S hi-Cheng Lin,Dian-Na Liu,Xiang-NanZhou,Yao-Xue Zhuang, Tian-Yu Liang, Xiao-FanWang, Kai-Wen Hu, Jing-Yi Sun, Quan-Wang Li?

Beijing University of Chinese Medicine, Beijing 100029, China; Department of Oncology, Dongfang Hospital, Beijing University of Chinese Medicine,Beijing 100078, China

ABSTRACT Objective: To study the regulatory effect of cryoablation on MAPK/ERK signaling pathway in mice with lung adenocarcinoma. Methods: Lewis mouse lung adenocarcinoma cell line was used to establish subcutaneous transplanted tumor model in C57BL / 6 mice. Ten mice were randomly divided into two groups: sham operation group and cryoablation group, with 5 mice in each group. The cryoablation group was treated with double circulation-rewarming ablation, and the sham operation group was treated with incision and suture at the transplanted tumor. The tumor tissues were taken 14 days after operation. Detect the effect of cryoablation on MAPK/ERK pathway related proteins by Western blot, such as KRAS, RAF1, MEK1,ERK1/2, P-RAF1, P-MEK1, P-ERK1/2. The expression of KRAS gene was further verified by qRt-PCR. Results: Compared with the sham operation group, the phosphorylated proteins P-RAF1, P-MEK1 and P-ERK1/2 in tumor tissue after cryoablation were decreased (P＜0.05 ), and the key molecule KRAS in MAPK/ERK pathway was decreased in protein and gene expression (P＜ 0.05 ). Conclusion: Cryoablation can negatively regulate MAPK / ERK signaling pathway by down-regulating KRas expression.

Keywords:Cryoablation MAPK/ERK pathway Lung adenocarcinoma Mice Mechanism

1. Introduction

According to the latest global cancer burden data in 2020, lung cancer is the most lethal malignant tumor, with the death rate of 1.8 million in 2020, far exceeding other malignant tumors[1]. Surgical resection is the preferred method for early lung cancer. However,the onset of lung cancer is hidden, and most of the lung cancer is late at the time of discovery. In recent years, cryoablation as a new targeted therapy has been proved to be effective in a variety of tumors, including lung cancer. In addition to physical direct tumor killing effect, cryoablation therapy also can reduce tumor invasion,tumor metastasis and recurrence rate, and activate anti-tumor immunity. However, there is still a lack of further research on its specific mechanism and molecular level changes. Mitogen-activated protein kinase ( MAPK ) is an important downstream of epidermal growth factor receptor ( EGFR ), which receives extracellular growth and proliferation signals and transmits them to the nucleus through MAPK / ERK signaling pathway, leading to cell growth,survival, repair and proliferation[2]. Studies have shown that MAPK/ ERK signal is abnormally activated in renal cell carcinoma, liver cancer, lung cancer and other malignant tumors [3-5]. In this study,the effect of cryoablation on MAPK / ERK signaling pathway was investigated by establishing subcutaneous xenograft tumor model in Lewis lung adenocarcinoma mice.

2. Information and methodology

2.1 Experimental animals and cells

10 male C57BL / 6 mice, aged 6-8 weeks, weighing ( 20 ± 2 )g, were purchased from Beijing Sbeford Biotechnology Co., Ltd.,license number : SCXK (Beijing) 2019-0010. All the experimental animals in this experiment were fed in the experimental animal center of Oriental Hospital of Beijing University of Traditional Chinese Medicine, SPF breeding environment. Animal experiments were approved by the Experimental Animal Ethics Committee of Oriental Hospital of Beijing University of Traditional Chinese Medicine, batch number was 202020. Lewis cell line of mouse lung cancer was purchased from Jiangsu Kaiji Biotechnology Co., Ltd.

