Low temperature upregulating HSP70 expression to mitigate the paclitaxel-induced damages in NHEK cell

Cell culture and experimental design

Normal human epidermal keratinocytes (NHEK) were purchased from Shanghai Guandao Biological Engineering Co. Ltd (Shanghai, China). For all the experiments, NHEKs were used at passages 1∼10 to ensure maximum proliferation capacity (Kleszczyński, Zillikens & Fischer, 2016). The NHEKs were cultured in a DMEM culture containing 10% fetal bovine serum. The control group was cultured at 37 °C and the low temperature group was cultured at 22 °C for 2 h (McCarthy et al., 2013). The set temperature of cell incubator is changed to 22 °C. Taxol powder (Shanghai Aladdin Biological Technology Shanghai City, China) was dissolved in 1:1 castor oil and ethanol to make 6 µg/ml mother liquor was stored at −20 °C, and paclitaxel mother liquor was diluted with stroke-physiological saline solution. NHEK cells were treated with paclitaxel for 2 h.

RNA extraction

A TRIzol kit (Invitrogen, Waltham, MA, USA) was used to purify the RNA from NHEK cells (Amirouche et al., 2021). one mL TRIzol was added to the six well plates with 5 × 10⁶ cells. The plate was shaken for 5 min. Then 250 µL trichloromethane was added. The plate was allowed to stand for 15 min and centrifuged for 15 min at 4 °C. The supernatant was removed and 500 µL of Isopropyl alcohol was added. Handling the other plate with the same procedure as above.

MRNA library preparation and sequencing

Genomic DNA was removed by using DNase I (Takara, Shiga, Japan), and RNA quality was determined by 2100 Bioanalyser. mRNA enriched with poly-A tail was first enriched from 1ug of total RNA by oligo (dT) beads and then fragmented with fragmentation buffer. Finally, double stranded cDNA was synthesized using Super Script double-stranded cDNA synthesis Kit (Invitrogen, Waltham, MA, USA) with random hexamer primers. After quantified by TBS380, the double terminal RNA-seq sequencing library was sequenced by using Illumina HiSeq xten sequencer (Zhou et al., 2013), which was conducted by Meiji Biological Co., Ltd (Toyko, Japan).

RNA-seq data analysis

FastQC (v0.11.9) was used for quality control of sequencing data. Adapters and reads with an average quality score of less than 20 after trimming were discarded by Trimmomatic (v0.39). Feature Counts (v1.6.5) was used for gene quantification (Zimmerli et al., 2020). The RNA-seq data described here are accessible via BioProject accession numbers PRJNA872966 (https://www.ncbi.nlm.nih.gov/bioproject/872966). The statistical power of this experimental design was calculated in cluster Profiler Power is q value ≤ 0.05.

Differential gene expression and functional annotation analysis

DEGs between two different samples were identified by calculating the expression level of each transcript according to the transcripts per million reads (TPM) method. The differential expression analysis is to use the Q value ≤ 0.05, — log2fc —>1 as an expression gene that is considered to be significantly different. The Kyoto Encyclopedia of genes and genomes (KEGG) database was used to enrich the biological functions of DEGs. GO functional enrichment and KEGG pathway analysis were performed by goatools (https://github.com/tanghaibao/Goatools) and kobas (http://kobas.cbi.pku.edu.cn/home.do). P-value ≤ 0.05 and FDR ≤ 0.05 was considered as statistically significant (Lanceta et al., 2021).

Reactive oxygen species (ROS) measurement

The oxidative fluorescent dye dihydroethidium (20 µM DHE; Sigma-Aldrich, St. Louis, USA) was used to assess ROS production in the NHEK cells according to the manufacturer’s instructions. The 10⁴ cells were planted in 96 well plates overnight, paclitaxel solution was added, and incubated at 37 °C. A DHE working solution was added and incubated for 20 min. The plate was washed three times with a serum-free cell culture solution. The fluorescence spectrophotometer (Biotek Synergy H1; Agilent, Santa Clara, CA, USA) was used to detect fluorescence intensity with 488 nm excitation wavelength and 525 nm emission wavelength.

