IL-6 up-regulates the expression of IL-6R through JAK2/STAT3 signaling pathway to promote HCC progression

IL-6 up-regulates the expression of IL-6R through JAK2/STAT3 signaling pathway to promote HCC progression | 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 Article IL-6 up-regulates the expression of IL-6R through JAK2/STAT3 signaling pathway to promote HCC progression Li Song, Ruyue Xu, Wenpeng Cai, Jiaojiao Liang, Niandie Cao, Jiafeng Gao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2111577/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Background The progression of hepatocellular carcinoma (HCC) involves multi-factor, multi-step interactions. It has been reported that the high expression of interleukin-6 receptor (IL-6R) plays an important role in the occurrence and development of tumors, but the regulation mechamismof IL-6R expression and its function in HCC have not been adequately reported. Methods Western Blot was used to evaluate the phosphorylation of key kinases in JAK2/STAT3 pathway and the protein expression levels of related proliferative molecules, migration molecules and apoptotic molecules. The anti-apoptosis, migration and proliferation abilities of cells of each group were analyzed using JC-1 measures cell apoptosis, EdU method to detect cell apoptosis, clone formation experiment and Transwell. Result The expression of IL-6R in HCC cells (HepG2, Huh7 and SK-Hep1) was higher than that in normal hepatocytes (THLE-2 and THLE-5), and the protein expression of IL-6R was relatively highest in SK-Hep1 and relatively lowest in HepG2. Compared with the HepG2 IL − 6 cell line, the protein levels of apoptotic molecules c-Caspase7 and c-Caspase3 were lower, while the protein levels of proliferative molecules p-P70S6K and migration molecules MMP9 and MMP2 were higher, showing stronger anti-apoptosis, proliferation and migration abilities. Compared with SK-Hep1 in SK-Hep1 TCZ and SK-Hep1 IL − 6R− , the protein levels of apoptotic molecules c-Caspase7 and c-Caspase3 were higher, while the protein levels of proliferative molecules p-P70S6K and migration molecules MMP9 and MMP2 were lower. It showed strong apoptotic ability and low proliferation and migration ability. Interestingly, IL-6 up-regulated the expression of IL-6R by activating JAK2/STAT3 signaling pathway. The expression of IL-6R protein was also down-regulated after lentivirus knockdown of STAT3. In subcutaneous tumor-bearing experiments in nude mice, compared with SK-Hep1 group, the up-regulation of IL-6R expression after JAK2/STAT3 signaling pathway activation by IL-6 in SK-Hep1 IL − 6 group significantly improved the tumor growth ability. However, the expression of IL-6R protein was down-regulated and the terminal tumor volume was significantly down-regulated in the lentiviral STAT3 knockdown group, which inhibited the tumor growth ability. Conclusions The results showed that IL-6 regulated the transcription of IL-6R through the activation of JAK2/STAT3 signaling pathway, thereby promoting the progression of HCC. The result are expected to provide experimental basis for IL-6R as a potential therapeutic target for HCC. IL-6R IL-6 JAK2/STAT3 HCC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction HCC is the sixth most common malignant tumor in the world, with the 6th morbidity and 3rd mortality in China, respectively[ 1 ]. surgical resection is the standard treatment for HCC in the early stage. Unfortunately, more than 80% of HCC patients miss the opportunity of surgery or liver transplantation due to the late detection. HCC patients can only prolong their survival by local radiofrequency ablation, arterial chemoembolization, radiotherapy and chemotherapy, but the efficacy is very limited[ 2 , 3 ]. With the development of molecular targeted therapy for HCC, sorafenib (tyrosine kinase inhibitor), a first-line drug for advanced HCC approved by the FDA since 2008, has significantly improved the overall survival rate of patients with unresectable HCC and has become one of the most promising drugs for the treatment of HCC[ 4 ]. However, HCC, as a disease with high molecular and clinical heterogeneity, is also prone to develop drug resistance to such multienzyme inhibitors, thus greatly hindering the treatment of HCC[ 5 ]. Therefore, it is of great significance to clarify the important mechanism of HCC progression and find a effective therapeutic target to prevent tumor progression. IL-6R is one of the main drivers of carcinogenesis. IL-6 binds to its receptor IL-6R, a multipotent pro-inflammatory cytokine, to regulate a variety of biological functions. High expression of IL-6R in tumor cells of patients is often associated with increased risk of tumor development and poor prognosis [ 6 ]. Multiple tumor cellsexpressing high levels of IL-6R (such as colorectal cancer, gastric cancer, multiple myeloma, prostate cancer and breast cancer) have been proved to be associated with poor prognosis of tumors [ 7 – 11 ]. A recent study confirmed that hepatic stellate cells increased the viability, migration capacity and stem of cancer cells through the IL-6R/STAT3 pathway, suggesting that the high expression of IL-6R may promote the progression of tumor diseases [ 12 ]. JAK2/STAT3 is a common signaling pathway to transfer signals from extracellular to intracellular receptors after cytokines or growth factors binding to extracellular binding domains [ 13 ]. JAK2/STAT3 signaling pathway is involved in pathological processes such as cancer and inflammation, and promotes tumorigenesis, tumor growth, survival and metastasis of cancer cells[ 14 ]. In view of the correlation among IL-6R,JAK2/STAT3 signaling pathway and the occurrence and development of cancer, The purpose of this study was to elucidate the biological effects of IL-6R on HCC cells and its expression mechanism. HCC cell line SK-Hep1 with high expression of IL-6R was selected in this study, and the expression of IL-6R in SK-Hep1 was knocked down by RNAi technology. The effects of IL-6R on the proliferation, migration and apoptosis resistance of HCC were discussed. Furthermore, Western Blot and RNAi techniques were used to discuss the JAK2/STAT3 signaling pathway involved in regulation mechanism of IL-6R. Our study provides important insights into the modulated mechanisms and biological functions of IL-6R in HCC. Result IL-6R is highly expressed in HCC cell lines To determine whether IL-6R is overexpressed in HCC cell lines, total proteins of THLE-2, THLE-5, HepG2, Huh7 and SK-Hep1 cells were extracted and quantified by Western Blot. As shown in Fig. 1 A, IL-6R expression was higher in HCC cells than in normal liver cells. Among HCC cell lines, the expression level of IL-6R was highest in SK-Hep1 cells, while it was lower in HepG2 cells. HepG2 was stimulated with different concentrations of IL-6 for 24h, and the expression of IL-6R peaked at 25ng/ mL, as shown in Fig. 1 B. And IL-6R expression level in HepG2 was the highest and remained stable at 24h after 25ng/mL IL-6 stimulation(Fig. 1 C). We then used immunocytochemistry to characterize and target IL-6R in THLE-2, THLE-5, HepG2, Huh7 and SK-Hep1.As shown in Fig. 1 D, the expression of IL-6R in SK-Hep1 was higher than that in HepG2, which was consistent with western blot results. Il-6r Enhances The Anti-apoptotic Ability Of Hcc Cells The anti-apoptotic ability of each cell group was detected under the stimulation of the same concentration of sorafenib. As shown in Fig. 2 A, the expression of apoptotic molecules c-Caspase3 and c-Caspase7 was down-regulated whenIL-6 (25ng/mL,) stimulated for 24h compared with HepG2 cells. PLVE3494 significantly down-regulated the expression level of IL-6R in SK-Hep1 compared with CTRL group(Fig. 2 B). Figure 2 C (hematoxylin-eosin staining) shows that lentivirus interference with IL-6R does not cause morphological changes in cells. It can be concluded from Fig. 2 D that compared with SK-Hep1, inhibiting IL-6R by Tocilizumab(20ng/mL, 24h) upregulated the levels of apoptotic molecules c-caspase3 and c-Caspase7. Compared with SK-Hep1 CTRL , SK-Hep1 IL − 6R− up-regulated the levels of apoptotic molecules c-caspase3 and c-Caspase7. In JC-1 apoptosis test(Fig. 2 E), the transition from red fluorescence to green fluorescence of JC-1, is an indicator of early apoptosis. Compared with HepG2 cell lines, green fluorescence of HepG2 IL − 6 was less and weaker after treatment with 1µM sorafenib, suggesting strong anti-apoptosis ability. Compared with SK-Hep1, SK-Hep1 TCZ had more green fluorescence and weaker anti-apoptosis ability. However, compared with SK-Hep1 CTRL , the green fluorescence of SK-Hep1 IL − 6R− group is significantly enhanced, suggesting that the anti-apoptosis ability of SK-Hep1 IL − 6R− cells is weakened. In conclusion, the expression level of IL-6R may be positively correlated with the anti-apoptotic ability of HCC cells. Il-6r Enhance The Proliferation Of Hcc Cells Sk-hep1 IL − 6R− was obtained by transfection of lentivirus. As indicatedin Fig. 3 A, compared with HepG2, the expression level of IL-6R was increased when the cells stimulated with IL-6(25ng/ml) for 24h, and the level of proliferating molecule p-P70S6K was also up-regulated. While, the p-P70S6K level was down-regulated in SK-Hep1 TCZ cells with inhibitor Tocilizumab(20ng/ml,24h) compared to SK-Hep1 cells(Fig. 3 B). Compared with SK-Hep1 CTRL , IL-6R expression level, as well as the level of p-P70S6K, was down-regulated in SK-Hep1 IL − 6R− . In the EdU proliferation test(Fig. 3 C), the number of red positive cells represents the strength of the cell proliferation ability. Compared with HepG2, The number of EdU positive cells in HepG2 IL − 6 increased, suggesting that the proliferation ability of HepG2 IL − 6 was enhanced. Compared with SK-Hep1, the positive cells in SK-Hep1 TCZ decreased, and its proliferation ability was weakened. Compared with SK-Hep1 CTRL , the decrease of positive cells in SK-Hep1 IL − 6R− indicated that the proliferation ability of SK-Hep1 IL − 6R− was weakened. Similarly, the results of clonal formation experiment in Fig. 3 D showed that, hepG2 IL − 6 group had more clonal cell masses compared with HepG2 group, indicating its stronger clonal formation ability. On the contrary, when TCZ was added to inhibit IL-6R in SK-Hep1, the number of clones was reduced, suggesting that the cloning ability of the cells was down-regulated after IL-6R inhibition. Compared with SK-Hep1 CTRL , sk-Hep1 IL − 6R − cells also had fewer clones, suggesting that silencing IL-6R weakened its cloning ability. In conclusion, IL-6R enhances the proliferation of HCC cells. Il-6r Promote The Migration Of Hcc Cells SK-Hep1 IL − 6R− stable cell lines were obtained by lentivirus transfection. As shown in Fig. 4 A, compared with HepG2, the expression level of IL-6R,as well as the levels of MMP2 and MMP9 in HepG2 IL − 6 stimulated with activator IL-6 (25ng/mL, 24h) were also up-regulated. This view was also verified in Fig. 4 B, which showed that compared with SK-Hep1, the expression levels of MMP2 and MMP9 were down-regulated after the addition of IL-6R inhibitor Tocilizumab (20ng/mL, 24h). Compared with SK-Hep1 CTRL , the expression levels of MMP2 and MMP9 in SK-Hep1 IL − 6R− were also down-regulated. Results of Transwell experiments test( Figure. 