Anti-proliferative mechanism of Huaier extract in human thyroid carcinoma cells

Anti-proliferative mechanism of Huaier extract in human thyroid carcinoma cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Anti-proliferative mechanism of Huaier extract in human thyroid carcinoma cells Jingni He, Ying Zhang, Lidong Wang, Yifang Yu, Baiyu Yao, Zhong Tian This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-70026/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Thyroid cancer is the most common endocrine tumor and typically has a good prognosis; however, some patients still present with local or distant metastases. Huaier is a traditional Chinese medicine reported as effective in treating certain types of tumor, but the effect of Huaier on thyroid cancer has not yet been reported. Methods The thyroid cancer cell lines, B-CPAP and C643, were treated with increasing concentrations of Huaier extract and the therapeutic effect was measured using a cell counting kit 8 (CCK-8) and flow cytometry. High-throughput sequencing was further performed to identify differentially expressed genes (DEGs) in Huaier-treated B-CPAP cells. Moreover, quantitative real-time PCR (RT-qPCR) was carried out to verify the selected RNAs. Finally, the dual luciferase detection kit was used to detect gene activity. Results Proliferation of B-CPAP and C643 cells was significantly suppressed by treatment with Huaier extract in a concentration- and time-dependent manner. Huaier extract also induced cell cycle arrest and apoptosis according to flow cytometry ( p < 0.05). High-throughput sequencing observed 7,979 significantly altered transcripts. Gene Ontology (GO) analysis showed that 270 genes were enriched in upregulated terms, while 171 genes were enriched in downregulated terms ( p < 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that there were 47 enriched pathways associated with DEGs ( p < 0.05). The expression levels of chosen lncRNAs (SNHG7, MIR181A2HG, ILF3-AS1, and CTA-29F11.1) and their corresponding mRNAs (BBC3, CTSL, GADD45A, and DDIT3) were verified to be overexpressed in Huaier-treated B-CPAP cells by RT-qPCR ( p < 0.05). Following transduction, the CCK-8 results showed that the proliferative capacity was increased in the shRNA group as compared with that in the Ctrl and Scr groups. According to flow cytometry, the number of cells in the G0/G1 phase was decreased in the shRNA group ( p < 0.01) and the apoptosis rate was lower ( p < 0.05). The shRNA-treated group had significantly reduced Huaier-induced apoptosis as compared with the Scr-treated group ( p < 0.05). Moreover, the number of cells in the G0/G1 phase in the shRNA-treated group was significantly lower than that in the Scr-treated group ( p < 0.05). The results of the dual luciferase reporter gene experiment showed that the activity in the GADD45A WT + miR-301a-3p(+) group was significantly reduced as compared with that in the GADD45A WT + miR-301a-3p(+) NC group ( p < 0.01). Further, the activity in the ILF3-AS1 WT + miR-301a-3p(+) group was significantly lower than that in the ILF3-AS1 WT + miR-301a-3p(+) NC group ( p < 0.05). Conclusions The present study demonstrates that Huaier extract inhibits the proliferation of thyroid cancer cells via changes in the expression levels of a multitude of genes. In particular, a decrease in GADD45A expression enhances the proliferative ability of thyroid cancer cells, the levels of which can be increased by Huaier treatment, thus regulating the cell cycle and apoptosis. Huaier can inhibit the proliferation of thyroid cancer cells through the ILF3-AS1/hsa-miR-301a-3p/GADD45A ceRNA axis. Cancer Biology Huaier extract thyroid cancer anti-proliferative mechanism high-throughput sequencing differentially expressed genes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights Huaier extract inhibits the proliferation of thyroid cancer cells via changes in the expression levels of a multitude of genes a decrease in GADD45A expression enhances the proliferative ability of thyroid cancer cells, the levels of which can be increased by Huaier treatment, thus regulating the cell cycle and apoptosis Huaier can inhibit the proliferation of thyroid cancer cells through the ILF3-AS1/hsa-miR-301a-3p/GADD45A ceRNA axis 1. Background Thyroid cancer (TC) is a common malignant tumor, accounting for 94.5% of all malignant endocrine tumors [ 1 ]. The incidence rate of TC ranks first among the various types of head and neck cancers [ 2 ], and an increasing prevalence has been demonstrated recently [ 3 ]. Following systematic treatment, such as thyroidectomy, radioiodine ablation, and thyroid hormone suppressive therapy [ 4 ], individuals diagnosed with TC typically have an excellent prognosis, with an overall 10-year survival rate greater than 97% [ 5 ]. However, 5 − 20% of patients still experience local recurrence, disease progression, and metastasis [ 6 ]; thus, it is of great importance to discover appropriate adjuvant therapies for TC. Trametes robiniophila Murr (Huaier), a traditional Chinese medicinal herb, has been used as an adjuvant anti-tumor therapy for 1600 years. Moreover, Huaier particles have been used clinically to treat patients with malignant tumors (China Food and Drug Administration approval number, Z20000109; http://app1.sfda.gov.cn/datasearch/face3/base.jsp ). It has been reported that Huaier is effective in treating diverse cancers such as hepatocellular carcinoma, breast cancer, lung cancer, gastric cancer, and cervical cancer [ 7 – 11 ], but the effect of Huaier on TC remains unknown. A large number of novel RNAs have been found based on the human genome project, and there is increasing interest in the mechanisms by which these RNAs participate in cellular regulation and transcription [ 12 , 13 ]. Therefore, we explored the role of RNA in the anti-tumor effect of Huaier in TC. 2. Materials and Methods 2.1. Cell lines and reagents The TC cell lines, B-CPAP and C643, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and were routinely cultured in RMPI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, USA) in an atmosphere of 5% CO 2 at 37℃. An electuary ointment of Huaier extract was donated by Qidong Gaitianli Pharmaceutical Co., Ltd. (Jiangsu, China). Huaier extract contained four monosaccharides and 18 amino acids; its main active ingredients were polysaccharides, named TP-1 (fucose, arabinose, galactose, glucose, xylopyranose, mannose, at molar ratios of 3.2:2.0:33.5:5.5:1.2:50.4), TP-2 (arabinose, galactose, glucose, xylopyranose, mannose, at molar ratios of 7.4:4.5:2.7:9.4:1.9), TP-3 (fucose, arabinose, galactose, glucose, xylopyranose, mannose, at molar ratios of 0.1:1.0:5.4:4.4:2.1: 7.0), and TP-4 (arabinose, galactose, glucose, xylopyranose, mannose, at molar ratios of 8.9:1.6:3.4:7.4:1.3). 2.2. Preparation of Huaier aqueous extract A mass of 2 g Huaier ointment was dissolved in 20 mL complete medium, passed through a 22-µm filter, and stored at -20℃ for future use. 2.3. Determination of cell viability The viability of B-CPAP and C643 cells in the presence of increasing concentrations of Huaier was measured using a cell counting kit-8 (CCK-8) kit (DOJINDO, Japan). An experimental group, control group, and blank group consisted of five replicate wells each. B-CPAP and C643 cells were seeded on 96-well plates at a density of 2 × 10 3 cells/well. Following synchronization, 200 µL medium containing 1, 2, 4, 8, 12, or 16 mg/mL Huaier was added, and the cells were cultured for 24 h, 48 h, or 72 h. Subsequently, 100 µL medium containing 10% CCK-8 was added to the cells and incubated for 3 h, following which the absorbance was measured at 450 nm using a microplate reader (Bio Tek, USA). The experiment was repeated three times. 2.4. Evaluation of apoptosis and the cell cycle Cell apoptosis assay (Propidium iodide (PI)-Annexin V staining): The proportion of apoptotic cells was assessed using a BD Pharmingen™ PE Annexin V apoptosis detection kit (DOJINDO, Japan). Briefly, after treatment with 8 mg/mL Huaier extract for 48 h, B-CPAP and C643 cells were harvested, washed twice with PBS, and centrifuged at 1000 rpm for 3 min. Subsequently, 5 µL Annexin V-FITC and 5 µL PI solution were mixed with 1 × Annexin V binding solution (100 µL) containing cells at a density of 1 × 10 5 /mL. Following incubation for 15 min in the dark at room temperature, another 400 µL 1 × Annexin V binding solution was added prior to analysis by FACScan flow cytometry (Becton-Dickinson, USA). The experiment was repeated three times. Cell cycle assay: The cell cycle phases were evaluated using a cell cycle detection kit (KeyGen China). The experimental group was treated with 8 mg/mL Huaier extract, while the control group was incubated with complete medium. Cells were harvested after 48 h, rinsed twice with PBS, and centrifuged at 1000 rpm for 5 min. Subsequently, cells were fixed in cold 70% ethanol overnight, washed once with PBS the following day, resuspended in 100 µL RNaseA, and placed in water at 37℃ for 30 min. A volume of 400 µL PI staining solution was added and cells were analyzed by FACScan flow cytometry after incubation for 30 min at 4℃ in the dark. Data were analyzed using the ModFitLT V2.0 software (Becton-Dickinson). The experiment was repeated three times. 