Hydrogen suppresses cervical cancerScientific Research

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Mechanism of hydrogen on cervical cancer suppression revealed by high‑throughput RNA sequencing


Xiaojun Liu, Zhijun Jin, Zhifeng Wang,  Jianhong Dang, Guoqiang Chen, Lingling Li, Jing Wang, Jinghai Gao, Jing Chu


Cervical cancer is considered one of the diseases with the highest mortality rate in women and limited treatment options. Hydrogen (H2) inhalation has been reported to have many tumor-inhibiting effects, but the exact mechanism remains unclear. In the present study, H2-treated HeLa cervical cancer cells and HaCaT keratinocytes and H2-inhaled HeLa xenograft mouse models were established. TUNEL, Cell Counting Kit-8, and Ki67 staining assays were used to detect apoptosis and proliferation. Oxidative stress was determined based on the levels of reactive oxygen species, malondialdehyde, and superoxide dismutase. Tumor growth was recorded every 3 days, and resected tumors were stained with hematoxylin and eosin. High-throughput RNA sequencing and subsequent Gene Ontology (GO) enrichment analysis was performed in HeLa-treated and untreated HeLa cells. The expression of hypoxia-inducible factor (HIF)-1α and NF-κB p65 was verified by western blot, immunohistochemistry and reverse transcription quantitative PCR. The results showed that in H2-treated HeLa cells, the rate of apoptosis was increased, and cell proliferation and oxidative stress were decreased, but not in HaCaT cells. Likewise, decreased tumor growth and cell proliferation and increased apoptosis were observed in H2-treated HeLa tumors.RNA-sequencing and GO analysis indicated that downregulation of HIF1A (HIF-1α mRNA) and RelA (NF-κB-p65) levels and reduced NF-κB signaling were associated with the antitumor activity of H2. Finally, decreased expression of HIF-1α and NF-κB p65 was observed at both transcriptional and translational levels in H2-treated HeLa cells and HeLa-derived tumors. In conclusion, this study demonstrates a novel mechanism of H2 against cervical cancer and may serve as a potential therapeutic target in clinical practice.


H2 intervention reduces HeLa cell proliferation and promotes apoptosis and oxidative stress in a selective and dose-dependent manner
First, the effect of H2 treatment on HeLa cells was examined in vitro. As shown in Figure 1A and B, the proportion of TUNEL-positive cells was significantly increased in all H2-treated Hela cells compared with the control group (P<0.01). Treatment with 66.7% H2 significantly increased the rate of apoptosis. Importantly, 5 µg/mL cisplatin (IC50 = 12 µg/mL) treatment induced an increase in the rate of apoptosis in HeLa cells consistent with the effect of 66.7% H2 treatment. Notably, no significant difference in apoptosis rate was observed in HaCaT cells treated with or without hydrogen. The CCK-8 assay showed a gradual decrease in cell proliferation after treatment with 33.3% H2 and 66.7% H2, and further decreased proliferation of HeLa cells but not HaCaT cells over time (Fig. 1C). Furthermore, the effect of time was significantly associated with cell proliferation in HeLa cells and HaCaT cells. These results suggest that H2 has a specific concentration-dependent inhibitory effect on HeLa cells but not non-tumor HaCaT cells. Therefore, H2 was used for further testing at a concentration of 66.7%.

Figure 1. – Decreased cell proliferation, and increased cell death and oxidative stress level in H2-treated HeLa cervical cancer cells. (A) TUNEL staining of HeLa and HaCaT cells in the control, 33% H2 and 66.7% H2 groups. Furthermore, 5 µg/ml cisplatin treatment was used as a positive control (scale bar, 200 µm). (B) Statistical results of TUNEL-positive HeLa and HaCaT cells in the control, 33% H2 and 66.7% H2 groups (n=4 per group). **P<0.01, ns indicates no significant difference. (C) Cell proliferation curves of the control, 33% H2 and 66.7% H2 groups in HeLa and HaCaT cells during different time periods (n=3 per group). *P<0.05, **P<0.01 vs. indicated group, and #P<0.05, ##P<0.01 vs. control group at day 5, ns indicates no significant difference. (D) Fluorescent staining of ROS in control and 66.7% H2-treated HeLa cells (scale bar, 75 µm). (E) Statistical results of ROS area (%) in the control and 66.7% H2 groups (n=4 per group). **P<0.01. Statistical results of (F) malondialdehyde (MDA) and (G) superoxide dismutase (SOD) levels in the control and 66.7% H2 treatment groups (n=5 per group). *P<0.05, **P<0.01. H2, hydrogen; ROS, reactive oxygen species.

