- Opting for Local Region in SHOX2 Promoter as a DNA Methylation Biomarker for Lung Cancer Diagnosis. - Abstract: Epigenetic alterations play a main role in the initiation and progression of lung cancer. - CpG methylation in the promoter of the Short Stature Homeobox 2 (SHOX2) gene has been evaluated and validated at different stages of this malignant disease using quantitative methylation-specific PCR (qMSP) method. - In this study, formalin-fixed, paraffin-embedded (FFPE) tissue samples were collected from 30 lung cancer patients and 30 patients suffering from non-cancerous pulmonary diseases. - The methylation level of SHOX2 was evaluated in two CpG-riched regions of the promoter by using qMSP. - The SHOX2 methylation level of both regions in lung cancer was significantly higher than that in non-cancerous lung diseases (23.62% versus 0.23%, and 8.52%. - versus 0.65%, respectively), indicating that SHOX2 methylation could be conferred as a potential biomarker to lung cancer.. - DNA methylation occurring at CpG dinucleotides that frequently locate in promoter regions is well known as an epigenetic regulation mechanism for transcriptionally silencing gene expression [1].. - https://doi.org vnunst.5333. - Currently, commercially available IVD tests by the type of methylation-based biomarker have been applied for diagnosis, prognosis, and predictive of various types of cancers such as lung, breast, cervical, colorectal, prostate, and even cancers of the unknown primary site [6].. - Lung cancer is the leading cause of cancer-related mortality worldwide [7]. - most biomarkers widely used for lung cancer diagnosis is serum biomarker and low-dose CT screening, which have the high false-positive rates [8]. - Therefore, investigating DNA methylation as a biomarker for lung cancer detection has been extensively investigated.. - Currently, several valuable DNA methylation markers have been evaluated and validated at different stages of lung cancer and across ethnicities [9, 10]. - Lung cancer is also the leading cause of cancer-related mortality and can reach double incidence in 2025 [12]. - Therefore, investigating and evaluating DNA methylation as a powerfully auxiliary biomarker for cancer in general and lung cancer, in particular, is urgently needed.. - Preliminary research on qualitative DNA methylation of genes involved in breast, colorectal cancer has been previously described to Vietnamese patients. - The most important ensuring the clinic value of DNA methylation marker is primer sets using for quantitatively specific methylation real-time PCR (qMSP-PCR) reaction must be designed based on CpG riched sequences whose methylation level should be significantly altered in cancer as comparison with noncancerous or healthy subjects [14].. - In this study, using the qMSP-PCR method we investigated the quantitative methylation level at the SHOX2 promoter gene in Vietnamese patients who suffered from cancer and non-cancerous lung diseases. - The SHOX2 gene consisted of three promoters and three CpG islands, one of which overlaps with the first exon and whose methylation level has. - been extensively investigated in order to developing a biomarker for lung cancer detection . - This study aims at opting for CpG riched sequences in the SHOX2 promoter region and evaluating their methylation levels in these tissues.. - Furthermore, the comparison of the SHOX2 methylation profiles in lung cancer and non-cancerous lung tissues will highlight the potential value of epigenetic biomarkers to contribute to the effective lung cancer diagnosis in our country.. - Out of 30 lung cancer, 24 were derived from early stage of lung cancer (stage I/II). - Subsequently, genomic DNAs were subjected to bisulfite conversion using the EZ DNA Methylation-Gold kit (Zymo Research). - Primer sets for methylation specific PCR method were designed for measuring the methylation level of the SHOX2 promoter region (NG-047079 positions 7750-7730). - region overlaps with the first exon and contains the CpG island whose methylation was altered in lung cancer . - Specific primers that are complementary to the sense strand of the bisulfite converted SHOX2 were designed using the Methyl Primer Express Software v1.0. - Two reverse primers were derived from two consecutive sequences on the SHOX2 promoter. - One forward primer was separately combined with two reverse primers in the qPCR reactions to amplify