2.5. RNA extraction, library preparation, sequencing, and data processing Following treatment with 8 mg/mL Huaier extract for 48 h, B-CPAP cells were harvested for high-throughput sequencing. Total RNA was isolated according to the manufacturer’s instructions. The quality and quantity of extracted RNA were examined using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and an RNA 6000 Nano LabChip kit (Agilent Technologies, USA). The preparation of whole transcriptome libraries and deep sequencing were performed by Beijing Ori-Gene Science and Technology Corp., Ltd. (Beijing, China). The libraries were constructed using the Ribo-Zero Magnetic Gold kit (Illumina, USA) and the NEBNext® Ultra™ RNA Library Prep kit for Illumina (New England Biolabs) according to the manufacturer’s manual. Sequence reads were aligned to the human genome (GRCh38) using the TopHat 2.0 program, and the resulting alignment files were reconstructed by Cufflinks. Following prediction of mRNAs and ncRNAs, histograms and box plots were used to represent the transcripts. A Pearson heat map was drawn to represent the difference between the two groups of samples. The differential genes were plotted using volcanoes and cluster analysis. GO and KEGG analyses were performed, and the co-expression network and ceRNA mechanistic diagram were constructed. Total RNA was extracted, and cDNA was synthesized using a PrimeScript TM RT reagent kit with gDNA Eraser (TaKaRa, Japan) according to the manufacturer's instructions. Subsequently, RT-qPCR was performed in three independent wells using SYBR Premix Ex Taq (TaKaRa, Japan), and relative RNA expression was calculated using the 2 −ΔΔCt method. The primer sequences are shown in Supplementary Table 1. 2.6. shRNA lentiviral vector transduction and verification The lentiviral vector was constructed by Shanghai Hanheng Technology Co., Ltd. A total of 30,000 cells/well were inoculated with lentiviral infection solution (MOI = 25), culturing to a fusion degree of 60 − 70%. After successful transduction, cells were screened to establish stable cell lines. RT-qPCR was used to verify the expression of GADD45A. The specific primer sequences were as follows: human GADD45A forward: 5'-ACTGTCGGGGTGTACGAAG-3'; human GADD45A reverse: 5'-ATCAGGGTGAAGTGGATCTGC-3'; human GAPDH forward: 5'-GAAGGTGAAGGTCGGAGTC-3'; and human GAPDH reverse: 5'-GAAGATGGTGATGGGATTTC-3'. Protein expression was verified by western blotting. Total cellular protein was extracted and quantitated using the Coomassie brilliant blue method. A 20-µL protein sample was separated by SDS-PAGE and transferred to PVDF membrane at 80 V for 1.5 h. The membrane was sequentially incubated in skimmed milk, specific primary antibody, and corresponding secondary antibody. Protein bands were visualized using C300ECL luminaire. 2.7. Evaluation of cell proliferation B-CPAP cells were divided into control, B-CPAP-Scr (Scr), and B-CPAP-sh-GADD45A (shRNA) groups. Cell proliferation was evaluated using a CCK-8 assay, and PI single-staining and Annexin V-PE/7-AAD double-staining were used to assess the cell cycle and apoptosis, respectively, by flow cytometry. For treatment with Huaier extract, B-CPAP cells were divided into B-CPAP-Scr (Scr), B-CPAP-Scr treated with 8 mg/mL Huaier extract (Scr-treated), and B-CPAP-sh-GADD45A treated with 8 mg/mL Huaier extract (shRNA-treated) groups. Cell cycle and apoptosis were evaluated as described above. 2.8. Dual-luciferase assay GADD45A and ILF3-AS1 WT and mutant dual-luciferase plasmids were constructed. The dual-luciferase detection kit (Promega, USA) was used to measure gene activity according to the manufacturer’s instructions. Both the Firefly and Renilla values were measured and the Firefly/Renilla ratio was calculated. Each experiment was repeated three times. 2.9. Statistical analysis Statistical analysis was performed using the SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA) software. The results are expressed as the mean ± standard deviation of at least three independent experiments in at least triplicate. One-way analysis of variance was used to assess significant differences between the groups; p < 0.05 was considered statistically significant. 3. Results 3.1. Huaier extract inhibits B-CPAP and C643 cell proliferation B-CPAP and C643 cells were treated with increasing concentrations of Huaier extract for 24 h, 48 h, or 72 h and subjected to the CCK-8 assay to assess the effect on cell proliferation. As shown in Fig. 1 A, a low concentration of Huaier extract may have slightly promoted cell proliferation; however, concentrations of 8 mg/mL and higher markedly inhibited cell proliferation in a concentration- and time-dependent manner. The IC 50 of Huaier extract in B-CPAP and C643 cells was 5.604 mg/mL and 8.330 mg/mL, respectively, following a 48-h incubation (Fig. 1 B), showing the most marked inhibitory effect on cell proliferation and suggesting optimal conditions for further experiments. To explore the potential anti-proliferative mechanism of Huaier extract in depth, the apoptotic rate and cell cycle distribution were assessed by flow cytometry following treatment. As shown in Fig. 1 C, the apoptotic ratio was 8.64 ± 1.60% in B-CPAP cells and 5.37 ± 0.35% in C643 cells following treatment with Huaier extract (8 mg/mL) for 48 h, which was significantly different from that in the control group ( p < 0.05). In addition, as shown in Fig. 1 D, the proportion of B-CPAP cells in the G0/G1 phase was increased from 63.50 ± 0.96% in the control group to 72.67 ± 1.07% ( p < 0.01) following incubation with Huaier extract for 48 h, while the proportion of C643 cells was increased from 47.77 ± 4.23% to 64.20 ± 3.34% ( p < 0.01). Moreover, the number of cells in the S and G2/M phases decreased ( p < 0.05). 3.2. Whole genome RNA-sequencing data and pathway analysis results A total of 65.38 − 103.71 million raw data reads were obtained for each sample. After filtering, 89.1 − 91.8% quality control data reads were obtained with an average length of 145.87 nucleotides, which corresponded to the reference human genome (Ensembl GRCh38 main assembly). As shown in Fig. 2 A, 16,159 known transcripts and 54,772 novel transcripts were acquired. A total of 7,979 differentially expressed transcripts were found, of which 2,185 were upregulated and 5,794 were downregulated. A total of 5,717 genes were differentially expressed, among which 869 lncRNAs, 1 circRNA, and 1,179 mRNAs were upregulated and 3,781 lncRNAs, 1 circRNA, and 921 mRNAs were downregulated (Fig. 2 B). The box plot shows the distribution of the expression of various transcripts (Fig. 2 C). The heat map demonstrates the expression levels of total transcripts among the different groups; the difference was significant among groups but not within groups (Fig. 2 D). The volcano map (Fig. 2 E) displays all the differentially expressed transcripts and cluster analysis (Fig. 2 F) shows the difference in expression of each transcript in the sample. These differentially expressed genes (DEGs) are valuable for elucidating the mechanism of action of Huaier in thyroid carcinoma cells. To further understand the DEGs, GO and KEGG analyses were performed. The significantly enriched GO terms are shown in Fig. 2 G; a total of 270 genes were enriched in upregulated terms, while 171 genes were enriched in downregulated terms ( p < 0.05). The top three enriched GO biological terms were integrin binding (GO: 0005178), coenzyme binding (GO: 0050662), and endoplasmic reticulum lumen (GO: 0005788). According to KEGG analysis, a total of 47 significantly enriched pathway terms were associated with the DEGs ( p < 0.05), the top 20 of which are presented in Fig. 2 H. 3.3. Gene relationship analysis and RNA validation Next, proliferation-related DEGs were chosen to build a coding − noncoding co-expression network (Fig. 3 A). Four lncRNAs (SNHG7, MIR181A2HG, ILF3-AS1, CTA-29F11.1) were selected from the proliferation-related pathways for the construction of the ceRNA network (Fig. 3 B). According to the selected lncRNAs, the corresponding mRNAs were determined and RT-qPCR was performed to verify the expression levels. As shown in Fig. 3 C, lncRNAs (SNHG7, MIR181A2HG, ILF3-AS1, CTA-29F11.1) and mRNAs (BBC3, CTSL, GADD45A, DDIT3) were over-expressed in the Huaier-treated group ( p < 0.05), which is consistent with the RNA-sequencing results. 3.4. Effect of GADD45A knockdown on the proliferation of human thyroid cancer cells The transduction efficiency was 90% according to fluorescent microscopy (Fig. 4 A), and the mRNA and protein expression of GADD45A was decreased in the shRNA group (Fig. 4 B) ( p < 0.01). With respect to proliferation, the shRNA group showed a time-dependent increase as compared with the Ctrl and Scr groups (Fig. 4 C). Moreover, flow cytometry revealed that the number of cells in the G0/G1 phase was decreased in the shRNA group as compared with that in the Scr group (47.77 ± 4.23% vs. 67.20 ± 4.28%, p < 0.01). The number of cells in the S phase was increased in the shRNA group (39.44 ± 1.56% vs. 22.49 ± 3.42%, p < 0.01), but there was no significant difference in the G2/M phase (12.79 ± 2.81% vs. 11.30 ± 4.07%, p = 0.6306) (Fig. 4 D). Further, the shRNA group had a lower apoptotic rate (3.97 ± 0.34% vs. 5.56 ± 0.18%, p = 0.0218) and a higher survival rate (81.46 ± 0.86% vs. 78.63 ± 1.08%, p = 0.0495) than those in the Scr group (Fig. 4 E). 