Mechanism Of Huaier' Effect On The Proliferation Of Human Thyroid Carcinoma Cells

Objective: Thyroid cancer is the most common endocrine tumor.Papillary thyroid cancer,follicular thyroid cancer,collectively referred to as differentiated thyroid cancer,which contains most of the thyroid malignant tumors,is accounting for 80%.Differentiated thyroid cancer has become the fastest growing solid tumor in the past decade.Differentiated thyroid cancer usually has a good prognosis.Through surgical resection,radioiodine therapy,and thyroid suppression therapy,the 10-year survival can reach more than 97%.However,5-20% of patients still relapse after treatment,with local or distant organ metastases.Patients with high risk factors still need more active diagnosis and treatment.Huaier is a traditional Chinese medicine.Many studies have reported that Huaier is effective in treating liver cancer,breast cancer,gastric cancer,lung cancer,cervical cancer,colon cancer,kidney cancer,prostate cancer,ovarian cancer,melanoma and other carcinomas.However,the effect of Huaier on thyroid cancer has not been reported.High-throughput sequencing can realize differential gene screening,and subsequent analysis can predict the pathways of differential gene enrichment and the possible targeting mi RNAs for differentially expressed lnc RNA and m RNA.Methods:1.Human thyroid papillary cancer cell line B-CPAP and human thyroid cancer undifferentiated cancer cell line c643 were cultured and passaged normally.Huaier extract solution was prepared and stored frozen.Afer cells synchronization,changed the media containing different concentrations of Huaier cultured for another 24 h,48h,and 72 h.The absorbance at 450 nm was used to calculate the inhibition rate and IC50 was analysed.Then the cells in the experimental group were added with medium at a concentration of 8mg /ml Huaier,and the control group was added with complete medium,after 48 hours,cell apoptosis(Annexin V-FITC / PI double staining method)and cycle changes(PI single Dyeing method)were done.After 48 hours of culture,the cells in two groups were counted and placed in the Transwell chamber.After 18 h for C643 cells,24 h for B-CPAP cell,stoped the migration and fixed the cells and stained.In the same way,cell cultures in the experimental group and the control group were placed in the Transwell chamber containing Matrigel for 24 hours,and then fixed and stained.2.High-throughput sequencing was performed using human thyroid papillary cancer cells B-CPAP.The two groups were normal cells group and Huaier-treat group.Total cellular RNA was extracted to construct a transcriptome library.Use the Qubit Fluorometer to detect the concentration of DNA in the library.If it is greater than 1.0 ng/?L,it is qualified.The constructed library was sequenced with Illumina Hiseq2000 /2500.Using cufflinks to estimate the abundance of all transcripts,the cuffdiff command screens for differential genes.Use boxplots,density plots,and other images to show the distribution of transcripts and gene expression values in each sample.The differential transcripts were visualized by cluster analysis and volcano map.GO analysis was performed to construct gene annotations.P and q values are used to test the reliability of the analysis.The major biological functions of differentially expressed genes can be determined by GO functional significance enrichment analysis.KEGG analysis can identify the most important biochemical metabolic pathways and signal transduction pathways involved in differentially expressed genes through Pathway significant enrichment.Use Pearson correlation to analyze correlations between differential genes.Select the RNA with correlation between> 0.99 or <-0.99,and drawed the nc RNA-m RNA co-expression network diagram.Circ Interactome,Target Scan,and mi RWalk 3.0 were used to predict mi RNAs that might be targeted to nc RNAs.At the same time,the target genes of these mi RNAs were predicted,and the ce RNA network was mapped.q PCR was used to validate the picked genes.3.The sh RNA vector of GADD45 A gene was constructed,transfected into human thyroid papillary cells B-CPAP,and GADD45 A gene was silenced.Infection solution was prepared according to the optimal MOI value(MOI = 20)obtained from the pre-experiment,and the expression of the reporter gene GFP green fluorescent protein was observed at 72 hours after infection to establish a stable cell line.q PCR and Western-blot were used to detect the GADD45 A gene silencing efficiency.The CCK-8 method was used to test the changes of B-CPAP cell proliferation after GADD45 A gene silencing.Flow cytometry was used to detect apoptosis and cycle change.A scramble group,a drug-added scramble group and a drug-transfected