Supplementary Materialsijms-20-01697-s001. findings suggested that syn-miR-143 acted as a tumor suppressor through the impairment of KRAS networks including the DDX6. 0.001. 2.2. Expression Levels of KRAS and Downstream Molecules Were Up-Regulated in HER2-Positive Gastric Cancer Cell Lines We investigated the expression level of HER2 in the gastric cell lines by performing Western blotting (WB). As shown in Figure 1B, the expression of HER2 was extremely high in MKN-7 cells, which display HER2 gene amplification, and in KATO-III cells, in which FGFR2 gene amplification occurs, when compared with the expression in MKN-74 cells, having no gene amplification of receptor of tyrosine kinases including HER2. In addition, the expression levels of downstream molecules such as KRAS, AKT, and ERK were Rabbit Polyclonal to CKS2 up-regulated in MKN-7 PROTAC Sirt2 Degrader-1 and KATO-III cells compared with those of the other gastric cancer cell lines examined (Figure 1B). Regarding KRAS mutation, both MKN-7 and KATO-III cells do not harbor any mutation of KRAS. Compared with that in HER2-positive breast cancer cell line SKBR-3, the expression levels of HER2 in HER2-positive gastric cancer cell lines MKN-7 and KATO-III were considerably lower. (Supplementary Figure S1). However, the expression level of KRAS in HER-2 gastric cancer cell lines was higher than that in the SKBR3 cell line (Supplementary Figure S1). The inverse correlation between miR-143 and HER2 or KRAS was not significant, but there was a tendency for such a correlation (Supplementary Figure S1). Since we elucidated the relationship between HER2 overexpression PROTAC Sirt2 Degrader-1 and the downstream transduction via miR-143, we focused on MKN-7 and KATO-III cells for further PROTAC Sirt2 Degrader-1 study. 2.3. Ectopic Expression of miR-143 Inhibited the Growth of MKN-7 and KATO-III Cells by Targeting KRAS and Its Related Signaling Molecules To investigate the effect of miR-143 on HER2-positive gastric cancer cells, we transfected MKN-7 and KATO-III cells with syn-miR-143. The ectopic expression of miR-143 in both cell lines significantly reduced the number of viable cells (Figure 2A). These results suggested that miR-143 functioned as a tumor suppressor microRNA (TS-miR) in HER2-positive gastric cancer. We considered that this inhibition of cell growth was due to suppression of KRAS networks by miR-143. Therefore, we next examined the expression levels of KRAS by performing WB and qRT-PCR. The expression level of KRAS protein in both cell lines was down-regulated by the transfection with syn-miR-143 (Figure 2B). In addition, in MKN-7 cells the down-regulation of KRAS was observed PROTAC Sirt2 Degrader-1 even at the mRNA level, which did not occur in the KATO-III cells (Figure 2B). Subsequently, we examined the expression levels of the effector molecules of KRAS by performing WB. The down-regulation of AKT, ERK, and c-MYC proteins was observed in MKN-7 and KATO-III cells (Figure 2C). The expression levels of pAKT and benefit had been up-regulated in MKN-7, however, not in KATO-III, cells (Shape 2C). Concerning SOS1, the manifestation degree of its proteins was also reduced in MKN-7 and KATO-III cells. Therefore, these findings were much like those manufactured in the entire case of cancer of the colon cells [15]. Open in another window Shape 2 Ectopic manifestation of miR-143 in gastric tumor cells MKN-7 and KATO-III. (A) Cell viability at 72 h after transfection of MKN-7 and KATO-III cells with control RNA or man made miR-143 syn-miR-143. (B) Traditional western blot evaluation and qRT-PCR of KRAS manifestation at 72 h after transfection with control RNA (20 nM) or syn-miR-143 (5 nM, 20 nM). Densitometric ideals of KRAS/-actin had been calculated, as well as the ideals of settings are indicated as 1. (C) Traditional western blot analysis from the PROTAC Sirt2 Degrader-1 expression degrees of AKT, pAKT, ERK1/2, benefit1/2, C-MYC, and SOS1 at 72 h after transfection with control RNA (20 nM) or.