.Therefore,this study also explored whether Wnt/β-catenin signaling pathway can regulate the expression of bFGF,which in turn affects chemoresistance induced by CSC.Therefore,this study explores the factors and mechanisms of Wnt/β-catenin pathway regulation of CSCs from the perspective of bFGF.Part I Effect of Wnt/p-catenin signaling pathway on tumor stem cell ratio and invasion ability in non-small cell lung cancer under tumor hypoxia microenvironment(in vitro study)Objectives:Hypoxia is an important feature of the tumor microenvironment and can affect a variety of malignant biological behaviors in non-small cell lung cancer.This part of the study focused on the effect of Wnt/β-catenin signaling pathway on tumor stem cell ratio and invasion ability in non-small cell lung cancer under tumor hypoxic microenvironment.Non-small cell lung cancer mainly includes lung adenocarcinoma and lung squamous cell carcinoma.A large number of basic and clinical studies have demonstrated that lung adenocarcinoma and lung squamous cell carcinoma have different biological properties and gene/molecular expression.Therefore,we conducted related studies on lung adenocarcinoma A549 cells and lung squamous cell carcinoma H520 cells.Methods:1.Lung adenocarcinoma A549 cells and lung squamous cell carcinoma H520 cells were cultured under normoxia(21%02,5%CO2)and hypoxia(1%02,5%C02 and 94%N2 mixed gas).After 48 hours of culture,the total protein was extracted and the expression of β-catenin was detected by western blot.2.Lung adenocarcinoma A549 cells and lung squamous cell carcinoma H520 cells were cultured under normoxia and hypoxia,respectively.Treat and incubate cells as required by the ALDEFLUORTM ASSAY kit instructions.The ratio of ALDH+ cells in A549 cells and H520 cells was detected by flow cytometry.The main research is the effect of hypoxia on the proportion of ALDH+ cells.3.A549 cells and H520 cells were cultured under normoxic conditions.When the cells were grown to a suitable state,the Wnt pathway inhibitor XAV939 was added to treat the cells.The expression of β-catenin was detected by western blot,and the ratio of ALDH+ cells was detected by ALDEFLUORTM ASSAY.The lung adenocarcinoma A549 cells and lung squamous cell carcinoma H520 cells were cultured under normoxia and hypoxia,respectively.The effect of hypoxia on the invasive ability of A549 and H520 cells was detected by Matrigel Matrigel-coated Transwell chamber system.5.HIF family genes(HIFla,HIF2a,etc.)are key regulators of cellular responses to hypoxia.A549 cells and H520 cells were cultured under normoxia and hypoxia,respectively.After the culture,the cellular RNA was extracted,and the expression levels of HIF la and HIF2a mRNA were detected by qRT-PCR.Cellular proteins were extracted and Western blot was used to detect changes in HIF 1α protein expression.6.A specific inhibitor XAV939 was added to A549 cells and H520 cells cultured under normoxic conditions to inhibit the Wnt pathway.The expression of HIF 1α protein was detected by western blot.Results:1.Lung adenocarcinoma A549 cells were cultured under normoxia and hypoxia for 48 h,and western blot analysis showed that the expression of β-catenin of Wnt/β-catenin signaling was significantly down-regulated.The results of ALDEFLUO ASSAY showed that the proportion of ALDH+ cells in lung adenocarcinoma A549 cells decreased significantly after 48 h of hypoxia culture(P<0.05);2.On the other hand,lung squamous cell carcinoma H520 cells cultured under hypoxia for 48 h showed significantly increased β-catenin expression compared to H520 cells cultured under normoxia,andthe proportion of ALDH+ cells in lung squamous cell carcinoma H520 cells also significantly increased(P<0.05).This indicates that hypoxia has different regulatory effects on the ratio of ALDH+ cells in lung adenocarcinoma and lung squamous cell carcinoma.3.After adding Wnt pathway inhibitor XAV939 to A549 cells and H520 cells under normoxic conditions,the expression of β-catenin was significantly decreased.The results of ALDEFLUO ASSAY showed that the ratio of ALDH+ cells was significantly decreased in both A549 cells and H520 