Measurement of mitochondrial membrane potential (MMP)

The mitochondrial membrane potential detection kit (Solarbio, Beijing, China) uses JC-1 as a fluorescent probe to quickly and sensitively detect the changes of MMP. The cells were planked in 96 well plates overnight, paclitaxel solution was added, and incubated at 37 °C. 100 µL JC-1 working solution was added after cleaning. This was followed by incubation for 20 min at 37 °C. The plate was washed twice with JC-1 dye buffer, and 100 µL culture medium was added. The fluorescence spectrophotometer was used to detect fluorescence intensity with 490 nm excitation wavelength and 530 nm emission wavelength.

Enzyme-linked immunosorbent assay (ELISA)

The HSP70 ELISA kit was purchased from Shanghai Frankel Industrial Co., Ltd. (Shanghai, China). Each group had 10⁴ cells in a centrifuge tube. The extract was added to the tube. The cells were broken by ultrasound. The tube was centrifuged at 8,000 g 4 °C for 10 min, take the supernatant. 40 µL diluent and 10 µl supernatant were added into the enzyme labeled coating plate, following 30 min of incubation at 37 °C. The plate was washed five times. This was followed by incubation for 30 min at 37 °C using enzyme labeled reagent. Following the same washing procedure as above, the color developing solutions A and B were applied for 10 min. The absorbance at 450 nm was measured following the application of the stopping solution.

Cell survival under the synergistic effect of concentrations and cooling temperatures

In order to determine the activity, the results of the cell activity experiment are necessary, the relationship between the activity and the concentration of the drug (Ribba et al., 2005): ${\displaystyle S=a+\frac{1}{b+k_{d}C}.}$

For each temperature group, the parameters a, b and k_d were fitted respectively. For each parameter, the function related to temperature is assumed and fitted (Ribba et al., 2005). The fitting result was shown in Fig. 1A. Although only four temperature data points were used, the fitting was meaningful. Due to the limited number of data points, the fitting results must be used carefully.

Figure 1A showed that cell survival was dependent on the concentration of paclitaxel. The higher the concentration of paclitaxel, the lower the cell survival rate. The lower the temperature, the more obvious the protective effect on cells. These results showed that low temperature had a significant protective effect on cells treated with paclitaxel. As shown in Fig. 1B, the isoactivity lines ranged from high concentration at low temperature to low concentration at higher temperature. With the temperature increased, the isoactivity curve showed an S-shaped curve. Caution must be exercised in interpreting the results outside the temperature range of the graph (10 °C < T <  37 °C). It had not been verified experimentally at a temperature below 10 °C. It was expected that the isoactivity line would increase when the temperature was lower than 10 °C.

Analysis of differential gene expression

There were three high-quality total transcriptome sequencing samples in each group tested in this study. HCA analysis of total transcriptome sequencing data showed that samples from different groups were well separated and had good biological repeatability. Principal component analysis (PCA) provided an overall view of gene expression profile analysis and showed significant differences between all groups.

In the cell samples of the control group and low temperature treatment group, a total of 104 DEGs responsed to low temperature treatment were observed, 59 genes were up-regulated and 45 genes were downregulated in NHEK cells (Figs. 2A and 2E). This indicated that the cells responded to low temperature treatment and a certain amount of DEGs. After low temperature treatment, the expression of HSPA8, HSPA1A, HSPA1B, PLK2 and TXNIP changed significantly (Table S1). In addition, we used DEG for GO analysis and KEGG pathway analysis, as well as gene function enrichment.

GO and KEGG pathway analysis of the DEGs

We performed GO enrichment analysis of DEGs to identify biological processes associated with low temperature. The significant (FDR < 0.05) GO enrichment of DEG in NHEK cells was shown in Figs. 2B–2D. These GO terms included biological process (BP), cellular component (CC) and molecular function (MF) terms.

