HTR1D regulates the PI3K/Akt signaling pathway to impact hepatocellular carcinoma development and resistance to sorafenib

HTR1D regulates the PI3K/Akt signaling pathway to impact hepatocellular carcinoma development and resistance to sorafenib | 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 HTR1D regulates the PI3K/Akt signaling pathway to impact hepatocellular carcinoma development and resistance to sorafenib Yingai Zhang, Yuting Zhang, Shuai Zhou, Mujeeb Ur Rehman, Fankai Lin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4447882/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Hepatocellular carcinoma (HCC) is a form of cancer with high incidence rates and low survival rates worldwide. Oral sorafenib is a promising therapy for advanced HCC, but resistance to the drug limits its effectiveness. HTR1D, a gene that is highly expressed in HCC, plays a crucial role in the development, drug resistance, and prognosis of the disease. Methods Firstly, the correlation between HTR1D and hepatocellular carcinoma was analyzed by TCGA database, and the expression level of HTR1D in clinical samples was detected by qPCR. Then the siRNA was transfected into HuH-7 and HEP3B cells, and the cell proliferation ability, colony formation ability, migration and invasion ability were detected with or without sorafenib. And the expression of PI3K/Akt pathway was detected by Western Blot. Finally, the potential of HTR1D as a predictive marker for patient prognosis was evaluated by immunohistochemistry Results Analysis of TCGA data showed that methylation of the HTR1D gene was associated with cancer status. Clinical samples confirmed significant differences in HTR1D expression between HCC and adjacent tissues, with higher expression correlating with poorer patient prognosis. Interference with HTR1D gene expression demonstrated its role in promoting HCC proliferation, migration, and drug resistance through the PI3K/Akt pathway. These findings were validated in a mouse model. Immunohistochemical analysis of clinicopathological samples suggested HTR1D could be a valuable prognostic marker for HCC. Conclusion HTR1D is highly expressed in hepatocellular carcinoma tissues, and it can influence hepatocellular carcinoma development and resistance to sorafenib by regulating the PI3K/Akt signaling pathway. In addition, HTR1D has potential as a prognostic indicator. Hepatocellular carcinoma HTR1D Sorafenib resistance PI3K/Akt Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Hepatocellular carcinoma (HCC) is one of the most common cancers in the word. It is associated with high morbidity and mortality, and is a serious threat to human life [ 1 , 2 ]. Over the past decades, hepatectomy, ablation, and liver transplantation have been the most likely curative strategies, but they need to be performed in the early stages of HCC [ 3 , 4 ]. Unfortunately, despite careful surveillance, a large proportion of patients with HCC are determined to have intermediate to advanced cancer and are often ineligible for radical treatment [ 5 ]. In these patients, systemic therapy remains essential [ 6 ]. Oral sorafenib, a kinase inhibitor, is the most promising treatment strategy in advanced cases [ 7 ]. However, less than one-third of patients benefit from treatment, and resistance develops within six months of initiating therapy [ 8 , 9 ]. Therefore, identifying the regulatory networks critical for sorafenib's antitumor efficacy will help to rationally design novel therapeutic approaches for HCC. The HTR1D gene encodes a protein called 5-hydroxytryptamine receptor 1D (5-HTR1D), which is known to be a specific receptor for the neurotransmitter 5-hydroxytryptamine (5-HT), which plays a critical role in the human nervous system [ 8 , 10 , 11 ]. Studies have shown that HTR1D may play an important role in the tumorigenesis, development, and prognosis in various types of cancer[ 12 – 14 ]. In addition, HTR1D has been found to be highly expressed in HCC, with the expression level closely associated with patient prognosis [ 15 ]. However, research into the factors influencing HTR1D in the pathogenesis of HCC is still limited. Therefore, it is important to explore the mechanism of the HTR1D gene in HCC. The phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway is important in tumor regulation, affecting cancer cell survival, proliferation, invasion, metastasis, and drug resistance [ 16 ]. Studies have shown that the PI3K/Akt signaling pathway is associated with acquired resistance to sorafenib in HCC and that inhibition of this pathway can reverse sorafenib-derived chemoresistance in HCC [ 17 , 18 ]. Additionally, it has been suggested that HTR1D promotes malignant regression of pancreatic cancer through the PI3K-AKT signaling pathway [ 13 ]. However, the association of HTR1D with either the PI3K-AKT signaling pathway or sorafenib resistance has not been reported in HCC. This study aims to explore the potential of HTR1D as a prognostic target for HCC patients and investigate its role in the development of HCC. The study will also investigate the impact of changes in HTR1D expression on HCC at the cellular and animal level, as well as its effect on sorafenib resistance in HCC cells. The potential mechanism of how HTR1D is involved in regulating sorafenib resistance in HCC will be explored. 2 Materials and methods 2.1 TCGA data collection and analysis The TCGA database was utilized to gather information on HTR1D gene expression in hepatocellular carcinoma tissues. The data was then analyzed to determine the correlation between HTR1D gene expression and various clinicopathological factors of HCC patients, including age, gender, TNM stage classification, TP53 mutation status, etc. Additionally, the correlation between HTR1D gene expression and patients' survival time was also examined. 2.2 Clinical sample collection Four liver cancer samples and four paraneoplastic control samples were collected from fresh tissues during surgeries at the Department of Hepatobiliary Surgery, Haikou People's Hospital, between June 2020 and February 2023. The samples were promptly stored in liquid nitrogen after resection. Pathologists evaluated each sample independently to determine whether it was HCC or paraneoplastic control tissue. Clinicopathological information on the patients, such as age, sex, TNM stage, and survival time, was retrieved from hospital records. In addition, 88 paraffin-embessed tissue sections were obtained for further analysis. 2.3 Immunohistochemical analysis Human liver microarrays (HLivH180Su30) were purchased from Shanghai Xinchao Biotechnology Co. Immunohistochemical (IHC) staining with a 5HT1D antibody was then performed on the microarrays using a staining kit from Bios Biological Technology Co., Ltd. 2.4 Quantitative real-time fluorescent PCR (qRT-PCR) Total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific), and RNA concentration and quality were assessed using NanoDrop 2000 (Thermo Fisher Scientific). RNA was reverse transcribed using the PrimeScript RT Kit (Takara, Japan), and the cDNA region of interest was amplified using TB Green™ Fast qPCR Mix (Takara). After normalization to GAPDH protein levels, relative gene expression levels were determined using the 2-ΔCt method. 2.5 Cell culture HuH-7 cells and HEP3B cells were purchased from Guangzhou Gineo Biotechnology Co. Cells were cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 1% penicillin-streptomycin (Thermo Fisher Scientific, USA), and 5 µg/mL plasmocin (InvivoGen, France). Cells were maintained at 37°C in a 5% CO 2 atmosphere. 2.6 Cell transfection Cells were collected and transferred to cell culture vessels and added to 6-well plates at a rate of 2 mL per well. The inoculated 6-well plate was then placed in a cell culture environment. When the cell count of approximately 60%-70% is observed, the transfection process can begin. The first step is to mix the transfection reagent, small RNA preservation solution, and transfection medium according to the product guide to form the transfection compound solution, then gently drop the compound solution into the wells to be transfected one by one, and then place the cells back into the cell culture incubator to continue incubation, and then wait for 48–72 h, and then decide on the next step according to the needs of the experiment. 2.7 CCK8 assay for cell viability Cells were continuously monitored for 0, 24, 48, 72 and 96 h after treatment. At the end of the assay, all the culture medium was removed, the cells were washed once with PBS, 10 µL of CCK8 solution and 90 µL of complete culture medium were added and incubated for 30 minutes at 37°C. At the end of the incubation, the OD value of the cells was detected by enzyme labeling at 450 nm. The proliferation rate of the cells or the multiplicity of the NC group was calculated, and then the proliferation curve was plotted 2.8 Flow cytometry to detect apoptosis Flow cytometry was used to detect apoptosis. The cells were treated according to the experimental protocol and then removed from the incubator. The supernatant was discarded and the cells were rinsed with PBS three times to remove surface material. The cells were digested (following the steps outlined in section 3.2.3, using 0.25% trypsin without EDTA), and then collected by centrifugation at 4°C for 10 minutes at 1000 r/min. After discarding the supernatant, the cells were washed with PBS twice. The cells were then centrifuged again at 1000 r/min for 10 minutes at 4°C, the supernatant was removed, and the cells were resuspended in flow buffer. The cell density was adjusted to approximately 5×10 4 /mL. A mixture of 5 µL Annexin V-FITC staining solution and 5 µL PI staining solution was added to the cell suspension, which was then incubated in a dark, room temperature environment on a centrifuge tube rack for 15 minutes to complete the staining. A total of 50,000 cells were analyzed using a flow cytometer, following the specific excitation channel recommended in the kit, to determine the apoptotic percentage of each group. 