MiR-100 Is Involved In Gain-of-function Mutant SHP-2 -enhanced MEFs Malignant Transformation Induced By As₂O₃

Tumor is a serious, life-threatening disease. Recently, the incidence and mortality have gradually increased in general population, and the newly established early onset trend is even more worrisome. Tumorigenesis is thought to be a long-term process that is influenced by environmental, genetic and epigenetic factors. Therefore, exploring the underlying mechanisms of tumorigenesis is of great significance.SHP-2, a tyrosine phosphatase encoded by PTPN11 gene, is involved in the activation of PI3K, MAPK, JAK/STAT signaling pathway. It’s mutants was fistly found in Noonan Syndrome,researchers have shown that SHP-2 mutants have a significant role in leukemia and solid tumors. D61G/D61Y mutant is common in tumors which our laboratory has devoted to.Arsenic, existing in water and soil,is a recognized chemical carcinogen which plays an important role in skin, lung and other cancers. Previous study has shown that activated mutations of SHP-2 tyrosine phosphatase enhance the risk of MEFs malignant transformation by arsenic. To explore it’s mechanism, we treated MEF with AS2O3 for 48h,then we found that the expression of miR-100 was reversely correlated with SHP-2D61G/+mutation and As2O3 treatment.MiR-100 is a small non-coding RNA, which has been implicated in the occurrence and development of lung cancer, breast cancer, cervical cancer, lymphoma and other tumors. But it is still unclear how miR-100 interacts with environmental carcinogens and genetic mutations. We hypothesize that miR-100 is involved in the gain-of-function mutant SHP-2-enhanced malignant transformation of MEFs induced by AS2O3.To test this hypothesis, we first used RT-PCR to detect the expression of miR-100 in SHP-2+/+、SHP-2D61G/+、SHP-2+/+-40 and SHP-2D61G/+-40 cells. The result showed that the expression of miR-100 was reversely correlated with the mutation and the As2O3 treatment, which implies that miR-100 is involved in the process. To further confirm the hypothesis and further understand the causal relationship, we manipulated the expression of miR-100 in indicated cells by transfection with miR-100 inhibitor or miR-100mimics. The results showed that miR-100 may reduce the malignant biological behavior, such as proliferation, colony forming ability, anchor independent growth and migration in vitro. Furthermore, the dual luciferase reporter assay demonstrated that both IGF1Rβ and mTOR are likely the target gene of miR-100. IGF1R expression in MEFs cells increased with the induction of As2O3 and the up-regulation was more significance in SHP-2D61G/+ background. Finally, similar results were observed in stable cell lines established by lentiviruses that expresse miR-100 inhibitor or miR-100mimics. The above results show that miR-100 is involved in the gain-of-function mutant SHP-2-enhanced malignant transformation of MEFs induced by As2O3,and acts as a suppressor.