Study On The Mutation Of BRAF V600E Gene In Thyroid Carcinoma And The Expression Of Key Genes In Tumor Invasion And Metastasis

Background:Thyroid cancer is the most common tumor of the head and neck endocrine system.During its development,BRAF V600 E gene mutation is a relatively common and well-studied genetic event.This mutation only occurs in papillary thyroid carcinoma and undifferentiated carcinoma.The occurrence of the BRAF V600 E mutation impairs the function of the sodium/iodine symporter,affects iodine uptake and results in radioiodine-refractory thyroid cancer,and has a poor prognosis.Multiple studies have shown that lymph node metastasis often occurs in patients with papillary carcinoma and BRAF V600 E gene mutation is associated with lymph node metastasis and extracapsular infiltration,and is a high risk factor for recurrence.It is known that CD44V6,CDH13,MMP2,TIMP1,and NME1 are found to be significantly associated with the occurrence of lymph node metastasis and infiltration of tumors in gastric cancer,non-small cell lung cancer,thyroid cancer,and other cancers.The above genes have some meaning of diagnosing clinical and pathological features.The incidence of BRAF V600 E gene mutation in thyroid cancer is about 45%,but its molecular mechanism is still unclear.The related literature reported that KRT19,SLC26A4,GDF15,and TPD52L1 are the main genes differentially expressed in BRAF V600 E mutant and wild-type patients.Their expression changes may have a significant relationship with the carcinogenic mechanism of BRAF V600 E gene,so the above-mentioned ten key gene was selected in this study.Objective:The purpose of this study was to investigate the expression of KRT19,SLC26A4,GDF15,TPD52L1,CD44V6,CDH13,MMP2,TIMP1,NME1,and HPSE key genes in patients with papillary thyroid carcinoma between BRAF V600 E mutant and wild type.The relationship between the expression of ten key genes and lymph node metastasis and extracapsular infiltration in patients was analyzed to provide a theoretical basis for the further study of the carcinogenic molecular mechanism of BRAF V600 E gene mutation and to find markers related to clinical pathological features,and provide stronger evidence for early diagnosis and prognosis assessment.Methods:Thirty-seven fresh tissue specimens collected from Jilin University Second Hospital from November 2014 to June 2017 were collected and immediately frozen in a refrigerator at-80°C.Pathological types of paraffin-embedded tissues were diagnosed by experienced pathologists.The basic clinical information of 37 patients including gender,age,tumor size,and relevant pathological data including lymph node metastasis and extracapsular infiltration were collected.A part of the fresh surgical tissue was extracted for DNA and the BRAF V600 E gene mutation was detected by RT-PCR.The patients were divided into two groups according to the gene mutation detection.The BRAF V600 E mutant was the experimental group and the wild type was the control group.The other part of the tissue was extracted total RNA and reversely transcribed into cDNA.Expression levels in experimental and control groups of ten key genes(KRT19,SLC26A4,GDF15,TPD52L1,CD44V6,CDH13,MMP2,TIMP1,HPSE,NME1)and the reference gene GAPDH were detected by real-time fluorescence quantitative PCR.Statistical method: Fisher's exact probability method was used to analyze the relationship between BRAF V600 E gene mutation and clinical data(including gender,age,tumor size,lymph node metastasis,and capsular infiltration)in the enrolled patients;The relative expression levels of the ten key genes in BRAF V600 E mutant and wild-type tissues were calculated using the 2-??Ct algorithm based on RT-PCR data.The same kind of algorithm was used to compare the relative expression levels of ten key genes between lymph node metastasis and non-metastatic patients,and extracapsular infiltration and non-invasive patients.Results:1.Detection rate of BRAF V600 E gene mutation: the detection rate of BRAF V600 E gene mutation was 81.08%(30/37)in 37 papillary thyroid cancer patients.Domestic literature shows that the incidence of BRAF V600 E gene mutation in papillary thyroid cancer is about 40% to 60%.However,foreign literature shows that there are differences in the detection rate due to different detection methods such as Sanger sequencing and fluorescence quantification.PCR and allele-specific amplification PCR,the overall range of 37% to 66%.The mutation detection rate in this study was 81.08%,which was relatively high.There may be a certain relationship between the detection results and the small sample size.The sample size should be further expanded to improve the accuracy of the results.2.Analysis of age,sex,tumor size,and BRAF V600 E gene mutations in patients with papillary thyroid cancer: this study showed that the BRAF V600 E mutation rate was 76.2%(16/21)in 37 patients aged 45 years or older,and the BRAF V600 E mutation rate was 87.5%(14/16)in patients aged <45 years.There was no statistically significant difference between the BRAF V600 E mutation and age(P>0.05).The mutation rate of BRAF V600 E in male patients was 100%(7/7),and the mutation rate in female patients was 76.7%(23/30).There was no significant difference between the two groups.The BRAF V600 E mutation and gender were not statistically significant by Fisher's exact test.