2.9 Transwell Transwell assays were performed to assess cell migration capacity. Specifically, cells were transfected with different plasmids and then inoculated into the top compartment with serum-free medium, while medium containing 10% FBS was added to the lower compartment. After 48 h of incubation, the remaining cells in the top compartment were removed with a cotton swab and the cells in the lower compartment were stained with 0.1% crystal violet for 10 minutes. Finally, the migrated cells were measured and photographed under a microscope. 2.10 Assessment of Clone Formation Ability The gelatin-coated 6-well plate was seeded with 10,000 cells per well, followed by transfection once cells had adhered to the plate. Cells were cultured for 7 days with a second transfection performed on day 4 to inhibit the HTR1D gene. Afterward, cells were washed with PBS, fixed with 4% paraformaldehyde, stained with crystal violet, and photographed. Cell counting was done using image J software. 2.11 Western Blot Analysis HCC cells were lysed in RIPA buffer containing protease inhibitors on ice for 30 minutes. The lysates were centrifuged, and the supernatant was collected for protein concentration measurement using the Micro BCA Protein Assay Kit. The supernatant was then boiled with 5x Sample Buffer, separated by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with skimmed milk, incubated with primary antibodies overnight, followed by secondary antibodies the next day. Protein signals were visualized using ECL reagents and Bio-Rad Image Lab. Antibodies used include Anti-RhoA, Anti-Cyclin D1, GSK3β, GAPDH, Phospho-Akt(Ser473), and Phospho-PI3K P85 α/β/P55γ. 2.12 Animal Models The HEP3B cell line was revived, expanded, and cultured. When the cell count reached the required tumorigenic level, the cells were adjusted to logarithmic growth phase and then harvested, resuspended in saline, and adjusted to a concentration of 1 × 10 7 cells/mL. Injections were administered in the axillary subcutaneous region of the upper limb of nude mice in a 200 µL volume, with 5 mice in each group. (1) NC group: Intratumoral injection of sh-NC every 2 days at 10 nmol/each. (2) sh-HTR1D group: Intratumoral injection of sh-HTR1D every 2 days at 10 nmol/each. (3) Sorafenib group: Intratumoral injection of Sorafenib (2 mg/kg) every 2 days. (4) sh-HTR1D/Sorafenib group: Intratumoral injection of si-HTR1D (2 mg/kg) and sh-HTR1D (10 nmol/each) every 2 days. 2.13 Statistical Analysis All experimental data were presented as mean ± standard deviation and statistical analysis was conducted using SPSS 19.0 software. T-test was used for comparison between two groups, while one-way analysis of variance was used for comparison among multiple groups. A P -value of less than 0.05 was considered statistically significant and indicative of a significant difference. 3 Results and Discussion 3.1 Correlation between HTR1D and hepatocellular carcinoma analyzed by TCGA database A systematic analysis of the correlation between the HTR1D gene and HCC was conducted using data from the TCGA database. The data on HCC were downloaded from the TCGA website and a bioinformatics analysis was carried out. The analysis revealed a significant increase in the expression of the HTR1D gene in 371 HCC tissues compared to normal tissues (Fig. 1 A). Kaplan-Meier curves were used to assess the association between the HTR1D gene expression and the prognosis of HCC patients. The results showed that patients with high expression of the HTR1D gene (n = 92) had a poorer overall survival rate compared to those with low/medium expression (n = 273), with a statistically significant difference (Fig. 1 B). These findings suggest that the HTR1D gene may play an important role in initiating or progressing of HCC. To further investigate the role of the HTR1D gene in HCC, the correlation between the gene and various clinicopathological factors of the disease was analyzed. The results indicated that there was no significant correlation between the HTR1D gene and conventional factors such as age, gender, and body weight of the patients (Fig. 1 C-E, Fig. S1 A-B). Surprisingly, there was also no significant correlation between the HTR1D gene and the stage grade of HCC, presence of metastasis, or the presence of TP53 gene mutation. This is in contrast to other types of tumors, such as colorectal cancer, where a correlation between the expression of the HTR1D gene and metastasis has been observed [ 8 ](Fig. 1 F-H). The regulatory effect on the HTR1D gene was also investigated, revealing that the promoter methylation level of the gene was lower in HCC tissues (n = 377) compared to normal control tissues (n = 50) (Fig. 1 I). Interestingly, there was no significant difference in the promoter methylation level of the HTR1D gene among cancer tissues with different tumor grades, stages, or with or without metastasis (Fig. S2 A-C). This suggests that the methylation regulation of the HTR1D gene is primarily associated with the occurrence of cancer. 3.2 HTR1D gene promotes proliferation, invasion and migration of HCC cells Based on the results of the TCGA database data analysis, it was determined that the HTR1D gene plays a crucial role in the clinical samples of HCC. To further investigate the potential mechanism of the HTR1D gene, a human liver tissue microarray (HLivH180Su30) was purchased from Shanghai Xinchao Biological Company for immunohistochemistry experiments. The results revealed significant differences in the expression of the HTR1D gene in hepatocellular carcinoma and paracarcinoma tissues, with higher expression correlating with poorer prognostic survival rates of patients (Fig. S3A and B). Additionally, qPCR analysis showed significantly higher mRNA expression levels of HTR1D in HCC tissues compared to paracancerous tissues (Fig. 2 A). These findings suggest that the HTR1D gene may contribute to the progression of HCC. To confirm this hypothesis, three pairs of siRNAs were synthesized and transfected into HuH-7 and HEP3B cells. HTR1D-si2 (Fig.S4), which effectively inhibited HTR1D expression in both cell lines, was chosen for further experiments. Functional assays demonstrated that interfering with HTR1D led to a decrease in the proliferative ability of HuH-7 and HEP3B cells, as shown by CCK8 and colony formation assays (Fig. 2 B-E). Flow cytometry results indicated an increase in apoptotic ratio in these cells upon HTR1D interference (Fig. 2 F, Fig.S5), suggesting HTR1D's role in promoting proliferation by decreasing apoptosis. Transwell assays showed a significant reduction in migration and invasion rates in HuH-7 and HEP3B cells following HTR1D interference (Fig. 2 G and H, Fig.S6). In conclusion, the HTR1D gene plays a promotional role in the proliferation and migration of hepatocellular carcinoma cells. 3.3 Impact of the HTR1D gene on sorafenib resistance We investigated whether HTR1D affects drug resistance in HCC cells, in addition to its role in regulating HCC tumor growth. Therefore, we knocked down HTR1D expression in HuH-7 and HEP3B cells, which were then treated with sorafenib, a multikinase inhibitor that is the only clinical standard of care for patients with advanced HCC, to determine whether reduction of HTR1D expression influenced the sensitivity of HCC cells to it. The CCK8 results showed that after knocking down HTR1D expression, both HuH-7 and HEP3B cells were more sensitive to sorafenib (Fig. 3 A and B). Transwell and colony formation assays also showed that HTR1D silencing enhanced the ability of sorafenib to inhibit the proliferation and migration of HCC cells (Fig. 3 C and D, Fig. S7A-D). Previous studies have shown that HTR1D promotes malignant outcomes in pancreatic cancer through the PI3K-AKT signaling pathway[ 13 ]. To investigate whether the effect of the HTR1D gene on sorafenib resistance is related to the PI3K-AKT signaling pathway, we simultaneously detected the effect of HTR1D knockdown on the PI3K/Akt/RhoA signaling pathway. The results showed that the expression of p-PI3K, p-Akt, RhoA, and CyclinD1 was suppressed after HTR1D knockdown, while GSK3β expression increased (Fig. 3 E and F). Furthermore, treatment of the cells with the PI3K agonist 740Y-P restored biological functions (Fig. 3 I), indicating that HTR1D promotes the proliferation, migration, and drug resistance of HCC cells through the PI3K/Akt pathway. 3.4 In vivo activity studies In vivo activity studies were conducted to verify the activity of the HTR1D gene. Initially, the tumorigenicity of hepatocellular carcinoma cells was observed in vivo by creating an animal subcutaneous tumorigenic model with varying levels of HTR1D expression. The results aligned with the cellular level, showing a reduction in in vivo tumorigenic efficiency after HTR1D expression was suppressed (Fig. 4 A-D). Furthermore, subcutaneous tumor formation in mice was examined after HTR1D knockdown in combination with sorafenib. The findings indicated that the synergistic effect significantly decreased the development of subcutaneous tumors compared to sorafenib alone (Fig. 4 E-H). Additionally, the expression of the PI3K/Akt pathway was evaluated in different treatment groups. It was evident that the HTR1D knockdown group exhibited lower levels of p-PI3K, p-Akt, RhoA, CyclinD1, and higher levels of GSK3β, regardless of whether sorafenib was included or not, compared to the control group (Fig. 4 I-L). This provided further evidence that HTR1D could impact the occurrence and progression of hepatocellular carcinoma through the PI3K/Akt pathway, thereby underscoring its potential as a target for hepatocellular carcinoma treatment. 