Vermani Litika, Kumar Rajeev, Senthil Kumar Nachimuthu
Biotechnology, Mizoram University, Aizawl, IND.
Research, Cachar Cancer Hospital and Research Centre, Silchar, IND.
Cureus. 2020 Dec 10;12(12):e12020. doi: 10.7759/cureus.12020.
Background The overwhelming majority of published articles have taken colon and rectal cancer as a single group, i.e., colorectal cancer, when normalizing gene expression data with housekeeping genes (HKG) in quantitative polymerase chain reaction (qPCR) experiments though there are published reports that suggest the differential expression pattern of genes between the colon and rectal cancer groups and hence the current experiment was attempted to find out the optimal set of housekeeping genes from the list of common HKG for rectal tumor gene expression analysis. Methods The expression of five potential housekeeping genes GAPDH, RPNI, PUM1, B2M, and PMM1 was analyzed through qPCR and Bestkeeper software (http://www.wzw.tum.de/gene-quantification/bestkeeper.html) in 20 stage II-IV rectal cancer samples to check for uniformity in their expression pattern. Cancer stem cell (CSC) marker ALDH1 and epithelial-mesenchymal transition marker (EMT) markers E cadherin, vimentin, Twist, and SNAI2 expression were evaluated in conjunction with the two optimal reference genes in 10 rectal cancers as part of validation. Results The standard deviation of the cycle threshold value of GAPDH was found the lowest at 0.65 followed by RPN1 at 0.88, PUM1 at 0.94, PMM1 at 0.94, and B2M at 1.21 when analyzed with BestKeeper software. Using GAPDH and PUM1 as the reference gene for the validation phase, rectal cancer patients with stage III/IV showed a 4.79-fold change (P=0.006) in ALDH1 expression, and an 11.76-fold change in Twist expression (P=0.003) with respect to stage II rectal tumor when normalized with GAPDH and PUM1. Conclusion GAPDH and PUM1 can be used as an optimal set of housekeeping genes for gene expression-related experiments in rectal tumors. ALDH1 and Twist were found significantly overexpressed in stage III/IV rectal tumors in comparison to stage II rectal cancer. Genes associated with cancer stem cells and EMT markers could be optimally analyzed by normalizing them with GAPDH and PUM1 as housekeeping genes.
在定量聚合酶链反应（qPCR）实验中，绝大多数已发表的文章在使用管家基因（HKG）对基因表达数据进行标准化时，将结肠癌和直肠癌视为一个单一的组，即结直肠癌，尽管有已发表的报告表明结肠癌组和直肠癌组之间基因的差异表达模式，因此当前实验试图从常见管家基因列表中找出用于直肠肿瘤基因表达分析的最佳管家基因集。方法：通过qPCR和Bestkeeper软件（http://www.wzw.tum.de/gene - quantification/bestkeeper.html）分析20例II - IV期直肠癌样本中五个潜在管家基因GAPDH、RPNI、PUM1、B2M和PMM1的表达，以检查其表达模式的一致性。作为验证的一部分，在10例直肠癌中结合两个最佳参考基因评估癌症干细胞（CSC）标志物ALDH1和上皮 - 间质转化标志物（EMT）标志物E - cadherin、波形蛋白、Twist和SNAI2的表达。结果：使用BestKeeper软件分析时，发现GAPDH的循环阈值标准差最低，为0.65，其次是RPN1为0.88，PUM1为0.94，PMM1为0.94，B2M为1.21。在验证阶段，以GAPDH和PUM1作为参考基因，与II期直肠肿瘤相比，III/IV期直肠癌患者的ALDH1表达变化了4.79倍（P = 0.006），Twist表达变化了11.76倍（P = 0.003）。结论：GAPDH和PUM1可作为直肠肿瘤中与基因表达相关实验的最佳管家基因集。与II期直肠癌相比，发现ALDH1和Twist在III/IV期直肠肿瘤中显著过表达。通过将与癌症干细胞和EMT标志物相关的基因用GAPDH和PUM1作为管家基因进行标准化，可以对其进行最佳分析。