(P>0.05);The BRAF V600 E mutation rate was 86.4%(19/22)in patients with tumor diameter ?1cm,and the BRAF V600 E mutation rate was 73.3%(11/15)in patients with diameter <1cm.Fisher's exact test method did not calculate the BRAF V600 E mutation and tumor diameter.Statistical significance(P>0.05);There is no correlation between BRAF V600 E gene mutation and patient age,gender,and tumor size at home and abroad,which is consistent with the results of this study.3.Outcomes of papillary thyroid carcinoma extracapsular infiltration and detection of BRAF V600 E gene mutation: the incidence of BRAF V600 E mutation was 71.4%(5/7)in 37 patients with extracapsular infiltration and 83.3%(25/30)in patients without extracapsular infiltration.Fisher's exact test Computation of BRAF V600 E and extracapsular infiltration was not statistically significant(P>0.05).Domestic and foreign related literature shows that there are still different views on the relationship between the two,and the number of patients enrolled in this study should be increased to improve the credibility of the results.4.Analysis of the relationship between BRAF V600 E gene mutation and lymph node metastasis in patients with papillary thyroid cancer: the incidence of BRAF V600 E mutation was 83.3%(10/12)in 37 patients with lymph node metastasis and 80.0%(20/25)in those without lymph node metastasis.Fisher's exact probability method was used to calculate the difference between them.There was no statistical significance(P>0.05).A large number of studies have shown that the occurrence of lymph node metastasis in patients with BRAF V600 E gene mutation is higher,but a small part of the study also pointed out that there is no statistical significance between the two,so it is particularly important to increase the number of patients into the group to further verify.5.Analysis of BRAF V600 E gene mutation and key gene expression in papillary thyroid carcinoma: in 37 patients after RT-PCR detection,the results of this study showed that the relative expression levels of SLC26A4,GDF15,HPSE,TIMP1,NME1,and MMP2 were associated with BRAF V600 E gene mutation,SLC26A4(P<0.01),GDF15(P<0.01),HPSE(P<0.01)and TIMP1(P<0.01)increased significantly in mutant PTC tissues,and NME1(P<0.01)and MMP2(P<0.05)decreased.This suggests that the above genes may interact with the BRAF V600 E gene during the progression of cancer,affect the progression of cancer,and provide a solid foundation for further research on the molecular mechanism of BRAF V600 E carcinogenesis.6.Analysis of the relationship between the expression of key genes and extracapsular infiltration in papillary thyroid cancer: out of 37 cases of thyroid papillary carcinoma,7 cases of tumor infiltration outside the capsule occurred,and 30 cases did not infiltrate.Among the patients with extracapsular infiltration,the relative expression levels of CD44V6(P<0.01),TPD52L1(P<0.01),HPSE(P<0.01),GDF15(P<0.05),and TIMP1(P<0.05)were significantly higher than not infiltrated.The difference was statistically significant.The results suggest that the above genes have a very important role in the identification of the pathological features of extracapsular infiltration.7.Analysis of the relationship between key gene expression and lymph node metastasis in papillary thyroid cancer: of the 37 patients with thyroid papillary carcinoma,12 had lymph node metastasis and 25 had no metastasis.The relative expression of GDF15(P<0.01),HPSE(P<0.01),MMP2(P<0.05),and TIMP1(P<0.05)was significantly increased in lymph node metastasis tissues compared with non-metastasis,SLC26A4(P<0.05)was decreased.The results suggest that the above genes can be combined with BRAF V600 E as a molecular marker to identify lymph node metastases.Conclusion:This study demonstrated that the incidence of BRAF V600 E mutations in 37 cases of papillary thyroid cancer was 81.08%.The BRAF V600 E gene mutation is closely related to the expression of GDF15 and SLC26A4,indicating that it is of great significance in the carcinogenic process of BRAF V600 E mutation.It can be used as the main target gene for the further study of the molecular pathogenesis of the BRAF V600 E gene in papillary thyroid carcinoma.HPSE is highly expressed in patients with lymph node metastasis and extracapsular infiltration of tumors.Therefore,the combined detection of BRAF V600 E and HPSE is expected to be a new tumor marker for the diagnosis,treatment and prognosis evaluation of thyroid cancer,and provide new clinical pathological features for judgment ideas.