3.5 Immunohistochemical analysis of HTR1D based on clinicopathologic samples Immunohistochemical analysis of HTR1D was conducted on clinicopathologic samples to explore its potential role in the development of HCC. Using previously collected clinical case samples, we aimed to determine if HTR1D could serve as a predictive marker for patient prognosis in liver tissues. Normal liver tissues did not show any expression of HTR1D, while liver tissues with partial steatosis exhibited weakly positive expression in the cytoplasm and nucleus in the steatotic region (Fig. 5 A and B). This suggests a potential correlation between cytoplasmic expression of HTR1D and patient prognosis. Further analysis of paracancerous tissues collected after surgical resection of cirrhosis revealed high expression of HTR1D in the nucleus of cirrhotic tissues, with varying levels of cytoplasmic expression (Fig. 5 C). A comparison between paracancerous cirrhotic tissues and cancerous tissues indicated that cirrhotic tissues had higher cytoplasmic positivity (Fig. 5 D). A correlation was observed between the results and the clinical information of the patients. It was observed that cirrhotic patients with low or weak cytoplasmic expression of HTR1D had better postoperative outcomes and responded well to chemotherapy. On the other hand, patients with higher cytoplasmic positivity may develop drug resistance, impacting the efficacy of chemotherapy and potentially leading to recurrence. These findings suggest that HTR1D could serve as a prognostic indicator in cirrhotic and hepatocellular carcinoma patients, highlighting its potential utility in clinical practice. 4 Discussion Hepatocellular Carcinoma (HCC) is one of the most common gastrointestinal malignancies globally, and its high incidence and low survival rate are among the highest in the world[ 1 , 2 ]. Oral sorafenib, a kinase inhibitor, is the most promising treatment strategy in advanced cases [ 7 ]. However, less than one-third of patients benefit from treatment, and resistance develops within six months of initiating therapy [ 8 , 9 ]. Therefore, identifying the regulatory networks critical for sorafenib's antitumor efficacy will help to rationally design novel therapeutic approaches for HCC. Although there have been findings that overexpression of HTR1D may lead to decreased overall survival and increased risk of reoccurrence in patients with HCC[ 15 ], the reasons why HTR1D is highly expressed in HCC and the relationship with sorafenib resistance in hepatocellular carcinoma cells remain unclear. In this study, we analyzed the correlation between HTR1D and hepatocellular carcinoma using data from the TCGA database. The results of this study indicate methylation regulation of the HTR1D gene was found to be associated with the presence of cancer. However, there is no significant correlation between the HTR1D gene and traditional factors such as age, gender, and body weight. Surprisingly, we also found no significant correlation between the HTR1D gene and liver cancer stage grade, metastasis, or TP53 gene mutation. Whereas in other types of tumors such as colorectal cancer, there is a strong correlation between HTR1D gene expression and metastasis [ 12 ]. In addition, in the study of Zuo X et al, HTR1D gene expression was correlated with TNM staging and stage grading of hepatocellular carcinoma[ 15 ]. The former may be because the signals involved in the regulation of the HTR1D gene vary greatly in different cancers, thus playing different roles in cancer metastatic behaviors, such as interfering with the EMT process and influencing the expression of MMP enzymes[ 19 ]. The discrepancy between the present study and that reported by Zuo X et al. may be due to differences in sample size and sample selection. The results of the clinical samples confirmed the high expression of the HTR1D gene in hepatocellular carcinoma tissues, which led to the speculation that HTR1D may play a role in promoting the development of hepatocellular carcinoma. To test this hypothesis, we conducted siRNA transfections in HuH-7 and HEP3B cells and observed a significant decrease in proliferation, colony formation, migration, and invasion abilities following HTR1D interference. This is consistent with other reported results [ 12 – 14 ]. Furthermore, we investigated the impact of HTR1D interference on sorafenib resistance and found that both cell lines exhibited increased sensitivity to sorafenib. This suggests that knockdown of HTR1D increases the sensitivity of hepatocellular carcinoma cells to sorafenib. Further experiments demonstrated that HTR1D stimulates proliferation, migration, and drug resistance in HCC cells via the PI3K/Akt pathway. These results were also validated in in vivo experiments in mice. In exploring the role of HTR1D in the development of hepatocellular carcinoma, we are also committed to verifying its potential value in clinical applications. Cirrhosis is one of the important risk factors for hepatocellular carcinoma development, and this study conducted a preliminary investigation on the prognosis of hepatocellular carcinoma caused by cirrhosis[ 20 , 21 ]. Firstly, by testing steatotic liver tissues and cirrhotic tissues, it was observed that the expression of HTR1D in fatty liver tissues was irregular, while in cirrhotic tissues, most of the nuclei showed positive expression of HTR1D with different levels of cytoplasmic expression, which showed differential expression. This demonstrates to some extent the potential of HTR1D as a prognostic indicator, but this study was only validated in a few samples and there was no quantification of HTR1D expression or patient prognosis. The expansion of the number of biological samples and the quantification of HTR1D expression are still needed to evaluate the potential of HTR1D as a clinical prognostic indicator for hepatocellular carcinoma in a more comprehensive manner. 5 Conclusion Analysis of the correlation between HTR1D and hepatocellular carcinoma through the TCGA database revealed that methylation regulation of the HTR1D gene was only associated with the occurrence of cancer. We verified the high expression of HTR1D gene in hepatocellular carcinoma tissues by clinical samples, and the proliferative, colony-forming, migratory, and invasive abilities of both types of hepatocellular carcinoma cells were significantly decreased after HTR1D was disrupted. In addition, we found that both HuH-7 and HEP3B cells were more sensitive to sorafenib after HTR1D expression was knocked down. By WB assay, we found that HTR1D promoted the proliferation, migration and drug resistance of hepatocellular carcinoma cells through the PI3K/Akt pathway. This result was also verified by in vivo experiments in mice. Finally, we performed immunohistochemical detection of HTR1D on collected clinical case samples and found that HTR1D has the potential to be used as a predictive marker for patient prognosis. Declarations Acknowledgments This study was supported by the Key Science and Technology project of Hainan Province (ZDXM2015082, ZDYF2018174), the Haikou science and technology plan project (2022-032) and Hainan academician platform research project (YSPTZX202027). Funding This study was supported by the Key Science and Technology project of Hainan Province (ZDXM2015082, ZDYF2018174), the Haikou science and technology plan project (2022-032) and Hainan academician platform research project (YSPTZX202027). Author Contribution Yingai Zhang, Funding acquisition, conceived and designed the experiments, performed the experiments, authored and reviewed drafts of the paper. Yuting Zhang, Data curation, Methodology,Writing & editing. Shuai Zhou, Resources, Writing -review & editing. Mujeeb Ur Rehman,Writing -review & editing. Fankai Lin, Prepared figures and/or tables. Jianquan Zhang, Funding acquisition, Writing –review & editing. Hailong Zhou, Conceptualization, Funding acquisition, Supervision, Writing – review & editing. Ethics approval and consent to participate This study was conducted under informed consent from all patients and approval from the Ethics Committee of Haikou People's Hospital (approval number: 2023-(Lunshen)-358), and it was implemented following the Helsinki Declaration. Consent for publication Not applicable Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Liu CY, Chen KF, Chen PJ: Treatment of Liver Cancer . Cold Spring Harbor perspectives in medicine 2015, 5 (9):a021535. Wang W, Wei C: Advances in the early diagnosis of hepatocellular carcinoma . Genes & diseases 2020, 7 (3):308-319. Anwanwan D, Singh SK, Singh S, Saikam V, Singh R: Challenges in liver cancer and possible treatment approaches . Biochimica et biophysica acta Reviews on cancer 2020, 1873 (1):188314. Yang C, Zhang H, Zhang L, Zhu AX, Bernards R, Qin W, Wang C: Evolving therapeutic landscape of advanced hepatocellular carcinoma . Nature reviews Gastroenterology & hepatology 2023, 20 (4):203-222. Yang JD, Hainaut P, Gores GJ, Amadou A, Plymoth A, Roberts LR: A global view of hepatocellular carcinoma: trends, risk, prevention and management . Nature reviews Gastroenterology & hepatology 2019, 16 (10):589-604. Couri T, Pillai A: Goals and targets for personalized therapy for HCC . Hepatology international 2019, 13 (2):125-137. Donne R, Lujambio A: The liver cancer immune microenvironment: Therapeutic implications for hepatocellular carcinoma . Hepatology (Baltimore, Md) 2023, 77 (5):1773-1796. Pan J, Xu Y, Song H, Zhou X, Yao Z, Ji G: Extracts of Zuo Jin Wan, a traditional Chinese medicine, phenocopies 5-HTR1D antagonist in attenuating Wnt/β-catenin signaling in colorectal cancer cells . BMC complementary and alternative medicine 2017, 17 (1):506. Tang W, Chen Z, Zhang W, Cheng Y, Zhang B, Wu F, Wang Q, Wang S, Rong D, Reiter FP et al : The mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects . Signal transduction and targeted therapy 2020, 5 (1):87. Xu P, Huang S, Zhang H, Mao C, Zhou XE, Cheng X, Simon IA, Shen DD, Yen HY, Robinson CV et al : Structural insights into the lipid and ligand regulation of serotonin receptors . Nature 2021, 592 (7854):469-473. Okaty