3.5. Effect of GADD45A on the Huaier-induced reduction in proliferation of human thyroid cancer cells Following downregulation of GADD45A , the number of cells in the G0/G1 phase was significantly lower in the shRNA-treated group than that in the Scr-treated group ( p < 0.05), and the number of cells in the S phase was increased ( p = 0.049); however, the number of cells in the G2/M phase did not change significantly ( p = 0.9572) (Fig. 5 A,B). The shRNA-treated group had a significantly reduced apoptotic rate as compared with that in the Scr-treated group (Fig. 5 C,D, p < 0.05). 3.6. ILF3-AS1 and GADD45A competitively inhibit hsa-miR-301a-3p to exert the anti-proliferative effect of Huaier on human thyroid cancer cells The ceRNA network predicts that ILF3-AS1 and GADD45A may exert their effects through the competitive inhibition of miRNA. Starbase predicts that ILF3-AS1 and GADD45A may have binding sites within hsa-miR-301a-3p (Fig. 6 A). The results of the dual luciferase reporter gene assay (Fig. 6 B,C) show that the GADD45A WT + miR-301a-3p(+) group had significantly reduced gene activity as compared with the GADD45A WT + miR-301a-3p(+) NC group ( p < 0.01), while the gene activity in the GADD45A Mut + miR-301a-3p(+) and GADD45A Mut + miR-301a-3p(+) NC groups was not significantly different ( p = 0.5352). The gene activity in the ILF3-AS1 WT + miR-301a-3p (+) group was significantly lower than that in the ILF3-AS1 WT + miR-301a-3p(+) NC group ( p < 0.05), while the gene activity in the ILF3-AS1 Mut + miR − 301a-3p(+) and ILF3-AS1 Mut + miR-301a-3p(+) NC groups was not significantly different ( p = 0.3694). 4. Discussion Thyroid cancer (TC) is the most common endocrine tumor; papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC), collectively referred to as differentiated thyroid cancer (DTC), account for 80% of TC. In recent years, the incidence of DTC has increased [ 14 – 17 ], with PTC constituting greater than 90% of cases. PTC typically has a good prognosis; however, patients with locally invasive and/or distant metastases have a markedly lower overall survival (OS) rate [ 18 ]. The mean OS duration in patients with advanced PTC ranges from less than 6 months to approximately 5 years; therefore, it is imperative that an effective treatment is found. The Huaier ointment used in the present study was a hot water extraction product of Huaier fungi fermentation, the main active ingredients of which are polysaccharides. Huaier extract has been used in China for approximately 1600 years [ 19 ] and the anti-tumor effects have been recently confirmed. Previous studies have noted that Huaier exerts anti-tumor effects by inhibiting tumor cell proliferation and reducing metastasis and angiogenesis. It has been reported that Huaier granules can decrease the rate of recurrence following curative resection of hepatic cell carcinoma [ 20 ]. Another study demonstrated that Huaier extract inhibits the growth and metastasis of gastric cancer through the c-Myc-Bmi1 axis [ 9 ]. Moreover, Huaier polysaccharide (HP-1) in combination with sunitinib can regulate the PI3K/Akt/VEGFR pathway by reducing the overexpression of certain proteins involved in clear cell renal cell carcinoma (cc RCC) [ 21 ]. Nevertheless, the effect of Huaier on TC remains unknown. Our experimental data demonstrates for the first time that Huaier extract inhibited the proliferation of TC cells. An increasing number of studies have revealed that RNAs participate in carcinogenesis and tumor progression [ 22 ]. Accordingly, our high-throughput sequencing results reveal thousands of DEGs in Huaier-treated TC cells. Specifically, DMBT1 was the most downregulated transcript, with a 133.4784-fold reduction, and ZNF780B was the most upregulated transcript, with the greatest fold change of 1657.641. Additionally, it has been reported that the overexpression of DMBT1 is related to biliary carcinoma [ 23 ] and colorectal cancer [ 24 ]. Moreover, ZNF780B expression has been correlated with hepatocellular carcinoma [ 25 ] and primary osteoarthritis [ 26 ]. Here, the DEGs involved in the proliferation pathway were chosen for verification and construction of ceRNA regulatory networks. SNHG7 has been demonstrated to promote the proliferation, migration, and invasion of lung cancer along with the inhibition of apoptosis; additionally, it is documented to promote the progression and growth of glioblastoma via the inhibition of miR-5095 [ 27 , 28 ]. CTA-29F11.1 is involved in recurrent myocardial infarction events [ 29 ]. ILF3-AS1 has been shown to play a role in the proliferation, invasion, and metastasis of many tumors, such as gastric cancer [ 30 ], prostate cancer [ 31 ], cervical cancer [ 32 ], and colon cancer [ 33 ] through the ceRNA mechanism. Our RT-qPCR results confirm that following Huaier treatment, the expression levels of these lncRNAs and corresponding mRNAs were upregulated, which is consistent with the high-throughput sequencing results. Our experiments confirm an inhibitory effect of Huaier on the proliferation of the PTC cell line, B-CPAP, and show that the expression of GADD45A was simultaneously increased. In humans, the family of growth-arrest and DNA damage-inducible proteins (GADD45 family) is composed of three 18-kD highly acidic proteins that are widely expressed in the nucleus and cytoplasm: GADD45A, GADD45B, and GADD45G [ 34 ]. The expression of GADD45 is involved in regulation of the cell cycle, apoptosis, and DNA repair processes [ 35 ]. Previous studies have also confirmed that GADD45A is involved in the regulatory mechanism of proliferation and metastasis in various tumors. Upregulation of GADD45A leads to cycle inhibition in human bladder cancer cells [ 36 ]. The interaction between BRCA1 and GADD45 may stimulate NER, increase genomic stability, and thus play a role in the pathogenesis of breast cancer [ 37 ]. Our data show that knockdown of GADD45A increased the proliferative capacity of cells in a time-dependent manner, indicating that increased expression of GADD45A may inhibit the proliferation of TC cells. At the same time, the number of cells in the G0/G1 phase of the shRNA group was decreased and the number of cells in the S phase was increased. Following knockdown of GADD45A , more cells entered the synthesis phase to complete subsequent mitosis, increasing proliferation. Moreover, knockdown of GADD45A weakened the effect of Huaier on apoptosis, suggesting that Huaier may inhibit the proliferation of TC cells by upregulating GADD45A , which may serve as a novel target for the treatment of TC. Through Starbase, ILF3-AS1 and GADD45A were predicted to possess binding sites for many miRNAs. The dual-luciferase reporter assay detected the binding of ILF3-AS1 and GADD45A to hsa-miR-301a-3p and confirmed the ceRNA mechanism. Huaier may play a role in inhibiting cell proliferation via the ILF3-AS1/hsa-miR-301a-3p/GADD45A axis. Previous studies have shown that upregulated ILF3-AS1 binds to miR-212 and promotes the progression of osteosarcoma by regulating SOX5 [ 38 ], which is not completely concordant with our experimental results. We demonstrate that Huaier treatment upregulated the expression of ILF3-AS1, which is consistent with the trend of GADD45A. Follow-up experiments are required to verify the effect of ILF3-AS1 in TC, in addition to elucidating whether GADD45A is directly related to ILF3-AS1. 5. Conclusions In summary, this is the first study to comprehensively identify the potential mechanism of Huaier extract in the treatment of TC in vitro . The present study provides evidence that Huaier extract inhibits cell proliferation and induces apoptosis and cell cycle arrest. Moreover, high-throughput sequencing demonstrates alterations in the expression levels of novel lncRNAs and related mRNAs. Our findings suggest that Huaier extract may improve TC via regulation of the ILF3-AS1/hsa-miR-301a-3p/GADD45A axis. Abbreviations TC, Thyroid cancer; Huaier, Trametes robiniophila Murr;CCK-8, cell counting kit 8; DEGs, differentially expressed genes ; RT-qPCR, quantitative real-time PCR; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request. Competing interests The authors declare that no competing interests exist. Funding This work was supported by Liaoning Natural Science Funds (Grant No. 20170541017). Authors’ contributions He JN and Tian Z conceived and designed the present study. He JN, Wang LD and Yao BY performed the experiments. Zhang Y, Yu YF and He JN analyzed the data. 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Characterization of dysregulated lncRNA-mRNA network based on ceRNA hypothesis to reveal the occurrence and recurrence of myocardial infarction. Cell Death Discov. 2018;4:35. Ren Z, Shang G, Wu K, Hu C, Ji T. WGCNA Co-Expression Network Analysis Reveals ILF3-AS1 Functions as a CeRNA to Regulate PTBP1 Expression by Sponging miR-29a in Gastric Cancer. Frontiers in genetics. 2020;11:39. Ye G, Guo L, Xing Y, Sun W, Yuan M. Identification of prognostic biomarkers of prostate cancer with long non-coding RNA-mediated competitive endogenous RNA network. Experimental therapeutic medicine. 2019;17(4):3035–40. Wu W, Sui J, Liu T, Yang S, Xu S, Zhang M, et al. Integrated analysis of two-lncRNA signature as a potential prognostic biomarker in cervical cancer: a study based on public database. PeerJ. 2019;7:e6761. Zhou M, Hu L, Zhang Z, Wu N, Sun J, Su J. Recurrence-Associated Long Non-coding RNA Signature for Determining the Risk of Recurrence in Patients with Colon Cancer. Molecular therapy Nucleic acids. 