group were set up to detect the apoptosis rate and cell cycle change after the effect of 8 mg / ml Huaier for 48 hours.Construction of GADD45 A and ILF3-AS1 wild-type and mutant dual-luciferase plasmids.According to the instructions,the dual luciferase detection kit was used to detect the dual luciferase activity.Results: 1,Huaier extract inhibited human thyroid cancer cell proliferation.Huaier inhibited the proliferation of thyroid cancer cells B-CPAP at a concentration above 2 mg/ml,and inhibited the proliferation of C643 at a concentration above 4 mg/ml.It had a dose-and time-dependent manner.The results showed that the inhibition effect of B-CPAP and C643 by Huaier extract was obvious at 24 and 48 hours with the concentration of 8 mg/ml.The IC50 values of B-CPAP and C643 at 48 hours were 5.604 mg/ml and 8.330 mg/ml,respectively.After thyroid cancer cells B-CPAP and C643 were treated with 8mg/ml for 48 hours,the ratio of G0/G1 phase in normal B-CPAP group was 63.50% ± 0.96,the percentage of S phase was 21.04% ± 0.45,and the ratio of G2/M phase was 15.45% ± 1.35.The ratio of G0 /G1 phase in the experimental group was 72.67% ± 1.07,the percentage of S phase was 16.88% ± 1.91,and the ratio of G2/M phase was 9.59% ± 1.16.The G0 / G1 phase ratio in the C643 normal group was 47.77% ± 4.23,the S phase percentage was 39.44% ± 1.56,and the G2 /M phase ratio was 12.79% ± 2.81.The G0 / G1 phase ratio in the experimental group was 64.2% ± 3.34,the percentage in the S phase was 28.34% ± 3.88,and the G2 / M phase ratio was 7.45% ± 1.08.It can be seen that the proportion of cells in the G0 / G1 phase increased,and the proportion of cells in the S phase and G2 / M phase decreased.There was a significant difference between the experimental group and the control group(p <0.05).Flow cytometry showed that the apoptosis rate of the B-CPAP control group was 4.37% ± 0.54,8 mg/ml Huaier treating cells B-CPAP after 48 h,the apoptotic rate was 8.64% ± 1.6.C643 control group had an apoptotic rate of 5.37% ± 0.35,while the apoptosis rate in the experimental group was 10.47% ± 1.24.The difference between the experimental group and the control group was significant(p <0.05).The effect of Huaier extract on the migration and invasion ability of human thyroid cancer cells B-CPAP and C643 was observed by transwell assay.By counting the number of migrating cells,the B-CPAP cells were not statistically significant compared with the control group(305 ± 35.36 vs 315.8 ± 30.96,p = 0.6781),but the C643 cells in treat group significantly reduced the number of migrating cells than the control group(125.6 ± 17.36 vs 212.6 ± 14.89,p <0.01).Similarly,further counting of invading cells by matrigel transwell experiment,the number of B-CPAP cells in the treating group was not statistically significant comparing the control group(188.8 ± 17.08 vs 204.2 ± 20.62,p = 0.5871),while compared with the control group,the number of C643 cells was significantly reduced(94.8 ± 6.98 vs 161.81 ± 11.80,p <0.01).2,The concentration and purity of the total RNA extracted from each group of samples were confirmed by a spectrophotometer Nano Drop? ND-1000.The RNA quality inspection was qualified,and downstream experiments can be performed.16159 known transcripts and 54772 novel transcripts were obtained by high-throughput sequencing,and 2185 transcripts were up-regulated and 5794 transcripts were down-regulated.Of the 24,741 genes obtained by sequencing,5717 genes were eligible for differential expression.Compared with the control group,the expression of 869 lnc RNA,1 circ RNA and 1179 m RNA was up-regulated in the experimental group,while the expression of 3781 lnc RNA,1 circ RNA and 921 m RNA decreased.Data analysis DMBT1 was the most obvious down-regulated transcript with a fold change of 133.4784,and ZNF780 B expression was most significantly up-regulated with a fold change of 1565.641.According to GO analysis,270 genes were enriched in the up-regulated term and 171 genes were enriched in the down-regulated term.KEGG analysis results showed that a total of 47 pathways related were significantly enriched to DEG(p <0.05).It can be seen from the co-expression network diagram that these nc RNAs may be involved in tumor functions in multiple pathways,including immune response,cell cycle,angiogenesis and other biological processes.A ce RNA network was constructed to show the regulatory relationship between lnc RNA,mi RNA and m RNA.q RT-PCR verified the expression levels of the selected lnc RNA and m RNA,and the results showed that the expression trends of lnc RNA(SNHG7,MIR181A2 HG,ILF3-AS1,CTA-29F11.1)and m RNA(BBC3,CTSL,GADD45 A,DDIT3)were consistent(p < 0.05)with the results of high-throughput sequencing.3,The vector of GADD45 