This paper verifies that the miR-17-5p’s expression level in A549/G + was lower than that in A549/G-. We first verified that RRM2 was one of miR-17-5p’s target genes in A549/G + cells with double luciferase gene report experiment, and then verified this conclusion by qRT-PCR.
CCK-8 assay and clone formation survival experiment showed that overexpression of miR-17-5p or low-expression of RRM2 in A549/G + cells, the cell survival rate decreased, IC50 decreased, clone formation ability decreased, and the cells became sensitive to gemcitabine. On the contrary, low-expression of miR-17-5p or overexpression of RRM2 in A549/G- cells, the survival rate of cells increased, IC50 increased, clonogenic ability increased, and the cells became drug-resistant. This suggests that the tolerance of A549 to gemcitabine is related to miR-17-5p/RRM2 pathway.
Coincidentally, Duxbury et al.  studies suggest that over expression of RRM2 in pancreatic cancer cells can cause resistance to gemcitabine chemotherapy. When siRNA inhibited RRM2 expression, it could enhance the cytotoxicity induced by gemcitabine and improve the sensitivity of cells to gemcitabine; It coincides with our results. Similarly, Mah et al.  used tissue microarray analysis to conclude that RRM2 is a very useful disease-specific predictor, which can predict the survival outcome of different subtypes of non-small cell lung cancer. High levels of RRM2 expression were associated with poor survival outcomes. This is the same as the result of "RRM2 is highly expressed and cells become drug resistant".
Similar studies as Grolmusz et al  found gemcitabine can strongly inhibit the proliferation of ACC and increase their apoptosis. However, after a period of treatment, RRM2 was up-regulated, resulting in cell resistance to gemcitabine, which was consistent with the clinical therapeutic effect of ACC. This also shows that the high expression of RRM2 can make cells develop drug resistance. In addition, Jin et al  found that expression level of RRM2 was much higher in tumor tissues of patients with lung adenocarcinoma than that in normal tissues; The high expression of RRM2 can boost A549 cells proliferate. On the contrary, inhibiting the expression of RRM2 can also inhibit A549 cells proliferate. This is consistent with our results.
The results of flow cytometry showed that overexpression of miR-17-5p or low-expression of RRM2 in drug-resistant cells A549/G+, the number of G1 phase cells rose and the number of S phase cells declined. Instead, when interfering with the expression of miR-17-5p or overexpressing RRM2 in sensitive cells A549/G-, the number of G1 phase cells dropped and the number of S phase cells went up. Based on the previous results, under the same miR-17-5p (or RRM2) expression conditions, when the number of G1 phase increases, the cells become sensitive; When the number of G1 phase decreased, the corresponding cells became resistant. This suggests that the tolerance of A549 to gemcitabine is related to the cell cycle.
Then, we selected the G1/S related proteins CCNE1, CCNA2 F and P21 for Western blotting detection. These results presented that expression levels of CCNE1, CCNA2 and P21 down-regulated at overexpression of miR-17-5p or low-expression of RRM2 in drug-resistant A549/G+. On the contrary, the expression levels of CCNE1, CCNA2 and P21 up-regulated at low-expression of miR-17-5p or overexpression of RRM2 in sensitive cells A549/G-. This indicates that the tolerance of A549 to gemcitabine is related to cell cycle related proteins CCNE1, CCNA2 and P21.
CCNE1 appears in the late stage of G1 phase, and its expression can be increased by cytokines and certain tumors. CCNE1 can promote the G1/S phase transition, shorten the G1 phase, and reduce the cell’s requirement for growth factors [15, 16]. CyclinE-CDK2 complex is a key kinase for cells to enter S phase from G1 phase [17–19]. It can phosphorylate its substrate proteins, such as retinoblastoma protein (Rb), Rb family members P107, CDC6, etc. CCNA2 is mainly expressed in S phase and mediates initiation of the S phase , which is a key cell cycle regulator and essential for DNA synthesis and G1/S transition . Therefore, when miR-17-5p is overexpressed (or low-expression of RRM2), when the expression level of CCNE1 decreases, it is difficult for cells to complete G1/S transformation, resulting in cell arrest in G1 phase, cell proliferation is blocked, and cells become sensitive to gemcitabine; However, when the expression level of miR-17-5p (or RRM2) is opposite, the cells become resistant to gemcitabine.
Then we detected the expression level of PI3K/AKT signaling pathway protein. Firstly, Western blotting suggested that PTEN and PI3K expressed higher level in A549/G + cells than did in A549/G- cells, but the level of p-PTEN (phosphorylated PTEN) was lower. This shows that the activated PTEN (p-PTEN) is at a low level in A549/G + cells and has a weak inhibitory effect on AKT. AKT is activated and can activate downstream signal molecules and play its physiological function, including cell growth, survival and proliferation, etc. All these help tumor cells survive better under drug pressure and show drug resistance [22, 23]. Secondly, PI3K level and PTEN level did not change, but p-PTEN level increased and p-AKT (phosphorylated AKT) level decreased at overexpression of miR-17-5p in A549/G + cells. When miR-17-5p was inhibited in A549/G- cells, the levels of PI3K and PTEN remained unchanged; The level of p-PTEN decreased and the level of p-AKT increased. This suggested that the miR-17-5p’s expression level was consistent with that of p-PTEN, but opposite to that of p-AKT. However, it had no change the expression level of PI3K and PTEN. That is to say that miR-17-5p could positively regulate p-PTEN, while p-PTEN negatively regulates p-AKT. After overexpression of miR-17-5p in A549/G + cells, the level of p-PTEN increased and inhibited the level of p-AKT, thus inhibiting the downstream signal pathway function of AKT, inhibiting the drug resistance of cells, and finally the cells became sensitive. Otherwise, knocking down the expression of miR-17-5p in A549/G- cells, the level of p-PTEN also decreases, and the effect on p-AKT is weakened. p-AKT can start the function of downstream signal pathway and promote the resistance of cells to drugs [23, 24]. When we use RRM2 instead of miR-17-5p for restorative experiment, we can reproduce the previous experimental results. This indicates that miR-17-5p/RRM2 is involved in the regulation of PI3K/AKT signaling pathway and regulates the cell response to drugs by regulating PI3K/AKT pathway. Such similar results were also reported in the study of Shah et al. Their experiments have shown that high expression of AKT can induce breast cancer cells to resist tamoxifen. It is also indicated that knockdown of RRM2 expression in AKT induced tamoxifen resistant breast cancer cells can inhibit the proliferation of these breast cancer cells under tamoxifen, that is, the reduction of RRM2 expression can reverse AKT induced tamoxifen resistance. This indicates that AKT induced breast cancer cell tamoxifen is closely related to RRM2. RRM2 and PI3K/AKT pathway may play an important role not only in drug resistance of lung cancer, but also in drug resistance of other tumors.