2018;12:518–29. Sultan F, Sweatt J. The role of the Gadd45 family in the nervous system: a focus on neurodevelopment, neuronal injury, and cognitive neuroepigenetics. Adv Exp Med Biol. 2013;793:81–119. Camilleri-Robles C, Serras F, Corominas M. D-GADD45Role of in JNK-Dependent Apoptosis and Regeneration in. Genes. 2019;10(5). Han N, Yuan F, Xian P, Liu N, Liu J, Zhang H, et al. GADD45a Mediated Cell Cycle Inhibition Is Regulated By P53 In Bladder Cancer. OncoTargets therapy. 2019;12:7591–9. Pietrasik S, Zajac G, Morawiec J, Soszynski M, Fila M, Blasiak J. Interplay between BRCA1 and GADD45A and Its Potential for Nucleotide Excision Repair in Breast Cancer Pathogenesis. International journal of molecular sciences. 2020;21(3). Hu X, Dai J, Shang H, Zhao Z, Hao Y. SP1-mediated upregulation of lncRNA ILF3-AS1 functions a ceRNA for miR-212 to contribute to osteosarcoma progression via modulation of SOX5. Biochemical and biophysical research communications. 2019;511(3):510–7. Supplementary Files graphabstract.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-70026","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":1952178,"identity":"1c6bd99c-ae3c-4271-8771-68ffa4fe6389","order_by":0,"name":"Jingni He","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingni","middleName":"","lastName":"He","suffix":""},{"id":1952179,"identity":"ce87d65e-ed11-42f4-ab11-273949db0efa","order_by":1,"name":"Ying Zhang","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Zhang","suffix":""},{"id":1952180,"identity":"8bf3291a-f07a-465b-8c95-7814298001c0","order_by":2,"name":"Lidong Wang","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lidong","middleName":"","lastName":"Wang","suffix":""},{"id":1952181,"identity":"7870293c-cea0-4bed-a841-8feb018c39cd","order_by":3,"name":"Yifang Yu","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yifang","middleName":"","lastName":"Yu","suffix":""},{"id":1952182,"identity":"f14184f4-57c7-4e45-9652-76c3c52bc559","order_by":4,"name":"Baiyu Yao","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Baiyu","middleName":"","lastName":"Yao","suffix":""},{"id":1952183,"identity":"15854179-b66f-46bf-99d3-f85a45e2001a","order_by":5,"name":"Zhong Tian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIiWNgGAWjYDACZiBmbAAS7A0MBmAGSOQBHh08cC08B2BagEIJ+LQwwLRIJEAZDAS02LMzP3v4c4dNnnzk8wcFjDts7M3Z+Q9+SGCwyZd3wOUwNnMDyTNpxYa3cwwMGM+kJe5sZmYG2phmufEATr+YSRi2HU7cODuHwfhv2+EEg8PMIEceNjBswKWF/ZtEIkjLzOMPDBjbDtsDtTD/wK+Fx0ziIFDLfAkGA5AWxg2HmdnAtsjj8D7PYZ4yyca2tMQNPCC/tIH9YmaRYJBmYIBDC3v/8W2SP9tsEue3H38G1AIMMf6Dj298qLAxkMfhMDgwOMDABjYXYrgBWAQ/AJrJ/AChBSIyCkbBKBgFowAEAEtOVWEn5eBPAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-7641-0944","institution":"China Medical University Second Hospital: Shengjing Hospital of China Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zhong","middleName":"","lastName":"Tian","suffix":""}],"badges":[],"createdAt":"2020-09-01 11:06:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-70026/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-70026/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":2234667,"identity":"894c7394-d812-4e92-86ed-3665e4ee398b","added_by":"auto","created_at":"2020-09-03 21:41:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":86376,"visible":true,"origin":"","legend":"Huaier inhibits the proliferation of TC cells. (A−B) CCK-8 assays were performed to analyze the viability of B-CPAP and C643 cells. The inhibitory rates represent the percentage of cells with impaired growth. The histogram represents the absorbance of B-CPAP and C643 cells at OD450 following treatment with increasing concentrations of Huaier for 24 h, 48 h, or 72 h. (C) Apoptosis was analyzed using PI-Annexin V staining following incubation with Huaier for 48 h. The histogram represents apoptotic rates. (D) Cell cycle distribution was analyzed by flow cytometry in B-CPAP and C643 cells following treatment with Huaier for 48 h. The histogram represents the number of cells in different phases of the cell cycle. Results are presented as the mean ±SD. *p \u003c 0.05 and **p \u003c 0.01.","description":"","filename":"pic1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70026/v1/pic1.jpg"},{"id":2234668,"identity":"dcd27ff1-ed41-4ce9-b36e-733b66da2598","added_by":"auto","created_at":"2020-09-03 21:41:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":47394,"visible":true,"origin":"","legend":"Results of high-throughput sequencing. (A) Comparison of all RNA transcripts. The known transcripts are highlighted in blue and the novel transcripts in red. The Y axis represents the number of transcripts. (B) Comparison of differentially expressed transcripts. The upregulated transcripts are highlighted in blue and the downregulated transcripts in red. The Y axis represents the number of transcripts. (C) Box plot displaying the distribution of transcript expression in the normal and treated groups. (D) Heat map showing the correlation within samples. Numbers in the diagram represent Pearson’s correlation coefficient. (E) Volcano plots indicating transcripts that were differently expressed between the normal and treated groups. Blue points refer to significantly differentially expressed transcripts (p \u003c 0.05). (F) Cluster diagram showing the top 100 transcripts with distinguishable differences among six samples. Red represents increased expression and blue represents decreased expression. (G) Results of GO analysis. The X axis represents the percentage of DEGs and the Y axis represents GO classification. (H) KEGG bubble diagram containing the top 20 pathways. The X axis represents the gene ratio and the Y axis represents the related pathways. The size of the bubble represents the number of DEGs and the color represents the p value. T: treated group, N: normal group. ","description":"","filename":"pic2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70026/v1/pic2.jpg"},{"id":2234669,"identity":"f796fac0-f12f-4349-a6ea-1ba6aa5810e5","added_by":"auto","created_at":"2020-09-03 21:41:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75876,"visible":true,"origin":"","legend":"Analysis of potential lncRNAs and mRNAs. (A) The co-expression network demonstrates a correlation between lncRNAs and mRNAs based on the expression of DEGs. (B) The ceRNA network demonstrates a relationship among lncRNAs, mRNAs, and miRNAs. (C) The expression levels of lncRNAs (SNHG7, MIR181A2HG, ILF3-AS1, CTA-29F11.1) and mRNAs (BBC3, CTSL, GADD45A, DDIT3). The histogram represents the expression of RNA. Results are presented as the mean ± SD. *p \u003c 0.05, **p \u003c 0.01.","description":"","filename":"pic3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70026/v1/pic3.jpg"},{"id":2234670,"identity":"0a6c8f01-e520-4756-8687-eb564214d544","added_by":"auto","created_at":"2020-09-03 21:41:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68810,"visible":true,"origin":"","legend":"The effect of GADD45A knockdown on the function of human thyroid cancer cells. (A) The effect of lentiviral transduction in B-CPAP. B: bright field; G: fluorescence field. (B) Changes in the mRNA and protein expression levels of GADD45A following lentiviral transduction. Scr: blank transduction group, shRNA: transduction group. #p \u003c 0.01. (C) The absorbance and proliferation curves of CCK-8 at different time points. Cell proliferation is most obvious in the shRNA group. (D) Comparison of the cell cycle phases according to flow cytometry. (E) Comparison of apoptosis according to flow cytometry. #p \u003c 0.05, ##p \u003c 0.01.","description":"","filename":"pic4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70026/v1/pic4.jpg"},{"id":2234671,"identity":"e87fec47-b1c7-448c-b7c1-81c99d61d1ec","added_by":"auto","created_at":"2020-09-03 21:41:44","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":88616,"visible":true,"origin":"","legend":"GADD45A participates in the inhibition of human thyroid cancer cell proliferation by Huaier. (A) Cell apoptosis results according to flow cytometry. (B) Comparison of apoptosis among the different groups. #p \u003c 0.05, ##p \u003c 0.01. (C) Cell cycle results according to flow cytometry. (D) Comparison of cell number at each cell stage. Scr group: blank transfection group, Scr-treated group: blank treated group, shRNA-treated group: transfection group. #p \u003c 0.05, ##p \u003c 0.01.","description":"","filename":"pic5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70026/v1/pic5.jpg"},{"id":2234672,"identity":"a5f6aba9-06ca-448a-9a9e-acf18ceb5ea1","added_by":"auto","created_at":"2020-09-03 21:41:44","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":58765,"visible":true,"origin":"","legend":"ILF3-AS1 and GADD45A competitively inhibit hsa-miR-301a-3p. (A) Prediction of the binding sites of ILF3-AS1 and GADD45A with hsa-miR-301a-3p. (B) Luciferase activity is significantly reduced in the GADD45A WT + miR-301a-3p(+) group as compared with that in the GADD45A WT + miR-301a-3p(+) NC group (p \u003c 0.01). (C) Luciferase activity is significantly reduced in the ILF3-AS1 WT + miR-301a-3p(+) group as compared with that in the ILF3-AS1 WT + miR-301a-3p(+) NC group (p \u003c 0.05). #p \u003c 0.05, ##p \u003c 0.01.","description":"","filename":"pic6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70026/v1/pic6.jpg"},{"id":13587531,"identity":"02cc5612-de8c-4eec-9796-e290a9606ee2","added_by":"auto","created_at":"2021-09-17 