A gene was constructed and transfected into human thyroid papillary cell B-CPAP.A stable transfected cell line was established.q PCR and Western-blot were used to detect changes of GADD45 A m RNA and protein levels in B-CPAP cells after transfection.Compared with the blank control group B-CPAP-Scr group(Scr group),B-CPAP-sh-GADD45 A group(sh-RNA group)showed higher knockdown efficiency(p <0.05).The protein expression in sh-RNA group was significantly decreased(p <0.05).CCK-8 experiments were performed to detect the absorbance at 450 nm at 24 h,48h,and 72 h and the cell proliferation curve was drawed.Compared with the negative control group(Ctrl group)and Scr group,the cell proliferation ability of the sh-RNA group increased and showed in a time-dependent manner.Flow cytometry results showed that compared with the Scr group,the number of cells in G0 / G1 phase in the sh RNA group was reduced(47.77% ± 4.23 vs 67.20% ± 4.28,p <0.01),and the number of cells in the S phase was increased(39.44% ± 1.56 vs 22.49% ± 3.42,p <0.01),but there was no statistically significant change in cell number at G2 / M phase(12.79% ± 2.81 vs 11.30% ± 4.07,p = 0.6306).This indicated that down-regulating GADD45 A may promote B-CPAP cell proliferation by increasing S-phase cells.Compared with the Scr group,the apoptosis rate of sh-RNA group(3.97% ± 0.34 vs 5.56% ± 0.18,p <0.05)and survival rate(81.46% ± 0.86 vs 78.63% ± 1.08,p = 0.0495)were statistically significant(p <0.05).After the cells were treated with 8mg / ml Huaier for 48 hours,the results showed that when the expression of GADD45 A was down-regulated,compared with the Scr-treated group,the sh RNA-treated group significantly reduced the apoptosis induced by Huaier.The apoptotic rate in the sh RNA-treated group was 10.44% ± 1.67,and the apoptotic rate in the Scr-treated group was 16.45% ± 0.91.The two groups were statistically significant(p <0.01).The survival rate of the sh RNA-treated group was 74.28% ± 3.90,and the survival rate of the Scr-treated group was 55.05% ± 11.22.The two groups were statistically significant(p <0.05).The number of cells in the G0 / G1 phase in sh RNA-treated group was significantly lower than that in the Scr-treated group(69.11% ± 2.90 vs 76.34% ± 2.26,p <0.05),and the number of cells in the S phase increased between the two groups(22.07% ± 3.83 vs 14.91% ± 2.25,p = 0.049),which is statistically significant.There was no significant change in the number of cells in the G2 / M phase(8.90% ± 3.28 vs 8.74% ± 3.29,p = 0.9572).The results of the dual luciferase reporter gene experiment showed that there was statistic significance between GADD45 A Wt + mi R-301a-3p(+)group and GADD45 A Wt + mi R-301a-3p(+)NC group(p <0.01),while The luciferase activity between GADD45 A Mut + mi R-301a-3p(+)group and GADD45 A Mut + mi R-301a-3p(+)NC group was not statistically significant(p = 0.5352).The luciferase activity of ILF3-AS1 Wt + mi R-301a-3p(+)group was significantly lower than that of ILF3-AS1 Wt + mi R-301a-3p(+)NC group(p <0.05),while there was no statistically significance between ILF3-AS1 Mut + mi R-301a-3p(+)group and ILF3-AS1 Mut + mi R-301a-3p(+)NC group(p = 0.3694).Conclusions: Huaier extract inhibits the proliferation of human thyroid cancer cells in a time-and dose-dependent manner.Huaier extract can affect the human thyroid cell cycle and pause cells in the stage of G0-G1 phase.Huaier extract inhibits human thyroid cancer cell proliferation by inducing apoptosis.Huaier extract can inhibit the invasion and metastasis of human thyroid cancer cell C643.High-throughput sequencing qualified samples for RNA quality inspection.16159 known transcripts and 54772 new transcripts were obtained by high-throughput sequencing.5717 differentially expressed genes were screened,including protein-coding RNA,long-chain non-coding RNA,and circular RNA.These differentially expressed genes are involved in molecular functions,cellular components,biological processes,and so on,and they participate in a number of important signaling pathways.By constructing a co-expression network diagram and a ce RNA diagram,multiple lnc RNAs and m RNAs may participate in Huaier's effect on human thyroid cancer cells through endogenous competitive inhibition.After the GADD45 A gene was knocked down,the proliferation of thyroid cancer cells increased.The low expression of GADD45 A reduced the cells' number in the G0/G1 phase and lets more cells going into the synthetic phase.Down-regulated GADD45 A reduced apoptosis of thyroid cancer cells.Huaier inhibits thyroid cancer cell proliferation through regulating the expression of GADD45 A.Huaier inhibites thyroid cancer cell proliferation through ILF3-AS1 / hsa-mi R-301a-3p / GADD45 A ce RNA mechanism.