4C ) showed, compared with HepG2, the migration ability of HepG2 IL − 6 was enhanced, but decreased in SK-Hep1 TCZ and SK-Hep1 IL − 6R− cells compared with SK-Hep1 cell line. The results of the scratch healing experiment showed ( Figure. 4D ) that, compared with HepG2, hepG2 IL − 6 cell line had narrower scar width between cells at 24h and 48h, indicating its strong migration ability. However, compared with SK-Hep1, SK-Hep1 TCZ cells at 24h and 48h had slower wound healing rate and lower mobility, suggesting that the migration ability of the cells was weak. Compared with SK-Hep1 CTRL , SK-Hep1 IL − 6R− had slower healing rate and lower mobility at 24h and 48h, indicating that the cell migration capacity was weak. In conclusion, the expression level of IL-6R is positively correlated with the migration ability of HCC cells. IL-6 regulates the expression of IL-6R through JAK2/STAT3 signaling pathway. The proteins of SK-Hep1 and HepG2 cells were extracted respectively. Western Blot results(Fig. 5 A) showed that the phosphorylation level of JAK2/STAT3 signaling pathway was significantly higher in SK-Hep1 with high IL-6R expression, suggesting that the high expression of IL-6R may be related to the abnormal activation of JAK2/STAT3 signaling pathway, and STAT3 may be a transcription factor regulating IL-6R. In order to detect the influence of JAK2/STAT3 signaling pathway on IL-6R. HepG2 cell were stimulated for 24h with IL-6(25ng/mL), the activator of JAK2/STAT3 signaling pathway, and than proteins were extracted, The levels of p-JAK2 and p-STAT3 were increased, and the expression of IL-6R was also up-regulated(Fig. 5 B). We used RNAi technology to knock down STAT3 on SK-Hep1, and found that the protein level of IL-6R was down-regulated after the decrease of STAT3 and p-STAT3 protein levels(Fig. 5 C). In order to further explore the influence of JAK2/STAT3 signaling pathway on IL-6R expression. In Fig. 5 D, the expression level of p-JAK2 was up-regulated after 24h of il-6 stimulation in SK-Hep1 and then interfered with STAT3. The protein expression levels of p-stat3 and STAT3 were down-regulated, while the expression level of IL-6R was not up-regulated. These results suggest that IL-6 regulates IL-6R expression through JAK2/STAT3. IL-6 regulates the expression of IL-6R and affects the tumorigenesis ability of nude mice in vivo To investigate whether IL-6 can regulate the expression of IL-6R through JAK2/STAT3 in vivo, we acclimated nude mice in our laboratory for one week. Nude mice were treated with SK-Hep1, SK-Hep1 IL − 6 , SK-Hep1 STAT3− and SK-Hep1 IL − 6+STAT3− . Figure 6 A shows that the body weight of the mice did not differ significantly with the treatment of the drug. According to the tumor volume results in Fig. 6 B, compared with the SK-Hep1 group, the tumor volume in the SK-Hep1 IL − 6 group was significantly increased, and the tumor volume in the SK-Hep1 STAT3− group was significantly down-regulated. The tumor volume of SK-Hep1 IL − 6+STAT3− group was also significantly down-regulated. Extract protein from tumor tissue. As can be seen in Fig. 6 C, the activation level of JAK2/STAT3 signaling pathway was up-regulated under IL-6 (25ng/ml) stimulation, and the protein expression of il-6r was also up-regulated. After STAT3 interference, the activation level of JAK2/STAT3 signaling pathway was down-regulated, and the protein expression of IL-6R was also down-regulated. However, the expression of IL-6R was not up-regulated after adding JAK2/STAT3 signaling pathway activator IL-6 to interfere with STAT3. These results further demonstrated that IL-6 promoted the expression of IL-6R through JAK2/STAT3 in vivo and enhanced the tumor-inducing ability of nude mice. Discussion As a kind of malignant tumor, HCC is an urgent medical problem to be solved [ 15 ]. Nowadays, although molecular targeted therapy has achieved some success in the clinical treatment of cancer, the therapeutic effect still needs to be improved [ 16 , 17 ]. Therefore, new therapeutic targets for HCC are urgently needed. It has been reported that IL-6R is highly expressed in colorectal cancer, gastric cancer, multiple myeloma, prostate cancer and breast cancer and is closely associated with the development of these tumors [ 18 – 23 ]. IL-6R can promote EMT and stem transformation of tumor cells, and promote invasion and metastasis, thus enhancing the viability of tumor cells [ 24 ]. However, the effect of IL-6R in HCC cells and its modulated expression mechanism remains unclear, which is particularly important for the treatment of HCC. The tumor microenvironment of hepatocellular carcinoma (HCC) is a complex system involving mutual promotion and inhibition between cells and molecules. It leads to changes in inflammatory factors, notably the characteristic elevation of IL-6. After the synthesis and release of IL-6, HCC is stimulated by the binding of IL-6 to IL-6R. However, the regulatory mechanism between IL-6 and IL-6R remains unclear. The results of this study showed that compared with normal hepatocytes THLE-2 and THLE-5, the expression level of IL-6R was increased in SK-Hep1, but not in HepG2. This result was further confirmed by immunocytochemistry. In addition, 25ng/mL recombinant IL-6 protein can stably stimulate sustained high IL-6R expression in HepG2 at 24h. These results suggest that the high expression of IL-6R may be correlated with the occurrence and development of HCC, and that IL-6R is involved in the development of HCC through different expression patterns. When IL-6R transduces IL-6 signal, IL-6 binds to its receptor to induce anti-apoptotic effect of cells. According to relevant studies, miaRNA-451a increases apoptosis in multiple myeloma by inhibiting IL-6R [ 27 ]. In this study, compared with HepG2 group, cell membrane potential of HepG2 IL − 6 group did not decrease significantly after treatment with 1µM sorafenib. Similarly, inhibition of IL-6R expression by Tocilizumab in SK-Hep1 cells was achieved by competitively binding IL-6R to lentivirus. These results suggest that IL-6/IL-6R can enhance the anti-apoptotic ability of HCC cells. Western Blot showed that compared with the control group, the expression levels of pro-apoptotic molecules c-Caspase7 and c-Caspase3 were lower in HepG2 IL − 6 cell line. The expression levels of c-Caspase7 and c-Caspase3 were higher in SK-Hep1 TCZ and SK-Hep1 IL − 6R− cell lines. The results suggest that IL-6R may enhance the anti-apoptotic ability of cells by decreasing the expression levels of pro-apoptotic molecules c-Caspase7 and c-Caspase3. It has been reported that IL-6R is also associated with the proliferation and migration of various tumors. For example, mesotheliin overexpression promotes autocrine IL-6/ sIL-6R to stimulate breast cancer cell proliferation [ 28 ]. P53 activation in colorectal cancer cells interferes with IL-6-induced invasion and migration through down-regulation of miR-34a-dependent IL-6R expression, and the IL-6R/STAT3/ miR-34a feedback loop promotes EMT-mediated invasion and metastasis of colorectal cancer [ 29 ]. Similarly, in this study, we found that the high expression of IL-6R can promote the proliferation and migration of HCC cells. Further molecular mechanism analysis revealed that up-regulation of IL-6R expression promoted the protein expression levels of p-P70S6K, mmp9 and mmp2 in HepG2 cells. In SK-Hep1 cells, the levels of p-P70S6K and mmp9 and mmp2 was down-regulated by TCZ competitive binding to IL-6R and lentivirus inhibition of IL-6R expression. These results suggest that IL-6R may regulate the proliferation and migration of HCC cells by regulating the levels of proliferating molecule p-P70S6K and migration molecule mmp9 and mmp2. IL-6 /IL-6R and transcription factor 3 (STAT3) signaling pathways are involved in a variety of physiological processes, including cell growth, differentiation, and immune regulation. Many studies have shown that abnormal IL-6/STAT3 signaling pathway plays a crucial role in the occurrence and development of liver cancer, lung cancer, breast cancer and gastric cancer [ 30 ]. Our results showed that IL-6R was highly expressed in SK-Hep1 cells, and p-JAK2 and p-STAT3 protein levels were higher than HepG2 cells. The abnormal activation of JAK2/STAT3 signaling pathway in SK-Hep1 cell line may be related to the high expression of IL-6R. Therefore, in order to explore whether IL-6 regulates the expression of IL-6R through JAK2/STAT3 signaling pathway, we used recombinant IL-6 protein to stimulate HepG2 cells and found that the JAK2/STAT3 signaling pathway in HepG2 cells was abnormally activated and the protein expression of IL-6R was increased [ 31 ]. Subsequently, SK-Hep1 cells interfered with STAT3 expression down-regulates the activation level of JAK2/STAT3 signaling pathway, as well as the expression of IL-6R. To further test this idea, we added IL-6 and then interfered STAT3 in SK-Hep1 cells, and detected the changes of related signaling pathways and downstream IL-6R protein expression levels. It was found that the expression level of p-JAK2 protein was increased, while the expression of IL-6R was consistent with the down-regulation of p-STAT3 and STAT3.This suggests that IL-6 may promote IL-6R expression through activation of JAK2/STAT3 pathway. The nuclear transcription factor STAT3 is a member of the STAT family of signal transduction and transcriptional activators. Our study showed that the expression of IL-6R may be regulated by the transcription factor STAT3, but it has not been further explored. In the follow-up study, we will improve the experiment and conduct analysis. In conclusion, our study suggests that IL-6 may enhance the proliferation, migration and anti-apoptotic ability of HCC cells by up-regulating the expression of IL-6R through JAK2/STAT3 signaling pathway, thus promoting the progression of HCC. The results may provide new targets and theoretical basis for the treatment of HCC. Material And Method Cell lines and reagents Human HCC Cell lines SK-Hep1, HepG2 and Huh-7 and human immortalized liver Cell lines THLE-2 and THLE-5 were purchased from the National Collection of Authenticated Cell Culyures. HCC cell lines were grown in RPMI-1640 (Hyclone, Salt Lake City, Utah, USA) containing 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials, Hangzhou, China). Human immortalized liver cells were cultured in RPMI-1640 containing 15% fetal bovine serum. Stored in 5%CO 2 , 37℃ constant temperature incubator for culture. The main reagents used in this study are as follows: IL-6protein was purchased from Sino Biological LNC, It is soluble in MP Biomedicals (CA, USA) at 100mg/mL. Tocilizumab was purchased from MedChemExpress, it goes into dimethyl sulfoxide. Sorafenib purchased from MedChem Express (Monmouth Junction, NJ, USA), dissolved in dimethyl sulfoxide. IL-6R antibody was purchased from Thermofisher. Cleaved Caspase-7, Caspase-7, Cleaved Caspase-3, Caspase-3, phospho-P70S6K, P70S6K, MMP9, MMP2, phospho-JAK2, JAK2, Phospho-Stat3, STAT3, β -actin and Secondary horseradish peroxidase (HRP) Conjugated goat anti-rabbit antibodies are purchased from Cell Signal Technology (Danvers, MA, USA). Western Blot The cells were placed in the culture dish