The Mechanism Research About The Regulation Of ZWINT In The Proliferation And Metastasis In Non-Small Cell Lung Cancer

Background : Lung cancer is the most common malignant tumor originating originally in the lung,with a high incidence rate and mortality rate,and the incidence rate is increasing year by year,which has brought a serious impact on people’s health.Lung cancers which is originating from bronchial epithelial cells or alveolar epithelial cells mostly classified histologically as non-small-cell lung cancer(NSCLC),accounting for more than 80%.Early-stage lung cancer can still be treated by surgery.Because many patients of lung cancer have entered the middle to advanced stage of the disease at the time of obtaining their initial diagnosis,and the tumor has already undergone local tissue invasion or distant metastasis.In this case,operation is difficult,and tumor metastasis is the main reason for the high mortality rate of lung cancer,which can cause the survival rate of lung cancer patients dropped sharply.As the understanding of the molecular mechanisms and potential biological markers of lung cancer has gradually improved,individualized treatment options such as immunotherapy and molecular targeted therapy are gradually applied in clinical practice.In recent years,specific genetic abnormalities have been found in some lung cancer patients,which has enabled the rapid development of individualized targeted therapy and opened up new ideas for the treatment of lung cancer patients.ZW 10 binding factor(Zeste White 10 Interactor,ZWINT),located between the chromosome and the spindle,is an important regulatory protein that can affect chromosome movement and the process of mitosis,thus regulating the cell life cycle.ZWINT is expressed in a variety of human malignancies and predicts a poor prognosis.Previous studies have reported that ZWINT can be overexpressed in NSCLC,but the effects of ZWINT on NSCLC and the underlying molecular mechanisms have not been elucidated.The study of ZWINT found that its downstream pathway enrichment prediction showed that its main regulation was cell division-related pathway,and the direct interacting proteins were BUB1 and NUF2.BUB1 is a spindle detection point protein that plays an important role in maintaining proper chromosome segregation and reducing alloploid production during mitosis.BUB1 and its kinase activity are an essential component of the TGFβsignaling pathway and promote the formation of TGFBRI/II receptor complexes,in which all three interact simultaneously and mediate TGFβ-dependent EMT,cell migration and invasion.Aberrant expression of NUF2,which is a gene encoding components of the Ndc 80 centromeric complex,promotes the progression of human cancer.NUF2 deficiency induces apoptosis by inducing cell cycle arrest in the G0/G1 phase and results in altered cell cycle distribution.Some researches have suggested that NUF2 may be involved in regulating EMT.The combined analysis of the histological database of TCGA and GTEx was able to identify the presence of ZWINT over expression in several human cancers,suggesting its important role in carcinogenesis.GO analysis revealed that ZWINT genes are not only involved in the cell cycle development of NSCLS,but also in cell division,chromosome segregation,and nucleoplasmic and cell peripheral development.The results of KEGG analysis further confirmed the effect of ZWINT on the cell cycle progression of NSCLC.Therefore,the aim of this study is to investigate the possible mechanisms of ZWINT involved in NSCLC proliferation and metastasis.Objective: By analyzing the expression level of ZWINT in NSCLC tissues and its relationship with clinicopathological factors such as tumor differentiation,lymph node metastasis,and TNM stage,to explore the role and molecular mechanism of ZWINT in the proliferation and metastasis of NSCLC lung cancer.Methods1.TCGA and GTEx were used in online databases to search clinical and postoperative sample data of lung cancer,compare the expression difference of ZWINT between cancer tissues and adjacent tissues of lung adenocarcinoma and lung squamous cell carcinoma,explore the relationship between ZWINT and various clinical pathological factors,and evaluate the clinical application value of ZWINT for the prognosis of lung cancer patients.2.Fifty fresh NSCLC tissue samples were collected,and ZWINT gene and protein expression levels were measured by fluorescence quantitative PCR and Western Blot,respectively.3.Another paraffin-embedded specimens of 60 cancerous and adjacent tissues(5cm away from tumor)of NSCLC patients who had not received chemoradiotherapy before surgery were collected.There were 40 lung adenocarcinoma(LUAD)and 20 lung squamous cell carcinoma(LUSC).The expression of ZWINT and EMT-related markers(E-cadherin,Vimentin,N-cadherin and Slug)in cancer tissues and adjacent tissues of 60 NSCLC patients was evaluated by immunohistochemistry,and the correlation between