Aging mitochondria produce ROS due to overproduction of free radicals and reduced antioxidant defenses (26,27). The present study showed that ROS levels were significantly reduced in H2-treated HeLa cells compared with controls (P<0.01; Figure 1D and E), which is consistent with the previously reported antioxidative stress activity of H2 gas. 10). Furthermore, MDA levels, an indicator of lipid peroxidation, were also decreased in H2-treated HeLa cells (Fig. 1F), whereas in the H2 group, by contrast, elevated SOD levels, which are antioxidants, were observed An important representative of enzymes. Compared with the control group (P<0.01; Fig. 1G). These results suggest that H2 intervention induces HeLa cell apoptosis, inhibits cell proliferation, and reduces the level of oxidative stress.

Reduced tumor growth is observed in a H2-induced xenograft in vivo model

A total of 1 x 10 7 HeLa cells were injected subcutaneously in the left axilla of nude mice, and tumor volumes were recorded every 3 days starting from 7 days post-injection (Figure 2A). On days 31 and 34, the tumor volume of mice in the high H2 environment was significantly smaller than that of the control group (P<0.05 on day 31 and P<0.01 on day 34), consistent with the change in tumor weight on day 34 (P<0.01). 0.01; Figure 2B and C).

Figure 2. – H2 intervention reduces tumor growth in tumor-bearing mice. (A) Tumor volume curves from 7 to 34 days after cell injection in the control and H2 treatment tumor-bearing groups (n=9 per group). *P<0.05, **P<0.01. (B) Images of the excised tumors in control and H2 tumor-bearing mice. (C) Statistical results of tumor weight (units, g) in the control and H2 groups (n=9 per group). **P<0.01. (D) Hematoxylin and eosin staining (H&E) and immunohistochemistry for TUNEL and Ki67 staining in control tumor sections or sections after treatment with H2 (scale bar, 200 or 400 µm). Black arrows indicate nucleus fragmentation. Percentage of (E) TUNEL-positive cells or (F) Ki67-positive cells in control and H2-treated tumors (n=5 per group). **P<0.01. H2, hydrogen.

H&E staining showed increased tumor cell mortality in the H2 xenograft group with cell swelling and nuclear fragmentation and decay (Figure 2D; black arrows). Furthermore, H2 treatment significantly increased the apoptotic rate and decreased cell proliferation in HeLa cells (Figure 2E and F). Taken together, these data suggest that a high H2 environment suppresses tumor growth in tumor-bearing mice.

Bulk RNA sequencing of H2-treated HeLa cells and control cells

To investigate the underlying mechanism by which H2 inhibits tumor cell growth, whole-genome sequencing was performed (Figure 3). As shown in Figure S1A-C, 11 up-regulated and 16 down-regulated circular RNAs (circRNAs) were determined in H2-treated HeLa cells by the HiSeq4000 platform. In addition, 2 long non-coding RNAs (lncRNAs) were found to be up-regulated in H2-treated HeLa cells, while 6 lncRNAs were down-regulated (Fig. S2A-C). A total of 12 hsa microRNAs (miRNAs or miRs) were found to be up-regulated and 2 miRNAs were down-regulated, including hsa-miR-431-5p and hsa-miR-762 (Fig. S3A-C). Importantly, mRNA sequencing revealed that 10 mRNAs were up-regulated and 4 mRNAs were down-regulated in H2-treated HeLa cells, including HIF1A, miR23B, miR4651, and RelA (Figure 4A-C).miR23B and miR4651 are noncoding RNAs, while HIF1A and RelA are genes encoding the p65 subunit of HIF-1α and NF-κB, respectively. These results indicated that HIF-1α and NF-κB p65 subunit mRNA levels were reduced in H2-treated HeLa cells compared to controls.

Figure 3. – Scheme of whole-genome (RNA) sequencing. RNA sequencing libraries were generated from 4 mg total RNA samples isolated from control or hydrogen-treated HeLa cells using TruSeq Stranded Total RNA Library Prep kit. Sequencing was carried out on Illumina NovaSeq 6000 and HiSeq X Ten platforms. After quality control, the clean reads of circular RNA, long non-coding RNA, microRNA and mRNA were then mapped to the human genome by HISAT2 software, and the analysis was performed using an DESeq2 software. Differential expression analysis was conducted with the DESeq2 platform (n=3 per group). Gene Ontology enrichment analysis of mRNA was performed using DAVID bioinformatics tool.