BW, Commons KG, Dymecki SM: Embracing diversity in the 5-HT neuronal system . Nature reviews Neuroscience 2019, 20 (7):397-424. Zeng C, Chen Y: HTR1D, TIMP1, SERPINE1, MMP3 and CNR2 affect the survival of patients with colon adenocarcinoma . Oncology letters 2019, 18 (3):2448-2454. Wu W, Li Q, Zhu Z, Li C, Lu P, Zhou X, Huang Y, Liu Y, Wang M, Gong J: HTR1D functions as a key target of HOXA10-AS/miR-340-3p axis to promote the malignant outcome of pancreatic cancer via PI3K-AKT signaling pathway . International journal of biological sciences 2022, 18 (9):3777-3794. Jin X, Li H, Li B, Zhang C, He Y: Knockdown and inhibition of hydroxytryptamine receptor 1D suppress proliferation and migration of gastric cancer cells . Biochem Biophys Res Commun 2022, 620 :143-149. Zuo X, Chen Z, Cai J, Gao W, Zhang Y, Han G, Pu L, Wu Z, You W, Qin J et al : 5-Hydroxytryptamine Receptor 1D Aggravates Hepatocellular Carcinoma Progression Through FoxO6 in AKT-Dependent and Independent Manners . Hepatology (Baltimore, Md) 2019, 69 (5):2031-2047. Glaviano A, Foo ASC, Lam HY, Yap KCH, Jacot W, Jones RH, Eng H, Nair MG, Makvandi P, Geoerger B et al : PI3K/AKT/mTOR signaling transduction pathway and targeted therapies in cancer . Molecular cancer 2023, 22 (1):138. Yang J, Nie J, Ma X, Wei Y, Peng Y, Wei X: Targeting PI3K in cancer: mechanisms and advances in clinical trials . Molecular cancer 2019, 18 (1):26. He Y, Sun MM, Zhang GG, Yang J, Chen KS, Xu WW, Li B: Targeting PI3K/Akt signal transduction for cancer therapy . Signal transduction and targeted therapy 2021, 6 (1):425. Qin G, Luo M, Chen J, Dang Y, Chen G, Li L, Zeng J, Lu Y, Yang J: Reciprocal activation between MMP-8 and TGF-β1 stimulates EMT and malignant progression of hepatocellular carcinoma . Cancer letters 2016, 374 (1):85-95. Moon AM, Singal AG, Tapper EB: Contemporary Epidemiology of Chronic Liver Disease and Cirrhosis . Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association 2020, 18 (12):2650-2666. Singal AG, Zhang E, Narasimman M, Rich NE, Waljee AK, Hoshida Y, Yang JD, Reig M, Cabibbo G, Nahon P et al : HCC surveillance improves early detection, curative treatment receipt, and survival in patients with cirrhosis: A meta-analysis . Journal of hepatology 2022, 77 (1):128-139. Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformationfrom2.docx SupplementaryinformationfromBMC.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4447882","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":307972927,"identity":"1716a441-5d33-4efc-91f0-2156282186f3","order_by":0,"name":"Yingai Zhang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Yingai","middleName":"","lastName":"Zhang","suffix":""},{"id":307972931,"identity":"87bff8a3-97c1-479b-8e10-201a22db568b","order_by":1,"name":"Yuting Zhang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Yuting","middleName":"","lastName":"Zhang","suffix":""},{"id":307972932,"identity":"f4515d81-b93d-45e3-bc1b-ea4f1e5cd360","order_by":2,"name":"Shuai Zhou","email":"","orcid":"","institution":"Affiliated Haikou Hospital of Xiangya Medical College, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Zhou","suffix":""},{"id":307972933,"identity":"aafb7fbd-3d09-4b1d-9f2c-364f861c1aa2","order_by":3,"name":"Mujeeb Ur Rehman","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Mujeeb","middleName":"Ur","lastName":"Rehman","suffix":""},{"id":307972934,"identity":"8ee9a6a6-1230-4a61-8d34-bfc4a6d9d8be","order_by":4,"name":"Fankai Lin","email":"","orcid":"","institution":"Affiliated Haikou Hospital of Xiangya Medical College, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Fankai","middleName":"","lastName":"Lin","suffix":""},{"id":307972935,"identity":"c3c5c43e-48af-4f7e-a3a3-85e52c64db86","order_by":5,"name":"Jianquan Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYBACxmYYSwJEVEjI8ZOo5YyFsWQD0faBtDC2VSRuIKSFuZ352YO3bXZ58rObnz38Ok+CcQMD88NHN/A6jM3ccG5bcjHjnGPmxrLbJJjNGdiMjXPwamEwk+ZtY05slkgwk5bcJsFm2cDDJo1fC/s3oJb6xDaJ9G/SknMkeAwOENTCA7LlcGKPRI6Z5McGCQlitJRJzjl3PHGGRE6ZNMMxCQPJZgJ+Mew/vk3iTVl14vwZ6dskf9TU1fezNz98jFdLA5DggXKYwQxmPMpBQJ4BSQvjDwKqR8EoGAWjYGQCAJYNRB8RhT4RAAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Haikou Hospital of Xiangya Medical College, Central South University","correspondingAuthor":true,"prefix":"","firstName":"Jianquan","middleName":"","lastName":"Zhang","suffix":""},{"id":307972936,"identity":"a6b0277c-3675-4c23-b88d-771dec5ab024","order_by":6,"name":"Hailong Zhou","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Hailong","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2024-05-20 08:53:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4447882/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4447882/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57943072,"identity":"469e9adf-5440-4453-ba2a-e23ba0f37a01","added_by":"auto","created_at":"2024-06-07 19:02:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":185493,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of HTR1D gene expression in hepatocellular carcinoma and paracarcinoma tissues. \u003c/strong\u003eA. HTR1D gene expression levels in HCC tissues compared to paracarcinomas. B. Correlation between HTR1D gene expression and prognostic survival in HCC patients. Association of HTR1D gene expression with patient demographics including C. Race, D. Age, and E. Weight. Association of HTR1D gene expression with F. Nodal metastasis status, G. Cancer stages, and H. TP53 mutation status. I. Promoter methylation levels of HTR1D in LIHC. * indicates statistical significance with \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4447882/v1/2c35dc7ac6c358c0d13382ab.jpg"},{"id":57943070,"identity":"5510d38c-489d-4bed-9d3c-a69db5054013","added_by":"auto","created_at":"2024-06-07 19:02:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":337857,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHTR1D gene enhances proliferation, invasion and migration of hepatocellular carcinoma (HCC) cells.\u003c/strong\u003e A. Quantitative PCR (qPCR) analysis showing HTR1D expression in clinical samples. B-C. Cell Counting Kit-8 (CCK8) assay demonstrating the proliferative capacity of HEP3B and HuH-7 cells after small interfering RNA (siRNA) transfection. D. Representative images and E. quantification of colony formation in HuH-7 and HEP3B cells. F. Flow cytometry analysis of apoptosis in HuH-7 and HEP3B cells. G-H. Transwell assay measuring migration abilities of HuH-7 and HEP3B cells. *\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":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4447882/v1/ccdcdec53dea0c86e76bff81.jpg"},{"id":57943068,"identity":"0ca14a3c-6fbc-46f5-82ab-0fed8736c1c0","added_by":"auto","created_at":"2024-06-07 19:02:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":348528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHTR1D affects cellular resistance to sorafenib via PI3K/Akt pathway. \u003c/strong\u003eA-B. CCK8 assay showing the proliferative capacity of HEP3B and HuH-7 cells after siRNA and sorafenib treatment. C. Representative images of colony formation assay for HuH-7 and HEP3B after siRNA transfection. D. Quantification of colony formation assay for HEP3B. E-F. Western blot analysis showing theexpression levels of p-PI3K, RhoA, p-Akt, CyclinD1, and GSK3β in HEP3B cells. G-H. CCK8 assay showing the proliferative capacity of HEP3B and HuH-7 cells after siRNA and 740Y-P treatment. I. CCK8 assay showing the proliferative capacity of HEP3B cells after siRNA,740Y-P, and sorafenib treatment. ** indicates \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, and *** indicates \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4447882/v1/a974e48c88b2d70b4a6735f8.jpg"},{"id":57943066,"identity":"34c1f6cb-866f-40e6-abfe-59a841419776","added_by":"auto","created_at":"2024-06-07 19:02:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":484744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHTR1Ddeletion enhances sorafenib-mediated inhibition of HCC tumor growth in vivo. A-B.\u003c/strong\u003e Images showing tumor size in mice treated with saline and shHTR1D. \u003cstrong\u003eC. \u003c/strong\u003eTumor weight after 28 days of treatment. \u003cstrong\u003eD.\u003c/strong\u003e Tumor volume progression over 28 days of treatment. \u003cstrong\u003eE-F.\u003c/strong\u003e Images displaying tumor size in mice treated with saline, shHTR1D, and sorafenib. \u003cstrong\u003eG.\u003c/strong\u003e Tumor weight after 28 days of treatment. \u003cstrong\u003eH.\u003c/strong\u003e Tumor volume progression over 28 days of treatment. \u003cstrong\u003eI-L.\u003c/strong\u003e Western blot analysis of p-PI3K, RhoA, p-Akt, CyclinD1, and GSK3β expression levels. *** indicatesstatistical significance at \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4447882/v1/0b45d7541015655b50a4dcc7.jpg"},{"id":57943064,"identity":"4f6f1b00-cea7-4b3f-9996-c17eefbd7049","added_by":"auto","created_at":"2024-06-07 19:02:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":505027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical analysis of HTR1D in different liver tissues. \u003c/strong\u003eA. Representative images showing staining of\u003cstrong\u003e \u003c/strong\u003eHTR1D in normal liver tissue. \u003cstrong\u003eB. \u003c/strong\u003eStaining of HTR1D in steatotic liver tissue. \u003cstrong\u003eC. \u003c/strong\u003ePathologic findings of cirrhotic tissues from various patients. \u003cstrong\u003eD.