ZWINT and common clinicopathological features of lung cancer and the correlation of ZWINT protein and EMT-related proteins were analyzed.4.The expression of ZWINT in the lung cancer cell lines A549 and H1299 was determined by q RT-PCR and Western Blot.H1299 cell lines transfected by ZWINT plasmid,and A549 cell lines interfered by ZWINT-si RNA,were constructed,and the transfection efficiency was verified by q RT-PCR and Western Blot.Cell proliferation assay,scratch assay,migration assay,and flow cytometry were used to analyze the effects of ZWINT over expression and ZWINT knockdown on H1299 and A549 in proliferation,migration,invasion,and cell apoptosis.5.Western Blot and immunofluorescence experiments were used to detect the expression of the major proteins associated with EMT in H1299 and A549,respectively.6.Gene expression and protein content of BUB1 and NUF2 in each group(H1299,si-NC,si-ZWINT,vector,ZWINT-OE)were measured by PCR and Western Blot.7.H1299 lung cancer cell lines were transfected with BUB1 and NUF2 plasmid and interfered with BUB1-si RNA and NUF2-si RNA to construct H1299 with BUB1 and NUF2 over expression and interference,respectively,and verified by q RT-PCR and Western Blot.The effects of BUB1 and NUF2 on lung cancer cell line H1299 in proliferation,migration,invasion and apoptosis were examined by cell proliferation,scratch,migration,and flow cytometry.8.Western Blot and immunofluorescence experiments were used to observe the changes of EMT-related proteins during the interference and transfection of BUB1 and NUF2.Results:I.ZWINT was highly expressed in NSCLC tissues and was associated with a poor prognosis1.As shown by TCGA and GTEx,the ZWINT gene was differently expressed in the carcinoma and adjacent tissues of NSCLC and is highly expressed in the tumors.2.Pathway enrichment analysis revealed that the cell cycle gene ZWINT was significantly associated with many classical tumor-related pathways,suggesting an intrinsic link between the cell cycle and biological behaviors such as tumor immune microenvironment,metabolic reprogramming,cell death,and angiogenesis.3.GO analysis revealed that the ZWINT gene was not only involved in the cell cycle development of NSCLC,but also participated in cell division,chromosome segregation,the develpoment of nucleoplasm and cell periphery.4.Immune correlation studies showed there was significant association between ZWINT gene and the multiple infiltrating forms of immune cells.5.The ZWINT gene has a stronger effect on membrane development and stability in LUAD than on the extracellular matrix and some immunomodulatory responses in LUSC.6.The ZWINT gene has a stronger effect on the single nucleotide variant(SNV)and copy number variant(CNV)mutations in LUAD than that in LUSC.7.Gene expression of ZWINT in 50 fresh NSCLC tissues and their paired adjacent frozen tissues,45 of which showed significantly higher relative ZWINTm RNA expression in NSCLC tissues compared to adjacent normal tissues,and the median value of Normal group was 1.241(1.013-2.575).The median value in the NSCLC group was 6.821(2.304-12.486),and the difference was statistically significant(P<0.001).8.According to the results of immunohistochemistry,ZWINT showed positive staining mainly in the cytoplasm.Of the 50 cases of NSCLC,42 cases had high expression of ZWINT protein,and the expression rate was 84%.Compared with the adjacent control group,the high expression rate of ZWINT was significantly higher in the NSCLC group(P <0.001).9.Among 60 NSCLC patients,there was no significant difference in the expression of the ZWINT protein among sex,age,pathological classification and the varying degrees of tumor differentiation(P>0.05).Of the 40 NSCLC cases of stage I + II,25 had high ZWINT protein expression.Of the 20 NSCLC cases of stage III + IV,18 had high ZWINT protein expression.High expression rates were 62.5% and 90%respectively,while ZWINT was significantly higher in stage III + IV than that in stage I + II,and the difference was statistically significant(P<0.05).In terms of lymph node metastasis,24 of 28 cases had high expression,while only 18 of 32 cases which had nolymphatic metastasis showed high expression,with high expression rates of85.7% and 56.3%,respectively,with statistically significant differences(P<0.05).10.Statistical analysis of the correlation of ZWINT protein with E-cadherin,Vimentin and Slug in NSCLC tissues,the expression of ZWINT and E-cadherin expression was negatively related(r=-0.4122,P=0.034),the expression of ZWINT and Vimentin was positive correlation(r=0.386,P=0.031),and the expression of ZWINT and Slug was positive correlation either(r=0.408,P=0.024).II Function and mechanism study of ZWINT in NSCLC cells(I)Effection of ZWINT on EMT,migration,invasion and metastasis of NSCLC cells1.ZWINT m RNA expression in A549 and H1299 was measured by q RT-PCR,and the expression of ZWINT m RNA in A549 was higher than that in H1299,with statistically