for about 3 days, and the cells were basically covered at the bottom of the cell culture dish, when there were about 1.0*10 7 cells. The cells were placed in protein phosphatase and loading buffer mixed at a ratio of 50:1. The cells were first fully lysed on a shaker for about half an hour, then centrifuged in a low-temperature centrifuge for 30min. Finally, 5× Loading buffer was added and mixed, and then the cells were placed in a water bath at 99 ° C and fully heated for about half an hour. Cool naturally at room temperature, centrifuge and store in the refrigerator at minus 20 degrees. Remove when in use. Whole cell lysates of different treatment groups were prepared, and the extracted total protein was dissolved in 10% SDS-PAGE and transferred to PVDF membrane (EMD Millipore). The membrane was blocked in 5% skim milk at 37°C for 1h. After blocking, the membrane was incubated with specific antibody at 4°C overnight, and the secondary antibody (diluted 1:2000) was incubated at room temperature for 1h. Bands were visualized and exposed with the ECL Luminescence Kit (EMD Millipore), and ImageJ 1.44P software (National Institutes of Health, National Institutes of Health) was used for immunoblot quantification. The gray values of different imprinted signals were compared with the control group to analyze the expression of target gene protein. Immunocytochemistry The cells with a concentration of about 2.0×10 6 cells/mL were planted in 24-well plates, and the cells were observed under a microscope when the cell density reached about 80% after 24h growth in the incubator. Wash with PBS twice and fix in 4% paraformaldehyde for 15 min, then seal with H 2 O 2 for 15 min. Incubate with closed serum at room temperature for 20min. 200 µL primary antibody (IL-6R, 1:100, PA5-102425) was added to each well and incubated overnight in a refrigerator at 4°C. Add the second antibody working solution and incubate for 30min at room temperature. The streptomycin working solution was added with horseradish peroxidase and incubated at room temperature for 30 min. DAB chromogenic solution (Beyotime Biotechnology, Shanghai,China) was used for dark staining for 15min, and hematoxylin (Beyotime Biotechnology, Shanghai,China) was used for nuclear staining for 10min. Finally, it was dehydrated with 95% alcohol and sealed with neutral resin for two seconds before being photographed under a microscope. H2O2, serum, secondary antibody and horseradish labeled streptomycin working solution were all products of SP kit (SP-9000, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.). Edu Method To Detect Cell Viability About 1.0×10 5 cells were planted in 24-well plates, and the experiment was carried out when the cells stuck to the wall and the growth basically covered the bottom of the petri dish for about two days. First replace the culture medium, add 0.5 microliter EdU reagent and then put it into the incubator for incubation for about 3 hours. Then take it out and wash it with PBS washing solution for 3 times for about 3 to 5 minutes each time. Apply 4% paraformaldehyde for about 15 minutes. The percentage of EdU positive cells was observed under a fluorescence microscope to measure cell proliferation. Jc-1 Measures Cell Apoptosis 1.0×10 6 cell suspension was prepared and planted on a 24-well plate and incubated in an incubator. The cells were adherent for 24h and then treated with 1µM sorafenib. 48h later, the slipper was removed for experiment. Fix with 4% paraformaldehyde for 15min. It was then stained with JC-1 reagent for about 30 minutes. The nuclei were stained with DAPI for 10 minutes. The apoptosis level was determined by observing the intensity of red-green fluorescence under fluorescence microscope. Transwell After the cells were starved in serum-free medium for 24h, about 10 5 ~10 6 200µ L cell suspensions were taken and planted in Transwell cells in the middle and upper chambers of 24-well plates. 750µL medium containing 15% serum was added to the lower chamber of Transwell and placed in an incubator for 24h before absorption of the medium. Remove the chamber and transfer it to PBS for cleaning twice. Methanol was fixed at room temperature for 15 min, 0.1% crystal violet was dyed at room temperature for 15 min. Finally, the film was sealed with gum and photographed with fluorescence microscope. The number of cells is a measure of how invasive a cell is. Scratch Healing Experiment The cells were measured with a ruler and marked with a "horizontal line" on the back of the petri dish before inoculation. The cells were diluted to a certain extent and then planted in a petri dish and cultured in an incubator until they were spread to the bottom of the dish and then removed, scratch the line perpendicular to the ground with the 10 microliter point of the ruler. The scar widths of 0h, 24h and 48h were photographed by microscope and cell mobility was calculated. Immunofluorescence About 2.0×10 5 cells were planted on a 24-well plate. After incubation for 24h in a constant temperature incubator of 37℃ and 5%CO 2 , fixation with 4% paraformaldehyde for 20min. Dye with hematoxylin for 15 minutes. Eosin was stained for 5min. Finally, the film was sealed with neutral gum and photographed under a positive fluorescence microscope. Clone Formation Experiment About 1,000 cells are planted evenly in a six-well plate, Culture in 37℃ constant temperature incubator for 10 days. When the clone group in the orifice are visible to the naked eye. The cells were fixed with 4% paraformaldehyde for 15min at room temperature. After fixation, it was stained with crystal violet for 15min and photographed. Xenotransplantation Of Nude Mice All experiments were performed in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines and in accordance with the principles and procedures approved by the Animal Experiment Ethics Committee of Anhui University of Science and Technology (Anhui Province, People's Republic of China). Nude mice (female, 6 weeks old) were provided by Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. SK-Hep1, SK-Hep1 IL − 6 , SK-Hep1 STAT3− and SK-Hep1 IL − 6+STAT3− were implanted into the subcutaneous ribs of nude mice, respectively. The body weight and tumor volume of the mice were recorded three days later, and the mice were sacrificed on day 30. Statistical analysis Experimental data were obtained through at least three independent experiments. Data are expressed as mean ± standard deviation. Statistical differences between the two groups were analyzed using the T-test. Analysis of variance (ANOVA) was used to compare differences between multiple groups. Tukey test to compare the mean values of multiple experimental groups. P < 0.05 is considered a statistically significant difference. GraphPad 8 statistical software was used for all analyses. Declarations Consent for publication Not applicable. Ethics approval and consent to participate All animal experiments were approved by the Ethics Committee of Medical College of Anhui University of Science and Technology and performed following the animal care and use guidelines of the National Institutes of Health of USA. Funding Statement This work was supported by grants from the University Natural Science Research Project of Anhui Province(grant nos. YJS20210407), National Natural Science Fund of China (NO. 82071862, 81872017), Research Foundation of the Institute of Environment-friendly Materials and Occupational Health (Wuhu), Anhui University of Science and Technology (ALW2020YF11), University Natural Science Research Project of Anhui Province (NO. KJ2019A0093, KJ2020A0340), Teaching Research Project of Anhui University of Science and Technology(NO. 2021xjjy59) and 2021 provincial quality engineering “Four new” research and reform practice project (NO.2021sx032). Disclosure The author reports no conflicts of interest in this work. Authors’ contributions Li Song, Ruyue Xu and Wenpeng Cai designed and performed this research; Jiaojiao Liang, Niandie Cao, Jiafeng Gao and Xiaolong Tang analysed data; Li Song, Ruyue Xu, Wenpeng Cai and Xiaolong Tang draughted the manuscript; all authors approved the manuscript. Data Availability Statement Not applicable. References Foerster, F, Gairing, SJ, Muller, L, Galle, PR. NAFLD-driven HCC: Safety and efficacy of current and emerging treatment options. J Hepatol,.2022; 76(2): p. 446-457. Wang W, Wei C. Advances in the early diagnosis of hepatocellular carcinoma. Genes Dis. 2020;7(3):308-319. Wei L, Lee D, Law CT, Zhang MS, Shen J, Chin DW, Zhang A, Tsang FH, Wong CL, Ng IO, Wong CC, Wong CM. Genome-wide CRISPR/Cas9 library screening identified PHGDH as a critical driver for Sorafenib resistance in HCC. Nat Commun, 2019 ;10(1): 4681. Tang W, Chen Z, Zhang W, Cheng Y, Zhang B, Wu F, Wang Q, Wang S, Rong D, Reiter FP, De Toni EN, Wang X. The mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects. Signal Transduct Target Ther; 2020; 5(1): 87. Keating, GM, Santoro, Sorafenib: a review of its use in advanced hepatocellular carcinoma. Drugs. 2009; 69(2): 223-240. Zhang M, Zhang S, Yang Z, Hu J, Hu W, Sun P, Wu L, Han B. Association between the expression levels of IL-6 and IL-6R in the hepatocellular carcinoma microenvironment and postoperative recurrence. Oncol Lett. 2018;16(6):7158-7165. Shi X, Kaller M, Rokavec M, Kirchner T, Horst D, Hermeking H. Characterization of a p53/miR-34a/CSF1R/STAT3 Feedback Loop in Colorectal Cancer. Cell Mol Gastroenterol Hepatol. 2020;10(2):391-418. Mishra, AK, Dingli D. Metformin inhibits IL-6 signaling by decreasing IL-6R expression on multiple myeloma cells. Leukemia. 2019; 33(11): 2695-2709. Santer FR, Malinowska K, Culig Z, Cavarretta IT. Interleukin-6 trans-signalling differentially regulates proliferation, migration, adhesion and maspin expression in human prostate cancer cells. Endocr Relat Cancer. 2010;17(1):241-253. Weng YS, Tseng HY, Chen YA, Shen PC, Al Haq AT, Chen LM, Tung YC, Hsu HL. MCT-1/miR-34a/IL-6/IL-6R signaling axis promotes EMT progression, cancer stemness and M2 macrophage polarization in triple-negative breast cancer. Mol Cancer. 2019;18(1):42. Bharti R, Dey G, Das AK, Mandal M. Differential expression of IL-6/IL-6R and MAO-A regulates invasion/angiogenesis in breast cancer. Br J Cancer. 2018;118(11):1442-1452. Xu J, Lin H, Wu G, Zhu M, Li M. IL-6/STAT3 Is a Promising Therapeutic Target for Hepatocellular Carcinoma. Front Oncol. 2021;11:760971. Mengie Ayele T, Tilahun Muche Z, Behaile Teklemariam A, Bogale Kassie A, Chekol Abebe E. Role of JAK2/STAT3 Signaling Pathway in the Tumorigenesis, Chemotherapy Resistance, and Treatment of Solid Tumors: A Systemic Review. J Inflamm Res. 2022;15:1349-1364. Jaśkiewicz A, Domoradzki T, Pająk B. Targeting the JAK2/STAT3 Pathway-Can We Compare It to the Two Faces of the God Janus? Int J Mol Sci. 2020;21(21):8261. Yim SH, Chung YJ. An Overview of Biomarkers and Molecular Signatures in HCC. Cancers (Basel). 2010;2(2):809-823. Wang T, Xu L, Jia R, Wei J. MiR-218 suppresses the metastasis and EMT of HCC cells via targeting SERBP1. Acta Biochim Biophys Sin (Shanghai). 2017;49(5):383-391. Samant H, Amiri HS, Zibari GB. Addressing the worldwide hepatocellular carcinoma: epidemiology, prevention and management. J Gastrointest Oncol. 