\u003c/strong\u003e Pathologic findings of cancerous and paracancerous cirrhotic tissues. Scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4447882/v1/b5f4d577f8c114078371b7bd.jpg"},{"id":58108786,"identity":"a2a33281-ae7d-42cc-bc86-23aa2e056a93","added_by":"auto","created_at":"2024-06-11 08:44:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2972373,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4447882/v1/96c41d5f-b738-4f0c-a801-24faf306e83c.pdf"},{"id":57943071,"identity":"905004b5-0fd2-4047-a1ed-fa0d26330275","added_by":"auto","created_at":"2024-06-07 19:02:44","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":769265,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformationfrom2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4447882/v1/6ed808e8c61a400e86c2b8ec.docx"},{"id":57943058,"identity":"ab594a61-02b6-4506-b5e8-963aa83a7e4e","added_by":"auto","created_at":"2024-06-07 19:02:40","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3037275,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationfromBMC.docx","url":"https://assets-eu.researchsquare.com/files/rs-4447882/v1/a61d43572055a3b091dfcfea.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"HTR1D regulates the PI3K/Akt signaling pathway to impact hepatocellular carcinoma development and resistance to sorafenib","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is one of the most common cancers in the word. It is associated with high morbidity and mortality, and is a serious threat to human life [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Over the past decades, hepatectomy, ablation, and liver transplantation have been the most likely curative strategies, but they need to be performed in the early stages of HCC [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Unfortunately, despite careful surveillance, a large proportion of patients with HCC are determined to have intermediate to advanced cancer and are often ineligible for radical treatment [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In these patients, systemic therapy remains essential [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Oral sorafenib, a kinase inhibitor, is the most promising treatment strategy in advanced cases [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, less than one-third of patients benefit from treatment, and resistance develops within six months of initiating therapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, identifying the regulatory networks critical for sorafenib's antitumor efficacy will help to rationally design novel therapeutic approaches for HCC.\u003c/p\u003e \u003cp\u003eThe HTR1D gene encodes a protein called 5-hydroxytryptamine receptor 1D (5-HTR1D), which is known to be a specific receptor for the neurotransmitter 5-hydroxytryptamine (5-HT), which plays a critical role in the human nervous system [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Studies have shown that HTR1D may play an important role in the tumorigenesis, development, and prognosis in various types of cancer[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition, HTR1D has been found to be highly expressed in HCC, with the expression level closely associated with patient prognosis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, research into the factors influencing HTR1D in the pathogenesis of HCC is still limited. Therefore, it is important to explore the mechanism of the HTR1D gene in HCC.\u003c/p\u003e \u003cp\u003eThe phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway is important in tumor regulation, affecting cancer cell survival, proliferation, invasion, metastasis, and drug resistance [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Studies have shown that the PI3K/Akt signaling pathway is associated with acquired resistance to sorafenib in HCC and that inhibition of this pathway can reverse sorafenib-derived chemoresistance in HCC [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, it has been suggested that HTR1D promotes malignant regression of pancreatic cancer through the PI3K-AKT signaling pathway [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the association of HTR1D with either the PI3K-AKT signaling pathway or sorafenib resistance has not been reported in HCC.\u003c/p\u003e \u003cp\u003eThis study aims to explore the potential of HTR1D as a prognostic target for HCC patients and investigate its role in the development of HCC. The study will also investigate the impact of changes in HTR1D expression on HCC at the cellular and animal level, as well as its effect on sorafenib resistance in HCC cells. The potential mechanism of how HTR1D is involved in regulating sorafenib resistance in HCC will be explored.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 TCGA data collection and analysis\u003c/h2\u003e \u003cp\u003eThe TCGA database was utilized to gather information on HTR1D gene expression in hepatocellular carcinoma tissues. The data was then analyzed to determine the correlation between HTR1D gene expression and various clinicopathological factors of HCC patients, including age, gender, TNM stage classification, TP53 mutation status, etc. Additionally, the correlation between HTR1D gene expression and patients' survival time was also examined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Clinical sample collection\u003c/h2\u003e \u003cp\u003eFour liver cancer samples and four paraneoplastic control samples were collected from fresh tissues during surgeries at the Department of Hepatobiliary Surgery, Haikou People's Hospital, between June 2020 and February 2023. The samples were promptly stored in liquid nitrogen after resection. Pathologists evaluated each sample independently to determine whether it was HCC or paraneoplastic control tissue. Clinicopathological information on the patients, such as age, sex, TNM stage, and survival time, was retrieved from hospital records. In addition, 88 paraffin-embessed tissue sections were obtained for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Immunohistochemical analysis\u003c/h2\u003e \u003cp\u003eHuman liver microarrays (HLivH180Su30) were purchased from Shanghai Xinchao Biotechnology Co. Immunohistochemical (IHC) staining with a 5HT1D antibody was then performed on the microarrays using a staining kit from Bios Biological Technology Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Quantitative real-time fluorescent PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific), and RNA concentration and quality were assessed using NanoDrop 2000 (Thermo Fisher Scientific). RNA was reverse transcribed using the PrimeScript RT Kit (Takara, Japan), and the cDNA region of interest was amplified using TB Green\u0026trade; Fast qPCR Mix (Takara). After normalization to GAPDH protein levels, relative gene expression levels were determined using the 2-ΔCt method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell culture\u003c/h2\u003e \u003cp\u003eHuH-7 cells and HEP3B cells were purchased from Guangzhou Gineo Biotechnology Co. Cells were cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 1% penicillin-streptomycin (Thermo Fisher Scientific, USA), and 5 \u0026micro;g/mL plasmocin (InvivoGen, France). Cells were maintained at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cell transfection\u003c/h2\u003e \u003cp\u003eCells were collected and transferred to cell culture vessels and added to 6-well plates at a rate of 2 mL per well. The inoculated 6-well plate was then placed in a cell culture environment. When the cell count of approximately 60%-70% is observed, the transfection process can begin. The first step is to mix the transfection reagent, small RNA preservation solution, and transfection medium according to the product guide to form the transfection compound solution, then gently drop the compound solution into the wells to be transfected one by one, and then place the cells back into the cell culture incubator to continue incubation, and then wait for 48\u0026ndash;72 h, and then decide on the next step according to the needs of the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 CCK8 assay for cell viability\u003c/h2\u003e \u003cp\u003eCells were continuously monitored for 0, 24, 48, 72 and 96 h after treatment. At the end of the assay, all the culture medium was removed, the cells were washed once with PBS, 10 \u0026micro;L of CCK8 solution and 90 \u0026micro;L of complete culture medium were added and incubated for 30 minutes at 37\u0026deg;C. At the end of the incubation, the OD value of the cells was detected by enzyme labeling at 450 nm. The proliferation rate of the cells or the multiplicity of the NC group was calculated, and then the proliferation curve was plotted\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Flow cytometry to detect apoptosis\u003c/h2\u003e \u003cp\u003eFlow cytometry was used to detect apoptosis. The cells were treated according to the experimental protocol and then removed from the incubator. The supernatant was discarded and the cells were rinsed with PBS three times to remove surface material. The cells were digested (following the steps outlined in section 3.2.3, using 0.25% trypsin without EDTA), and then collected by centrifugation at 4\u0026deg;C for 10 minutes at 1000 r/min. After discarding the supernatant, the cells were washed with PBS twice. The cells were then centrifuged again at 1000 r/min for 10 minutes at 4\u0026deg;C, the supernatant was removed, and the cells were resuspended in flow buffer. The cell density was adjusted to approximately 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e /mL. A mixture of 5 \u0026micro;L Annexin V-FITC staining solution and 5 \u0026micro;L PI staining solution was added to the cell suspension, which was then incubated in a dark, room temperature environment on a centrifuge tube rack for 15 minutes to complete the staining. A total of 50,000 cells were analyzed using a flow cytometer, following the specific excitation channel recommended in the kit, to determine the apoptotic percentage of each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Transwell\u003c/h2\u003e \u003cp\u003eTranswell assays were performed to assess cell migration capacity. Specifically, cells were transfected with different plasmids and then inoculated into the top compartment with serum-free medium, while medium containing 10% FBS was added to the lower compartment. After 