A single nuclear polymorphism in let‐7g binding site affects the doubling time of thyroid nodule by regulating KRAS‐induced cell proliferation

As an indicator for the malignancy of thyroid nodules (TN), the doubling time of TN was studied in this study to evaluate the effect of rs712 polymorphism on the progression of TN. In addition, we aimed to study the potential molecular mechanisms underlying the pathological effect of rs712 polymorphism upon TN. A Taqman method was used to genotype the patients according to their rs712 polymorphism. Real‐time polymerase chain reaction, western blot, Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium (MTT) assay was conducted to study the correlation between KRAS expression and the pathological effect of rs712 polymorphism. In‐silicon analysis and luciferase assay were utilized to establish the regulatory relationship between let‐7g and KRAS. KRAS messenger RNA (mRNA)/protein levels in the GG group were upregulated with a decreased apoptosis index. KRAS mRNA was validated to be a virtual target of let‐7g. In addition, the mRNA/protein level of KRAS as well as cell proliferation index was decreased in primary thyroid cancer cells genotyped as TT/TG and transfected with KRAS small interfering RNA (siRNA)/let‐7g precursors. The cell apoptosis index was evidently elevated in the KRAS siRNA/let‐7g precursors group compared with that in the scramble controls. Moreover, KRAS mRNA/protein only showed slight reduction when GG‐genotyped primary thyroid cancer cells were transfected by let‐7g precursors. Additionally, let‐7g precursors exhibited no significant effect on cell proliferation index or cell apoptosis in GG cells. Rs712 polymorphism T>G in the 3′‐untranslated region of KRAS interrupts the interactions between let‐7g and KRAS mRNA, leading to a higher cell proliferation index and reduced doubling time of TN. In this study, we demonstrated that the rs712 located in KRAS 3′‐untranslated region (3′‐UTR) was involved in the doubling time of thyroid nodules (TN) by affecting KRAS‐induced cell proliferation. We also identified KRAS as a target gene of let‐7g. The rs712 located in KRAS 3′‐UTR can disrupt the let‐7 binding site and finally result in the dysregulation of KRAS. For example, the rs712 TT genotype enhanced the binding between let‐7g and KRAS and inhibited KRAS expression, thus resulting in cell apoptosis and extended doubling time of TN in subjects genotyped as TT+TG. Therefore, the rs712 located in KRAS 3′‐UTR may act as a novel biomarker of the doubling time of TN with the presenc