2.2 Main experimental materials and equipment

DMEM high glucose medium purchased from Hyclone ; fetal bovine serum purchased from Merck ; trypsin and Streptomycin mixture purchased from GIBCO ;RIPA (strong) pyrolysis liquid purchased from Biyuntian Company ; BCA protein quantitative kit purchased from Jiangsu Kaiji Biotechnology Co., Ltd. ; reveraid First Strand cDNA Synthesis Kit purchased from Semerfeld ;sYBR qPCR SuperMix Plus kit was purchased from Novoprotein ;nucleoZol RNA extraction reagent purchased from Gene Company; kRAS, RAF1, ERK1 / 2, P-ERK1 / 2 polyclonal antibody were purchased from proteintech company, P-RAF1, P-MEK1 were purchased from Abcam company, MEK1 polyclonal antibody were purchased from santa cruz company, goat anti-rabbit (mouse ) IgG secondary antibody were purchased from proteintech company; argon-helium cryosurgery system and 1.2 mm cryo scalpel(Endocare,USA) ; fluorescence quantitative PCR instrument (ABI 7300, USA), small vertical electrophoresis transfer system (BIORAD 1658033, USA ).

2.3 Cell culture and establishment of animal models

Lewis cells were cultured in DMEM high glucose medium containing 10 % fetal bovine serum and 1 % double antibody at 37℃. The cells were cultured in 5 % CO2 incubator, and the medium was changed every 2 days. When the cell adherent area reached 70%, the cells were subcultured.

After one week of adaptive feeding, Lewis cells in logarithmic phase were digested with trypsin and resuspended. The cells containing 2 × 106cells in 0.2 mL cell suspension of each mouse were inoculated subcutaneously in the medial right leg of the mouse.After one week, the tumor diameter was measured by vernier caliper and the experiment was started.

2.4 Mouse freezing scheme and grouping

The successfully modeled mice were randomly divided into sham operation group and cryoablation group, with 5 mice in each group.Cryoablation group mice anesthesia, fixation, skin preparation,disinfection, skin incision, exposure of tumor, 1.2 mm cryoablation scalpel inserted into the tumor center. The dual-cycle freezing method was adopted : the temperature of the blade tip was rapidly reduced to -120℃ for 10s by releasing argon, and then the helium was released to rewarm to 15℃, and the above freezing ablation cycle was repeated once. The mice in the sham operation group were anesthetized and fixed, the skin was cut, the tumor was exposed and sutured directly. The mice in the two groups were fed after anesthesia and resuscitation, and the mice were sacrificed at the same time after 14 days.

2.5 Western blot detection of MAPK/ERK pathway related protein expression in tumor tissues

Take 50mg of the stripped mouse tumor tissue, cut the tissue into a glass homogenizer, add 500ul RIPA lysis solution, repeatedly grind to fully lyse the tissue protein, centrifuge the supernatant to obtain the total protein, and use the BCA protein concentration determination method to detect Sample protein content, and adjust the total protein concentration of all samples to 3ug/ul. The sample protein is denatured by boiling at 100℃ for 8min, and then subjected to SDS-PAGE electrophoresis. Take 30ug sample and load the sample, 100V constant voltage electrophoresis to the bottom of the strip, 100mA constant current electrophoresis for 100min, remove the converted PVDF membrane and add TBST to rinse for 5min×3 times, 10% skimmed milk powder blocked the reaction sites of unrelated proteins on the PVDF membrane for 1 h, rinsed with TBST for 5 min × 3 times, and added rabbit (mouse) polyclonal protein primary antibody working solution KRAS (1:5000), RAF1( 1:3000), MEK1 (1:200), ERK1/2 (1:3000), P-RAF1 (1:3000),P-MEK1 (1:3000), P-ERK1/2 (1:3000), Incubate with GAPDH(1:50 000) at room temperature for 1.5h, rinse with TBST for 5min×3 times, add goat anti-rabbit (mouse) secondary antibody (1:10 000), incubate at room temperature for 1h, rinse with TBST for 5min×3 times, use hypersensitivity Incubate the PVDF membrane with ECL luminescent solution, adjust the exposure conditions for development, save the picture, and use Imagel J software to analyze the band gray value.