04:51:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4643567,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-70026/v1/cd67f99e-eb03-4a16-a814-a5af46102d49.pdf"},{"id":2234674,"identity":"4f994b7a-5f95-499b-9a0d-5887905559a3","added_by":"auto","created_at":"2020-09-03 21:41:45","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10024580,"visible":true,"origin":"","legend":"","description":"","filename":"graphabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-70026/v1/graphabstract.tif"}],"financialInterests":"","formattedTitle":"Anti-proliferative mechanism of Huaier extract in human thyroid carcinoma cells","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n\u003cli\u003eHuaier extract inhibits the proliferation of thyroid cancer cells via changes in the expression levels of a multitude of genes\u003c/li\u003e\n\u003cli\u003ea decrease in GADD45A expression enhances the proliferative ability of thyroid cancer cells, the levels of which can be increased by Huaier treatment, thus regulating the cell cycle and apoptosis\u003c/li\u003e\n\u003cli\u003eHuaier can inhibit the proliferation of thyroid cancer cells through the ILF3-AS1/hsa-miR-301a-3p/GADD45A ceRNA axis\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Background","content":" \u003cp\u003eThyroid cancer (TC) is a common malignant tumor, accounting for 94.5% of all malignant endocrine tumors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The incidence rate of TC ranks first among the various types of head and neck cancers [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and an increasing prevalence has been demonstrated recently [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Following systematic treatment, such as thyroidectomy, radioiodine ablation, and thyroid hormone suppressive therapy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], individuals diagnosed with TC typically have an excellent prognosis, with an overall 10-year survival rate greater than 97% [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, 5\u0026thinsp;\u0026minus;\u0026thinsp;20% of patients still experience local recurrence, disease progression, and metastasis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]; thus, it is of great importance to discover appropriate adjuvant therapies for TC.\u003c/p\u003e \u003cp\u003eTrametes robiniophila Murr (Huaier), a traditional Chinese medicinal herb, has been used as an adjuvant anti-tumor therapy for 1600\u0026nbsp;years. Moreover, Huaier particles have been used clinically to treat patients with malignant tumors (China Food and Drug Administration approval number, Z20000109; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://app1.sfda.gov.cn/datasearch/face3/base.jsp\u003c/span\u003e\u003c/span\u003e). It has been reported that Huaier is effective in treating diverse cancers such as hepatocellular carcinoma, breast cancer, lung cancer, gastric cancer, and cervical cancer [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], but the effect of Huaier on TC remains unknown. A large number of novel RNAs have been found based on the human genome project, and there is increasing interest in the mechanisms by which these RNAs participate in cellular regulation and transcription [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, we explored the role of RNA in the anti-tumor effect of Huaier in TC.\u003c/p\u003e "},{"header":"2. Materials and Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell lines and reagents\u003c/h2\u003e \u003cp\u003eThe TC cell lines, B-CPAP and C643, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and were routinely cultured in RMPI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 100 U/mL penicillin, and 100\u0026nbsp;\u0026micro;g/mL streptomycin (Gibco, USA) in an atmosphere of 5% CO \u003csub\u003e2\u003c/sub\u003e at 37℃.\u003c/p\u003e \u003cp\u003eAn electuary ointment of Huaier extract was donated by Qidong Gaitianli Pharmaceutical Co., Ltd. (Jiangsu, China). Huaier extract contained four monosaccharides and 18 amino acids; its main active ingredients were polysaccharides, named TP-1 (fucose, arabinose, galactose, glucose, xylopyranose, mannose, at molar ratios of 3.2:2.0:33.5:5.5:1.2:50.4), TP-2 (arabinose, galactose, glucose, xylopyranose, mannose, at molar ratios of 7.4:4.5:2.7:9.4:1.9), TP-3 (fucose, arabinose, galactose, glucose, xylopyranose, mannose, at molar ratios of 0.1:1.0:5.4:4.4:2.1: 7.0), and TP-4 (arabinose, galactose, glucose, xylopyranose, mannose, at molar ratios of 8.9:1.6:3.4:7.4:1.3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of Huaier aqueous extract\u003c/h2\u003e \u003cp\u003eA mass of 2\u0026nbsp;g Huaier ointment was dissolved in 20\u0026nbsp;mL complete medium, passed through a 22-\u0026micro;m filter, and stored at -20℃ for future use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Determination of cell viability\u003c/h2\u003e \u003cp\u003eThe viability of B-CPAP and C643 cells in the presence of increasing concentrations of Huaier was measured using a cell counting kit-8 (CCK-8) kit (DOJINDO, Japan). An experimental group, control group, and blank group consisted of five replicate wells each. B-CPAP and C643 cells were seeded on 96-well plates at a density of 2\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e cells/well. Following synchronization, 200\u0026nbsp;\u0026micro;L medium containing 1, 2, 4, 8, 12, or 16\u0026nbsp;mg/mL Huaier was added, and the cells were cultured for 24\u0026nbsp;h, 48\u0026nbsp;h, or 72\u0026nbsp;h. Subsequently, 100\u0026nbsp;\u0026micro;L medium containing 10% CCK-8 was added to the cells and incubated for 3\u0026nbsp;h, following which the absorbance was measured at 450\u0026nbsp;nm using a microplate reader (Bio Tek, USA). The experiment was repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Evaluation of apoptosis and the cell cycle\u003c/h2\u003e \u003cp\u003eCell apoptosis assay (Propidium iodide (PI)-Annexin V staining): The proportion of apoptotic cells was assessed using a BD Pharmingen\u0026trade; PE Annexin V apoptosis detection kit (DOJINDO, Japan). Briefly, after treatment with 8\u0026nbsp;mg/mL Huaier extract for 48\u0026nbsp;h, B-CPAP and C643 cells were harvested, washed twice with PBS, and centrifuged at 1000\u0026nbsp;rpm for 3\u0026nbsp;min. Subsequently, 5\u0026nbsp;\u0026micro;L Annexin V-FITC and 5\u0026nbsp;\u0026micro;L PI solution were mixed with 1\u0026thinsp;\u0026times;\u0026thinsp;Annexin V binding solution (100\u0026nbsp;\u0026micro;L) containing cells at a density of 1\u0026thinsp;\u0026times;\u0026thinsp;10 \u003csup\u003e5\u003c/sup\u003e/mL. Following incubation for 15\u0026nbsp;min in the dark at room temperature, another 400\u0026nbsp;\u0026micro;L 1\u0026thinsp;\u0026times;\u0026thinsp;Annexin V binding solution was added prior to analysis by FACScan flow cytometry (Becton-Dickinson, USA). The experiment was repeated three times.\u003c/p\u003e \u003cp\u003eCell cycle assay: The cell cycle phases were evaluated using a cell cycle detection kit (KeyGen China). The experimental group was treated with 8\u0026nbsp;mg/mL Huaier extract, while the control group was incubated with complete medium. Cells were harvested after 48\u0026nbsp;h, rinsed twice with PBS, and centrifuged at 1000\u0026nbsp;rpm for 5\u0026nbsp;min. Subsequently, cells were fixed in cold 70% ethanol overnight, washed once with PBS the following day, resuspended in 100\u0026nbsp;\u0026micro;L RNaseA, and placed in water at 37℃ for 30\u0026nbsp;min. A volume of 400 \u0026micro;L PI staining solution was added and cells were analyzed by FACScan flow cytometry after incubation for 30\u0026nbsp;min at 4℃ in the dark. Data were analyzed using the ModFitLT V2.0 software (Becton-Dickinson). The experiment was repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. RNA extraction, library preparation, sequencing, and data processing\u003c/h2\u003e \u003cp\u003eFollowing treatment with 8\u0026nbsp;mg/mL Huaier extract for 48\u0026nbsp;h, B-CPAP cells were harvested for high-throughput sequencing. Total RNA was isolated according to the manufacturer\u0026rsquo;s instructions. The quality and quantity of extracted RNA were examined using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and an RNA 6000 Nano LabChip kit (Agilent Technologies, USA). The preparation of whole transcriptome libraries and deep sequencing were performed by Beijing Ori-Gene Science and Technology Corp., Ltd. (Beijing, China). The libraries were constructed using the Ribo-Zero Magnetic Gold kit (Illumina, USA) and the NEBNext\u0026reg; Ultra\u0026trade; RNA Library Prep kit for Illumina (New England Biolabs) according to the manufacturer\u0026rsquo;s manual. Sequence reads were aligned to the human genome (GRCh38) using the TopHat 2.0 program, and the resulting alignment files were reconstructed by Cufflinks.\u003c/p\u003e \u003cp\u003eFollowing prediction of mRNAs and ncRNAs, histograms and box plots were used to represent the transcripts. A Pearson heat map was drawn to represent the difference between the two groups of samples. The differential genes were plotted using volcanoes and cluster analysis. GO and KEGG analyses were performed, and the co-expression network and ceRNA mechanistic diagram were constructed.