2021;12(Suppl 2):S361-S373. Zhong Q, Fang Y, Lai Q, Wang S, He C, Li A, Liu S, Yan Q. CPEB3 inhibits epithelial-mesenchymal transition by disrupting the crosstalk between colorectal cancer cells and tumor-associated macrophages via IL-6R/STAT3 signaling. J Exp Clin Cancer Res. 2020;39(1):132. Simondurairaj C, Krishnakumar R, Sundaram S, Venkatraman G. Interleukin-6 Receptor (IL-6R) Expression in Human Gastric Carcinoma and its Clinical Significance. Cancer Invest. 2019;37(7):293-298. Camacho X, Perroni C, Machado CL, de Godoi Carneiro C, de Souza Junqueira M, Faria D, García MF, Fernández M, Oddone N, Benech J, Buchpiguel CA, Cerecetto H, Chammas R, Riva E, Cabral P, Gambini JP. 99mTechnetium- or Cy7-Labeled Fab(Tocilizumab) as Potential Multiple Myeloma Imaging Agents. Anticancer Agents Med Chem, 2021. 21(14): p. 1883-1893. Armakolas A, Dimakakos A, Loukogiannaki C, Armakolas N, Antonopoulos A, Florou C, Tsioli P, Papageorgiou E, Alexandrou TP, Stathaki M, Spinos D, Pektasides D, Patsouris E, Koutsilieris M. IL-6 is associated to IGF-1Ec upregulation and Ec peptide secretion, from prostate tumors. Mol Med, 2018. 24(1): p. 6. Libertini SJ, Chen H, al-Bataina B, Koilvaram T, George M, Gao AC, Mudryj M. The interleukin 6 receptor is a direct transcriptional target of E2F3 in prostate tumor derived cells. Prostate. 2012;72(6):649-660. Bharti R, Dey G, Ojha PK, Rajput S, Jaganathan SK, Sen R, Mandal M. Diacerein-mediated inhibition of IL-6/IL-6R signaling induces apoptotic effects on breast cancer. Oncogene. 2016 Jul 28;35(30):3965-3975. Herzog AE, Warner KA, Zhang Z, Bellile E, Bhagat MA, Castilho RM, Wolf GT, Polverini PJ, Pearson AT, Nör JE. The IL-6R and Bmi-1 axis controls self-renewal and chemoresistance of head and neck cancer stem cells. Cell Death Dis. 2021;12(11):988. Taher MY, Davies DM, Maher J. The role of the interleukin (IL)-6/IL-6 receptor axis in cancer. Biochem Soc Trans. 2018;46(6):1449-1462. Kampan NC, Xiang SD, McNally OM, Stephens AN, Quinn MA, Plebanski M. Immunotherapeutic Interleukin-6 or Interleukin-6 Receptor Blockade in Cancer: Challenges and Opportunities. Curr Med Chem. 2018;25(36):4785-4806. Zhong L, Xu Z, Jin X, He Y, Zhang J, Jiang T, Chen J. miR-451a suppression of IL-6R can inhibit proliferation and increase apoptosis through the JAK2/STAT3 pathway in multiple myeloma. Oncol Lett. 2020;20(6):339. Bharadwaj U, Marin-Muller C, Li M, Chen C, Yao Q. Mesothelin overexpression promotes autocrine IL-6/sIL-6R trans-signaling to stimulate pancreatic cancer cell proliferation. Carcinogenesis. 2011;32(7):1013-1024. Rokavec M, Öner MG, Li H, Jackstadt R, Jiang L, Lodygin D, Kaller M, Horst D, Ziegler PK, Schwitalla S, Slotta-Huspenina J, Bader FG, Greten FR, Hermeking H. IL-6R/STAT3/miR-34a feedback loop promotes EMT-mediated colorectal cancer invasion and metastasis. J Clin Invest. 2014;124(4):1853-1867. Hirano T. IL-6 in inflammation, autoimmunity and cancer. Int Immunol. 2021;33(3):127-148. Johnson DE, O'Keefe RA, Grandis JR. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat Rev Clin Oncol. 2018;15(4):234-248. Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.png Graphical abstract Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions 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. 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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-2111577","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":149906517,"identity":"aa9f16bd-e92f-46b7-963d-2409727a260d","order_by":0,"name":"Li Song","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Song","suffix":""},{"id":149906518,"identity":"cdc3e618-0fb1-4b7b-a84a-e7b4d6e77b31","order_by":1,"name":"Ruyue Xu","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruyue","middleName":"","lastName":"Xu","suffix":""},{"id":149906519,"identity":"a17152c4-f133-4e1d-bd13-a2b0308031a1","order_by":2,"name":"Wenpeng Cai","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenpeng","middleName":"","lastName":"Cai","suffix":""},{"id":149906520,"identity":"850237e5-8d7d-4de0-ae35-3e6a5ae1f980","order_by":3,"name":"Jiaojiao Liang","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiaojiao","middleName":"","lastName":"Liang","suffix":""},{"id":149906521,"identity":"1db7db31-44d1-421b-8af5-9c7c6e24ff18","order_by":4,"name":"Niandie Cao","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Niandie","middleName":"","lastName":"Cao","suffix":""},{"id":149906522,"identity":"5c5dd393-0fe9-4f5e-bca1-f6c3c0aebe63","order_by":5,"name":"Jiafeng Gao","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiafeng","middleName":"","lastName":"Gao","suffix":""},{"id":149906523,"identity":"1adbed31-7f0b-4d73-9daa-f7cfb142a70d","order_by":6,"name":"Xiaolong Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYFAC5gYDngoJHn72xsYHH4jRwMPACNRyxkZOsudws+EMYrUw8LalGRvMSG+T5iBGi71EYkPBG7bDiRskHzZIMzDYyek2ELKF52CD4Ryew4nbpRMbjAsYko3NDhDSwt7YYMwjcThx5+zEhuQZDAcStxHUwswI1GIAdNjNgw2HeYjSArYlAej9G4yNzcRpOQPyywFQICc2M84wIMIv7DOSjxm8/QeKyuPPf3yosJMjqAUI2AwQbAPcypAB8wPi1I2CUTAKRsGIBQAOtUUFM3O7uwAAAABJRU5ErkJggg==","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiaolong","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2022-09-28 07:59:20","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-2111577/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-2111577/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":28751312,"identity":"d0187be9-7631-440c-a7d3-08e9f8f1fa75","added_by":"auto","created_at":"2022-11-07 14:55:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":362068,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of IL-6R in different cells was detected. \u003cstrong\u003eA\u003c/strong\u003e: The expression of IL-6R in different cells was analyzed by Western Blot. \u003cstrong\u003eB\u003c/strong\u003e: HepG2 was treated with different concentrations of IL-6. After 24h, the protein was extracted and the expression level of IL-6R was observed. \u003cstrong\u003eC\u003c/strong\u003e: HepG2 was treated with 25 ng/mL IL-6 for different times, and the level of IL-6R protein was detected by western blot. D: The expression of IL-6R in different cells was detected by immunocytochemistry. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2111577/v2/0f086e68af5a9bff335a5a27.png"},{"id":28751314,"identity":"b746ee7a-14ed-4b29-8c2d-b1d0ca485fda","added_by":"auto","created_at":"2022-11-07 14:55:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1143435,"visible":true,"origin":"","legend":"\u003cp\u003eIL-6R enhances the anti-apoptotic ability of cells. \u003cstrong\u003eA\u003c/strong\u003e: Western Blot assay was used to detect the correlation between IL-6R and apoptosis. \u003cstrong\u003eB\u003c/strong\u003e: Western Blot was used to screen the efficiency of interference with IL-6R by lentivirus. \u003cstrong\u003eC\u003c/strong\u003e: Morphological differences of cell lines after lentivirus transfection were observed by HE staining. \u003cstrong\u003eD\u003c/strong\u003e: The correlation between IL-6R and apoptosis was detected by western blot. E: JC-1 assay was used to evaluate the difference in apoptotic ability of each cell line. SFB: Sorafenib; TCZ: Tocilizumab; *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05,**\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2111577/v2/55e4ba85845a17af31df1571.png"},{"id":28753388,"identity":"43df8ec4-655e-4518-be7c-12c8aadbbd72","added_by":"auto","created_at":"2022-11-07 15:11:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1267411,"visible":true,"origin":"","legend":"\u003cp\u003eIL-6R promotes cell proliferation. \u003cstrong\u003eA\u003c/strong\u003e、\u003cstrong\u003eB\u003c/strong\u003e: The relationship between IL-6R expressions and the proliferative molecule p-P70S6K was demonstrated by Western Blot.\u003cstrong\u003e C\u003c/strong\u003e: The EdU assay was used to evaluate the proliferative capacity of different cells expressing IL-6R. \u003cstrong\u003eD\u003c/strong\u003e: Clone formationexperiments was used to evaluate the cloning ability of different cells. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05,**\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2111577/v2/4d9d931171f53494fc7627e6.png"},{"id":28751318,"identity":"50adecc6-0863-4006-87f5-bc5253bac5a3","added_by":"auto","created_at":"2022-11-07 14:55:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1456770,"visible":true,"origin":"","legend":"\u003cp\u003eIL-6R promotes cell migration. \u003cstrong\u003eA\u003c/strong\u003e,\u003cstrong\u003eB\u003c/strong\u003e: Western Blot analysis of MMP2 and MMP9 protein levels after activation and interference with IL-6R. \u003cstrong\u003eC\u003c/strong\u003e: Transwell assay was used to observe the changes of invasiveness of IL-6R after high expression and interference. \u003cstrong\u003eD\u003c/strong\u003e: The difference of migration ability was verified by scratch test. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05,**\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2111577/v2/c69fc44773dc7d79951307b9.png"},{"id":28751317,"identity":"e84fccd1-275a-44da-b37c-fe813710901e","added_by":"auto","created_at":"2022-11-07 14:55:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":440500,"visible":true,"origin":"","legend":"\u003cp\u003eIL-6 regulates the expression of IL-6R through JAK2/STAT3 signaling pathway. \u003cstrong\u003eA\u003c/strong\u003e: Differences in JAK2/STAT3 activation levels in cell lines. \u003cstrong\u003eB\u003c/strong\u003e: The JAK2/STAT3 signaling pathway on HepG2 was activated by IL-6, and the activation level of the signaling pathway and the expression level of IL-6R were observed by western blot. \u003cstrong\u003eC\u003c/strong\u003e: Western Blot was used to screen the interference efficiency of STAT3 and the protein level of IL-6R.\u003cstrong\u003eD\u003c/strong\u003e: The activation level of JAK2/STAT3 signaling pathway and il-6r protein expression after 24h il-6 treatment interfered with STAT3. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05,**\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01,***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2111577/v2/9fb89a2a527f68c5f707504e.png"},{"id":28752538,"identity":"202321da-23bf-43ac-b85c-2154fc23e0ba","added_by":"auto","created_at":"2022-11-07 15:03:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":406361,"visible":true,"origin":"","legend":"\u003cp\u003eIl-6 promotes the expression of IL-6R and enhances tumorigenicity of nude mice in vivo. \u003cstrong\u003eA\u003c/strong\u003e: The body weight of nude mice was recorded from subcutaneous tumor-bearing to end. \u003cstrong\u003eB\u003c/strong\u003e: Subcutaneous tumor volume in nude mice. \u003cstrong\u003eC\u003c/strong\u003e: SK-Hep1, SK-Hep1\u003csup\u003eIL-6\u003c/sup\u003e, SK-Hep1\u003csup\u003eSTAT3-\u003c/sup\u003e, SK-Hep1\u003csup\u003eIL-6+STAT3-\u003c/sup\u003e tumor tissue proteins were extracted to evaluate the changes of JAK2/STAT3 signaling pathway and IL-6R protein levels. