In Vitro Validation Study by Real-Time PCR and Western Blotting

To further validation of the RNA sequencing data generated by the above-mentioned parameters, the differential expression of top five genes including two upregulated (GADD45G and ATF3) and three downregulated (BUB1B, CCNA2 and CDK1) were evaluated on all treated and non-treated samples in mRNA and protein levels. As Figure 6A shows, the expression of BUB1B, CCNA2 and CDK1 is significantly down-expressed in treated cells. Accordingly, the data illustrated that ATF3 (*p* value < 0.05) and GADD45G are upregulated in Gemini-Cur treated cells, although this was not significant for GADD45G. These modulations were confirmed in protein level as shown in Figure 6B.

**Table 5.** The top significantly gene ontology (GO) categories (BP: biological process; CC: cellular component; MF; molecular function) based on upregulated genes with the threshold of |log fold change (FC)| ≥ 2 and a Bonferroni *p* < 0.05. Padj: adjusted *p* value.


**Figure 6.** Validation of the expression of selected genes by real-time PCR and Western blotting. Nearly all genes except GADD45A validate findings from RNA seq. data. (**A**) The data show relative expression of genes compared to non-treated controls. (**B**) Blots illustrate protein expression in control and treated cells.

#### **4. Discussion**

Despite recent advances in early diagnosis and proper treatments, colorectal cancer (CRC) is still considered as the third leading cause of cancer-related deaths worldwide [1,2]. Gemini-Cur is one of the latest nano-formulations of curcumin with a significant anticancer effect that has recently been developed by our group [15]. We have already reported that Gemini-Cur inhibits the proliferation of different cancer cells through the induction of apoptosis [13–16]. Due to the limited information on the global effect of curcumin on transcriptome profiling, we employed RNA sequencing to uncover the differentially expressed genes and cellular pathways that are affected by Gemini-Cur in colorectal cancer cells. Here, our data not only confirm the suppressive effect but also demonstrate that numerous genes in different cellular pathways are modulated by Gemini-Cur in HCT-116 cells.

Along with all the steps of quality control and RNA-seq raw data normalization, the DEA (Differentially Expressed Analysis) process introduced about 3892 genes as DEGs (padj < 0.01) with 244 upregulated (padj < 0.01, log2 FC ≥ 2), and 198 downregulated genes (padj < 0.01, log2 FC ≤ −2). The PPI network was also created by Cytoscape to better figure out the possible correlations between DEGs. Consequently, three of the top significant downregulated genes including cyclin-dependent kinase 1 (CDK; padj = 1.08 × <sup>10</sup><sup>−</sup>06, log2 FC = −3.15), cyclin A2 (CCNA2; padj = 2.87 × <sup>10</sup><sup>−</sup>05, log2 FC = −3.056) and BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B; padj = 2.72 × <sup>10</sup>−06, log2 FC = −2.99) as well as two upregulated genes including growth arrest and DNA damage inducible gamma (GADD45G; padj = 1.47 × <sup>10</sup>−06, log2 FC = 4) and activating transcription factor 3 (ATF3; padj = 1.03 × <sup>10</sup><sup>−</sup>19, log2 FC = 4) were selected from top ten DEG lists for further validation of RNA sequencing results.

Numerous reports have shown that ATF3 and GADD45G play critical roles in cancer. Inoue et al. illustrated that the ATF3 transcription factor inhibits the migration and invasion of HCT-116 cells. ATF3 is also involved in the process of cellular stress response [18]. Here, RNA sequencing and experimental studies show that transcription factor ATF3 as a key regulator of cellular stress response is upregulated in cancer cells after treatment with Gemini-Cur. These findings demonstrate increasingly the potential of ATF3 as a therapeutic candidate in colorectal cancer. In another work, Guo et al. demonstrated that the overexpression of GADD45G acts as tumor suppressor in AML [19]. Although RNA sequencing showed that GADD45G is upregulated in treated cells, this overexpression was not detected in PCR and Western blotting. This contradictory finding may be due to the presence of different RNA reads including RNAs from paralogous genes and pseudogenes. However, PCR and Western blotting only detect main predominant variant of GADD45G.

Accordingly, CCNA2, BUB1B, and CDK1 genes have been downregulated in Gemini-Cur treated cells versus the non-treated group. According to the gene-pathway annotated network, it is revealed that CDK1 is involved in the cell cycle and associated with other CRC-related genes (CCNA2, BUB1B, GADD45G, ATF3). CDK1 contributes to the cell proliferation, apoptosis, and cell migration [20,21]. It was shown that the upregulation of CDK1 leads to poor prognosis in patients with CRC [22]. Zhu et al. and Thoma et al. indicated that the downregulation of CDK1 inhibited fluorouracil-resistant CRC cell proliferation [23,24].

Regarding the BUB1B and CCNA2, both genes have been reported as upregulated genes in CRC [25–27]. Gan et al. indicated that the CCNA2 knockdown could significantly suppress CRC cell growth by impairing cell cycle progression and inducing cell apoptosis [20]. Ding et al. also reported that the upregulation of BUB1B, CDK1, and CCNA2 genes contribute to the progression of tumor growth and metastasis of CRC [28]. These results further validate our analysis methods and the accuracy of RNA sequencing for exploring the novel top up/downregulated genes in different conditions.

The joint cooperation of GADD45G and CDK1 in the p53 activity regulation pathway is another attractive result of this study. GADD45G plays a role in the activation of S and G2/M checkpoints during p53-related DNA damage responses [29,30]. The modulatory effect of Gemini-Cur on these genes may highlight their collaborative role in cell stress response.
