Cell line selection
The rates of expression of B7H6 siRNA were compared utilizing real-time PCR in MKN-45 and KATO-III cell lines after RNA extraction and cDNA synthesis. According to the data presented in Figure 1, significant increases were seen in the MKN-45 cell line. in B7H6 gene expression among the two cell lines examined. The MKN-45 cell line was selected for further investigations.
MTT test results for determination of IC50 of FU-5 on MKN-45 cell line
To determine the inhibitory concentrations of 5-FU, MKN-45 cells were given various doses of this chemotherapeutic drug, and the MTT test was used to measure cell viability. As seen in Figure 2, MKN-45 cells were treated with 5-FU at various doses ranging from 1 to 100 µg / mL, which resulted in a continuous drop in cell survival rates. 5-FU treatment of the cells (38.025 µg / mL) resulted in cell viability decrease down to 50%, compared to the control ones. As a result, this concentration was used as the IC50 of 5-FU in subsequent analyses.
Transfection of B7H6 siRNA into MKN-45 cells
Following transfection of MKN-45 cells with a variety of B7H6 siRNA dosages at various time points, we used qRT-PCR to find the optimal situation for B7H6 suppression. As a consequence, our data demonstrated that B7H6 mRNA expression was dose-dependently downregulated due to transfection of MKN-45 cells with B7H6 siRNA relative to the control cells. Additionally, 48 hours after transfection, a more efficient reduction of B7H6 was found. As a result, the next tests used 60 pmol of siRNA as the optimal transfection dosage at 48 h (Figure 3).
The B7H6-siRNA enhanced MKN-45 cells chemosensitivity to 5-FU.
Our data imply that combining B7H6 silencing with 5-FU (IC50) induced a considerable decrease in MKN-45 cell line viability in comparison with monotherapy with 5-FU (IC50) (Figure 4). Thus, silencing the B7H6 gene increases the chemosensitivity of MKN-45 cells to 5-FU and decreases the amount of 5FU needed to reduce the viability of MKN-45 cells.
The combined impact of B7H6 siRNA and 5-FU on MKN-45 cells' apoptosis
We utilized Annexin V-FITC/PI double staining assay to examine whether B7H6-siRNA alone or in addition with 5-FU induces apoptosis in MKN-45 cells. As shown in Figure 5 A and B, transfection of cells with B7H6-siRNA or treatment with the IC50/IC25 concentrations of 5-FU alone may substantially (p<0.05 and p<0.0001) trigger apoptosis in these cells relative to normal control. Consequently, combined therapy raised cell apoptosis rate as much as either siRNA targeting B7H6 or 5-FU treatment alone. Additionally, the apoptosis rate was considerably greater in the IC25/IC50+siRNA group than in cells treated with the IC25/IC50 of 5-FU, validating MTT findings that B7H6 knockdown might enhance the cytotoxic impact of 5-FU on MKN-45 cells by boosting apoptosis generated by 5-FU.
Expression changes of BAX, MMP3, Caspase-3, and Bcl-2 genes through combination therapy with B7H6/5-FU using real time-PCR
The rates of mRNA expression of key modulators of cell apoptosis, such as Bax, Bcl-2, MMP3, and Caspase-3, were measured using real-time PCR in order to validate these findings and examine the mechanism behind treatment-induced apoptosis. As shown in Figure 6, our findings indicated that the pro-survival gene Bcl-2 was considerably downregulated after B7H6 siRNA was transfected into cells, and they were treated with 5-FU medication (p<0.0001), compared to the control cells. Combination treatment resulted in the lowest MMP3 expression levels (p<0.01). Additionally, in contrast to individual treatments and control cells, the combined therapy of MKN-45 cells with B7H6 siRNA and 5-FU significantly enhanced the rates of expression of Bax and caspase-3 (p<0.001, p<0.01), which are critical inducers of apoptosis. As a consequence, these results suggest that silencing B7H6 might enhance 5-FU-induced apoptosis in MKN-45 cells via modifying the expression of genes involved in apoptosis.
Sub-G1 cell cycle arrest was achieved when B7H6 silencing was combined with 5-FU therapy.
To figure out whether 5-FU treatment, as well as a combination of B7H6 siRNA and 5-FU therapy, has an impact on cell growth, we examined the dispersion of MKN-45 cells throughout the cell cycle following 5-FU treatment and the combination of 5-FU therapy and B7H6 siRNA transfection. Flow cytometry analysis revealed that only 5-FU drug treatment increased the number of sub-G1 phase cells by 17.6 percent in IC25 treated groups and 20.1 percent in IC50 treated groups in relation to the control group. Moreover, MKN-45 cells were treated with combination therapy with 5-FU and B7H6 siRNA, which indicated that the number of sub-G1 phase cells increased from 17.6 to 28.5 (IC25+ siRNA group) and from 20.1 to 33.1 (IC50+ siRNA group) in comparison to treated groups (Fig.7).
The findings indicate the simultaneous impact of B7H6 siRNA and 5-FU on MKN-45 cell migration
The wound healing experiment was used to study the effects of B7H6 siRNA and 5-FU on MKN-45 cell migration. The qualitative test results were captured as a series of photographs, with the images in Figure 8 demonstrating a significant decrease in cell migration. The 5-FU treated groups were stronger and more powerful when B7H6 siRNA was used beside the drug dosage in groups simultaneously with transfected siRNA.
The interaction of B7H6 siRNA and 5-FU inhibited stemness characteristics in MKN-45 cells We developed a colony formation test to examine the influence of B7H6 siRNA/5-FU on stemness characteristics in MKN-45 cells. The findings suggest that when B7H6 siRNA and 5-FU are used once, there will be a decline in the number of colonies. in comparison to the control group (Fig.9). Additionally, combination treatment inhibited MKN-45 clonogenic potential more effectively than other monotherapies.