| Background and purpose of the study:The latest epidemiological results show that lung cancer incidence and mortality rank first in all malignancies,among which non-small cell lung cancer(NSCLC)accounts for about 80-85% of patients.In recent years,although significant progress has been made in the comprehensive treatment of lung cancer,the prognosis of NSCLC is still poor,and the 5-year survival rate is less than 20%.The main reasons leading to the failure of NSCLC treatment are relapse and metastasis,and chemistry resistance.Therefore,how to further elucidate the molecular mechanism of lung cancer metastasis and recurrence and identify new key factors that drive the shift is a major medical problem that needs urgent solution.The p53 gene is one of the most important tumor suppressor genes in the human body,and its function is very powerful.Wild-type p53 promotes cell cycle arrest,promotes apoptosis and senescence,and maintains genome stability by transcriptionally regulating many downstream target genes.P53 has mutations in more than 50% of human cancers.When p53 is mutated,the function of the tumor suppressor gene in wild-type p53 is lost.This is called the "loss of function" of mutp53.At the same time,mutp53 has acquired a series of functions similar to oncogene properties,such as the transcription of a series of target genes to accelerate the progression of cancer,enhance the chemo-resistance of cancer cells,and promote the occurrence of tumor metastasis.This process is called the “Gain of function”.For example,recent studies have shown that mutant p53-R273 H promotes tumor invasion and metastasis through transcriptional activation of target genes such as Notch1 in NSCLC.Although the important role of p53 mutations in the initiation and progression of tumors is well known,it remains unclear why p53 has such a high mutation rate in many tumors.The latest research results show that the abnormal expression of cytosine deaminase APOBEC3 B is closely related to the high mutation rate of p53.APOBEC3B(A3B)is a member of the apolipoprotein B m RNA-editing enzyme catalytic polypeptide(APOBEC)family and is a highly efficient cytosine nucleotide deaminase.A3 B and its family members have been shown to have a deaminating effect on cytidine on single-stranded DNA and RNA and can induce mutations in the genomic C>T or C>G by deacetylating cytosine on the target gene to become uracil(C>U).On the one hand,A3 B has antiviral innate immune activity on the one hand,and on the other hand,A3 B has a strong gene mutation effect on host genomic DNA.A large number of studies have shown that A3 B is consistently expressed at high levels in various tumors(including breast cancer,lung cancer,endometrial cancer,liver cancer,etc.),and A3 B expression has been widely confirmed by the association of mutations in classic oncogenes with cancer.The Burns team found that A3 B is up-regulated in most primary breast and breast cancer cell lines.At the same time,high A3 B expression increases the total gene mutation rate in tumors more than 2-fold,and it is more prone to mutations at multiple sites of p53.In a retrospective study,Shumei Yan et al found that A3 B is abnormally expressed in lung cancer,and its expression is closely related to the overall survival rate of lung cancer and TNM stage,suggesting that A3 B plays an important role in the development of lung cancer.However,the role and mechanism of A3 B in the development of lung cancer have only been reported sporadically by now.In order to explore the role of A3 B in the development and evolution of lung cancer,this study intends to elucidate the close relationship between the evolution of A3 B in lung cancer,especially metastasis,through cell models,animal experiments,and clinical specimen detection,in order to further elucidate the key factors driving lung cancer metastasis.Overcoming the transfer of lung cancer and developing new intervention strategies provide a theoretical basis.Research methods:1.RT-q PCR and western blot were used to detect the expression of A3 B in lung cancer cell lines and normal lung epithelial cells.The HCC827 cell line with relatively low A3 B expression was selected as the next study object.2.HCC827/B3 cells derived from HCC827 monoclonal were obtained by limiting dilution.Then the A3 B overexpression lentiviral vector was constructed by Cas9-SAM technology.The cells were first infected with Cas-VP64 lentivirus and screened by puromycin.The stable cells obtained were infected with the recombinant sg RNA-GV419 lentivirus and finally selected by G418.,RT-q PCR and western blot methods were used to verify A3 B expression level of A3 B overexpressing cell line B3/A3 B.3.A3 B converts cytosine to uracil through base deamination,breaks glycosidic linkage under UDG enzyme action,and then undergoes base excision repair to generate apurinic apyrimidinic site(AP site),The more AP site means the higher the in vitro deaminase activity of A3 B.The AP assay kit detects B3/A3 B cells to verify the in vitro activity of cytosine deaminase in overexpressing cell lines.Transwell invasion assay was used to detect the invasion and metastasis ability of A3B-overexpressing cells B3/A3 B and control cells B3/control.Scratches and Transwell metastasis experiments were used to detect the migration ability of cells.At the same time,a small animal model of lung cancer metastasis was constructed.Injected B3/A3 B and B3/control cells from the tail