2.6 qRt-PCR method to detect KRas gene expression in tumor tissues

According to the manufacturer’s instructions, take 50mg of tumor tissue and add 500ul of NucleoZol RNA extraction reagent,Grind thoroughly with a glass homogenizer, add 200ul of sterile enzymefree water, vortex for 15s and then stand at room temperature for 5min, centrifuge at 12000g for 15min, take the supernatant into another centrifuge tube, add 500ul isopropanol, and let stand at room temperature for 10min Centrifuge at 12000g for 10 minutes and discard the supernatant. At this time, RNA sinks to the bottom of the tube. Add 500ul of 75% ethanol and centrifuge at 8000g for 3 minutes to wash the RNA. Use a pipette to remove the upper layer of ethanol. Add 75% ethanol again to dry the RNA in the tube. Add 50ul sterile enzyme-free water to dissolve the extracted RNA. Measure OD260/280 by UV spectrophotometer,calculate the total RNA concentration of each group, refer to the product manual using Oligo (dT) 18 primer for reverse transcription to synthesize first-strand cDNA, reaction time: 42℃ 60min, 70℃ 5min. Use SYBR Green as the fluorescent dye,β-actin mRNA as the internal control, KRas primer upstream:5′-TGTGGACGAATATGATCCAACA-3′, downstream: 5′-GCAAATACACAAAGAAAGCCCT-3′; β-actin primer upstream:5′-CTACCTCATGAAGATCCTGACC-3′ , Downstream 5'-CACAGCTTCTCTTTGATGTCAC-3'. Use ABI 7300 qRt-PCR system to detect the expression of related mRNA. The reaction program is: 95℃ for 1min, 95℃ for 20s, 60℃ for 1min, 40 cycles in total. The fluorescence signal is recorded at the last step at 60℃, and the dissolution curve is set according to the program. The relative expression of KRas mRNA was calculated according to 2-ΔΔCt.

2.7 Statistical analysis

Statistical data use SPSS 20.0, graph construction use Graphpad Prism8.0, normal distribution measurement data are expressed as mean ± standard deviation (±s), two sets of samples with normal distribution and uniform variance are tested by two independent samples t-test, P＜0.05 indicates that the difference is statistically signifciant.

3. Results

3.1 The regulation of cryoablation on MAPK/ERK pathway related proteins

Western blot results showed that compared with the sham operation group, the expression of KRAS protein in the cryoablation group was reduced (P＜0.05), the expression of non-phosphorylated proteins RAF1, MEK1, and ERK1/2 was not statistically different.Phosphorylated proteins P-RAF1, P -MEK1 and P-ERK1/2 expression are all inhibited (P＜0.05), Figure 1A, B, Table 1.

Table1 Comparison of MAPK / ERK pathway related protein expression in tumor of mice between two groups

Figure 1 Relative expression of MAPK / ERK pathway related proteins in tumor tissues of mice in two groups

3.2 The effect of cryoablation on the expression of KRAS mRNA

The results of qRt-PCR showed that compared with mice in the sham-operated group, the expression of KRas mRNA in the tumor tissues of mouse lung adenocarcinoma tissues was reduced after cryoablation (1.000±0.073 vs. 0.645±0.053) (t=6.825, P＜0.05) )Figure 2.

Figure 2 Expression of KRas mRNA in tumor tissue of mice in two groups

4. Discussion

Lung cancer still ranks first in the incidence and mortality of malignant tumors in my country [6]. For most advanced patients who have lost the chance of surgical resection, cryoablation treatment has the characteristics of small side effects, local minimally invasive,safe and effective, which can better control the progression of lung cancer, improve the quality of life of patients, reduce pain and prolong survival. Cryo-ablation technology usually uses media such as argon or liquid nitrogen to rapidly form ice balls at -140℃ to -160℃ in the lesion, and quickly form ice crystals inside and outside the tumor cells. The ice crystals ablate during the process of gradual rewarming to 30℃, causing tumor cells to rupture And the physical and chemical denaturation of intracellular proteins, causing mechanical damage to tumor tissues [7]. In recent years, studies have found that tumor cell necrosis products can inhibit the progression of residual tumors and may affect distant metastases [8-10]. The effect of cryoablation on tumors may involve more complex molecular mechanisms in addition to direct mechanical damage. . This study explored the effect of cryoablation on MAPK/ERK, a classic tumor signaling pathway, through animal experiments, in order to provide clinical guidance for the combination therapy of cryoablation and molecular targeted drugs.