48 h of incubation, the remaining cells in the top compartment were removed with a cotton swab and the cells in the lower compartment were stained with 0.1% crystal violet for 10 minutes. Finally, the migrated cells were measured and photographed under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Assessment of Clone Formation Ability\u003c/h2\u003e \u003cp\u003eThe gelatin-coated 6-well plate was seeded with 10,000 cells per well, followed by transfection once cells had adhered to the plate. Cells were cultured for 7 days with a second transfection performed on day 4 to inhibit the HTR1D gene. Afterward, cells were washed with PBS, fixed with 4% paraformaldehyde, stained with crystal violet, and photographed. Cell counting was done using image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Western Blot Analysis\u003c/h2\u003e \u003cp\u003eHCC cells were lysed in RIPA buffer containing protease inhibitors on ice for 30 minutes. The lysates were centrifuged, and the supernatant was collected for protein concentration measurement using the Micro BCA Protein Assay Kit. The supernatant was then boiled with 5x Sample Buffer, separated by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with skimmed milk, incubated with primary antibodies overnight, followed by secondary antibodies the next day. Protein signals were visualized using ECL reagents and Bio-Rad Image Lab. Antibodies used include Anti-RhoA, Anti-Cyclin D1, GSK3β, GAPDH, Phospho-Akt(Ser473), and Phospho-PI3K P85 α/β/P55γ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Animal Models\u003c/h2\u003e \u003cp\u003eThe HEP3B cell line was revived, expanded, and cultured. When the cell count reached the required tumorigenic level, the cells were adjusted to logarithmic growth phase and then harvested, resuspended in saline, and adjusted to a concentration of 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells/mL. Injections were administered in the axillary subcutaneous region of the upper limb of nude mice in a 200 \u0026micro;L volume, with 5 mice in each group.\u003c/p\u003e \u003cp\u003e(1) NC group: Intratumoral injection of sh-NC every 2 days at 10 nmol/each.\u003c/p\u003e \u003cp\u003e(2) sh-HTR1D group: Intratumoral injection of sh-HTR1D every 2 days at 10 nmol/each.\u003c/p\u003e \u003cp\u003e(3) Sorafenib group: Intratumoral injection of Sorafenib (2 mg/kg) every 2 days.\u003c/p\u003e \u003cp\u003e(4) sh-HTR1D/Sorafenib group: Intratumoral injection of si-HTR1D (2 mg/kg) and sh-HTR1D (10 nmol/each) every 2 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and statistical analysis was conducted using SPSS 19.0 software. T-test was used for comparison between two groups, while one-way analysis of variance was used for comparison among multiple groups. A \u003cem\u003eP\u003c/em\u003e-value of less than 0.05 was considered statistically significant and indicative of a significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Correlation between HTR1D and hepatocellular carcinoma analyzed by TCGA database\u003c/h2\u003e \u003cp\u003eA systematic analysis of the correlation between the HTR1D gene and HCC was conducted using data from the TCGA database. The data on HCC were downloaded from the TCGA website and a bioinformatics analysis was carried out. The analysis revealed a significant increase in the expression of the HTR1D gene in 371 HCC tissues compared to normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Kaplan-Meier curves were used to assess the association between the HTR1D gene expression and the prognosis of HCC patients. The results showed that patients with high expression of the HTR1D gene (n\u0026thinsp;=\u0026thinsp;92) had a poorer overall survival rate compared to those with low/medium expression (n\u0026thinsp;=\u0026thinsp;273), with a statistically significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These findings suggest that the HTR1D gene may play an important role in initiating or progressing of HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the role of the HTR1D gene in HCC, the correlation between the gene and various clinicopathological factors of the disease was analyzed. The results indicated that there was no significant correlation between the HTR1D gene and conventional factors such as age, gender, and body weight of the patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B). Surprisingly, there was also no significant correlation between the HTR1D gene and the stage grade of HCC, presence of metastasis, or the presence of TP53 gene mutation. This is in contrast to other types of tumors, such as colorectal cancer, where a correlation between the expression of the HTR1D gene and metastasis has been observed [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e](Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003eThe regulatory effect on the HTR1D gene was also investigated, revealing that the promoter methylation level of the gene was lower in HCC tissues (n\u0026thinsp;=\u0026thinsp;377) compared to normal control tissues (n\u0026thinsp;=\u0026thinsp;50) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Interestingly, there was no significant difference in the promoter methylation level of the HTR1D gene among cancer tissues with different tumor grades, stages, or with or without metastasis (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-C). This suggests that the methylation regulation of the HTR1D gene is primarily associated with the occurrence of cancer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2 HTR1D gene promotes proliferation, invasion and migration of HCC cells\u003c/h2\u003e \u003cp\u003eBased on the results of the TCGA database data analysis, it was determined that the HTR1D gene plays a crucial role in the clinical samples of HCC. To further investigate the potential mechanism of the HTR1D gene, a human liver tissue microarray (HLivH180Su30) was purchased from Shanghai Xinchao Biological Company for immunohistochemistry experiments. The results revealed significant differences in the expression of the HTR1D gene in hepatocellular carcinoma and paracarcinoma tissues, with higher expression correlating with poorer prognostic survival rates of patients (Fig. S3A and B).\u003c/p\u003e \u003cp\u003eAdditionally, qPCR analysis showed significantly higher mRNA expression levels of HTR1D in HCC tissues compared to paracancerous tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These findings suggest that the HTR1D gene may contribute to the progression of HCC. To confirm this hypothesis, three pairs of siRNAs were synthesized and transfected into HuH-7 and HEP3B cells. HTR1D-si2 (Fig.S4), which effectively inhibited HTR1D expression in both cell lines, was chosen for further experiments. Functional assays demonstrated that interfering with HTR1D led to a decrease in the proliferative ability of HuH-7 and HEP3B cells, as shown by CCK8 and colony formation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-E). Flow cytometry results indicated an increase in apoptotic ratio in these cells upon HTR1D interference (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, Fig.S5), suggesting HTR1D's role in promoting proliferation by decreasing apoptosis. Transwell assays showed a significant reduction in migration and invasion rates in HuH-7 and HEP3B cells following HTR1D interference (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and H, Fig.S6). In conclusion, the HTR1D gene plays a promotional role in the proliferation and migration of hepatocellular carcinoma cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Impact of the HTR1D gene on sorafenib resistance\u003c/h2\u003e \u003cp\u003eWe investigated whether HTR1D affects drug resistance in HCC cells, in addition to its role in regulating HCC tumor growth. Therefore, we knocked down HTR1D expression in HuH-7 and HEP3B cells, which were then treated with sorafenib, a multikinase inhibitor that is the only clinical standard of care for patients with advanced HCC, to determine whether reduction of HTR1D expression influenced the sensitivity of HCC cells to it. The CCK8 results showed that after knocking down HTR1D expression, both HuH-7 and HEP3B cells were more sensitive to sorafenib (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Transwell and colony formation assays also showed that HTR1D silencing enhanced the ability of sorafenib to inhibit the proliferation and migration of HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D, Fig. S7A-D). Previous studies have shown that HTR1D promotes malignant outcomes in pancreatic cancer through the PI3K-AKT signaling pathway[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To investigate whether the effect of the HTR1D gene on sorafenib resistance is related to the PI3K-AKT signaling pathway, we simultaneously detected the effect of HTR1D knockdown on the PI3K/Akt/RhoA signaling pathway. The results showed that the expression of p-PI3K, p-Akt, RhoA, and CyclinD1 was suppressed after HTR1D knockdown, while GSK3β expression increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). Furthermore, treatment of the cells with the PI3K agonist 740Y-P restored biological functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), indicating that HTR1D promotes the proliferation, migration, and drug resistance of HCC cells through the PI3K/Akt pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4 In vivo activity studies\u003c/h2\u003e \u003cp\u003eIn vivo activity studies were conducted to verify the activity of the HTR1D gene. Initially, the tumorigenicity of hepatocellular carcinoma cells was observed in vivo by creating an animal subcutaneous tumorigenic model with varying levels of HTR1D expression. The results aligned with the cellular level, showing a reduction in in vivo tumorigenic efficiency after HTR1D expression was suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). Furthermore, subcutaneous