vein of the nude mice,mice were sacrificed 35 days later to obtain complete paraffin-embedded tissues for paraffin-embedded specimens.The metastasis of lung tissues was observed under HE staining.4.The Sanger sequencing method was used to sequence P53 exons 5-8 in the constructed A3 B overexpressing cell line to analyze the p53 mutation.74 specimens of non-small cell lung cancer tissue samples were collected.On the one hand,mutations of exon 5-8 of P53 gene were detected by sequencing.On the other hand,the corresponding specimens were subjected to immunohistochemistry to detect the expression of A3 B,and the correlation between p53 mutation and A3 B expression was analyzed.5.Using si RNA transient transfection technology to interfere with the expression of p53 in B3/A3 B cells,detect the invasion and metastasis of cells by scratch and chamber invasion assays,and reversely verify whether p53 participates in cell phenotype changes after A3 B overexpression.Process;A3B was over-expressed in P53-deleted cell line H1299 to examine the effect of A3 B on cell invasion and migration in the absence of p53.It further validated the changes in the levels of metastasis-associated proteins after different treatments of cells.6.Collect 249 cases of lung cancer patients from 2008 to 2016 in this hospital.The expression of A3 B was detected by immunohistochemistry and scored according to the degree of its expression.The level of A3 B expression and the difference between clinical data and prognosis were analyzed.relationship.Result:1.q-PCR and Western blot results showed that compared with normal lung epithelial cells,the expression of A3 B in non-small cell lung cancer cells such as H1975,A549,etc.was significantly increased,and only the expression of A3 B in HCC827 lung cancer cells was relatively low.2.The HCC827 monoclonal origin cell HCC827/B3 was obtained by limiting dilution method in this laboratory and was identified by cell STR.The genetics were consistent with the parental cell.A3B-overexpressing lung cancer cell line B3/A3 B and control cell B3/control were successfully constructed by Cas9-SAM technology.The m RNA expression of A3 B in B3/A3 B cells was increased by more than 20-fold compared with the control group.Similarly,the expression at the protein level was higher.The control group increased significantly.3.The AP assay detected more AP sites in B3/A3 B cells than B3/con(p<0.001).The difference was statistically significant and indirectly represented an increase in cell deaminase activity after overexpression of A3 B.4.After overexpressign A3 B in cells,the invasion ability was significantly increased compared with the control cells,p<0.0001;the metastatic capacity was significantly increased,p=0.0001.Scratch tests showed that B3/A3 B cells migrate faster.In the lung cancer metastasis model of nude mice,the number of lung tumor nodules increased in mice injected with B3/A3 B cells compared with the control group,and HE staining showed structural disorder of the cancer tissue.5.Overexpression of A3 B cell B3/A3 B was confirmed by sequencing of P53 exons 5-8.A3B-expressing cell lines showed multiple site mutations(c.521A>G;p.R174W),T(c.Missense mutations such as 573C>T;p.P191V),T(c.885C>T;p.P295V),etc.,P53 mutation rate(27/74)also existed in 74 cases of lung cancer pathological specimens and there are the following hot spots: Mutations: T(c.767C>G;p.T256R),T(c.722C>T;p.S241F),T(c.817C>T;p.R273C),T(c.722C>T;p(S241F),T(c.742C>T;p.R248W),T(c.859G>A;p.E287K).In 74 cases of lung cancer pathological tissue immunohistochemistry experiments,the overall expression of p53 mutant case A3 B was significantly higher than p53 wild type cases,p<0.05.6.After B3/A3 B interferes with p53 expression,the percentage of wound healing at 48 hours after cell scratches was 16.21± 10.19.The percentage of wound healing at 48 hours after interference with control cells was 66.95± 4.82,p=0.0108,and the difference was statistically significant.The number of cells invading into the inferior compartment of the Transwell chamber invasion experiment group and the control group were 198.7 ± 9.493 and 396 ± 19.97,respectively,p < 0.0001,suggesting that the interference of p53 can reverse the invasion and metastasis potential of B3/A3 B.Similarly,after overexpression of A3 B in p53 depleted cells H1299,there was no difference in the invasion and metastasis of cells.Finally,Western blot analysis of p53 and its downstream metastasis-associated factors revealed that B3/A3 B metastasis-associated factors such as Slug,MMP2,and MMP9 were significantly increased,whereas after interfering with p53,the above protein expression was relatively down-regulated.7.The immunohistochemical results of 249 lung cancers and 42 normal lung tissues showed that the expression of A3 B in lung cancer tissues was higher than that in normal lung tissues,with p<0.005.The difference was statistically significant;the expression of A3 B was correlated with the clinicopathological data of patients.The analysis showed that A3 B expression was positively correlated with lymph node metastasis and poor prognosis,and there was no statistical correlation with age,sex,tumor type,and other data.Conclusion:1.A3 B has the effect of promoting the invasion and metastasis of lung cancer.2.A3 B may mediate lung cancer metastasis by promoting p53 gene mutation.3.A3 B can be used as a marker for judging the prognosis of lung cancer. |
PDF Full Text Request