significant(P <0.01).2.The expression of ZWINT protein in A549 and H1299 was detected by Western blot,and ZWINT protein in A549 was higher than that in H1299,which was significant difference(P <0.01),consistent with ZWINT m RNA expression.3.Construct ZWINT over expressed H1299 and ZWINT silenced A549,the expression levels of ZWINT m RNA and protein were both up regulated in ZWINT over expressed H1299 cell lines and down regulated in ZWINT silenced A549 cell lines,which indicated that the NSCLC cell lines over expressing and knockdown ZWINT were successfully constructed.4.In the scratch assay,over expression of ZWINT promoted the scratch healing ability of H1299 cells,while knockdown of ZWINT weakened the scratch healing ability of A549 cells.5.In the Transwell without matrix paving and matrix paving experiments,over expression of ZWINT promoted the migration and invasion capacity of H1299 cells,while knockdown of ZWINT attenuated the migration and invasion ability of A549 cells.6.In the CCK-8 assay,over expression of ZWINT promoted the proliferation capacity of H1299 cells,while knockdown of ZWINT attenuated the proliferation capacity of A549 cells.7.In Flow detection of apoptosis experiments,over expression of ZWINT reduced apoptosis in H1299 cells,and knockdown of ZWINT promoted apoptosis in A549 cells.8.EMT-related markers were detected by Western blot method,the ZWINT over expression group showed high expression of Vimentin,N-cadherin,and Slug,and low expression of E-cadherin,while the ZWINT knockdown group showed high expression of E-cadherin and low expression of Vimentin,N-cadherin and Slug.9.EMT-related marker Vimentin was detected by Immunofluorescence,and it showed high immunofluorescence intensity in over expressing BUB1 and NUF2 group,and weak immunofluorescence intensity in knockdown BUB1 and NUF2 group,consistent with the Western blot results.(II)Discussion of the mechanism of ZWINT on the proliferation and metastasis of NSCLC cells1.The m RNA and protein expression levels of BUB1 and NUF2 were measured by RT-q PCR and Western blot,and the m RNA and protein expression of BUB 1 and NUF2 were increased in ZWINT over expressing cells,significantly different from the control group(P<0.05).Both BUB1 and NUF2 m RNA were down regulated in ZWINT knockdown cells,significantly different from the control group(P <0.05).2.Compared with the control group,the expression levels of BUB1 m RNA and protein were up regulated in H1299 cell lines over expressing BUB1 and down regulated in H1299 of BUB1 knockdown,indicating the successful construction of NSCLC cell lines over expressing and knocking down BUB1.Compared with the control group,the expression levels of NUF2 m RNA and protein were up regulated in H1299 cell lines over expressing NUF 2 and down regulated in H1299 with knockout NUF2,indicating the successful construction of NSCLC cell lines over expressing and knockdown NUF2.3.In the scratch assay,over expression of BUB1 and NUF2 promoted the scratch healing ability of H1299 cells,while knockdown of BUB1 and NUF2 attenuated the scratch healing ability of H1299 cells.4.In Transwell migration and invasion assay,over expression of BUB1 or NUF2 promoted the migration and invasion of H1299 cells,while knockdown of BUB1 or NUF2 attenuated the migration and invasion of H1299 cells.5.In CCK-8 experiments,over expression of both BUB 1 and NUF 2 promoted the proliferation capacity of H1299 cells,whereas knockdown of BUB1 and NUF2 attenuated the proliferation capacity of H1299 cells.6.In the flow detection of cell apoptosis experiment,over expression of both BUB1 and NUF2 reduced the apoptosis of H1299 cells,and knockdown of both BUB 1 and NUF2 promoted the apoptosis of H1299 cells.7.Western blot Methods is used to detect EMT related markers,BUB1 and NUF2 over expressed cells showed high expression of Vimentin,N-cadherin and Slug,and low expression of E-cadherin.Both BUB1 and NUF2 knockdown cells showed high expression of E-cadherin,while Vimentin,N-cadherin and Slug showed low expression.8.Immunofluorescence for EMT-related marker Vimentin,over expressing BUB1 and NUF2 group,immunofluorescence intensity and knockdown BUB1 and NUF2 group,weak immunofluorescence,consistent with the Western blot results.Conclusion:1.Bioinformatics analysis revealed that ZWINT expression was significantly up regulated in NSCLC.ZWINT was involved in cell cycle development and cell division in NSCLC,and it showed a significant association with immune cell infiltration.2.ZWINT was highly expressed in NSCLC tissues,and was associated with lymph node metastasis,and high TNM stage of lung cancer.3.ZWINT could promote EMT,proliferation,migration and invasion in NSCLC cells,and reduce cell apoptosis.4.ZWINT promoted tumor EMT progression by regulating the cell cycle-ralated proteins BUB1 and NUF2,and promoted the enhanced proliferation,migration and invasion ability of NSCLC cells,thus leading to its malignant progression.