the methylated SHOX2.1 and SHOX2.2 sequences, respectively. - In addition, the classical ΔΔCT approach using a calibrator reference was used for relative calculation of methylation level;. - The bisulfite converted ACTB and methylated SHOX2 sequences were amplified from bisulfite converted DNA extracted from lung cancer sample, purified by GeneJET PCR. - The SHOX2 methylation level was calculated by using the ΔΔCT method that requires a calibrator sample with a defined methylation level. - In this study, the defined methylation level of 10% was obtained by mixing linearized pACTB and pMe-SHOX plasmids. - A serial dilution of the linearized recombinant plasmids pACTB and pMe-SHOX containing bisulfite converted ACTB and methylated SHOX2 sequences, respectively were used for determination of cut-off value for the measurement of SHOX2 methylation level.. - Simple linear regression fits a straight line through Ct values of a serial concentration of plasmids to find the best-fit value of the slope and intercept. - Comparisons between two groups on the methylation level were assessed by using the Mann-Whitney U test and were graphed in the box-and-whisker plot format. - Specificity of the Designed Primers In order to confirm the accuracy of the methylated specific primer derived from the SHOX2 promoter sequences, the PCR product amplified by the SHOX-Me-F/SHOX-Me-R2 primer pair and successfully cloned into. - Nucleotide sequence of the insert in pMe-SHOX plasmid. - Nucleotides present in the primers were arrowed. - All cytosines in the CpG sites remain cytosines while the cytosines alone were converted to thymines.. - plasmid concentrations (from 10 1 to 10 4 copies/reaction), showing high amplification eficiency of qPCR, thus meaning that ΔΔCT calculation was suitable to analyze the methylated SHOX2 level of two SHOX2.1 and SHOX2.2 regions.. - Analytical performance of the SHOX2 qPCR assay. - A serial concentrations of the plasmids pACTB and pMe-SHOX were used as templates for qPCR reactions amplified with primer pair ACTB-F/ACTB-R (A), SHOX-Me-F/SHOX-Me-R1 (B) and SHOX-Me-F/SHOX-Me-R2 (C), respectively. - Analysis of SHOX2.1 and SHOX2.2 Methylation Levels in Patients with Lung Cancer and Non-cancerous Lung Diseases. - In order to investigate the methylation profile of SHOX2, two regions enriched CpGs on the promoter (positions NG-047079), SHOX2.1 and SHOX2.2, was chosen to analysis on 30 FFPE samples (30 patients with lung cancer versus 30 patients with non-cancerous lung diseases). - statistics of SHOX2.1 and SHOX2.2 methylation were shown in Table 2. - The methylation level of both regions was low in non-cancerous lung diseases (0.23 and 0.65) (Figure 3A) but significantly increased in lung cancer, and the methylation level of SHOX2.1 was significantly higher than that of SHOX2.2 (23.62 versus 8.52, respectively) (Figure 3B).. - Both regions were dramatically hypermethylated in lung cancer (Figure 3C, D).. - Descriptive statistics of SHOX2.1 and SHOX2.2. - LC: lung cancer. - Descriptive statistics SHOX2.1 SHOX2.2. - Different variance in SHOX2.1 and SHOX2.2 methylation could be explained by potential transcription factor binding sites for SHOX2 promoter [15] and epigenetic heterogeneity in cancer [18]. - thus, the precise location of clinically relevant methylated CpGs plays an important role in the development of a DNA methylation-based biomarker [20]. - Moreover, epigenetic variation between cancer cells within a tumor of the same patient (intratumor heterogeneity) is a remarkable tumor stages [21]. - Our result was in line with previous reports on SHOX2 hypermethylation in lung cancer. - Moreover, SHOX2.1 was higher methylated than SHOX2.2 in lung cancer, thus allowing better discrimination of lung cancer from non-cancerous lung diseases.. - SHOX2 methylation. - Methylation level between SHOX2.1 and SHOX2.2 in non-cancerous lung diseases (NC) (A) and in lung cancer (LC) (B).. - Hypermethylation was observed in SHOX2.1 (C) and SHOX2.2 (D) in NC and LC. - We have showed that hypermethylation in SHOX2.1 sequence could be conferred as a potential biomarker to lung cancer. - These encouraging results prompt us to extend the SHOX2.1 methylation analysis in noninvasive liquid biopsy samples, in the common effort to foster the use of DNA methylation analysis in biomarker development and clinical applications.. - 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