\u003c/p\u003e \u003cp\u003eTotal RNA was extracted, and cDNA was synthesized using a PrimeScript\u003csup\u003eTM\u003c/sup\u003eRT reagent kit with gDNA Eraser (TaKaRa, Japan) according to the manufacturer's instructions. Subsequently, RT-qPCR was performed in three independent wells using SYBR Premix Ex Taq (TaKaRa, Japan), and relative RNA expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. The primer sequences are shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. shRNA lentiviral vector transduction and verification\u003c/h2\u003e \u003cp\u003eThe lentiviral vector was constructed by Shanghai Hanheng Technology Co., Ltd. A total of 30,000 cells/well were inoculated with lentiviral infection solution (MOI\u0026thinsp;=\u0026thinsp;25), culturing to a fusion degree of 60\u0026thinsp;\u0026minus;\u0026thinsp;70%. After successful transduction, cells were screened to establish stable cell lines.\u003c/p\u003e \u003cp\u003eRT-qPCR was used to verify the expression of GADD45A. The specific primer sequences were as follows: human GADD45A forward: 5'-ACTGTCGGGGTGTACGAAG-3'; human GADD45A reverse: 5'-ATCAGGGTGAAGTGGATCTGC-3'; human GAPDH forward: 5'-GAAGGTGAAGGTCGGAGTC-3'; and human GAPDH reverse: 5'-GAAGATGGTGATGGGATTTC-3'.\u003c/p\u003e \u003cp\u003eProtein expression was verified by western blotting. Total cellular protein was extracted and quantitated using the Coomassie brilliant blue method. A 20-\u0026micro;L protein sample was separated by SDS-PAGE and transferred to PVDF membrane at 80\u0026nbsp;V for 1.5\u0026nbsp;h. The membrane was sequentially incubated in skimmed milk, specific primary antibody, and corresponding secondary antibody. Protein bands were visualized using C300ECL luminaire.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Evaluation of cell proliferation\u003c/h2\u003e \u003cp\u003eB-CPAP cells were divided into control, B-CPAP-Scr (Scr), and B-CPAP-sh-GADD45A (shRNA) groups. Cell proliferation was evaluated using a CCK-8 assay, and PI single-staining and Annexin V-PE/7-AAD double-staining were used to assess the cell cycle and apoptosis, respectively, by flow cytometry.\u003c/p\u003e \u003cp\u003eFor treatment with Huaier extract, B-CPAP cells were divided into B-CPAP-Scr (Scr), B-CPAP-Scr treated with 8\u0026nbsp;mg/mL Huaier extract (Scr-treated), and B-CPAP-sh-GADD45A treated with 8\u0026nbsp;mg/mL Huaier extract (shRNA-treated) groups. Cell cycle and apoptosis were evaluated as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Dual-luciferase assay\u003c/h2\u003e \u003cp\u003eGADD45A and ILF3-AS1 WT and mutant dual-luciferase plasmids were constructed. The dual-luciferase detection kit (Promega, USA) was used to measure gene activity according to the manufacturer\u0026rsquo;s instructions. Both the Firefly and Renilla values were measured and the Firefly/Renilla ratio was calculated. Each experiment was repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using the SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA) software. The results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of at least three independent experiments in at least triplicate. One-way analysis of variance was used to assess significant differences between the groups; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e "},{"header":"3. Results","content":" \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Huaier extract inhibits B-CPAP and C643 cell proliferation\u003c/h2\u003e \u003cp\u003eB-CPAP and C643 cells were treated with increasing concentrations of Huaier extract for 24\u0026nbsp;h, 48\u0026nbsp;h, or 72\u0026nbsp;h and subjected to the CCK-8 assay to assess the effect on cell proliferation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, a low concentration of Huaier extract may have slightly promoted cell proliferation; however, concentrations of 8\u0026nbsp;mg/mL and higher markedly inhibited cell proliferation in a concentration- and time-dependent manner. The IC\u003csub\u003e50\u003c/sub\u003e of Huaier extract in B-CPAP and C643 cells was 5.604\u0026nbsp;mg/mL and 8.330\u0026nbsp;mg/mL, respectively, following a 48-h incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), showing the most marked inhibitory effect on cell proliferation and suggesting optimal conditions for further experiments. To explore the potential anti-proliferative mechanism of Huaier extract in depth, the apoptotic rate and cell cycle distribution were assessed by flow cytometry following treatment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, the apoptotic ratio was 8.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60% in B-CPAP cells and 5.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35% in C643 cells following treatment with Huaier extract (8\u0026nbsp;mg/mL) for 48\u0026nbsp;h, which was significantly different from that in the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, the proportion of B-CPAP cells in the G0/G1 phase was increased from 63.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96% in the control group to 72.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) following incubation with Huaier extract for 48\u0026nbsp;h, while the proportion of C643 cells was increased from 47.77\u0026thinsp;\u0026plusmn;\u0026thinsp;4.23% to 64.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.34% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Moreover, the number of cells in the S and G2/M phases decreased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Whole genome RNA-sequencing data and pathway analysis results\u003c/h2\u003e \u003cp\u003eA total of 65.38\u0026thinsp;\u0026minus;\u0026thinsp;103.71\u0026nbsp;million raw data reads were obtained for each sample. After filtering, 89.1\u0026thinsp;\u0026minus;\u0026thinsp;91.8% quality control data reads were obtained with an average length of 145.87 nucleotides, which corresponded to the reference human genome (Ensembl GRCh38 main assembly). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, 16,159 known transcripts and 54,772 novel transcripts were acquired. A total of 7,979 differentially expressed transcripts were found, of which 2,185 were upregulated and 5,794 were downregulated. A total of 5,717 genes were differentially expressed, among which 869 lncRNAs, 1 circRNA, and 1,179 mRNAs were upregulated and 3,781 lncRNAs, 1 circRNA, and 921 mRNAs were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The box plot shows the distribution of the expression of various transcripts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The heat map demonstrates the expression levels of total transcripts among the different groups; the difference was significant among groups but not within groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The volcano map (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) displays all the differentially expressed transcripts and cluster analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) shows the difference in expression of each transcript in the sample. These differentially expressed genes (DEGs) are valuable for elucidating the mechanism of action of Huaier in thyroid carcinoma cells. To further understand the DEGs, GO and KEGG analyses were performed. The significantly enriched GO terms are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG; a total of 270 genes were enriched in upregulated terms, while 171 genes were enriched in downregulated terms (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The top three enriched GO biological terms were integrin binding (GO: 0005178), coenzyme binding (GO: 0050662), and endoplasmic reticulum lumen (GO: 0005788). According to KEGG analysis, a total of 47 significantly enriched pathway terms were associated with the DEGs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the top 20 of which are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Gene relationship analysis and RNA validation\u003c/h2\u003e \u003cp\u003eNext, proliferation-related DEGs were chosen to build a coding\u0026thinsp;\u0026minus;\u0026thinsp;noncoding co-expression network (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Four lncRNAs (SNHG7, MIR181A2HG, ILF3-AS1, CTA-29F11.1) were selected from the proliferation-related pathways for the construction of the ceRNA network (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). According to the selected lncRNAs, the corresponding mRNAs were determined and RT-qPCR was performed to verify the expression levels. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, lncRNAs (SNHG7, MIR181A2HG, ILF3-AS1, CTA-29F11.1) and mRNAs (BBC3, CTSL, GADD45A, DDIT3) were over-expressed in the Huaier-treated group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which is consistent with the RNA-sequencing results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Effect of \u003cem\u003eGADD45A\u003c/em\u003e knockdown on the proliferation of human thyroid cancer cells\u003c/h2\u003e \u003cp\u003eThe transduction efficiency was 90% according to fluorescent microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and the mRNA and protein expression of GADD45A was decreased in the shRNA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). With respect to proliferation, the shRNA group showed a time-dependent increase as compared with the Ctrl and Scr groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Moreover, flow cytometry revealed that the number of cells in the G0/G1 phase was decreased in the shRNA group as compared with that in the Scr group (47.77\u0026thinsp;\u0026plusmn;\u0026thinsp;4.23% vs. 67.20\u0026thinsp;\u0026plusmn;\u0026thinsp;4.28%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The number of cells in the S phase was increased in the shRNA group (39.