cells.4.The effect of hypoxia on the invasive ability of A549 and H520 cells by Transwell invasion assay showed that the invasive ability of lung adenocarcinoma A549 cells and lung squamous cell carcinoma H520 cells were significantly increased(P<0.05).5.Hypoxia significantly up-regulated the expression of HIFla mRNA in A549 cells and H520 cells,while the expression of HIF2a mRNA was not significantly different under normoxia and hypoxia.The results of western blot further confirmed that the expression level of HIFla protein was also significantly increased.6.After the specific inhibitor XAV939 was used to inhibit the expression of Wnt pathway,the expression of HIFla was also significantly decreased.This suggests that there is a mutual regulation of hypoxia and Wnt/β-catenin signaling pathways.Conclusions:Hypoxia significantly decreased the expression of β-catenin in lung adenocarcinoma A549 cells,and the proportion of ALDH+ cells also decreased significantly.However,the expression ofβ-catenin was significantly increased in lung squamous cell carcinoma H520 cells,and the proportion of ALDH+ cells was significantly increased,indicating that hypoxia has different regulatory effects on the ratio of ALDH+ cells in lung adenocarcinoma and lung squamous cell carcinoma.The expression of β-catenin was significantly decreased after the activation of Wnt pathway inhibitor in A549 cells and H520 cells,and the proportion of ALDH+ cells in A549 cells or H520 cells was significantly decreased.Hypoxia significantly promoted the invasive ability of lung adenocarcinoma A549 cells and lung squamous cell carcinoma H520 cells.Hypoxia significantly up-regulated the expression of HIFla mRNA and protein levels in A549 cells and H520 cells.The expression of HIFla was down-regulated after inhibition of Wnt pathway expression by specific inhibitors,suggesting that hypoxia and Wnt/β-catenin signaling pathways are mutually regulated.Part Ⅱ Correlation analysis of β-catenin and HIF-1α expression in pathological tissues of patients with non-small cell lung cancer and its influence on survival prognosis of patientsObjectives:The expression of β-catenin and HIF-1α in tumor tissues of patients with advanced NSCLC was detected by immunohistochemistry.The correlation between β-catenin and HIF-1αexpression was statistically analyzed.The survival outcome of patients with advanced NSCLC was followed up and whether P-catenin expression was associated with the survival prognosis of patients with NSCLC was also evaluated.The results from clinical specimen analyses would provide more proof for the experimental findings in the former part.Methods:1.The clinical and pathological data of 76 patients with advanced non-small cell lung cancer were collected,including gender,age,smoking history,,pathological type,differentiation degree and lymph node metastasis.The overall survival(OS)was followed up.Pathological tissue specimens of these patients were also collected.2.The expression of β-catenin and HIF-1α in 76 patients with advanced non-small cell lung cancer were detected by immunohistochemistry.The correlation between β-catenin and HIF-1αexpression was analyzed by chi-square test.3.Kaplan-Meier survival analysis was used to study the correlation between P-catenin expression and OS.Results:1.Immunohistochemical results showed that β-catenin has a high expression rate in the pathological tissues of patients with advanced non-small cell lung cancer(positive rate>85%).The expression of β-catenin was associated with TNM staging and had no significant correlation with other clinicopathological factors.HIF-la was expressed in different degrees in the pathological tissues of patients with non-small cell lung cancer.Statistical analysis did not find significant correlation with clinical and pathological factors.2.Spearman’s rank test analysis showed that β-catenin expression was correlated with HIF-1αexpression(P<0.001);3.Survival analysis showed that the survival prognosis of patients with advanced non-small cell lung cancer with high expression of P-catenin was significantly worse than that of low expression group(P<0.01).Conclusions:1.The