Studies have shown that Kushen can regulate immunity, reduce inflammation, and relieve cancer pain. Kushen also has a significant effect on anti-tumor activity [1]. The main effects of CKI on cancer cells are inhibiting the proliferation of cancer cells, inducing cell cycle arrest, promoting apoptosis, inhibiting angiogenesis, inducing cell differentiation, inhibiting cancer metastasis and invasion, and reversing multidrug resistance [8]. A recent study showed that matrine could suppress epithelial-mesenchymal transition signaling pathways and ultimately inhibit the migration and invasion of NSCLC [29]. Oxymatrine from Kushen, combined with paclitaxel, can inhibit lung cancer growth in vitro and in vivo[30]. In our study, Kushen treatment can significantly inhibit NSCLC cell growth. The cell morphology showed a noticeable change from IC30 concentration treatment, and IC50 concentration caused many cell deaths in the petri dish. We also concluded that IC10 and IC30 concentration treatment showed similar enrichment results from the functional enrichment of DEGs under different concentrations. In contrast, IC50 treatment shows some new different pathway enrichment, such as Aminoacyl-tRNA biosynthesis. In addition, IC30 treatment induced the highest EGR1 and PTEN expressions according to qPCR and Western blots results, while IC50 group began to decrease the expressions of EGR1 and PTEN. This might indicate that IC30 was the effective and safe dose for inhibiting NSCLC, while IC10 and IC50 were insufficient or excessive doses, respectively.
Interest in developing Chinese herbal medicines has continued to increase in recent years because they can affect multiple targets with few side effects. In TCM, Kushen has been used to treat tumors, inflammation, and other diseases for a long time. However, herbal medicine always contains thousands of compounds, and the therapeutic mechanisms of these compounds remain largely unknown. New high-throughput technologies have opened a novel avenue to study complex biological processes holistically with multi-omics data. Analysis of multi-omics data and clinical information is more conducive to understanding molecular function [31]. They help bridge the gap between phenotype and genotype in organism, thus improving disease prognostics and predictive accuracy. In the current study, seven essential genes we screened from the DEGs-DE miRs network by TCGA data analysis, Oncomine data analysis, and survival analysis. The results indicated that these seven genes (CCND2, KLF15, FOS, SH2D3C, FRY, KIAA1324L, and EGR1) are down-regulated in tumor samples, and their up-regulation shows a better overall survival rate.
miR-183, a member of miR-183 family (including miR-96, miR-182, and miR-183), is located on human chromosome 7. miR-183 is a proven onco-miRNA in several tumor types, including NSCLC [32]. Xu et al. showed that over-expression of miR-183 is an essential advanced progression and poor prognosis biomarker for patients with lung adenocarcinoma. It is associated with advanced clinical stage, lymph node metastasis, and poor OS rate of lung adenocarcinoma [33]. miR-183-5p is up-regulated in NSCLC and promotes cell proliferation, cell cycle, and cell migration of NSCLC. miR-183-5p promotes NSCLC by directly targeting phosphatase tensin (PTEN). PTEN is one of the most common tumor suppressors, which can suppress tumors by removing phosphate from PIP3, thereby inhibiting the activation of AKt phosphorylation [28]. A zinc-finger transcription factor early growth response gene 1 (EGR1), another important tumor suppressor, has been associated with NSCLC [34, 35]. Through bioinformatics analyses, miR-183 was found to target EGR1 mRNA to promote cell migration, and this specific interaction was validated in vitro in synovial sarcoma, rhabdomyosarcoma, and colon cancer cell lines [34]. In an NSCLC clinical study, compared with patients with low EGR1 levels, patients with higher levels of EGR1 had better OS and disease-free survival. EGR1 expression was strongly correlated with PTEN expression (p = 0.0001) [35]. EGR1 has been confirmed to directly regulate many tumor suppressors, including PTEN [36]. In our study, EGR1 was predicted to be the target of miR183-5p in A549 cells. After Kushen treatment, EGR1 was significantly up-regulated, and miR-183-5p was inhibited. Survival analysis revealed that EGR1 was also associated with a better OS rate, consistent with previous studies. However, miR-183-5p/EGR1 interaction has not been reported in NSCLC. Based on previous studies, we hypothesized that miR-183-5p functions as a potential oncogene in NSCLC by directly or indirectly regulating EGR1 and PTEN expression levels to activate AKt phosphorylation and promote cancer cells growth. Kushen treatment will reverse the effects by down-regulating miR-183 expression and up-regulating EGR1 and PTEN expression. To test our hypothesis, we transfected the mimic miR-183-5p into A549 cells, and EGR1 expression was significantly suppressed. However, we did not observe the changes in PTEN expression levels as expected. This might because that miR183-5p/EGR1 binding is much stronger than miR183-5p/PTEN in A549 cells. The low EGR1 expression level by miR-183-5p suppression in A549 cells cannot drive the PTEN expression up-regulation.
Many studies have demonstrated that the active component of Kushen can mediate lung cancer through the AKt pathways. Kurarinone, a flavonoid isolated from Kushen, has been reported to repress the activity of AKt and induce A549 cell apoptosis. The authors also performed in vivo experiments in a mouse model to validate the apoptosis-inducing effects of kurarinone [37]. Another crucial active component of Kushen, matrine has been reported to play tumor inhibitor effects in many cancers (e.g., breast cancer, thyroid cancer, and lung cancer) by inhibiting the AKt signaling pathways [38, 39]. Matrine-treated FTC-133 cells (human follicular thyroid cancer cells) show significant down-regulation of miR-21, up-regulation of PTEN and down-regulation of p-Akt at the mRNA and protein expression levels [39]. Qi An et al. suggested that matrine treatment (1.0 mg/mL) can inhibit cell proliferation and induce apoptosis via up-regulation of miR-126 expression in A549 cells [40]. Matrine inhibits the invasion and metastasis of lung cancer cells by elevating miR-133a expression via suppressing the activation of the EGFR/ AKt pathway in NCI-H1299 (NSCLC cell line) [41]. These studies suggested that Kushen component treatment inhibits cancer cell growth via the miRNA/target gene/PTEN/AKt signaling pathway. The present study found that Kushen can inhibit the miR-183-5p expression, and EGR1 is the direct target of miR-183-5p. EGR1 is significantly up-regulated in both mRNA and protein expression levels after Kushen treatment. EGR1 can transcriptionally up-regulate PTEN expression. Then, PTEN can inhibit the pAKt phosphorylation process and further inhibit the lung cancer cell growth (Fig. 10).