The sodium new houttuyfonate suppresses NSCLC via activating pyroptosis through TCONS‐14036/miR‐1228‐5p/PRKCDBP pathway

Abstract Several studies have suggested the potential value of Houttuynia cordata as a therapeutic agent in lung cancer, but direct evidence is still lacking. The study aimed to determine the regulatory impact of a major H. cordata constituent derivative (sodium new houttuyfonate [SNH]) on lncRNA networks in non‐small cell lung cancer (NSCLC) to identify new potential therapeutic targets. After exposing NSCLC cells to SNH, we analysed the following: cell death (via flow cytometry, TUNEL and ASC speck formation assays), immune factors (via ELISA), gene transcription (via RT‐qPCR), subcellular localisation (via FISH), gene–gene and gene‐protein interactions (via dual‐luciferase reporter and RNA immunoprecipitation assays, respectively) and protein expression and distribution (via western blotting and immunocytochemistry or immunohistochemistry). In addition, statistical analysis (via one‐way ANOVA or unpaired t‐tests) was performed. Exposure to SNH promoted NSCLC cell pyroptosis, concomitant with significant up‐regulation of TCONS‐14036, a novel lncRNA. Mechanistic research demonstrated that TCONS‐14036 functions as a competing endogenous (ce)RNA by sequestering microRNA (miR)‐1228‐5p, thereby up‐regulating PRKCDBP‐encoding transcript levels. Indeed, PRKCDBP promoted pyroptosis by activating the NLRP3 inflammasome, resulting in CASP1, IL‐1β and GSDMD cleavage. Our findings elucidate the potential molecular mechanisms underlying the ability of SNH to suppress NSCLC growth through activation of pyroptosis via the TCONS‐14036/miR‐1228‐5p/PRKCDBP pathway. Thus, we identify a new potential therapeutic targets for NSCLC.

Our findings elucidate the potential molecular mechanisms underlying the ability of SNH to suppress NSCLC growth through activation of pyroptosis via the TCONS-14036/miR-1228-5p/PRKCDBP pathway. Thus, we identify a new potential therapeutic targets for NSCLC.

| INTRODUCTION
Lung cancer is the one of the leading causes of cancer-related deaths in worldwide. 1 The latest statistics declared that despite the fact that lung cancer-related deaths have decreased rapidly in the last 26 years, the number of deaths caused by lung cancer outnumber those by breast cancer, prostatic cancer and colorectal cancer combined. 2 Pyroptosis is recognized as a highly inflammatory form of programmed cell death, 3,4 characterized by cell swelling and large bubbles emerging from the plasma membrane. 5,6 In pyroptosis, mature Caspase-1 proteolytically cleaves the members of gasdermin family, such as GSDMD, and activates inflammatory cytokines, such as IL-18 and IL-1β. 7 Pyroptosis plays a critical role in modulating the growth of cancer in vitro and in vivo. 8 Natural products (NPs) have always been used in traditional medicine to treat diseases. 9 According to one study of anti-cancer drugs in modern medicine, NPs were the source of 75% approved small molecules from 1981 to 2014. 10 Traditional Chinese medicine is a a library of NPs that has been confirmed clinically and passed down for thousands of years. The discovery of anti-cancer small molecules in traditional Chinese medicine may be both justified and efficient.
Houttuynia cordata Thunb. is a traditional Chinese medicine that has been used to treat lung disease in China. The potential value of Houttuynia cordata in lung cancer treatment was reported in several studies. [11][12][13] Research by Chen et al. declared that the Houttuynia cordata Thunb extract modulates G0/G1 phase arrest and Fas/CD95-dependent death receptor apoptotic cell death in human lung cancer A549 cells, but failed to mention the critical compound. 12 Han et al. characterized the polysaccharide structure of Houttuynia cordata and found that the Caspase-3 cleavage and cyclinB1 expression was upregulated in A549 cell line. 11 Lou et al. believed that the bioactive compound 2-undecanone, found in Houttuynia cordata, could suppress the lung tumorigenesis through activating the Nrf2-HO-1/NQO-1 signalling pathway. 13 Sodium new houttuyfonate (SNH) is a ramification of Sodium houttuyfonate (SH) derived from Houttuynia cordata. Recent papers reported that the regulation of SH was diverse, always referring to NF-κB signal pathways and displaying an relationship with inflammation. [14][15][16] Latest papers of SNH have solely focused on its antibacterial properties and identified the mechanism of regulating Quorum Sensing 17 or Ras1-cAMP-Efg1 Pathway. 18 In our previous study, we first suggested that SNH could suppresses metastasis in non-small cell lung cancer (NSCLC) by regulating EMT progression. 19 However, the mechanism by which SNH direct induces NSCLC cell death is still unclear.
Recently, an increasing number of lncRNAs were reported to modulate oncological downstream DNA, RNA and protein through chromatin remodelling, transcription and post-transcriptional regulation. [20][21][22] There are still some similarities between the multiple network regulation mechanism of lncRNA and the complex regulation mechanism of traditional Chinese medicine. As a result, we attempted to find the link between lncRNA and SNH.
Herein, we first demonstrated that SNH induced the cleavage of Caspase-1 and GSDMD, consequently inducing pyroptosis of NSCLC cells. The transcriptome sequencing screened out a novel lncRNA TCONS-14036, which was found to regulate pyroptosis. Further analysis revealed that the TCONS-14036 and the mRNA of PRKCDBP function as competing endogenous RNA (ceRNA) to miR-1228-5p.
Importantly, high PRKCDBP expression is associated with the transcriptional signature of activated pyroptosis in NSCLC, implying the involvement of PRKCDBP in the anti-oncogenic activity of pyroptosis.

