The Potential Role And Mechanism Of WSB2 In Proliferation And Migration Of Xuanwei Lung Cancer

Background and Objective:Lung cancer is a malignant tumor with high morbidity and mortality,and the number of deaths caused by lung cancer is high every year.At present,the specific mechanism of the occurrence and development of lung cancer is still not clear.Therefore,further study on the development of lung cancer is helpful for the diagnosis and treatment of lung cancer.Different from the high incidence of lung cancer in the world and other provinces of China,lung cancer in Yunnan presents a point-like regional distribution,mainly concentrated in Xuanwei and Gejiu.Lung cancer in Xuanwei is a malignant tumor with extremely high morbidity and mortality in China and even in the world,and it is a representative disease in the research of global pointlike regional high incidence of lung cancer.The so-called lung cancer in Xuanwei refers to the lung cancer patients with a high incidence in this area,who were born locally and lived for ≥3 generations.Generalized "xuanwei area" refers to the pearl river source region,including xuanwei,FuYuan County,Gu Lin county,malong county,zhanyi county,and southwest guizhou area,is located in the southwest and northeast yunnan WuMengShan District,local rich Tibetan mineral resources such as iron,copper,coal,and has a population of about 3.1 million,land area of 6069.88 square kilometers,particularly in the region of the most common for xuanwei(260 km from provincial capital of kunming,yunnan is the most populous county,is also one of the key coal-producing counties),Including Rongcheng,Jingwai,Laibin,Gunning,Ruishui 14 towns,8 townships,25 neighborhood committees and 331 administrative villages,the city’s population is about 1.297,500 people,more than are farmers,about the Han nationality.Local residents are used to using bituminous coal as the main living fuel,and most of the houses are wood structures of the ground floor and subsoil.Due to the lack of smoke exhaust equipment,indoor air circulation is not smooth all the year round,and the large amount of smoke and dust emitted during fuel combustion leads to strict indoor air Heavy pollution.Since the national tumor death survey in 1975 found that the lung cancer mortality rate of peasants in Xuanwei County,Yunnan Province was relatively high,thus leading to the research on Xuanwei lung cancer.Xuanwei lung cancer has attracted the attention of national and global researchers.The death rate from lung cancer in Xuanwei has risen steadily since the 1970s,according to three national review of causes of death.According to the data from the center for disease control and prevention of xuanwei city,yunnan province,from 1990 to 1992,the death rate of lung cancer in xuanwei city was 23.14 per 100,000,and the death rate of male and female was 4 times and 8 times of the national average level respectively.From 2014 to 2015,the death rate of lung cancer in the region reached 91.11 per 100,000,three times that of the national average,and six times that of women.Comparing the two time points,the death rate of lung cancer in the latter was nearly four times higher than in the 1990s.As for the current research on cancer,genetic change is an important factor in the pathogenesis of tumor,so looking for genes related to Xuanwei lung cancer is an important way to understand the pathogenesis of Xuanwei lung cancer,and to carry out early diagnosis and early treatment.According to its special structure,tryptophan aspartate repeat and C-terminal cytokine signaling inhibitor WSB2(WD repeat and SOCS box containing protein)is named as a gene containing WD repeat and SOCS box containing protein(WSB)structure.According to the number of WD motifs,WSB is divided into wsbl and WSB2.WD repeat is a kind of repeat that contains many tryptophan aspartate genes,and SOCS box is a kind of suppressor of cytokine signaling box(SOCS box)at C-terminal.These two structures play an important role in the formation and growth of cancer,and participate in the formation of various types of malignant tumors.The specific mechanism has not been clearly determined.Some studies believe that SOCS box protein can activate the related oncogenes and lead to tumorigenesis by promoting the formation of elonginb/c-cullin2 complex.Because WSB2 protein also contains SOCS box structure,it has attracted researchers’ attention.In this study,to download the expression level data of WSB2 in lung cancer through TCGA database,and analyze the expression level of WSB2 in