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Han et al. Cancer Drug Resist 2024;7:16 https://dx.doi.org/10.20517/cdr.2024.01 Page 21 of 25

Next, the genes and pathways through which isocuB acts were verified. A series of experiments confirmed
that isocuB inhibited the proliferation, migration, and invasion of U251 and U87 cells, and induced
apoptosis of U251 cells. Furthermore, the inhibitory effect of isocuB on U251 cells was significantly greater
than that of TMZ. We hypothesized that it functions through the MMP family. The MMP family is
associated with the survival, proliferation, apoptosis, invasion, and metastasis of cancer cells . The WB and
[25]
RT-qPCR results indicated a significant decrease in the mRNA and protein expression levels of MMP-2 and
MMP-9. EMT is a process in which cells acquire invasive mesenchymal potential by undergoing a series of
events such as loss of cell junctions, alteration of the cytoskeleton, and remodeling of the extracellular
matrix . The results showed a significant reduction in the protein levels of N-cadherin and Vimentin,
[26]
indicating that isocuB could inhibit EMT and thus inhibit glioma invasion. Several studies have shown that
the expression of MMP2/9 is regulated by the STAT3 signaling pathway in a variety of solid tumors,
suggesting that increased activation of STAT is the cause of upregulation of MMP2/9 expression in cancer
cells [32,33] . Our results further demonstrated that isocuB could impede glioma cell proliferation and migration
by inhibiting STAT3 to inhibit MMP2/9. For specific pathways, WB and RT-qPCR experiments were
performed for verification. Our study showed that isocuB could inhibit MAPK and STAT3 phosphorylation
and PI3K-AKT non-phosphorylated expression, but not PI3K-AKT phosphorylated expression. Our results
demonstrated that isocuB could inhibit U251 cells with the modulation of PI3K/AKT and MAPK signaling
pathways. To further investigate the mechanism of action of isocuB, we verified the expression of pdk1 and
Bcl-2 in vitro using RT-qPCR. The experiments in vitro showed that isocuB downregulated the protein and
mRNA expressions by inhibiting PDK1, RXRα, PPARα, and Bcl-2. Different PPARs have significant effects
on cancer : PPARα is implicated in promoting proliferation, whereas PPARγ exhibits the opposite effect.
[34]
[36]
Previous studies have linked PPARα to the promotion of the PI3K-AKT pathway . Furthermore, RXRα ,
[35]
PDK1 [37,38] , and Bcl-2 [39,40] genes are involved in the PI3K/AKT and MAPK signaling pathways. These results
further demonstrated that isocuB acts with the regulation of the PI3K/AKT and MAPK signaling pathways.
Hsa-mir-1268a is upregulated in both cells and exosomes of highly metastatic melanoma CSCs. In addition,
the OL-ScS-derived exosome hsa-mir-1268a, after being taken up by OL cells, promoted the transfer and
colonization ability of OL cells in vitro and in vivo. Therefore, inhibiting hsa-mir-1286a can inhibit tumor
[41]
metastasis and colonization . Then, through database search and literature review, we found that hsa-mir-
1286a has great research potential in glioma resistance , and hsa-mir-1268a was inhibited, and its
[42]
inhibitors enhanced adriamycin-induced liver cancer cell death . Our RT-qPCR experiment observed a
[43]
gradual reduction in hsa-mir-1286a RNA content with increasing drug concentration. After inhibiting the
expression of hsa-mir-1286a, the drug sensitivity of TMZ in U251 can be effectively improved. Then, we
further established the U251/TMZ resistant strains, and the CCK-8 test found that isocuB could inhibit the
growth of the resistant strains. In vivo experiments showed that isocuB inhibited tumor growth in nude
mice. Our results collectively suggested that isocuB inhibited glioma cell proliferation, migration, and
invasion, and induced apoptosis via the PI3K/AKT, MAPK, and STAT3 pathways. Our experiments showed
that isocuB could reduce the level of hsa-mir-1286a in glioma U251 cells and increase the drug sensitivity to
TMZ.

Although the above results exerted the inhibitory effect of isocuB on glioma, further study could be
conducted to explore more detail about the anti-glioma effect and mechanism of isocuB. In the first place,
the modulation of isocuB on hub genes in glioma is worthwhile to investigate further. We found the
potential core targets of isocuB through network pharmacology and molecular docking, and verified the
regulation of isocuB on hub genes via western blot and real-time PCR. However, the direct binding of
isocuB to the core target has not been confirmed yet. Cellular thermal shift assay and MicroScale
thermophoresis could be utilized for the confirmation of whether isocuB binds directly to the core target.