A total of 41 significant GO terms were obtained, 19 terms were involved in biological processes, 14 and eight terms belonged to cellular components and molecular functions. The main BP included cellular process (n = 59), biological regulation (n = 53), response to stimulus (n = 45), developmental process (n = 28) and metabolic process (n = 27). The results of GO enrichment analysis showed that under the condition of low temperature treatment, DEGs in the cells were mainly involved in response to stress and stimulus, which was consistent with the situation of low temperature treatment. HSPA8, HSPA1A, HSPA1B, PLK2 and TXNIP all responded to low temperature and participated in response to stimulus Table S2), which was consistent with the genes screened by volcano plot.

KEGG pathway analysis was used to characterize the functional consequences of changes in cell gene expression after low temperature treatment. KEGG pathway analysis showed that DEGs were mainly involved in the regulation of MAPK and TNF signaling pathway with low temperature treatment. HSPA8, HSPA1A and HSPA1B, which were all involved in the MAPK signaling pathway Table S3), proving that HSP70 had an impact on MAPK signaling pathway.

The effect of low temperature on HSP70, MMP and ROS of NHEK

HSP70 has the function of protecting the cells. In order to understand the effect of hypothermia on cellular HSP70, the concentration of HSP70 in the NHEK cells was tested using the HSP70 ELISA kit. The HSP70 level did not change over time in the control group. In Fig. 3A, the concentration of HSP70 was related to the time of low temperature treatment, initially increasing and then decreasing. When the cells were stimulated by low temperature, the HSP70 level in cells increased rapidly and resisted external stimuli. As the stress time lengthens, the cells gradually adapted to the cold, and the expression level of HSP70 decreased. With the extension of stress time, cells would gradually have cold adaptation and the expression level of HSP70 decreased. The concentrations in the control and 2 h groups were 88.351 ng/L and 264.298 ng/L respectively. After low temperature treatment for 2 h, the concentration of HSP70 was about 3 times that of the control group. When cells were stimulated by low temperature, HSP70 in cells increased rapidly, playing a role in resisting external stimuli.

The destruction of MMP could indicate mitochondrial dysfunction, which was the key to the pathway of mitochondrial death induced by chemotherapeutic drugs. To clarify the effect of low temperature on MMP, the MMP of NHEK cells was detected using the ELISA kit. Fig. 3B showed that after paclitaxel chemotherapy, the MMP increased significantly and the mitochondria produced obvious dysfunction. After the low-temperature treatment, the MMP was decreased significantly, but was still higher than that in the control group. The results showed that hypothermia could reduce the MMP and alleviate the mitochondrial dysfunction.

ROS can cause oxidative modification of cellular components such as proteins, nucleic acids and lipids, which can lead to programmed cell death. To prove the effect of low temperature on ROS, the ROS of cells was detected. Low temperature could not be significantly altered in the control group, but paclitaxel chemotherapy resulted in a significant increase in ROS levels. Low temperature treatment significantly decreased the level of ROS, as shown in Fig. 3C.

The effect of HSP70 inducer and inhibitor on cell survival rate and HSP70 of NHEK

In the paclitaxel treatment group at 37 °C, the HSP70 agonist TRC051384 was added, and the working concentration was 12.5 µM. The results showed that the cell survival rate was significantly improved, which was basically the same as that of the 22 °C paclitaxel treatment group. The HSP70 inhibitor KNK437 was added in paclitaxel treatment group at 22 °C, and the working concentration was 100 µM. The cell survival rate decreased significantly, reaching the same level as that of the 37 °C paclitaxel treatment group, as shown in Fig. 4A. The above results revealed that HSP70 was the key to the protection of cells by low temperature, but it still needed further verification. The concentration of HSP70 in the cell experimental group was evaluated by HSP70 detection kit. As shown in Fig. 4B, compared with 37 °C group, the concentration of HSP70 in 37 °C and HSP70 inducer group and 22 °C group increased significantly, which proved that both inducer and low temperature could promote the cell synthesis of HSP70. When HSP70 inhibitors were added to the 22 °C group, the concentration of HSP70 was slightly changed compared with that of the 37 °C group, but there was no significant change, which proved that HSP70 inhibitors could significantly inhibit the synthesis of HSP70 in cells. The synthesis of HSP70 was also inhibited even when the cells were under low temperature. HSP70 is a key factor of low temperature therapy.