| Immunocytochemistry
Cells were seeded on laser confocal petri dishes and exposure to indicated treatments. 5% goat serum and 0.3% Triton X-100 in phosphate-buffered saline (PBS) were used to block empty spaces.

| Statistical analysis
Dates for the experiments were presented as the mean ± SD of three independent experiments. Differences between groups were analysed by one-way ANOVA or unpaired t-tests. p < 0.05 was considered as statistically significant (*p < 0.05, **p < 0.01, ***p < 0.005 and ****p < 0.001).

| SNH promotes NSCLC cell pyroptosis
In our previous study, we demonstrated that SNH suppresses NSCLC cells in vitro. The IC50 of SNH on NSCLC cell lines was 87.45-94.27 μmol/l. 19 Herein, we tested SNH on NSCLC cell lines at the previously used dose.
To understand the condition of NCI-H1299 and NCI-H23, we conducted several phenotype experiments. Using flow cytometric analysis, we found that the death of NSCLC cells was increased dose-dependently with the increase in SNH concentration ( Figure 1A,B). Consistently, the TUNEL staining also revealed that the death rate of NSCLC cells was increased as the SNH dose increased ( Figure 1C). Next, we used ELISA to test IL-1β and IL-18, the two typical pyroptosis associated inflammatory factor.
Interestingly, we found that SNH increased the expression of IL-1β and IL-18 ( Figure 1D). Further experimentation revealed that a high dosage of SNH promoted ASC oligomerization and the formation of ASC speaks ( Figure 1E). Mechanism research demonstrated that SNH promoted the cleavage of Caspase-1, GSDMD and IL-1β ( Figure 1F). Immunofluorescence staining further revealed that SNH upregulated the expression of NLRP3 and was involved in its distribution in NSCLC cells ( Figure 1G).
Altogether, these findings suggest that the SNH induces NSCLC cell death through pyroptosis.

| TCONS-14036 is a potential target of SNH
To identify RNAs whose biological functions are dysregulated in NSCLC with SNH, NCI-H1299 samples were used to analyse the profile of lncRNA and mRNA transcriptome sequencing performed using SNH. In this analysis, nine unreported lncRNAs were screened out that were significantly regulated by SNH (Figure 2A). In our previous research, KEGG analysis was performed for SNH influenced mRNAs, and the inflammation associated pathways such as TNF signalling pathway, TGF-β signalling pathway and NF-κB signalling pathway were found to be promoted. 19 After performing a qRT-PCR verification on A549 and NCI-H1299, the TCONS-14036 was found to be the most significantly regulated unreported lncRNA ( Figure 2B,C). Thus, we suspected that TCONS-14036 was the main F I G U R E 5 Legend on next page. target regulated by SNH and the regulatory pathway may be associate with immunity. TCONS-14036 was found to be expressed at significantly lower levels in NCI-H1299 and NCI-H23 than in HBE cells in a diverse range of human NSCLC cells and bronchial epithelial cells (HBE), while it was found to be highly expressed in NCI-H2170 and SK-MES-1 cells ( Figure 2D). To further investigate the role of TCONS-14036 in NSCLC cells, we constructed a TCONS-14036 overexpression vector using PGMLV-6395 ( Figure 2E). We also used pGMLV-SC5 to construct three individual kinds of TCONS-14036-knockdown plasmids, named sh-TCONS-14036-1, sh-TCONS-14036-2 and sh-TCONS-14036-3.
In vitro testing revealed that the knockdown efficiency of sh-TCONS-14036-2 was achieving to 70% ( Figure 2F). Therefore, we used sh-TCONS-14036-2 to conduct the following experiments. Combined, these results demonstrated that the TCONS-14036 was required for inflammation-mediated pyroptosis in NSCLC cell death.