lung cancer,and the correlation between WSB2 expression level and clinical stage and prognosis of lung cancer.The role of WSB2 in the occurrence and development of Xuanwei lung cancer was studied in clinical samples.The effects of WSB2 on the proliferation,invasion,migration and apoptosis of Xuanwei lung cancer were studied in vivo and in vitro,and the regulatory role of WSB2 on Wnt/β-catenin signaling pathway and its role in Xuanwei lung cancer were discussed.Through the above studies,the role and mechanism of WSB2 in lung cancer in Xuanwei are discussed,so as to further develop new methods for the prevention,treatment and treatment of lung cancer in Xuanwei area.Part Ⅰ The expression and clinical significance of WSB2 in Xuanwei lung cancer tissueObjective:To analyze and detect the expression of WSB2 in cancer tissues and normal lung tissues of Xuanwei lung cancer patients.Method:1.WSB2 expression in lung cancer samples was downloaded from TCGA expression profile,and patients with complete clinical data and survival data and survival time greater than 2 months were screened for subsequent survival curve and Cox multivariate risk model ratio analysis.2.A total of 42 patients with lung cancer in Xuanwei were collected from the first department of thoracic surgery of the Third Affiliated Hospital of Kunming Medical University(Yunnan Cancer Hospital),and samples of cancer tissue and paired distal normal lung tissue were collected.The pathological morphology of lung cancer in Xuanwei was identified by hematoxylin-eosin(HE)staining.Immunohistochemistry(IHC)staining,quantitative real-time PCR(qRT-PCR)detection and Western Blotting analysis were used to detect the expression of WSB2 in Xuanwei lung cancer tissues and normal lung tissues.Results:1.Bioinformatics analysis showed that WSB2 was highly expressed in lung cancer tissues,and was significantly correlated with clinical stage and prognosis of lung cancer(P<0.05).2.Collection of kunming medical university cancer hospital(yunnan province)the third affiliated hospital of chest outside a row xuanwei of surgical treatment of lung cancer patients with carcinoma tissue and paired distal normal lung tissue samples,a total of 42 cases,using immunohistochemical staining(IHC)showed that xuanwei WSB2 in lung cancer tissue were higher than that of normal lung tissue,(83.3%vs.26.2%,p=0.007,<0.01).3.Meanwhile,Western Blotting was used to verify the expression level of WSB2 protein in Xuanwei lung cancer,and the results showed that the expression level of WSB2 protein in Xuanwei lung cancer tissue was significantly higher than that in normal lung tissue(P<0.01).4.At the same time,the above samples were tested by Q-PCR to verify the expression level of WSB2 gene in Xuanwei lung cancer.The results showed that the expression level of WSB2 gene in Xuanwei lung cancer tissue was significantly higher than that in normal lung tissue(P<0.01).5.The comparison of the positive rate of WSB2 in immunohistochemistry of Xuanwei lung cancer patients with basic clinical data and staging showed that the positive rate of tissue samples from patients with stage ⅢA was higher than that of patients with stage Ⅰ,with statistical significance(P<0.05).Conclusion:WSB2 is highly expressed in the lung cancer tissues of Xuanwei,which may play an important role in the occurrence and development of Xuanwei lung cancer,and its expression is closely related to the staging and survival rate of Xuanwei lung cancer patients.Part Ⅱ The biological function of WSB2 in vitro and in vivoObjective:In the first part of the study,WSB2 was found to be a highly expressed gene in Xuanwei lung cancer tissues,and the elevated protein expression indicated a poor prognosis of the patients.In order to confirm that the malignant biological behavior of WSB2 in Xuanwei lung cancer leads to such a series of clinicopathological features in patients,the effects of WSB2 on cell proliferation,migration,invasion and apoptosis in Xuanwei lung cancer cell line XWLC-05 were further explored.The effect of WSB2 on Xuanwei lung cancer in vivo was studied by mouse tumor transplantation experiment.The aim is to obtain reliable evidence that WSB2 promotes the malignant process such as the occurrence and development of lung cancer in Xuanwei,and to provide scientific basis for WSB2 to become a new target for the treatment of lung cancer in Xuanwei.Method:1.WSB2-shRNA lentiviral interference vector infection was used to construct WSB2-knockdown XWLC-05 stable cell line,so as to knock down WSB2 gene in XWLC-05 cells and