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56% vs. 22.49\u0026thinsp;\u0026plusmn;\u0026thinsp;3.42%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), but there was no significant difference in the G2/M phase (12.79\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81% vs. 11.30\u0026thinsp;\u0026plusmn;\u0026thinsp;4.07%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.6306) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Further, the shRNA group had a lower apoptotic rate (3.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34% vs. 5.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0218) and a higher survival rate (81.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86% vs. 78.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0495) than those in the Scr group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Effect of \u003cem\u003eGADD45A\u003c/em\u003e on the Huaier-induced reduction in proliferation of human thyroid cancer cells\u003c/h2\u003e \u003cp\u003eFollowing downregulation of \u003cem\u003eGADD45A\u003c/em\u003e, the number of cells in the G0/G1 phase was significantly lower in the shRNA-treated group than that in the Scr-treated group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the number of cells in the S phase was increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049); however, the number of cells in the G2/M phase did not change significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.9572) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,B). The shRNA-treated group had a significantly reduced apoptotic rate as compared with that in the Scr-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC,D, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003ch2\u003e3.6. ILF3-AS1\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eGADD45A\u003c/span\u003e \u003cb\u003ecompetitively inhibit hsa-miR-301a-3p to exert the anti-proliferative effect of Huaier on human thyroid cancer cells\u003c/h2\u003e \u003cp\u003eThe ceRNA network predicts that ILF3-AS1 and GADD45A may exert their effects through the competitive inhibition of miRNA. Starbase predicts that ILF3-AS1 and GADD45A may have binding sites within hsa-miR-301a-3p (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The results of the dual luciferase reporter gene assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB,C) show that the GADD45A WT\u0026thinsp;+\u0026thinsp;miR-301a-3p(+) group had significantly reduced gene activity as compared with the GADD45A WT\u0026thinsp;+\u0026thinsp;miR-301a-3p(+) NC group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the gene activity in the GADD45A Mut\u0026thinsp;+\u0026thinsp;miR-301a-3p(+) and GADD45A Mut\u0026thinsp;+\u0026thinsp;miR-301a-3p(+) NC groups was not significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.5352). The gene activity in the ILF3-AS1 WT\u0026thinsp;+\u0026thinsp;miR-301a-3p (+) group was significantly lower than that in the ILF3-AS1 WT\u0026thinsp;+\u0026thinsp;miR-301a-3p(+) NC group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the gene activity in the ILF3-AS1 Mut\u0026thinsp;+\u0026thinsp;miR \u0026minus;\u0026thinsp;301a-3p(+) and ILF3-AS1 Mut\u0026thinsp;+\u0026thinsp;miR-301a-3p(+) NC groups was not significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.3694).\u003c/p\u003e \u003c/div\u003e "},{"header":"4. Discussion","content":" \u003cp\u003eThyroid cancer (TC) is the most common endocrine tumor; papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC), collectively referred to as differentiated thyroid cancer (DTC), account for 80% of TC. In recent years, the incidence of DTC has increased [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], with PTC constituting greater than 90% of cases. PTC typically has a good prognosis; however, patients with locally invasive and/or distant metastases have a markedly lower overall survival (OS) rate [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The mean OS duration in patients with advanced PTC ranges from less than 6\u0026nbsp;months to approximately 5 years; therefore, it is imperative that an effective treatment is found.\u003c/p\u003e \u003cp\u003eThe Huaier ointment used in the present study was a hot water extraction product of Huaier fungi fermentation, the main active ingredients of which are polysaccharides. Huaier extract has been used in China for approximately 1600\u0026nbsp;years [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and the anti-tumor effects have been recently confirmed. Previous studies have noted that Huaier exerts anti-tumor effects by inhibiting tumor cell proliferation and reducing metastasis and angiogenesis. It has been reported that Huaier granules can decrease the rate of recurrence following curative resection of hepatic cell carcinoma [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Another study demonstrated that Huaier extract inhibits the growth and metastasis of gastric cancer through the c-Myc-Bmi1 axis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, Huaier polysaccharide (HP-1) in combination with sunitinib can regulate the PI3K/Akt/VEGFR pathway by reducing the overexpression of certain proteins involved in clear cell renal cell carcinoma (cc RCC) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Nevertheless, the effect of Huaier on TC remains unknown. Our experimental data demonstrates for the first time that Huaier extract inhibited the proliferation of TC cells.\u003c/p\u003e \u003cp\u003eAn increasing number of studies have revealed that RNAs participate in carcinogenesis and tumor progression [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Accordingly, our high-throughput sequencing results reveal thousands of DEGs in Huaier-treated TC cells. Specifically, DMBT1 was the most downregulated transcript, with a 133.4784-fold reduction, and ZNF780B was the most upregulated transcript, with the greatest fold change of 1657.641. Additionally, it has been reported that the overexpression of DMBT1 is related to biliary carcinoma [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and colorectal cancer [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Moreover, ZNF780B expression has been correlated with hepatocellular carcinoma [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and primary osteoarthritis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Here, the DEGs involved in the proliferation pathway were chosen for verification and construction of ceRNA regulatory networks. SNHG7 has been demonstrated to promote the proliferation, migration, and invasion of lung cancer along with the inhibition of apoptosis; additionally, it is documented to promote the progression and growth of glioblastoma via the inhibition of miR-5095 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. CTA-29F11.1 is involved in recurrent myocardial infarction events [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. ILF3-AS1 has been shown to play a role in the proliferation, invasion, and metastasis of many tumors, such as gastric cancer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], prostate cancer [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], cervical cancer [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and colon cancer [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] through the ceRNA mechanism. Our RT-qPCR results confirm that following Huaier treatment, the expression levels of these lncRNAs and corresponding mRNAs were upregulated, which is consistent with the high-throughput sequencing results.\u003c/p\u003e \u003cp\u003eOur experiments confirm an inhibitory effect of Huaier on the proliferation of the PTC cell line, B-CPAP, and show that the expression of GADD45A was simultaneously increased. In humans, the family of growth-arrest and DNA damage-inducible proteins (GADD45 family) is composed of three 18-kD highly acidic proteins that are widely expressed in the nucleus and cytoplasm: GADD45A, GADD45B, and GADD45G [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The expression of \u003cem\u003eGADD45\u003c/em\u003e is involved in regulation of the cell cycle, apoptosis, and DNA repair processes [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Previous studies have also confirmed that GADD45A is involved in the regulatory mechanism of proliferation and metastasis in various tumors. Upregulation of GADD45A leads to cycle inhibition in human bladder cancer cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The interaction between BRCA1 and GADD45 may stimulate NER, increase genomic stability, and thus play a role in the pathogenesis of breast cancer [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our data show that knockdown of \u003cem\u003eGADD45A\u003c/em\u003e increased the proliferative capacity of cells in a time-dependent manner, indicating that increased expression of GADD45A may inhibit the proliferation of TC cells. At the same time, the number of cells in the G0/G1 phase of the shRNA group was decreased and the number of cells in the S phase was increased. Following knockdown of \u003cem\u003eGADD45A\u003c/em\u003e, more cells entered the synthesis phase to complete subsequent mitosis, increasing proliferation. Moreover, knockdown of \u003cem\u003eGADD45A\u003c/em\u003e weakened the effect of Huaier on apoptosis, suggesting that Huaier may inhibit the proliferation of TC cells by upregulating \u003cem\u003eGADD45A\u003c/em\u003e, which may serve as a novel target for the treatment of TC.