The occurrence, development and metastasis of lung cancer is a multi-stage and multi-step process. In each stage of tumor progression, abnormal activation of cell signaling pathways plays an important role[11]. At present, the most researched pathways for lung cancer include MAPK pathway, PI3K/AKT pathway, DLL4-Notch1 pathway, TGFβ/Smad pathway, etc. Among them, MAPK signaling pathway is the main pathway of intracellular signal transduction and plays an important role in the progression of lung cancer. . The MAPK pathway has 4 main branch routes: ERK, JNK, p38/MAPK and ERK5. Among them, MAPK/ERK is mainly involved in the regulation of cell growth, differentiation, apoptosis, and migration.The upstream signal is the famous Ras and Raf proteins. Ras gene is a proto-oncogene and one of the earliest human oncogenes with the highest mutation rate. . KRas gene mutation is one of the most common gene mutations in lung cancer. About 32% of non-small cell lung cancers have KRas mutation[12]. This signal pathway is mainly activated by Ras combined with guanosine triphosphate under the stimulation of extracellular signals, thereby phosphorylation and activation of Raf protein. Raf then activates Mek, and Mek is phosphorylated and finally activates Erk. Only p-Erk has it. Active,p-Erk enters the nucleus through translocation, activates a variety of transcription factors and kinases, thereby completing the process of transmitting extracellular stimulus signals to the cell, causing a series of cellular responses, thereby regulating cell proliferation,differentiation, apoptosis, and metastasis Wait.

The results of this study show that cryoablation of Lewis lung cancer tumor-bearing mice can directly inhibit the transcription of KRas gene, reduce the expression of KRAS protein, and cause the downstream RAF1 and MEK1 protein phosphorylation activation process compared with the sham operation group. Inhibition ultimately inhibits the process of phosphorylation of ERK1/2 and may cause the inhibition of tumor cell proliferation, differentiation,apoptosis, and metastasis.

Under normal circumstances, the expression of KRas is transmitted by the upstream EGFR receptor to transmit extracellular information.The more common KRas gene mutation in lung cancer makes KRas still active and uncontrollable without EGFR exogenous signal stimulation. Continuous activation will cause tumors. Disorderly proliferation. Therefore, KRas gene mutation affects the efficacy of EGFR-TKI targeted therapy, and the results of this experiment suggest that cryoablation can inhibit the expression of KRas gene and inhibit the activation of MAPK/ERK signaling pathway in addition to the direct mechanical killing of tumor cells. Patients with KRas gene mutations may have better curative effects in combination with EGFR-TKI targeted medication after cryoablation.

In summary, cryoablation can down-regulate the expression of KRas gene and negatively regulate the MAPK/ERK signaling pathway, which may be related to the inhibition of tumor growth and metastasis by cryoablation. As the MAPK/ERK signaling pathway is an important downstream pathway of the epidermal growth factor receptor (EGFR), the combination therapy of cryoablation and EGFR-targeted drugs may have a sensitization effect and further improve the efficacy, which requires further research and exploration.

Author's contribution

The first Author: Lin Shicheng was responsible for designing experimental scheme, feeding mice, performing the cryoablations of mice, detecting experimental indicators and writing the article.

The second author: Liu Dianna improved the experimental scheme,guided the cryoablations of mice, and lead the relevant detection of experiments.

The third author: Zhou Xiangnan was assisted in the completion of testing laboratory indicators and parameters, meanwhile checked and modified the paper.

The fourth author:Zhuang Yaoxue gave assistance for cryoablation therapy and tissue sample operation of mice, also was assisted for extraction of total protein and RNA, and detection of WB and PCR.

The fifth author: Liang Tianyu supported mice cold melt,experimental sample acquisition and WB detection.The sixth author: Wang Xiaofan taked mutual help for freezing experiment and gaining sample of mice, and PCR detection also.

The seventh author: Hu Kaiwen taked charge of design and review of project.

The eighth author: Sun Jingyi was responsible for collecting experimental materials, detecting WB and PCR and collating data.

The corresponding author: Li Quanwang, project director, is leading for providing the overall ideas of the project, improving the experimental scheme, and auditing and revising the paper.

Author conflict of interest statement

All authors declare that they have no conflict of interest