| TCONS-14036 functions as a ceRNA and sponges miR-1228-5p in NSCLC cells
To confirm the location of TCONS-14036 in NSCLC cells, we conducted RNA florescent in situ hybridization (FISH) experiment.

| miR-1228-5p directly targets PRKCDBP
To further elucidate the molecular mechanism underlying how miR-1228-5p exerted its effect on pyroptosis in NSCLC cells, we used bioinformatics tools (psRNATarget, December 24, 2019) to identify downstream targets. Following inclusion criteria with an expectation score of ≤5 (maximum cut-off of score based on given scoring schema), we collected 158 potential targets (Table S5). QRT-PCR, with more stringent inclusion criteria with an expectation score of ≤2.5, was performed to verify the potential target (including eight genes, Figure 6A). Following the variation tendency of complementary base pairing, the expression of PRKCDBP mRNA was the most significantly changed ( Figure 6A). With rigorous binding site prediction between miR-1228-5p and PRKCDBP mRNA using bioinformatics, we constructed plasmids in the pmirGLO vector adding wild-type (WT) or mutated-type (MUT) binding regions of the firefly luciferase gene ( Figure 6B). In 293T, NCI-H1299 and NCI-H2170 cell lines, co-transfection with miR-1228-5p mimics and wildtype plasmids significantly reduced luciferase activity ( Figure 6C). The Cancer Genome Atlas (TCGA) analysis of lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) clinic samples demonstrated that the PRKCDBP transcripts per million (TPM) was low expressed in tumour tissues than in normal tissues ( Figure 6D,E). Next, western blotting showed the PRKCDBP was expression was upregulated by SNH, overexpression of TCONS-14036, and inhibition of miR-1228-5p ( Figure 6F). Combining these results, we conclude that the miR-1228-5p directly targets PRKCDBP in NSCLC cells.