reduce the expression of its coding protein.The expression of WSB2 was inhibited.2.CCK-8 method was used to detect cell proliferation,flow cytometry was used to detect cell apoptosis,cell scratch test and Transwell transfer chamber test were used to detect cell migration and invasion ability.Western Blotting detected the expression levels of proteins associated with the process of "EMT".3.To further verify whether the effect of WSB2 on XWLC-05 cells in vivo is consistent with that in vitro,XWLC-05 cell line knocked down by WSB2 was used to construct xenocolonization tumor model in nude mice.The effect of WSB2 knockdown on the tumorigenicity of XWLC-05 cells was detected.Results:1.In Xuanwei lung cancer cell line,WSB2 knockdown significantly inhibited the proliferation of XWLC-05 cells(P<0.05)2.In Xuanwei lung cancer cell lines,apoptosis rate was significantly increased after WSB2 knockdown(P<0.05);3.In Xuanwei lung cancer cell lines,the migration and invasiveness of WSB2 were significantly reduced after WSB2 knockdown(P<0.05).4.In Xuanwei lung cancer cell lines,the protein expression levels of N-cadherin and Vimentin were significantly decreased after WSB2 knockdown(P<0.01);The protein expression level of E-cadherin was significantly increased(P<0.01).5.In vivo experiments in nude mice,WSB2 knockdown significantly inhibited the tumorigenesis ability of XWLC-05 cells in nude mice(P<0.05).Conclusion:1.Downregulation of WSB2 can inhibit the proliferation of XWLC-05 cells,significantly decrease the migration and invasion ability of cells,and significantly increase the apoptosis rate.WSB2 plays a role in promoting the occurrence,development and invasion of Xuanwei lung cancer.2.WSB2 can also promote the growth of Xuanwei lung cancer in nude mice.Part Ⅲ The mechanism of WSB2 activating Wnt/β-catenin signaling pathway to promote the proliferation and migration of Xuanwei lung cancer cells and inhibit cell apoptosisObjective:the study WSB2 may have promote xuanwei lung cancer cell proliferation,migration,the role of inhibition of apoptosis,is proposed to construct XWLC WSB2 over expression-05 stable cell lines,and at the same time the Wnt/beta-Catenin signaling pathway inhibitor FH535 processing XWLC-05 cell comparison,preliminarily WSB2 promote xuanwei lung tumor cell proliferation,migration,molecular mechanism of inhibiting apoptosis may,of xuanwei lung cancer treatment for WSB2 to become the new target is to provide scientific theory basis.Method:XWLC-05 cell line overexpressing WSB2 was constructed,and XWLC-05 cells were treated with Wnt/β-catenin signaling pathway inhibitor FH535 for control.CCK-8 assay was used to detect cell proliferation,flow cytometry was used to detect cell apoptosis,cell scratch assay and Transwell transfer chamber assay were used to detect cell migration and invasion ability.The expression levels of "EMT" process and Wnt/β-catenin signaling pathway were detected by Western Blotting.To explore the role of Wnt/β-catenin signaling pathway in Xuanwei lung cancer.Results:1.The overexpression of WSB2 significantly increased the proliferation,migration and invasiveness of XWLC-05 cells(P<0.05),while the Wnt/β-catenin signaling pathway inhibitor FH535 significantly inhibited the promoting effect of WSB2(P<0.01).2.After WSB2 gene expression was knocked down,the protein levels of c-myc andβ-catenin were significantly decreased(P<0.01);However,after the overexpression of WSB2 gene,the protein levels of c-myc and β-catenin were significantly increased(P<0.01).WSB2 can regulate the expression levels of c-myc and β-catenin,two key proteins in Wnt/β-catenin signal transduction.3.After WSB2 overexpression,the protein expression levels of N-cadherin and Vimentin were significantly increased(P<0.01);The protein expression level of E-cadherin was significantly decreased(P<0.01).The protein expression levels of N-cadherin and Vimentin were significantly decreased after FH535 treatment(P<0.01).The protein expression level of E-cadherin was significantly increased(P<0.01).Conclusion:1.WSB2 regulates the growth and metastasis of Xuanwei lung cells by regulating Wnt/β-catenin signal transduction.2.Overexpression of WSB2 also leads to activation of Wnt/β-catenin signaling.In addition,Wnt/β-catenin signal transduction inhibitors can reduce lung cancer caused by WSB2 overexpression.3.WSB2 promotes the progression of lung cancer in Xuanwei area by activating the Wnt/β-catenin signaling pathway,and may become a therapeutic target for lung cancer in Xuanwei area,especially for lung cancer.