expression of β-catenin and HIF-1α can be detected in the pathological tissues of patients with advanced non-small cell lung cancer,and the expression of the former is related to TNM staging.2.Statistical analysis confirmed there was close correlation between between β-catenin expression and HIF-la expression;3.The expression of β-catenin was correlated with the prognosis of patients.The OS of patients with high expression of P-catenin was significantly lower than that with low β-catenin expression.4.The results of pathological examination of patients with non-small cell lung cancer further validate the results of the first part of the laboratory study,and provide more molecular indicators and basis for prognosis prediction of patients with non-small cell lung cancer.Part Ⅲ The effect of Wnt/β-catenin signaling pathway on the expression of bFGF on the characteristics of tumor stem cells in non-small cell lung cancer cell linesObjectives:To investigate whether Wnt/β-catenin signaling pathway affects the stem cell characteristics of non-small cell lung cancer(including OCT-4 expression,chemoresistance,etc.)by regulating the expression of bFGF.Methods:1.Wln pathway mutation activation plasmid pcDNA3-S33Y-β-catenin and control plasmid pcDNA3 were used to transfect lung cancer A549 cells,and transfected cells were screened by G418.Western blot was used to detect the expression of P-catenin,and it was verified whether the cell line overexpressing P-catenin was successfully established.2.qRT-PCR and western blot were used to detect the expression of bFGF in β-catenin overexpressing cell lines and control cell lines,and to explore whether Wnt/β-catenin signaling pathway can regulate bFGF expression.3.The expression of bFGF in A549 cells was knocked down by siRNA interference,and the knockdown effect was verified by qRT-PCR and western blot.4.After knockdown bFGF expression in A549 cells by specific siRNAs,the cell viability was analyzed by CCK-9 assay,and the cell apoptosis was detected by flow cytometry with Annexin V/PI kit.Hence,the effect of bFGF on A549 cell sensitivity to cisplatin sensitivity was investigated.5.bFGF siRNA down-regulated the expression of bFGF in A549 cells,and the clone formation assay tested its effect on the ability of A549 cells to clone.Western blot was used to detect the effect of OCT-4 on the key regulatory gene of CSCs.Results:1.The results of western blot analysis showed that the expression of β-catenin was significantly up-regulated compared with control and A549 cells,which indicated that β-catenin overexpressing cells A549 and control cells were successfully established.2.Compared with the control cell line,the expression of bFGF mRNA and protein levels inβ-catenin overexpressing cell line were significantly up-regulated,indicating that Wnt/β-catenin signaling pathway activation can promote bFGF expression;3.bFGF siRNA significantly inhibited the expression of bFGF in A549 cells,both at mRNA and protein levels;4.Compared with the control siRNA-treated group,bFGF siRNA significantly enhanced the inhibitory effect of different concentrations of cisplatin(2.5,5,and 10 μg/mL)on A549 cells.bFGF siRNA did not significantly affect the apoptosis rate of A549 cells,but it did significantly promot cisplatin-induced apoptosis(26.67%± 2.59%vs.9.05%± 1.20%,P<0.05).5.After bFGF siRNA down-regulated the expression of bFGF in A549 cells,the clone formation assay showed that the clone formation rate of A549 cells was significantly decreased(P<0.05),and the expression of OCT-4 was also down-regulated by western blot.Conclusions:Wnt/β-catenin signaling pathway significantly promoted the expression of bFGF in non-small cell lung cancer A549 cells,while bFGF promoted the proliferation of A549 cells and decreased their sensitivity to cisplatin.At the same time,bFGF also promoted the clonality of A549 cells and the expression of OCT-4.This indicates that Wnt/β-catenin signaling pathway can regulate the expression of bFGF in non-small cell lung cancer and further influence its stem cell characteristics such as colony forming ability,chemoresistance and OCT-4 expression.