Preliminary Study On The Expression Of LZTS1 Protein And Gene In Lung Cancer And LZTS1 Gene Methylation Of Promoter Region

Background:Within the scope of world,covering China,lung cancer ranks the topest in the list of cancer-related deaths.lt can be divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).NSCLC can be classifed on the basis of different histological types,including adenocarcinoma,squamous cell carcinoma,neuroendocrine cancer,adenosquamous carcinoma,and sarcomatoid carcinoma,etc.The leucine zipper putative tumor suppressor 1(LZTS1)is located near the short arm of the chromosome 8(8p)21.3 and encodes a 67kd leucine zipper protein.The LZTS1 protein is one of the super families of leucine-rich repeats,and most of them are located in the cytoplasm,and a small part is located in the nucleus.A number of studies have shown that the LZTS1 gene is ubiquitously expressed in many normal tissues,and the LZTS I gene has a certain anti-cancer effect in the development of many tumors,including prostate cancer,lung cancer,stomach cancer,head and neck cancer,and breast cancer.It has been reported in the literature that 27 of the 103 lung cancers have LZTS1 gene expression loss,and 43 LZTS1 gene expression is decreased.The results show that the LZTS1 gene expression level is positively correlated with tumor grade.Therefore,the LZTS1 gene plays an important role in the development of lung cancer,nonetheless its mechanism of inactivation as a tumor suppressor gene has not been studied clearly.The traditional view attributed the inactivation of tumor suppressor genes to gene mutations and chromosome deletions,but the aberrant methylation of DNA provides another possible explanation for the inactivation of the LZTS1 gene.Therefore,this experiment will detect the expression level of LZTS 1 protein and its gene in lung cancer,and compare the consistency of LZTS1 gene expression in lung cancer tissue and circulating tumor DNA(ctDNA),and analyze the relationship between LZTS1 protein and gene expression levels in the same lung cancer tissue samples.To elucidate the role of LZTS1 gene in the development of lung cancer,and to detect the presence of mutations in the coding region of LZTS1 gene and the methylation status of the promoter region of LZTS1 gene.Part One:Expression of LZTS1 protein in lung cancer tissuesMethods:Sixty specimens of surgically resected lung cancer were selected and confirmed by pathology.The expression of LZTS1 protein in lung cancer tissues was detected by immunohistochemistry(IHC).At the same time,the relationship between LZTS1 protein expression and clinicopathological features of lung cancer tissues was analyzed.Results:1.The expression of LZTS1 protein was changed in primary lung cancer tissues.The loss and/or decrease rate of LZTS1 protein expression in 60 lung cancer tissues was 65%(39/60).2.The later the TNM stage' and the higher the smoking index,the higher the rate of loss and/or decrease in LZTS1 protein expression.3.Decreased and/or deleted expression of LZTS1 protein is associated with progression of lung cancer and may result in tumor cells being more susceptible to metastasis.Part Two:Expression of LZTS1 in lung cancer tissues,normal lung tissues and ctDNAMethods:50 cases of lung cancer were selected(among them,23 cases were the same case as the sample of LZTS1 protein expression level detected in the first part)to extract total RNA from lung cancer tissues and normal lung tissues,reverse transcription PCR was used to detect the expression of LZTS1 gene in lung cancer tissues and normal lung tissues.CtDNA was extracted from plasma,the expression of LZTS 1 gene was detected by PCR amplification.The expression of LZTS1 gene in lung cancer tissues and plasma ctDNA was compared.Consistency,the relationship between LZTS1 gene expression and clinicopathological features of lung cancer tissues was analyzed,and the correlation between LZTS1 gene and LZTS1 protein expression in the same lung cancer tissue samples was compared.Results:1.The expression of LZTS1 gene in lung cancer tissues was changed.The expression of LZTS1 gene decreased(+/-)in 16 cases(16/50,32%),loss of expression(-)in 16 cases(16/50,32%),and normal expression(+)in 18 cases(18/50,36%).2.The higher the smoking index,the higher the incidence of loss or/and decrease in LZTS1 gene expression in lung cancer tissues.3.There was a good agreement between LZTS1 gene expression status in lung cancer tissues and plasma ctDNA(p=0.000,Kappa value=0.791,p<0.05 and 0.791>0.75).4.There was a significant correlation between LZTS1 gene and LZTS1 protein expression in lung cancer tissues.Part Three:LZTS1 gene coding region detectionMethods:Two samples were chosen,one case was selected in two cases with normal expression of LZTS1 gene but decreased expression of LZTS1 protein,another case was chosen from three samples with normal expression of LZTS1 gene but loss of expression LZTSI protein.The DNA in the lung cancer tissues was extracted.The LZTS1 genomic DNA coding region was sequenced by PCR amplification.Analyze the presence of genetic mutations.Results:1.The C?T mutation of the base station at position 922 of the LZTSI gene coding region does not change after the mutation,and the mutation is a synonymous mutation.2.The LZTS1 gene coding region 1168 base site G?Amutation,after the mutation,the amino acid does not change,the mutation is a synonymous mutation.3.G?A mutation at base station 1949 of the LZTSI gene coding region can result in a change in the amino acid encoded by the site,which is a missense mutation.Part Four:LZTS1 gene promoter region methylation detectionMethods:The genomic DNA of 6 lung cancer eell lines was extracted and treated with bisulfite.Then the genomic DNA was amplified by MSP method,2%agarose gel electrophoresis,and the gel electrophoresis PCR product was recovered and then ligated to T vector,transfected and identified.The methylation status of the promoter region of LZTS1 gene was detected by direct sequencing.Results:1.The degree of methylation in the promoter region of the LZTS1 gene of different tumor-derived cell lines in lung cancer cell lines was inconsistent.2.The promoter region of LZTS1 gene in lung cancer cell lines has a high degree of methylation,and the average methylation frequency is 66.67%.Conclusions:1.The expression of LZTS1 protein and gene was changed in primary lung cancer tissues.2.Decreased and/or deleted expression of LZTS1 protein is associated with progression of lung cancer and may result in tumor cells being more susceptible to metastasis.3.There was a good agreement between LZTS1 gene expression status in lung cancer tissues and plasma ctDNA(p=0.000,Kappa value=0.791,p<0.05 and 0.791>0.75).4.There was a significant correlation between LZTS1 gene and LZTS1 protein expression in lung cancer tissues.5.G?A mutation at base station 1949 of the LZTS1 gene coding region can result in a change in the amino acid encoded by the site.6.The promoter region of LZTS1 gene in lung cancer cell lines has a high degree of methylation,and the average methylation frequency is 66.67%.