tumor formation in mice was examined after HTR1D knockdown in combination with sorafenib. The findings indicated that the synergistic effect significantly decreased the development of subcutaneous tumors compared to sorafenib alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H). Additionally, the expression of the PI3K/Akt pathway was evaluated in different treatment groups. It was evident that the HTR1D knockdown group exhibited lower levels of p-PI3K, p-Akt, RhoA, CyclinD1, and higher levels of GSK3β, regardless of whether sorafenib was included or not, compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-L). This provided further evidence that HTR1D could impact the occurrence and progression of hepatocellular carcinoma through the PI3K/Akt pathway, thereby underscoring its potential as a target for hepatocellular carcinoma treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Immunohistochemical analysis of HTR1D based on clinicopathologic samples\u003c/h2\u003e \u003cp\u003eImmunohistochemical analysis of HTR1D was conducted on clinicopathologic samples to explore its potential role in the development of HCC. Using previously collected clinical case samples, we aimed to determine if HTR1D could serve as a predictive marker for patient prognosis in liver tissues. Normal liver tissues did not show any expression of HTR1D, while liver tissues with partial steatosis exhibited weakly positive expression in the cytoplasm and nucleus in the steatotic region (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). This suggests a potential correlation between cytoplasmic expression of HTR1D and patient prognosis. Further analysis of paracancerous tissues collected after surgical resection of cirrhosis revealed high expression of HTR1D in the nucleus of cirrhotic tissues, with varying levels of cytoplasmic expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA comparison between paracancerous cirrhotic tissues and cancerous tissues indicated that cirrhotic tissues had higher cytoplasmic positivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). A correlation was observed between the results and the clinical information of the patients. It was observed that cirrhotic patients with low or weak cytoplasmic expression of HTR1D had better postoperative outcomes and responded well to chemotherapy. On the other hand, patients with higher cytoplasmic positivity may develop drug resistance, impacting the efficacy of chemotherapy and potentially leading to recurrence. These findings suggest that HTR1D could serve as a prognostic indicator in cirrhotic and hepatocellular carcinoma patients, highlighting its potential utility in clinical practice.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eHepatocellular Carcinoma (HCC) is one of the most common gastrointestinal malignancies globally, and its high incidence and low survival rate are among the highest in the world[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Oral sorafenib, a kinase inhibitor, is the most promising treatment strategy in advanced cases [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, less than one-third of patients benefit from treatment, and resistance develops within six months of initiating therapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, identifying the regulatory networks critical for sorafenib's antitumor efficacy will help to rationally design novel therapeutic approaches for HCC. Although there have been findings that overexpression of HTR1D may lead to decreased overall survival and increased risk of reoccurrence in patients with HCC[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], the reasons why HTR1D is highly expressed in HCC and the relationship with sorafenib resistance in hepatocellular carcinoma cells remain unclear.\u003c/p\u003e \u003cp\u003eIn this study, we analyzed the correlation between HTR1D and hepatocellular carcinoma using data from the TCGA database. The results of this study indicate methylation regulation of the HTR1D gene was found to be associated with the presence of cancer. However, there is no significant correlation between the HTR1D gene and traditional factors such as age, gender, and body weight. Surprisingly, we also found no significant correlation between the HTR1D gene and liver cancer stage grade, metastasis, or TP53 gene mutation. Whereas in other types of tumors such as colorectal cancer, there is a strong correlation between HTR1D gene expression and metastasis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, in the study of Zuo X et al, HTR1D gene expression was correlated with TNM staging and stage grading of hepatocellular carcinoma[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The former may be because the signals involved in the regulation of the HTR1D gene vary greatly in different cancers, thus playing different roles in cancer metastatic behaviors, such as interfering with the EMT process and influencing the expression of MMP enzymes[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The discrepancy between the present study and that reported by Zuo X et al. may be due to differences in sample size and sample selection.\u003c/p\u003e \u003cp\u003eThe results of the clinical samples confirmed the high expression of the HTR1D gene in hepatocellular carcinoma tissues, which led to the speculation that HTR1D may play a role in promoting the development of hepatocellular carcinoma. To test this hypothesis, we conducted siRNA transfections in HuH-7 and HEP3B cells and observed a significant decrease in proliferation, colony formation, migration, and invasion abilities following HTR1D interference. This is consistent with other reported results [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, we investigated the impact of HTR1D interference on sorafenib resistance and found that both cell lines exhibited increased sensitivity to sorafenib. This suggests that knockdown of HTR1D increases the sensitivity of hepatocellular carcinoma cells to sorafenib. Further experiments demonstrated that HTR1D stimulates proliferation, migration, and drug resistance in HCC cells via the PI3K/Akt pathway. These results were also validated in in vivo experiments in mice.\u003c/p\u003e \u003cp\u003eIn exploring the role of HTR1D in the development of hepatocellular carcinoma, we are also committed to verifying its potential value in clinical applications. Cirrhosis is one of the important risk factors for hepatocellular carcinoma development, and this study conducted a preliminary investigation on the prognosis of hepatocellular carcinoma caused by cirrhosis[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Firstly, by testing steatotic liver tissues and cirrhotic tissues, it was observed that the expression of HTR1D in fatty liver tissues was irregular, while in cirrhotic tissues, most of the nuclei showed positive expression of HTR1D with different levels of cytoplasmic expression, which showed differential expression. This demonstrates to some extent the potential of HTR1D as a prognostic indicator, but this study was only validated in a few samples and there was no quantification of HTR1D expression or patient prognosis. The expansion of the number of biological samples and the quantification of HTR1D expression are still needed to evaluate the potential of HTR1D as a clinical prognostic indicator for hepatocellular carcinoma in a more comprehensive manner.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eAnalysis of the correlation between HTR1D and hepatocellular carcinoma through the TCGA database revealed that methylation regulation of the HTR1D gene was only associated with the occurrence of cancer. We verified the high expression of HTR1D gene in hepatocellular carcinoma tissues by clinical samples, and the proliferative, colony-forming, migratory, and invasive abilities of both types of hepatocellular carcinoma cells were significantly decreased after HTR1D was disrupted. In addition, we found that both HuH-7 and HEP3B cells were more sensitive to sorafenib after HTR1D expression was knocked down. By WB assay, we found that HTR1D promoted the proliferation, migration and drug resistance of hepatocellular carcinoma cells through the PI3K/Akt pathway. This result was also verified by in vivo experiments in mice. Finally, we performed immunohistochemical detection of HTR1D on collected clinical case samples and found that HTR1D has the potential to be used as a predictive marker for patient prognosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Key Science and Technology project of Hainan Province (ZDXM2015082, ZDYF2018174), the Haikou science and technology plan project (2022-032) and Hainan academician platform research project (YSPTZX202027).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Key Science and Technology project of Hainan Province (ZDXM2015082, ZDYF2018174), the Haikou science and technology plan project (2022-032) and Hainan academician platform research project (YSPTZX202027).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYingai Zhang, Funding acquisition, conceived and designed the experiments, performed the experiments, authored and reviewed drafts of the paper.\u003c/p\u003e\n\u003cp\u003eYuting Zhang, Data curation, Methodology,Writing \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eShuai Zhou, Resources, Writing -review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eMujeeb Ur Rehman,Writing -review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eFankai Lin, Prepared figures and/or tables.\u003c/p\u003e\n\u003cp\u003eJianquan Zhang, Funding acquisition, Writing \u0026ndash;review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eHailong Zhou, Conceptualization, Funding acquisition, Supervision, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted under informed consent from all patients and approval from the Ethics Committee of Haikou People\u0026apos;s Hospital (approval number: 2023-(Lunshen)-358), and it was implemented following the Helsinki Declaration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLiu CY, Chen KF, Chen PJ: \u003cstrong\u003eTreatment of Liver Cancer\u003c/strong\u003e. \u003cem\u003eCold Spring Harbor perspectives in medicine \u003c/em\u003e2015, \u003cstrong\u003e5\u003c/strong\u003e(9):a021535.