Figure 4. – Highly deregulated mRNAs in HeLa cells with or without H2 intervention. (A) A mRNA heatmap was generated based on the 10 maximum and 10 minimum FC values (n=3 per group). (B) Volcano plot for deregulated mRNAs [log2(FC) > 1 or < −1, adjusted P<0.05] between H2 and control groups. The area above the gray horizontal solid line indicates P<0.05. (C) Upregulated mRNAs (red) and downregulated mRNAs (aqua blue) are listed. H2, hydrogen; FC, fold-change.

GO enrichment analysis for HeLa cells with H2 intervention

To further confirm the above gene regulation in H2-treated HeLa cells, GO enrichment analysis was performed. As shown in Figure 5A, biological process (BP), cellular component (CC), and molecular function (MF) were analyzed. “Translation” and “NF-κB signaling” are the two most important processes in BP, and gene regulation mainly occurs in the nucleus and non-membrane-bound organelles. MF analysis showed that “DNA and RNA binding” predominated. The scatter plot of GO enrichment further indicated that the processes of “NF-κB signaling”, “protein transport” and “motility of multicellular organisms” were enriched in H2-treated HeLa cells (Fig. 5B). These results suggest that downregulation of NF-κB signaling may be an important target of H2 intervention in HeLa cells.

Figure 5. – GO enrichment analysis of H2-treated HeLa cells. (A) GO enrichment of BP, CC and MF analysis between H2-treated HeLa cells and controls. (B) GO enrichment scatterplot for BP, CC and MF progress between HeLa cells with or without H2 treatment. GO, Gene Ontology; BP, biological process, CC, cellular component; MF, molecular function; H2, hydrogen.

HIF-1α and NF-κB p65 expression levels are downregulated in H2-treated HeLa cells in vivo and in vitro.

Since the p65 subunit is the classically active type of the NF-κB family (28), this study validated the expression levels of NF-κB p65 and HIF-1α in HeLa cells. As expected, Western blot analysis indicated that HIF-1α and NF-κB p65 protein levels were significantly reduced in H2-treated HeLa cells compared with control HeLa cells (Fig. 6A-C). RT-qPCR analysis revealed that HIF-1α and NF-κB p65 (HIF1A and RelA) mRNA expression levels were increased in HeLa cells (Figure 6D and E).

Figure 6. – H2 treatment downregulates both the gene and post-transcriptional levels of HIF-1α and NF-κB in HeLa cells and HeLa tumors. (A) Western blotting of HIF-1α and NF-κB in control and H2-treated HeLa cells. Cytoplasmic (GAPDH) and nuclear (lamin B1) loading controls were used. (B and C) Statistical results of (B) HIF-1α and (C) NF-κB protein expression levels in HeLa cells treated with H2 or not (n=4 per group). **P<0.01. RT-qPCR analysis for (D) HIF-1α mRNA (HIF1A) and (E) NF-κB mRNA (RelA) levels in control and H2-treated HeLa cells (n=4 per group). **P<0.01, ***P<0.001. (F) Immunohistochemical analysis for determining the HIF-1α and NF-κB expression in tumor-bearing mice after H2 intervention and their controls (scale bar, 250 µm). Percentage of positive area of (G) HIF-1α and (H) NF-κB in an in vivo xenograft model with or without H2 intervention (n=9 per group). **P<0.01, ***P<0.001. (I and J) RT-qPCR analysis for (I) HIF1A and (J) RelA expression in vivo (n=9 per group). *P<0.05, ***P<0.001. H2, hydrogen; HIF, hypoxia inducible factor; RT-qPCR, reverse transcription-quantitative PCR.

The levels of HIF-1α and NF-κB p65 were then determined in HeLa cell-derived tumors. As shown in Figure 6F-H, IHC showed that the expression of HIF-1α and NF-κB p65 was significantly reduced in H2-treated HeLa tumors compared with controls. In addition, mRNA levels were also examined in vivo and showed that HIF1A and RelA expression was significantly reduced in H2-treated tumors compared to control tumors (Figure 6I and J). These results indicated that the expression levels of HIF-1α and NF-κB p65 were downregulated in both HeLa cells and HeLa tumors.



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