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05，**\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2111577/v2/ce2bdf9e460f4350a3db2e66.png"},{"id":29873153,"identity":"3fca8c87-ae2e-4974-b609-96eff679243a","added_by":"auto","created_at":"2022-12-04 16:14:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5328151,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2111577/v2/c0d84a2f-2ec5-4ac5-a134-cd032a869b99.pdf"},{"id":28751313,"identity":"5a86b4ed-584f-48af-8323-e27044dfe12f","added_by":"auto","created_at":"2022-11-07 14:55:45","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":314080,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-2111577/v2/19c2ccfa6c74ce1c60676cb4.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"IL-6 up-regulates the expression of IL-6R through JAK2/STAT3 signaling pathway to promote HCC progression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHCC is the sixth most common malignant tumor in the world, with the 6th morbidity and 3rd mortality in China, respectively[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. surgical resection is the standard treatment for HCC in the early stage. Unfortunately, more than 80% of HCC patients miss the opportunity of surgery or liver transplantation due to the late detection. HCC patients can only prolong their survival by local radiofrequency ablation, arterial chemoembolization, radiotherapy and chemotherapy, but the efficacy is very limited[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. With the development of molecular targeted therapy for HCC, sorafenib (tyrosine kinase inhibitor), a first-line drug for advanced HCC approved by the FDA since 2008, has significantly improved the overall survival rate of patients with unresectable HCC and has become one of the most promising drugs for the treatment of HCC[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, HCC, as a disease with high molecular and clinical heterogeneity, is also prone to develop drug resistance to such multienzyme inhibitors, thus greatly hindering the treatment of HCC[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, it is of great significance to clarify the important mechanism of HCC progression and find a effective therapeutic target to prevent tumor progression.\u003c/p\u003e \u003cp\u003eIL-6R is one of the main drivers of carcinogenesis. IL-6 binds to its receptor IL-6R, a multipotent pro-inflammatory cytokine, to regulate a variety of biological functions. High expression of IL-6R in tumor cells of patients is often associated with increased risk of tumor development and poor prognosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Multiple tumor cellsexpressing high levels of IL-6R (such as colorectal cancer, gastric cancer, multiple myeloma, prostate cancer and breast cancer) have been proved to be associated with poor prognosis of tumors [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A recent study confirmed that hepatic stellate cells increased the viability, migration capacity and stem of cancer cells through the IL-6R/STAT3 pathway, suggesting that the high expression of IL-6R may promote the progression of tumor diseases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eJAK2/STAT3 is a common signaling pathway to transfer signals from extracellular to intracellular receptors after cytokines or growth factors binding to extracellular binding domains [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. JAK2/STAT3 signaling pathway is involved in pathological processes such as cancer and inflammation, and promotes tumorigenesis, tumor growth, survival and metastasis of cancer cells[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn view of the correlation among IL-6R,JAK2/STAT3 signaling pathway and the occurrence and development of cancer, The purpose of this study was to elucidate the biological effects of IL-6R on HCC cells and its expression mechanism. HCC cell line SK-Hep1 with high expression of IL-6R was selected in this study, and the expression of IL-6R in SK-Hep1 was knocked down by RNAi technology. The effects of IL-6R on the proliferation, migration and apoptosis resistance of HCC were discussed. Furthermore, Western Blot and RNAi techniques were used to discuss the JAK2/STAT3 signaling pathway involved in regulation mechanism of IL-6R. Our study provides important insights into the modulated mechanisms and biological functions of IL-6R in HCC.\u003c/p\u003e"},{"header":"Result","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003eIL-6R is highly expressed in HCC cell lines\u003c/h2\u003e\n \u003cp\u003eTo determine whether IL-6R is overexpressed in HCC cell lines, total proteins of THLE-2, THLE-5, HepG2, Huh7 and SK-Hep1 cells were extracted and quantified by Western Blot. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, IL-6R expression was higher in HCC cells than in normal liver cells. Among HCC cell lines, the expression level of IL-6R was highest in SK-Hep1 cells, while it was lower in HepG2 cells. HepG2 was stimulated with different concentrations of IL-6 for 24h, and the expression of IL-6R peaked at 25ng/ mL, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB. And IL-6R expression level in HepG2 was the highest and remained stable at 24h after 25ng/mL IL-6 stimulation(Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). We then used immunocytochemistry to characterize and target IL-6R in THLE-2, THLE-5, HepG2, Huh7 and SK-Hep1.As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD, the expression of IL-6R in SK-Hep1 was higher than that in HepG2, which was consistent with western blot results.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eIl-6r Enhances The Anti-apoptotic Ability Of Hcc Cells\u003c/h3\u003e\n\u003cp\u003eThe anti-apoptotic ability of each cell group was detected under the stimulation of the same concentration of sorafenib. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, the expression of apoptotic molecules c-Caspase3 and c-Caspase7 was down-regulated whenIL-6 (25ng/mL,) stimulated for 24h compared with HepG2 cells. PLVE3494 significantly down-regulated the expression level of IL-6R in SK-Hep1 compared with CTRL group(Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC (hematoxylin-eosin staining) shows that lentivirus interference with IL-6R does not cause morphological changes in cells. It can be concluded from Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD that compared with SK-Hep1, inhibiting IL-6R by Tocilizumab(20ng/mL, 24h) upregulated the levels of apoptotic molecules c-caspase3 and c-Caspase7. Compared with SK-Hep1\u003csup\u003eCTRL\u003c/sup\u003e, SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6R\u0026minus;\u003c/sup\u003e up-regulated the levels of apoptotic molecules c-caspase3 and c-Caspase7. In JC-1 apoptosis test(Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE), the transition from red fluorescence to green fluorescence of JC-1, is an indicator of early apoptosis. Compared with HepG2 cell lines, green fluorescence of HepG2\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sup\u003e was less and weaker after treatment with 1\u0026micro;M sorafenib, suggesting strong anti-apoptosis ability. Compared with SK-Hep1, SK-Hep1\u003csup\u003eTCZ\u003c/sup\u003e had more green fluorescence and weaker anti-apoptosis ability. However, compared with SK-Hep1\u003csup\u003eCTRL\u003c/sup\u003e, the green fluorescence of SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6R\u0026minus;\u003c/sup\u003e group is significantly enhanced, suggesting that the anti-apoptosis ability of SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6R\u0026minus;\u003c/sup\u003e cells is weakened. In conclusion, the expression level of IL-6R may be positively correlated with the anti-apoptotic ability of HCC cells.\u003c/p\u003e\n\u003ch3\u003eIl-6r Enhance The Proliferation Of Hcc Cells\u003c/h3\u003e\n\u003cp\u003eSk-hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6R\u0026minus;\u003c/sup\u003e was obtained by transfection of lentivirus. As indicatedin Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, compared with HepG2, the expression level of IL-6R was increased when the cells stimulated with IL-6(25ng/ml) for 24h, and the level of proliferating molecule p-P70S6K was also up-regulated. While, the p-P70S6K level was down-regulated in SK-Hep1\u003csup\u003eTCZ\u003c/sup\u003e cells with inhibitor Tocilizumab(20ng/ml,24h) compared to SK-Hep1 cells(Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Compared with SK-Hep1\u003csup\u003eCTRL\u003c/sup\u003e, IL-6R expression level, as well as the level of p-P70S6K, was down-regulated in SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6R\u0026minus;\u003c/sup\u003e. In the EdU proliferation test(Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC), the number of red positive cells represents the strength of the cell proliferation ability. Compared with HepG2, The number of EdU positive cells in HepG2\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sup\u003e increased, suggesting that the proliferation ability of HepG2\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sup\u003e was enhanced. Compared with SK-Hep1, the positive cells in SK-Hep1\u003csup\u003eTCZ\u003c/sup\u003e decreased, and its proliferation ability was weakened. Compared with SK-Hep1\u003csup\u003eCTRL\u003c/sup\u003e, the decrease of positive cells in SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6R\u0026minus;\u003c/sup\u003e indicated that the proliferation ability of SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6R\u0026minus;\u003c/sup\u003e was weakened. Similarly, the results of clonal formation experiment in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD showed that, hepG2\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sup\u003e group had more clonal cell masses compared with HepG2 group, indicating its stronger clonal formation ability. On the contrary, when TCZ was added to inhibit IL-6R in SK-Hep1, the number of clones was reduced, suggesting that the cloning ability of the cells was down-regulated after IL-6R inhibition. Compared with SK-Hep1\u003csup\u003eCTRL\u003c/sup\u003e, sk-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6R \u0026minus;\u003c/sup\u003e cells also had fewer clones, suggesting that silencing IL-6R weakened its cloning ability. In conclusion, IL-6R enhances the proliferation of HCC cells.\u003c/p\u003e\n\u003ch3\u003eIl-6r Promote The Migration Of Hcc Cells\u003c/h3\u003e\n\u003cp\u003eSK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6R\u0026minus;\u003c/sup\u003e stable cell lines were obtained by lentivirus transfection. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, compared with HepG2, the expression level of IL-6R,as well as the levels of MMP2 and MMP9 in HepG2\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sup\u003e stimulated with activator IL-6 (25ng/mL, 24h) were also up-regulated. This view was also verified in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, which showed that compared with SK-Hep1, the expression levels of MMP2 and MMP9 were down-regulated after the addition of IL-6R inhibitor Tocilizumab (20ng/mL, 24h). Compared with SK-Hep1\u003csup\u003eCTRL\u003c/sup\u003e, the expression levels of MMP2 and MMP9 in SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6R\u0026minus;\u003c/sup\u003e were also down-regulated. Results of Transwell experiments test(\u003cstrong\u003eFigure. 