\u003c/p\u003e \u003cp\u003eThrough Starbase, ILF3-AS1 and GADD45A were predicted to possess binding sites for many miRNAs. The dual-luciferase reporter assay detected the binding of ILF3-AS1 and GADD45A to hsa-miR-301a-3p and confirmed the ceRNA mechanism. Huaier may play a role in inhibiting cell proliferation via the ILF3-AS1/hsa-miR-301a-3p/GADD45A axis. Previous studies have shown that upregulated ILF3-AS1 binds to miR-212 and promotes the progression of osteosarcoma by regulating SOX5 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], which is not completely concordant with our experimental results. We demonstrate that Huaier treatment upregulated the expression of ILF3-AS1, which is consistent with the trend of GADD45A. Follow-up experiments are required to verify the effect of ILF3-AS1 in TC, in addition to elucidating whether GADD45A is directly related to ILF3-AS1.\u003c/p\u003e "},{"header":"5. Conclusions","content":" \u003cp\u003eIn summary, this is the first study to comprehensively identify the potential mechanism of Huaier extract in the treatment of TC \u003cem\u003ein vitro\u003c/em\u003e. The present study provides evidence that Huaier extract inhibits cell proliferation and induces apoptosis and cell cycle arrest. Moreover, high-throughput sequencing demonstrates alterations in the expression levels of novel lncRNAs and related mRNAs. Our findings suggest that Huaier extract may improve TC via regulation of the ILF3-AS1/hsa-miR-301a-3p/GADD45A axis.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eTC, Thyroid cancer; Huaier, Trametes robiniophila Murr;CCK-8, cell counting kit 8; DEGs, differentially expressed genes ; RT-qPCR, quantitative real-time PCR; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes\u003c/p\u003e "},{"header":"Declarations","content":" \u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e \u003cp\u003eThe datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that no competing interests exist.\u003c/p\u003e \u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Liaoning Natural Science Funds (Grant No. 20170541017).\u003c/p\u003e \u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e \u003cp\u003eHe JN and Tian Z conceived and designed the present study. He JN, Wang LD and Yao BY performed the experiments. Zhang Y, Yu YF and He JN analyzed the data. He JN and Tian Z wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e \u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\u003cli\u003e \u003cspan\u003eCabanillas ME, McFadden DG, Durante C. Thyroid cancer. Lancet. 2016;388(10061):2783\u0026ndash;95.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eChen WQ, Li H, Sun KX, Zheng RS, Zhang SW, Zeng HM, et al. Report of cancer incidence and mortality in China, 2014. Zhonghua Zhong Liu Za Zhi. 2018;40(1):5\u0026ndash;13.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003ePellegriti G, Frasca F, Regalbuto C, Squatrito S, Vigneri R. Worldwide increasing incidence of thyroid cancer: update on epidemiology and risk factors. 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The role of the Gadd45 family in the nervous system: a focus on neurodevelopment, neuronal injury, and cognitive neuroepigenetics. Adv Exp Med Biol. 2013;793:81\u0026ndash;119.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eCamilleri-Robles C, Serras F, Corominas M. D-GADD45Role of in JNK-Dependent Apoptosis and Regeneration in. Genes. 2019;10(5).\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eHan N, Yuan F, Xian P, Liu N, Liu J, Zhang H, et al. GADD45a Mediated Cell Cycle Inhibition Is Regulated By P53 In Bladder Cancer. OncoTargets therapy. 2019;12:7591\u0026ndash;9.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003ePietrasik S, Zajac G, Morawiec J, Soszynski M, Fila M, Blasiak J. Interplay between BRCA1 and GADD45A and Its Potential for Nucleotide Excision Repair in Breast Cancer Pathogenesis. International journal of molecular sciences. 2020;21(3).\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eHu X, Dai J, Shang H, Zhao Z, Hao Y. SP1-mediated upregulation of lncRNA ILF3-AS1 functions a ceRNA for miR-212 to contribute to osteosarcoma progression via modulation of SOX5. Biochemical and biophysical research communications. 2019;511(3):510\u0026ndash;7.\u003c/span\u003e \u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Huaier extract, thyroid cancer, anti-proliferative mechanism, high-throughput sequencing, differentially expressed genes ","lastPublishedDoi":"10.21203/rs.3.rs-70026/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-70026/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eBackground\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThyroid cancer is the most common endocrine tumor and typically has a good prognosis; however, some patients still present with local or distant metastases. Huaier is a traditional Chinese medicine reported as effective in treating certain types of tumor, but the effect of Huaier on thyroid cancer has not yet been reported.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cem\u003eMethods\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe thyroid cancer cell lines, B-CPAP and C643, were treated with increasing concentrations of Huaier extract and the therapeutic effect was measured using a cell counting kit 8 (CCK-8) and flow cytometry. High-throughput sequencing was further performed to identify differentially expressed genes (DEGs) in Huaier-treated B-CPAP cells. Moreover, quantitative real-time PCR (RT-qPCR) was carried out to verify the selected RNAs. Finally, the dual luciferase detection kit was used to detect gene activity.\u003c/p\u003e\u003cp\u003e\u003cem\u003eResults\u003c/em\u003e\u003c/p\u003e\u003cp\u003eProliferation of B-CPAP and C643 cells was significantly suppressed by treatment with Huaier extract in a concentration- and time-dependent manner. Huaier extract also induced cell cycle arrest and apoptosis according to flow cytometry (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\u003cp\u003eHigh-throughput sequencing observed 7,979 significantly altered transcripts. Gene Ontology (GO) analysis showed that 270 genes were enriched in upregulated terms, while 171 genes were enriched in downregulated terms (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that there were 47 enriched pathways associated with DEGs (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The expression levels of chosen lncRNAs (SNHG7, MIR181A2HG, ILF3-AS1, and CTA-29F11.1) and their corresponding mRNAs (BBC3, CTSL, GADD45A, and DDIT3) were verified to be overexpressed in Huaier-treated B-CPAP cells by RT-qPCR (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e\u003cp\u003eFollowing transduction, the CCK-8 results showed that the proliferative capacity was increased in the shRNA group as compared with that in the Ctrl and Scr groups. According to flow cytometry, the number of cells in the G0/G1 phase was decreased in the shRNA group (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01) and the apoptosis rate was lower (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05). The shRNA-treated group had significantly reduced Huaier-induced apoptosis as compared with the Scr-treated group (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05). Moreover, the number of cells in the G0/G1 phase in the shRNA-treated group was significantly lower than that in the Scr-treated group (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05). The results of the dual luciferase reporter gene experiment showed that the activity in the GADD45A WT + miR-301a-3p(+) group was significantly reduced as compared with that in the GADD45A WT + miR-301a-3p(+) NC group (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01). Further, the activity in the ILF3-AS1 WT + miR-301a-3p(+) group was significantly lower than that in the ILF3-AS1 WT + miR-301a-3p(+) NC group (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e\u003cp\u003e\u003cem\u003eConclusions\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe present study demonstrates that Huaier extract inhibits the proliferation of thyroid cancer cells via changes in the expression levels of a multitude of genes. In particular, a decrease in GADD45A expression enhances the proliferative ability of thyroid cancer cells, the levels of which can be increased by Huaier treatment, thus regulating the cell cycle and apoptosis. Huaier can inhibit the proliferation of thyroid cancer cells through the ILF3-AS1/hsa-miR-301a-3p/GADD45A ceRNA axis.\u003c/p\u003e","manuscriptTitle":"Anti-proliferative mechanism of Huaier extract in human thyroid carcinoma cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-09-03 21:41:42","doi":"10.21203/rs.3.rs-70026/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bca44dfc-837e-4343-9663-ce5863070d53","owner":[],"postedDate":"September 3rd, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":431368,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2020-09-15T19:11:39+00:00","versionOfRecord":[],"versionCreatedAt":"2020-09-03 21:41:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-70026","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-70026","identity":"rs-70026","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}