| PRKCDBP overexpression confers pyroptosis via NLRP3 releasing
To investigate the mechanism of PRKCDBP in NSCLC, we tried to understand the significance of IL-1β and NLRP3 in clinical tissues from LUAD and LUSC. The R value expressed NLRP3 much more strongly ( Figure 7A). Later, we conducted a phenotypic experiment using Annexin V/PI flow cytometric analysis, which exhibited the overexpression of PRKCDBP aggravated cell death ( Figure 7B,C). Consistently, TUNEL and ASC immunofluorescence clearly demonstrated that the cell death was caused by pyroptosis, which was induced by PRKCDBP overexpression (Figure 7D,E). ELISA assay showed that the IL-1β and IL-18 expression was upregulated by PRKCDBP overexpression ( Figure 7F). Furthermore, we conducted western blotting, which showed the significantly high cleavage of Caspase-1, IL-1β and GSDMD by p-PRKCDBP ( Figure 7G). It was interesting that the stain of NLRP3 expression had a high overlap ratio to p-PRKCDBP transfection which may means indicate that NLRP3 and PRKCDBP are in close contact ( Figure 7H). A clinic analysis of TCGA revealed that PRKCDBP, Caspase-1, IL-1β, NLRP3 and Caspase-4 were expressed at low level in tumour tissues than in the normal tissues in LUAD and LUSC ( Figure 7I). Taken together, these results highlighted an important role for PRKCDBP as a promotor of pyroptosis and as a regulator of the release of NLRP3. biological functions, such as regulating enzyme activity, inhibiting virus activity and adjusting mRNAs and proteins in signalling pathways. 26,27 Because of its anti-inflammatory properties, we suspected that SNH had anti-tumour properties via an anti-inflammatory pathway. However, the SNH ELISA produced the opposite result. SNH upregulated the inflammatory factors. Since SNH induces NSCLC cell death, we suspected that it activates an inflammation-mediated cell death. Coincidentally, pyroptosis conformed to all characteristic. From the standpoint of phenotypic and mechanistic research, pyroptosis was confirmed to be activated by SNH. However, inflammasome sensors cannot ignore on pyroptosis. The immunofluorescence ferreted out the NLRP3, which assembled the NLRP3-ASC-Caspase-1 inflammasome complex, activated by SNH.
The results of our study provide novel insights into the critical role of SNH in inducing pyroptosis of NSCLC via the TCONS-14036/ miR-1228-5p/PRKCDBP pathway. Herein, we detected that SNH regulated a novel lncRNA TCONS-14036 and expressed a close relationship with inflammation pathways (Figure 2A-D). The TCONS-14036 was a 191-nt newfound lncRNA located at chromosome 14: 20343070-20343261 (hg38). The function of TCONS-14036 are not reported yet. However, with its sequence being screened, the whole length of TCONS-14036 was included in lnc-CCNB1IP1-1. 28 The CCNB1IP1 (also named HEI10) is short for cyclin B1 interacting protein 1, which is a protein coding gene and widely reported to regulate meiotic recombination. 29,30 The alterations of the CCNB1IP1 ubiquitin ligases correlated significantly with breast and lung cancer prognostic factors, especially in lung cancer. 31 Increasing evidence demonstrated that the proliferative deficiencies and transcriptional deregulation of CCNB1IP1 caused tumour mutations and invasion. 32,33 The progress free interval (PFI) of CCNB1IP1 demonstrated better prognosis in the NSCLC high expression group ( Figure S3A). However, as it is a fragment of CCNB1IP1, the function of TCONS-14036 is must be identified. By overexpression TCONS-14036, we could easily induce pyroptosis in cells. However, after conducting a knockdown, the transfection did not have an effect. This provided the evidence of TCONS-14036 taking part in pyroptosis of NSCLC.
Our results further revealed that TCONS-14036 promoted pyroptosis of NSCLC by disrupting the repressive effect of miR-1228-5p on PRKCDBP by sponging miR-1228-5p. High level miR-1228-5p always promoted cancer progress on proliferation and metastasis. [34][35][36] Survival probability of miR-1228-5p in LUAD and LUSC shows that decreased expression of miR-1228-5p improves the prognosis of NSCLC patients ( Figure S3B,C). 37 Interestingly, some research identified certain genes found in the p53 signal pathway as the targets of miR-1228-5p, 38,39 which proves to be consistent with the background of NCI-H1299 and NCI-H23 cells line in our study (The source of NCI-H1299 and NCI-H23 was lung cancer patient with p53 deletion). Based on the oncogenicity of miR-1228-5p and the bioinformatic prediction, we hypothesized that miR-1228-5p could be an important link between TCONS-14036 and pyroptosis. The ceRNA theory, which based on complementary base pairing, was substantiated on the mounting lncRNA research. 40 The dual-luciferase report assay, FISH and RIP, helped us validate the binding site and area between TCONS-14036 and miR-1228-5p. Consistently, the inhibition of miR-1228-5p induced pyroptosis in NSCLC cells.
The downstream regulation target of miR-1228-5p was also detected by combining bioinformatic prediction and binding site certification. After whole pathway verification, we believe that PRKCDBP is the target gene that regulate pyroptosis. PRKCDBP is a putative tumour suppressor whose alteration has been observed in several human cancers. 41 Experiments in the latest oncology research regarding PRKCDBP as a transcriptional target of TNF-α demonstrated that low expression of PRKCDBP forebode poor prognosis in tumour patients, including those with lung adenocarcinoma. 42,43 In colorectal cancers, PRKCDBP induction by TNF-α was confirmed to be disrupted when NF-κB signalling was blocked, and previous research revealed PRKCDBP is implicated in TNF-α-induced apoptosis. 41 However, in our opinion, the cell death induced by inflammation expressed a closer relationship with pyroptosis. It is worth noting that the frequency of hypermethylation was high for ASC and PRKCDBP in a previously conducted microarray analysis in lung cancers. 44 ASC oligomerization is a typical feature of pyroptosis. Thus, we evaluated the relationship between PRKCDBP and pyroptosis and successfully found that upregulation PRKCDBP activates pyroptosis in NSCLC cells. Using in vivo experimental approaches, we identified the key regulation points of TCONS-14036/miR-1228-5p/PRKCDBP pathway and that tumours were suppressed by SNH.
In summary, we believe that the SNH directly suppressed NSCLC through activating pyroptosis. Further research revealed a novel transcriptional and post-transcriptional network in which pyroptosis is induced by upregulation of TCONS-14036, downregulation of miR-1228-5p and activation of PRKCDBP ( Figure 8I). Activated factors include NLRP3 release, IL-1β/Caspase-1/GSDMD cleavage and ASC oligomerization. This research not only provide a theoretical basis for SNH usage in NSCLC in clinic, but also uncovered potential target for oncology therapy.