4C\u003c/strong\u003e) showed, compared with HepG2, the migration ability of HepG2\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sup\u003e was enhanced, but decreased in SK-Hep1\u003csup\u003eTCZ\u003c/sup\u003e and SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6R\u0026minus;\u003c/sup\u003e cells compared with SK-Hep1 cell line. The results of the scratch healing experiment showed (\u003cstrong\u003eFigure. 4D\u003c/strong\u003e) that, compared with HepG2, hepG2\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sup\u003e cell line had narrower scar width between cells at 24h and 48h, indicating its strong migration ability. However, compared with SK-Hep1, SK-Hep1\u003csup\u003eTCZ\u003c/sup\u003e cells at 24h and 48h had slower wound healing rate and lower mobility, suggesting that the migration ability of the cells was weak. Compared with SK-Hep1\u003csup\u003eCTRL\u003c/sup\u003e, SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6R\u0026minus;\u003c/sup\u003e had slower healing rate and lower mobility at 24h and 48h, indicating that the cell migration capacity was weak. In conclusion, the expression level of IL-6R is positively correlated with the migration ability of HCC cells.\u003c/p\u003e\n\u003ch3\u003eIL-6 regulates the expression of IL-6R through JAK2/STAT3 signaling pathway.\u003c/h3\u003e\n\u003cp\u003eThe proteins of SK-Hep1 and HepG2 cells were extracted respectively. Western Blot results(Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA) showed that the phosphorylation level of JAK2/STAT3 signaling pathway was significantly higher in SK-Hep1 with high IL-6R expression, suggesting that the high expression of IL-6R may be related to the abnormal activation of JAK2/STAT3 signaling pathway, and STAT3 may be a transcription factor regulating IL-6R. In order to detect the influence of JAK2/STAT3 signaling pathway on IL-6R. HepG2 cell were stimulated for 24h with IL-6(25ng/mL), the activator of JAK2/STAT3 signaling pathway, and than proteins were extracted, The levels of p-JAK2 and p-STAT3 were increased, and the expression of IL-6R was also up-regulated(Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). We used RNAi technology to knock down STAT3 on SK-Hep1, and found that the protein level of IL-6R was down-regulated after the decrease of STAT3 and p-STAT3 protein levels(Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). In order to further explore the influence of JAK2/STAT3 signaling pathway on IL-6R expression. In Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD, the expression level of p-JAK2 was up-regulated after 24h of il-6 stimulation in SK-Hep1 and then interfered with STAT3. The protein expression levels of p-stat3 and STAT3 were down-regulated, while the expression level of IL-6R was not up-regulated. These results suggest that IL-6 regulates IL-6R expression through JAK2/STAT3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIL-6 regulates the expression of IL-6R and affects the tumorigenesis ability of nude mice in vivo\u003c/strong\u003e To investigate whether IL-6 can regulate the expression of IL-6R through JAK2/STAT3 in vivo, we acclimated nude mice in our laboratory for one week. Nude mice were treated with SK-Hep1, SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sup\u003e, SK-Hep1\u003csup\u003eSTAT3\u0026minus;\u003c/sup\u003e and SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6+STAT3\u0026minus;\u003c/sup\u003e. Figure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA shows that the body weight of the mice did not differ significantly with the treatment of the drug. According to the tumor volume results in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB, compared with the SK-Hep1 group, the tumor volume in the SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sup\u003e group was significantly increased, and the tumor volume in the SK-Hep1\u003csup\u003eSTAT3\u0026minus;\u003c/sup\u003e group was significantly down-regulated. The tumor volume of SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6+STAT3\u0026minus;\u003c/sup\u003e group was also significantly down-regulated. Extract protein from tumor tissue. As can be seen in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, the activation level of JAK2/STAT3 signaling pathway was up-regulated under IL-6 (25ng/ml) stimulation, and the protein expression of il-6r was also up-regulated. After STAT3 interference, the activation level of JAK2/STAT3 signaling pathway was down-regulated, and the protein expression of IL-6R was also down-regulated. However, the expression of IL-6R was not up-regulated after adding JAK2/STAT3 signaling pathway activator IL-6 to interfere with STAT3. These results further demonstrated that IL-6 promoted the expression of IL-6R through JAK2/STAT3 in vivo and enhanced the tumor-inducing ability of nude mice.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs a kind of malignant tumor, HCC is an urgent medical problem to be solved [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Nowadays, although molecular targeted therapy has achieved some success in the clinical treatment of cancer, the therapeutic effect still needs to be improved [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, new therapeutic targets for HCC are urgently needed. It has been reported that IL-6R is highly expressed in colorectal cancer, gastric cancer, multiple myeloma, prostate cancer and breast cancer and is closely associated with the development of these tumors [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. IL-6R can promote EMT and stem transformation of tumor cells, and promote invasion and metastasis, thus enhancing the viability of tumor cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, the effect of IL-6R in HCC cells and its modulated expression mechanism remains unclear, which is particularly important for the treatment of HCC.\u003c/p\u003e \u003cp\u003eThe tumor microenvironment of hepatocellular carcinoma (HCC) is a complex system involving mutual promotion and inhibition between cells and molecules. It leads to changes in inflammatory factors, notably the characteristic elevation of IL-6. After the synthesis and release of IL-6, HCC is stimulated by the binding of IL-6 to IL-6R. However, the regulatory mechanism between IL-6 and IL-6R remains unclear. The results of this study showed that compared with normal hepatocytes THLE-2 and THLE-5, the expression level of IL-6R was increased in SK-Hep1, but not in HepG2. This result was further confirmed by immunocytochemistry. In addition, 25ng/mL recombinant IL-6 protein can stably stimulate sustained high IL-6R expression in HepG2 at 24h. These results suggest that the high expression of IL-6R may be correlated with the occurrence and development of HCC, and that IL-6R is involved in the development of HCC through different expression patterns.\u003c/p\u003e \u003cp\u003eWhen IL-6R transduces IL-6 signal, IL-6 binds to its receptor to induce anti-apoptotic effect of cells. According to relevant studies, miaRNA-451a increases apoptosis in multiple myeloma by inhibiting IL-6R [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this study, compared with HepG2 group, cell membrane potential of HepG2\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sup\u003e group did not decrease significantly after treatment with 1\u0026micro;M sorafenib. Similarly, inhibition of IL-6R expression by Tocilizumab in SK-Hep1 cells was achieved by competitively binding IL-6R to lentivirus. These results suggest that IL-6/IL-6R can enhance the anti-apoptotic ability of HCC cells. Western Blot showed that compared with the control group, the expression levels of pro-apoptotic molecules c-Caspase7 and c-Caspase3 were lower in HepG2\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sup\u003e cell line. The expression levels of c-Caspase7 and c-Caspase3 were higher in SK-Hep1\u003csup\u003eTCZ\u003c/sup\u003e and SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6R\u0026minus;\u003c/sup\u003e cell lines. The results suggest that IL-6R may enhance the anti-apoptotic ability of cells by decreasing the expression levels of pro-apoptotic molecules c-Caspase7 and c-Caspase3.\u003c/p\u003e \u003cp\u003eIt has been reported that IL-6R is also associated with the proliferation and migration of various tumors. For example, mesotheliin overexpression promotes autocrine IL-6/ sIL-6R to stimulate breast cancer cell proliferation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. P53 activation in colorectal cancer cells interferes with IL-6-induced invasion and migration through down-regulation of miR-34a-dependent IL-6R expression, and the IL-6R/STAT3/ miR-34a feedback loop promotes EMT-mediated invasion and metastasis of colorectal cancer [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, in this study, we found that the high expression of IL-6R can promote the proliferation and migration of HCC cells. Further molecular mechanism analysis revealed that up-regulation of IL-6R expression promoted the protein expression levels of p-P70S6K, mmp9 and mmp2 in HepG2 cells. In SK-Hep1 cells, the levels of p-P70S6K and mmp9 and mmp2 was down-regulated by TCZ competitive binding to IL-6R and lentivirus inhibition of IL-6R expression. These results suggest that IL-6R may regulate the proliferation and migration of HCC cells by regulating the levels of proliferating molecule p-P70S6K and migration molecule mmp9 and mmp2.\u003c/p\u003e \u003cp\u003eIL-6 /IL-6R and transcription factor 3 (STAT3) signaling pathways are involved in a variety of physiological processes, including cell growth, differentiation, and immune regulation. Many studies have shown that abnormal IL-6/STAT3 signaling pathway plays a crucial role in the occurrence and development of liver cancer, lung cancer, breast cancer and gastric cancer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Our results showed that IL-6R was highly expressed in SK-Hep1 cells, and p-JAK2 and p-STAT3 protein levels were higher than HepG2 cells. The abnormal activation of JAK2/STAT3 signaling pathway in SK-Hep1 cell line may be related to the high expression of IL-6R. Therefore, in order to explore whether IL-6 regulates the expression of IL-6R through JAK2/STAT3 signaling pathway, we used recombinant IL-6 protein to stimulate HepG2 cells and found that the JAK2/STAT3 signaling pathway in HepG2 cells was abnormally activated and the protein expression of IL-6R was increased [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Subsequently, SK-Hep1 cells interfered with STAT3 expression down-regulates the activation level of JAK2/STAT3 signaling pathway, as well as the expression of IL-6R. To further test this idea, we added IL-6 and then interfered STAT3 in SK-Hep1 cells, and detected the changes of related signaling pathways and downstream IL-6R protein expression levels. It was found that the expression level of p-JAK2 protein was increased, while the expression of IL-6R was consistent with the down-regulation of p-STAT3 and STAT3.This suggests that IL-6 may promote IL-6R expression through activation of JAK2/STAT3 pathway. The nuclear transcription factor STAT3 is a member of the STAT family of signal transduction and transcriptional activators. Our study showed that the expression of IL-6R may be regulated by the transcription factor STAT3, but it has not been further explored. In the follow-up study, we will improve the experiment and conduct analysis.