\u003c/li\u003e\n\u003cli\u003eWang W, Wei C: \u003cstrong\u003eAdvances in the early diagnosis of hepatocellular carcinoma\u003c/strong\u003e. \u003cem\u003eGenes \u0026amp; diseases \u003c/em\u003e2020, \u003cstrong\u003e7\u003c/strong\u003e(3):308-319.\u003c/li\u003e\n\u003cli\u003eAnwanwan D, Singh SK, Singh S, Saikam V, Singh R: \u003cstrong\u003eChallenges in liver cancer and possible treatment approaches\u003c/strong\u003e. \u003cem\u003eBiochimica et biophysica acta Reviews on cancer \u003c/em\u003e2020, \u003cstrong\u003e1873\u003c/strong\u003e(1):188314.\u003c/li\u003e\n\u003cli\u003eYang C, Zhang H, Zhang L, Zhu AX, Bernards R, Qin W, Wang C: \u003cstrong\u003eEvolving therapeutic landscape of advanced hepatocellular carcinoma\u003c/strong\u003e. \u003cem\u003eNature reviews Gastroenterology \u0026amp; hepatology \u003c/em\u003e2023, \u003cstrong\u003e20\u003c/strong\u003e(4):203-222.\u003c/li\u003e\n\u003cli\u003eYang JD, Hainaut P, Gores GJ, Amadou A, Plymoth A, Roberts LR: \u003cstrong\u003eA global view of hepatocellular carcinoma: trends, risk, prevention and management\u003c/strong\u003e. \u003cem\u003eNature reviews Gastroenterology \u0026amp; hepatology \u003c/em\u003e2019, \u003cstrong\u003e16\u003c/strong\u003e(10):589-604.\u003c/li\u003e\n\u003cli\u003eCouri T, Pillai A: \u003cstrong\u003eGoals and targets for personalized therapy for HCC\u003c/strong\u003e. \u003cem\u003eHepatology international \u003c/em\u003e2019, \u003cstrong\u003e13\u003c/strong\u003e(2):125-137.\u003c/li\u003e\n\u003cli\u003eDonne R, Lujambio A: \u003cstrong\u003eThe liver cancer immune microenvironment: Therapeutic implications for hepatocellular carcinoma\u003c/strong\u003e. \u003cem\u003eHepatology (Baltimore, Md) \u003c/em\u003e2023, \u003cstrong\u003e77\u003c/strong\u003e(5):1773-1796.\u003c/li\u003e\n\u003cli\u003ePan J, Xu Y, Song H, Zhou X, Yao Z, Ji G: \u003cstrong\u003eExtracts of Zuo Jin Wan, a traditional Chinese medicine, phenocopies 5-HTR1D antagonist in attenuating Wnt/\u0026beta;-catenin signaling in colorectal cancer cells\u003c/strong\u003e. \u003cem\u003eBMC complementary and alternative medicine \u003c/em\u003e2017, \u003cstrong\u003e17\u003c/strong\u003e(1):506.\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\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eThe mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects\u003c/strong\u003e. \u003cem\u003eSignal transduction and targeted therapy \u003c/em\u003e2020, \u003cstrong\u003e5\u003c/strong\u003e(1):87.\u003c/li\u003e\n\u003cli\u003eXu P, Huang S, Zhang H, Mao C, Zhou XE, Cheng X, Simon IA, Shen DD, Yen HY, Robinson CV\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eStructural insights into the lipid and ligand regulation of serotonin receptors\u003c/strong\u003e. \u003cem\u003eNature \u003c/em\u003e2021, \u003cstrong\u003e592\u003c/strong\u003e(7854):469-473.\u003c/li\u003e\n\u003cli\u003eOkaty BW, Commons KG, Dymecki SM: \u003cstrong\u003eEmbracing diversity in the 5-HT neuronal system\u003c/strong\u003e. \u003cem\u003eNature reviews Neuroscience \u003c/em\u003e2019, \u003cstrong\u003e20\u003c/strong\u003e(7):397-424.\u003c/li\u003e\n\u003cli\u003eZeng C, Chen Y: \u003cstrong\u003eHTR1D, TIMP1, SERPINE1, MMP3 and CNR2 affect the survival of patients with colon adenocarcinoma\u003c/strong\u003e. \u003cem\u003eOncology letters \u003c/em\u003e2019, \u003cstrong\u003e18\u003c/strong\u003e(3):2448-2454.\u003c/li\u003e\n\u003cli\u003eWu W, Li Q, Zhu Z, Li C, Lu P, Zhou X, Huang Y, Liu Y, Wang M, Gong J: \u003cstrong\u003eHTR1D functions as a key target of HOXA10-AS/miR-340-3p axis to promote the malignant outcome of pancreatic cancer via PI3K-AKT signaling pathway\u003c/strong\u003e. \u003cem\u003eInternational journal of biological sciences \u003c/em\u003e2022, \u003cstrong\u003e18\u003c/strong\u003e(9):3777-3794.\u003c/li\u003e\n\u003cli\u003eJin X, Li H, Li B, Zhang C, He Y: \u003cstrong\u003eKnockdown and inhibition of hydroxytryptamine receptor 1D suppress proliferation and migration of gastric cancer cells\u003c/strong\u003e. \u003cem\u003eBiochem Biophys Res Commun \u003c/em\u003e2022, \u003cstrong\u003e620\u003c/strong\u003e:143-149.\u003c/li\u003e\n\u003cli\u003eZuo X, Chen Z, Cai J, Gao W, Zhang Y, Han G, Pu L, Wu Z, You W, Qin J\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003e5-Hydroxytryptamine Receptor 1D Aggravates Hepatocellular Carcinoma Progression Through FoxO6 in AKT-Dependent and Independent Manners\u003c/strong\u003e. \u003cem\u003eHepatology (Baltimore, Md) \u003c/em\u003e2019, \u003cstrong\u003e69\u003c/strong\u003e(5):2031-2047.\u003c/li\u003e\n\u003cli\u003eGlaviano A, Foo ASC, Lam HY, Yap KCH, Jacot W, Jones RH, Eng H, Nair MG, Makvandi P, Geoerger B\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003ePI3K/AKT/mTOR signaling transduction pathway and targeted therapies in cancer\u003c/strong\u003e. \u003cem\u003eMolecular cancer \u003c/em\u003e2023, \u003cstrong\u003e22\u003c/strong\u003e(1):138.\u003c/li\u003e\n\u003cli\u003eYang J, Nie J, Ma X, Wei Y, Peng Y, Wei X: \u003cstrong\u003eTargeting PI3K in cancer: mechanisms and advances in clinical trials\u003c/strong\u003e. \u003cem\u003eMolecular cancer \u003c/em\u003e2019, \u003cstrong\u003e18\u003c/strong\u003e(1):26.\u003c/li\u003e\n\u003cli\u003eHe Y, Sun MM, Zhang GG, Yang J, Chen KS, Xu WW, Li B: \u003cstrong\u003eTargeting PI3K/Akt signal transduction for cancer therapy\u003c/strong\u003e. \u003cem\u003eSignal transduction and targeted therapy \u003c/em\u003e2021, \u003cstrong\u003e6\u003c/strong\u003e(1):425.\u003c/li\u003e\n\u003cli\u003eQin G, Luo M, Chen J, Dang Y, Chen G, Li L, Zeng J, Lu Y, Yang J: \u003cstrong\u003eReciprocal activation between MMP-8 and TGF-\u0026beta;1 stimulates EMT and malignant progression of hepatocellular carcinoma\u003c/strong\u003e. \u003cem\u003eCancer letters \u003c/em\u003e2016, \u003cstrong\u003e374\u003c/strong\u003e(1):85-95.\u003c/li\u003e\n\u003cli\u003eMoon AM, Singal AG, Tapper EB: \u003cstrong\u003eContemporary Epidemiology of Chronic Liver Disease and Cirrhosis\u003c/strong\u003e. \u003cem\u003eClinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association \u003c/em\u003e2020, \u003cstrong\u003e18\u003c/strong\u003e(12):2650-2666.\u003c/li\u003e\n\u003cli\u003eSingal AG, Zhang E, Narasimman M, Rich NE, Waljee AK, Hoshida Y, Yang JD, Reig M, Cabibbo G, Nahon P\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eHCC surveillance improves early detection, curative treatment receipt, and survival in patients with cirrhosis: A meta-analysis\u003c/strong\u003e. \u003cem\u003eJournal of hepatology \u003c/em\u003e2022, \u003cstrong\u003e77\u003c/strong\u003e(1):128-139.\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":"Hepatocellular carcinoma, HTR1D, Sorafenib resistance, PI3K/Akt","lastPublishedDoi":"10.21203/rs.3.rs-4447882/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4447882/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHepatocellular carcinoma (HCC) is a form of cancer with high incidence rates and low survival rates worldwide. Oral sorafenib is a promising therapy for advanced HCC, but resistance to the drug limits its effectiveness. HTR1D, a gene that is highly expressed in HCC, plays a crucial role in the development, drug resistance, and prognosis of the disease.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFirstly, the correlation between HTR1D and hepatocellular carcinoma was analyzed by TCGA database, and the expression level of HTR1D in clinical samples was detected by qPCR. Then the siRNA was transfected into HuH-7 and HEP3B cells, and the cell proliferation ability, colony formation ability, migration and invasion ability were detected with or without sorafenib. And the expression of PI3K/Akt pathway was detected by Western Blot. Finally, the potential of HTR1D as a predictive marker for patient prognosis was evaluated by immunohistochemistry\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnalysis of TCGA data showed that methylation of the HTR1D gene was associated with cancer status. Clinical samples confirmed significant differences in HTR1D expression between HCC and adjacent tissues, with higher expression correlating with poorer patient prognosis. Interference with HTR1D gene expression demonstrated its role in promoting HCC proliferation, migration, and drug resistance through the PI3K/Akt pathway. These findings were validated in a mouse model. Immunohistochemical analysis of clinicopathological samples suggested HTR1D could be a valuable prognostic marker for HCC.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHTR1D is highly expressed in hepatocellular carcinoma tissues, and it can influence hepatocellular carcinoma development and resistance to sorafenib by regulating the PI3K/Akt signaling pathway. In addition, HTR1D has potential as a prognostic indicator.\u003c/p\u003e","manuscriptTitle":"HTR1D regulates the PI3K/Akt signaling pathway to impact hepatocellular carcinoma development and resistance to sorafenib","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-07 19:02:21","doi":"10.21203/rs.3.rs-4447882/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":"0b347b20-b4b6-4a50-93f9-9fd743cbff2a","owner":[],"postedDate":"June 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-11T08:36:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-07 19:02:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4447882","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4447882","identity":"rs-4447882","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}