\u003c/p\u003e \u003cp\u003eIn conclusion, our study suggests that IL-6 may enhance the proliferation, migration and anti-apoptotic ability of HCC cells by up-regulating the expression of IL-6R through JAK2/STAT3 signaling pathway, thus promoting the progression of HCC. The results may provide new targets and theoretical basis for the treatment of HCC.\u003c/p\u003e"},{"header":"Material And Method","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and reagents\u003c/h2\u003e \u003cp\u003eHuman HCC Cell lines SK-Hep1, HepG2 and Huh-7 and human immortalized liver Cell lines THLE-2 and THLE-5 were purchased from the National Collection of Authenticated Cell Culyures. HCC cell lines were grown in RPMI-1640 (Hyclone, Salt Lake City, Utah, USA) containing 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials, Hangzhou, China). Human immortalized liver cells were cultured in RPMI-1640 containing 15% fetal bovine serum. Stored in 5%CO\u003csub\u003e2\u003c/sub\u003e, 37℃ constant temperature incubator for culture.\u003c/p\u003e \u003cp\u003eThe main reagents used in this study are as follows: IL-6protein was purchased from Sino Biological LNC, It is soluble in MP Biomedicals (CA, USA) at 100mg/mL. Tocilizumab was purchased from MedChemExpress, it goes into dimethyl sulfoxide. Sorafenib purchased from MedChem Express (Monmouth Junction, NJ, USA), dissolved in dimethyl sulfoxide. IL-6R antibody was purchased from Thermofisher. Cleaved Caspase-7, Caspase-7, Cleaved Caspase-3, Caspase-3, phospho-P70S6K, P70S6K, MMP9, MMP2, phospho-JAK2, JAK2, Phospho-Stat3, STAT3, β -actin and Secondary horseradish peroxidase (HRP) Conjugated goat anti-rabbit antibodies are purchased from Cell Signal Technology (Danvers, MA, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e\n\u003cp\u003eThe cells were placed in the culture dish for about 3 days, and the cells were basically covered at the bottom of the cell culture dish, when there were about 1.0*10\u003csup\u003e7\u003c/sup\u003e cells. The cells were placed in protein phosphatase and loading buffer mixed at a ratio of 50:1. The cells were first fully lysed on a shaker for about half an hour, then centrifuged in a low-temperature centrifuge for 30min. Finally, 5\u0026times; Loading buffer was added and mixed, and then the cells were placed in a water bath at 99 \u0026deg; C and fully heated for about half an hour. Cool naturally at room temperature, centrifuge and store in the refrigerator at minus 20 degrees. Remove when in use. Whole cell lysates of different treatment groups were prepared, and the extracted total protein was dissolved in 10% SDS-PAGE and transferred to PVDF membrane (EMD Millipore). The membrane was blocked in 5% skim milk at 37\u0026deg;C for 1h. After blocking, the membrane was incubated with specific antibody at 4\u0026deg;C overnight, and the secondary antibody (diluted 1:2000) was incubated at room temperature for 1h. Bands were visualized and exposed with the ECL Luminescence Kit (EMD Millipore), and ImageJ 1.44P software (National Institutes of Health, National Institutes of Health) was used for immunoblot quantification. The gray values of different imprinted signals were compared with the control group to analyze the expression of target gene protein.\u003c/p\u003e\n\u003ch3\u003eImmunocytochemistry\u003c/h3\u003e\n\u003cp\u003eThe cells with a concentration of about 2.0\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL were planted in 24-well plates, and the cells were observed under a microscope when the cell density reached about 80% after 24h growth in the incubator. Wash with PBS twice and fix in 4% paraformaldehyde for 15 min, then seal with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 15 min. Incubate with closed serum at room temperature for 20min. 200 \u0026micro;L primary antibody (IL-6R, 1:100, PA5-102425) was added to each well and incubated overnight in a refrigerator at 4\u0026deg;C. Add the second antibody working solution and incubate for 30min at room temperature. The streptomycin working solution was added with horseradish peroxidase and incubated at room temperature for 30 min. DAB chromogenic solution (Beyotime Biotechnology, Shanghai,China) was used for dark staining for 15min, and hematoxylin (Beyotime Biotechnology, Shanghai,China) was used for nuclear staining for 10min. Finally, it was dehydrated with 95% alcohol and sealed with neutral resin for two seconds before being photographed under a microscope. H2O2, serum, secondary antibody and horseradish labeled streptomycin working solution were all products of SP kit (SP-9000, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.).\u003c/p\u003e\n\u003ch3\u003eEdu Method To Detect Cell Viability\u003c/h3\u003e\n\u003cp\u003eAbout 1.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were planted in 24-well plates, and the experiment was carried out when the cells stuck to the wall and the growth basically covered the bottom of the petri dish for about two days. First replace the culture medium, add 0.5 microliter EdU reagent and then put it into the incubator for incubation for about 3 hours. Then take it out and wash it with PBS washing solution for 3 times for about 3 to 5 minutes each time. Apply 4% paraformaldehyde for about 15 minutes. The percentage of EdU positive cells was observed under a fluorescence microscope to measure cell proliferation.\u003c/p\u003e\n\u003ch3\u003eJc-1 Measures Cell Apoptosis\u003c/h3\u003e\n\u003cp\u003e1.0\u0026times;10\u003csup\u003e6\u003c/sup\u003e cell suspension was prepared and planted on a 24-well plate and incubated in an incubator. The cells were adherent for 24h and then treated with 1\u0026micro;M sorafenib. 48h later, the slipper was removed for experiment. Fix with 4% paraformaldehyde for 15min. It was then stained with JC-1 reagent for about 30 minutes. The nuclei were stained with DAPI for 10 minutes. The apoptosis level was determined by observing the intensity of red-green fluorescence under fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eTranswell\u003c/h3\u003e\n\u003cp\u003eAfter the cells were starved in serum-free medium for 24h, about 10\u003csup\u003e5\u003c/sup\u003e~10\u003csup\u003e6\u003c/sup\u003e 200\u0026micro; L cell suspensions were taken and planted in Transwell cells in the middle and upper chambers of 24-well plates. 750\u0026micro;L medium containing 15% serum was added to the lower chamber of Transwell and placed in an incubator for 24h before absorption of the medium. Remove the chamber and transfer it to PBS for cleaning twice. Methanol was fixed at room temperature for 15 min, 0.1% crystal violet was dyed at room temperature for 15 min. Finally, the film was sealed with gum and photographed with fluorescence microscope. The number of cells is a measure of how invasive a cell is.\u003c/p\u003e\n\u003ch3\u003eScratch Healing Experiment\u003c/h3\u003e\n\u003cp\u003eThe cells were measured with a ruler and marked with a \"horizontal line\" on the back of the petri dish before inoculation. The cells were diluted to a certain extent and then planted in a petri dish and cultured in an incubator until they were spread to the bottom of the dish and then removed, scratch the line perpendicular to the ground with the 10 microliter point of the ruler. The scar widths of 0h, 24h and 48h were photographed by microscope and cell mobility was calculated.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eAbout 2.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were planted on a 24-well plate. After incubation for 24h in a constant temperature incubator of 37℃ and 5%CO\u003csub\u003e2\u003c/sub\u003e, fixation with 4% paraformaldehyde for 20min. Dye with hematoxylin for 15 minutes. Eosin was stained for 5min. Finally, the film was sealed with neutral gum and photographed under a positive fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eClone Formation Experiment\u003c/h3\u003e\n\u003cp\u003eAbout 1,000 cells are planted evenly in a six-well plate, Culture in 37℃ constant temperature incubator for 10 days. When the clone group in the orifice are visible to the naked eye. The cells were fixed with 4% paraformaldehyde for 15min at room temperature. After fixation, it was stained with crystal violet for 15min and photographed.\u003c/p\u003e\n\u003ch3\u003eXenotransplantation Of Nude Mice\u003c/h3\u003e\n\u003cp\u003e All experiments were performed in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines and in accordance with the principles and procedures approved by the Animal Experiment Ethics Committee of Anhui University of Science and Technology (Anhui Province, People's Republic of China). Nude mice (female, 6 weeks old) were provided by Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. SK-Hep1, SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/sup\u003e, SK-Hep1\u003csup\u003eSTAT3\u0026minus;\u003c/sup\u003e and SK-Hep1\u003csup\u003eIL\u0026thinsp;\u0026minus;\u0026thinsp;6+STAT3\u0026minus;\u003c/sup\u003e were implanted into the subcutaneous ribs of nude mice, respectively. The body weight and tumor volume of the mice were recorded three days later, and the mice were sacrificed on day 30.\u003c/p\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eExperimental data were obtained through at least three independent experiments. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical differences between the two groups were analyzed using the T-test. Analysis of variance (ANOVA) was used to compare differences between multiple groups. Tukey test to compare the mean values of multiple experimental groups. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is considered a statistically significant difference. GraphPad 8 statistical software was used for all analyses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Ethics Committee of Medical College of Anhui University of Science and Technology and performed following the animal care and use guidelines of the National Institutes of Health of USA.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u003c/strong\u003eThis work was supported by grants from the University Natural Science Research Project of Anhui Province(grant nos. YJS20210407), National Natural Science Fund of China\u0026nbsp;(NO. 82071862, 81872017), Research Foundation of the Institute of Environment-friendly Materials and Occupational Health (Wuhu), Anhui University of Science and Technology (ALW2020YF11), University Natural Science Research Project of Anhui Province (NO. KJ2019A0093, KJ2020A0340), Teaching Research Project of Anhui University of Science and Technology(NO. 2021xjjy59) and 2021 provincial quality engineering \u0026ldquo;Four new\u0026rdquo; research and reform practice project (NO.2021sx032).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author reports no conflicts of interest in this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLi Song, Ruyue Xu and Wenpeng Cai designed and performed this research; Jiaojiao Liang, Niandie Cao, Jiafeng Gao and Xiaolong Tang analysed data; Li Song, Ruyue Xu, Wenpeng Cai and Xiaolong Tang draughted the manuscript; all authors approved the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFoerster, F, Gairing, SJ, Muller, L, Galle, PR. NAFLD-driven HCC: Safety and efficacy of current and emerging treatment options. J Hepatol,.2022; 76(2): p. 446-457.\u003c/li\u003e\n\u003cli\u003eWang W, Wei C. Advances in the early diagnosis of hepatocellular carcinoma. Genes Dis. 2020;7(3):308-319. \u003c/li\u003e\n\u003cli\u003eWei L, Lee D, Law CT, Zhang MS, Shen J, Chin DW, Zhang A, Tsang FH, Wong CL, Ng IO, Wong CC, Wong CM. Genome-wide CRISPR/Cas9 library screening identified PHGDH as a critical driver for Sorafenib resistance in HCC. Nat Commun, 2019 ;10(1): 4681.\u003c/li\u003e\n\u003cli\u003eTang W, Chen Z, Zhang W, Cheng Y, Zhang B, Wu F, Wang Q, Wang S, Rong D, Reiter FP, De Toni EN, Wang X. The mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects. Signal Transduct Target Ther; 2020; 5(1): 87.\u003c/li\u003e\n\u003cli\u003eKeating, GM, Santoro, Sorafenib: a review of its use in advanced hepatocellular carcinoma. Drugs. 2009; 69(2): 223-240.\u003c/li\u003e\n\u003cli\u003eZhang M, Zhang S, Yang Z, Hu J, Hu W, Sun P, Wu L, Han B. Association between the expression levels of IL-6 and IL-6R in the hepatocellular carcinoma microenvironment and postoperative recurrence. Oncol Lett. 2018;16(6):7158-7165.\u003c/li\u003e\n\u003cli\u003eShi X, Kaller M, Rokavec M, Kirchner T, Horst D, Hermeking H. Characterization of a p53/miR-34a/CSF1R/STAT3 Feedback Loop in Colorectal Cancer. Cell Mol Gastroenterol Hepatol. 2020;10(2):391-418. \u003c/li\u003e\n\u003cli\u003eMishra, AK, Dingli D. Metformin inhibits IL-6 signaling by decreasing IL-6R expression on multiple myeloma cells. Leukemia. 2019; 33(11): 2695-2709.\u003c/li\u003e\n\u003cli\u003eSanter FR, Malinowska K, Culig Z, Cavarretta IT. Interleukin-6 trans-signalling differentially regulates proliferation, migration, adhesion and maspin expression in human prostate cancer cells. Endocr Relat Cancer. 2010;17(1):241-253.\u003c/li\u003e\n\u003cli\u003eWeng YS, Tseng HY, Chen YA, Shen PC, Al Haq AT, Chen LM, Tung YC, Hsu HL. MCT-1/miR-34a/IL-6/IL-6R signaling axis promotes EMT progression, cancer stemness and M2 macrophage polarization in triple-negative breast cancer. Mol Cancer. 2019;18(1):42.\u003c/li\u003e\n\u003cli\u003eBharti R, Dey G, Das AK, Mandal M. Differential expression of IL-6/IL-6R and MAO-A regulates invasion/angiogenesis in breast cancer. Br J Cancer. 2018;118(11):1442-1452.\u003c/li\u003e\n\u003cli\u003eXu J, Lin H, Wu G, Zhu M, Li M. IL-6/STAT3 Is a Promising Therapeutic Target for Hepatocellular Carcinoma. Front Oncol. 2021;11:760971.\u003c/li\u003e\n\u003cli\u003eMengie Ayele T, Tilahun Muche Z, Behaile Teklemariam A, Bogale Kassie A, Chekol Abebe E. Role of JAK2/STAT3 Signaling Pathway in the Tumorigenesis, Chemotherapy Resistance, and Treatment of Solid Tumors: A Systemic Review. J Inflamm Res. 2022;15:1349-1364. \u003c/li\u003e\n\u003cli\u003eJaśkiewicz A, Domoradzki T, Pająk B. Targeting the JAK2/STAT3 Pathway-Can We Compare It to the Two Faces of the God Janus? Int J Mol Sci. 2020;21(21):8261. \u003c/li\u003e\n\u003cli\u003eYim SH, Chung YJ. An Overview of Biomarkers and Molecular Signatures in HCC. Cancers (Basel). 2010;2(2):809-823.\u003c/li\u003e\n\u003cli\u003eWang T, Xu L, Jia R, Wei J. MiR-218 suppresses the metastasis and EMT of HCC cells via targeting SERBP1. Acta Biochim Biophys Sin (Shanghai). 2017;49(5):383-391.\u003c/li\u003e\n\u003cli\u003eSamant H, Amiri HS, Zibari GB. Addressing the worldwide hepatocellular carcinoma: epidemiology, prevention and management. J Gastrointest Oncol. 2021;12(Suppl 2):S361-S373.\u003c/li\u003e\n\u003cli\u003eZhong Q, Fang Y, Lai Q, Wang S, He C, Li A, Liu S, Yan Q. CPEB3 inhibits epithelial-mesenchymal transition by disrupting the crosstalk between colorectal cancer cells and tumor-associated macrophages via IL-6R/STAT3 signaling. J Exp Clin Cancer Res. 2020;39(1):132.\u003c/li\u003e\n\u003cli\u003eSimondurairaj C, Krishnakumar R, Sundaram S, Venkatraman G. Interleukin-6 Receptor (IL-6R) Expression in Human Gastric Carcinoma and its Clinical Significance. Cancer Invest. 2019;37(7):293-298.\u003c/li\u003e\n\u003cli\u003eCamacho X, Perroni C, Machado CL, de Godoi Carneiro C, de Souza Junqueira M, Faria D, Garc\u0026iacute;a MF, Fern\u0026aacute;ndez M, Oddone N, Benech J, Buchpiguel CA, Cerecetto H, Chammas R, Riva E, Cabral P, Gambini JP. 99mTechnetium- or Cy7-Labeled Fab(Tocilizumab) as Potential Multiple Myeloma Imaging Agents. Anticancer Agents Med Chem, 2021. 21(14): p. 1883-1893.\u003c/li\u003e\n\u003cli\u003eArmakolas A, Dimakakos A, Loukogiannaki C, Armakolas N, Antonopoulos A, Florou C, Tsioli P, Papageorgiou E, Alexandrou TP, Stathaki M, Spinos D, Pektasides D, Patsouris E, Koutsilieris M. IL-6 is associated to IGF-1Ec upregulation and Ec peptide secretion, from prostate tumors. Mol Med, 2018. 24(1): p. 6.\u003c/li\u003e\n\u003cli\u003eLibertini SJ, Chen H, al-Bataina B, Koilvaram T, George M, Gao AC, Mudryj M. The interleukin 6 receptor is a direct transcriptional target of E2F3 in prostate tumor derived cells. Prostate. 2012;72(6):649-660.\u003c/li\u003e\n\u003cli\u003eBharti R, Dey G, Ojha PK, Rajput S, Jaganathan SK, Sen R, Mandal M. Diacerein-mediated inhibition of IL-6/IL-6R signaling induces apoptotic effects on breast cancer. Oncogene. 2016 Jul 28;35(30):3965-3975.\u003c/li\u003e\n\u003cli\u003eHerzog AE, Warner KA, Zhang Z, Bellile E, Bhagat MA, Castilho RM, Wolf GT, Polverini PJ, Pearson AT, N\u0026ouml;r JE. The IL-6R and Bmi-1 axis controls self-renewal and chemoresistance of head and neck cancer stem cells. Cell Death Dis. 2021;12(11):988.\u003c/li\u003e\n\u003cli\u003eTaher MY, Davies DM, Maher J. The role of the interleukin (IL)-6/IL-6 receptor axis in cancer. Biochem Soc Trans. 2018;46(6):1449-1462.\u003c/li\u003e\n\u003cli\u003eKampan NC, Xiang SD, McNally OM, Stephens AN, Quinn MA, Plebanski M. Immunotherapeutic Interleukin-6 or Interleukin-6 Receptor Blockade in Cancer: Challenges and Opportunities. Curr Med Chem. 2018;25(36):4785-4806.\u003c/li\u003e\n\u003cli\u003eZhong L, Xu Z, Jin X, He Y, Zhang J, Jiang T, Chen J. miR-451a suppression of IL-6R can inhibit proliferation and increase apoptosis through the JAK2/STAT3 pathway in multiple myeloma. Oncol Lett. 2020;20(6):339.\u003c/li\u003e\n\u003cli\u003eBharadwaj U, Marin-Muller C, Li M, Chen C, Yao Q. Mesothelin overexpression promotes autocrine IL-6/sIL-6R trans-signaling to stimulate pancreatic cancer cell proliferation. Carcinogenesis. 2011;32(7):1013-1024.\u003c/li\u003e\n\u003cli\u003eRokavec M, \u0026Ouml;ner MG, Li H, Jackstadt R, Jiang L, Lodygin D, Kaller M, Horst D, Ziegler PK, Schwitalla S, Slotta-Huspenina J, Bader FG, Greten FR, Hermeking H. IL-6R/STAT3/miR-34a feedback loop promotes EMT-mediated colorectal cancer invasion and metastasis. J Clin Invest. 2014;124(4):1853-1867. \u003c/li\u003e\n\u003cli\u003eHirano T. IL-6 in inflammation, autoimmunity and cancer. Int Immunol. 2021;33(3):127-148.\u003c/li\u003e\n\u003cli\u003eJohnson DE, O\u0026apos;Keefe RA, Grandis JR. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat Rev Clin Oncol. 2018;15(4):234-248.\u003c/li\u003e\n\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":"IL-6R, IL-6, JAK2/STAT3, HCC","lastPublishedDoi":"10.21203/rs.3.rs-2111577/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2111577/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\n\u003cp\u003eThe progression of hepatocellular carcinoma (HCC) involves multi-factor, multi-step interactions. It has been reported that the high expression of interleukin-6 receptor (IL-6R) plays an important role in the occurrence and development of tumors, but the regulation mechamismof IL-6R expression and its function in HCC have not been adequately reported.\u003c/p\u003e\n\u003ch2\u003eMethods\u003c/h2\u003e\n\u003cp\u003eWestern Blot was used to evaluate the phosphorylation of key kinases in JAK2/STAT3 pathway and the protein expression levels of related proliferative molecules, migration molecules and apoptotic molecules. The anti-apoptosis, migration and proliferation abilities of cells of each group were analyzed using JC-1 measures cell apoptosis, EdU method to detect cell apoptosis, clone formation experiment and Transwell.\u003c/p\u003e\n\u003ch2\u003eResult\u003c/h2\u003e\n\u003cp\u003eThe expression of IL-6R in HCC cells (HepG2, Huh7 and SK-Hep1) was higher than that in normal hepatocytes (THLE-2 and THLE-5), and the protein expression of IL-6R was relatively highest in SK-Hep1 and relatively lowest in HepG2. Compared with the HepG2\u003csup\u003eIL − 6\u003c/sup\u003e cell line, the protein levels of apoptotic molecules c-Caspase7 and c-Caspase3 were lower, while the protein levels of proliferative molecules p-P70S6K and migration molecules MMP9 and MMP2 were higher, showing stronger anti-apoptosis, proliferation and migration abilities. Compared with SK-Hep1 in SK-Hep1\u003csup\u003eTCZ\u003c/sup\u003e and SK-Hep1\u003csup\u003eIL − 6R−\u003c/sup\u003e, the protein levels of apoptotic molecules c-Caspase7 and c-Caspase3 were higher, while the protein levels of proliferative molecules p-P70S6K and migration molecules MMP9 and MMP2 were lower. It showed strong apoptotic ability and low proliferation and migration ability. Interestingly, IL-6 up-regulated the expression of IL-6R by activating JAK2/STAT3 signaling pathway. The expression of IL-6R protein was also down-regulated after lentivirus knockdown of STAT3. In subcutaneous tumor-bearing experiments in nude mice, compared with SK-Hep1 group, the up-regulation of IL-6R expression after JAK2/STAT3 signaling pathway activation by IL-6 in SK-Hep1\u003csup\u003eIL − 6\u003c/sup\u003e group significantly improved the tumor growth ability. However, the expression of IL-6R protein was down-regulated and the terminal tumor volume was significantly down-regulated in the lentiviral STAT3 knockdown group, which inhibited the tumor growth ability.\u003c/p\u003e\n\u003ch2\u003eConclusions\u003c/h2\u003e\n\u003cp\u003eThe results showed that IL-6 regulated the transcription of IL-6R through the activation of JAK2/STAT3 signaling pathway, thereby promoting the progression of HCC. The result are expected to provide experimental basis for IL-6R as a potential therapeutic target for HCC.\u003c/p\u003e","manuscriptTitle":"IL-6 up-regulates the expression of IL-6R through JAK2/STAT3 signaling pathway to promote HCC progression","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-11-07 14:55:40","doi":"10.21203/rs.3.rs-2111577/v2","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}},{"code":1,"date":"2022-10-03 18:08:55","doi":"10.21203/rs.3.rs-2111577/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":"aff0b3f1-1c43-44cb-952a-3966fc623b35","owner":[],"postedDate":"November 7th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-12-06T00:29:15+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-07 14:55:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-2111577","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2111577","identity":"rs-2111577","version":["v2"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}