C-type lectin domain family 4 member G(CLEC4G) is a negative marker for CD34 in the evolution of liver pathogenesis

doi:10.21203/rs.3.rs-1761623/v1

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C-type lectin domain family 4 member G(CLEC4G) is a negative marker for CD34 in the evolution of liver pathogenesis

https://doi.org/10.21203/rs.3.rs-1761623/v1

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Purpose: This study aimed to explore the expression of the C-type lectin domain family 4 member G (CLEC4G) gene in the liver sinusoidal endothelial cells (LSECs) during liver pathogenesis, and to evaluate its correlation with CD34 and clinical significance in hepatocellular carcinoma patients.

Methods: We conducted bioinformatics analysis of the differential expression of CLEC4G in in various human organs, carcinomatous and adjacent tissues. Then, the differential expression in carcinomatous and adjacent tissues was verified by real-time quantitative reverse transcription PCR (qRT-PCR) and immunohistochemical (IHC) analyses in clinical HCC samples. The expression of CLEC4G in normal liver tissues and cirrhotic tissues was further detected. Besides, mRNA and protein expression levels of CD34 in HCC samples were detected via qRT-PCR and IHC, respectively. ELISA was applied to detect serum levels of CLEC4G in healthy controls, liver fibrosis and HCC patients. The correlation between the CLEC4G and CD34 expression was analyzed using the Spearman test, whereas the Chi-square test was applied to analyze the correlation between CLEC4G and clinicopathological characteristics of HCC patients. Furthermore, the Kaplan-Meier method was used to estimate the relationship between CLEC4G serum levels and survival.

Results: The expressions of mRNA and protein levels of CLEC4G were higher in normal liver tissues, moderately expressed in cirrhotic and para-cancerous tissues (P<0.001), and lowest in HCC tissues (P<0.001). We also found high CD34 expression in late tumors, which was negatively correlated with CLEC4G at both mRNA and protein levels (R=-0.830, P<0.001; R=-0.817, P<0.001, respectively). Compared to the healthy controls, the CLEC4G levels in liver fibrosis patients, and HCC patients were gradually lower (P<0.001). Furthermore, CLEC4G was highly associated with tumor size (P=0.023), tumor stage (P=0.046), and vascular metastasis (P=0.008; Table 1) in HCC patients. Notably, HCC patients expressing low CLEC4G levels were characterized by a shorter survival time (P=0.032).

Conclusions: The low expression of CLEC4G is potentially correlated with LSEC capillarization and the appearance of micro-vessels. Such a phenomenon may exert an endogenous protective effect against the progression of liver disease and serve as a reliable diagnostic marker for hepatocellular carcinoma.

Carcinoma

hepatocellular

liver cirrhosis

LSECs capillarization

CD34

CLEC4G

Hepatic tumors are one of the most common tumor diseases worldwide and the leading cause of cancer death. According to reports, chronic liver disease and cirrhosis are the main risk factors for liver cancer, while chronic hepatitis transpires to precancerous lesions, causing hepatocellular carcinoma, a gradual multi-step process of carcinogenesis involving a series of gene expression and pathological structural changes⁽¹⁾. Numerous studies have been geared towards elucidating the potential association between cirrhosis, precancerous lesions, and hepatocellular carcinoma (HCC), attempting to evaluate the features and diagnostic points of precancerous lesions, and ultimately provide references for early clinical detection of precancerous lesions and HCC. According to existing reports, increased loss of chromosome heterozygosity, decreased length of telomerase, small cell dysplasia, and morphological changes in liver sinusoidal endothelial cells (LSECs) are suggestive of hepatic dysplasia and carcinogenesis^(2–4). Notably, LSEC capillarization, a crucial morphological change, may provide a satisfactory potential differential diagnosis between highly dysplastic nodules and HCC. It is therefore promising to serve as an important link in the process of liver disease⁽⁵⁾.

During LSECs capillarization, the liver is damaged by various acute or chronic stimuli. The sinusoidal endothelial cells lose their fenestrations to form an organized subcutaneous basement membrane, from which many liver diseases originate⁽⁶⁾. LSECs capillarization is associated with the regulation of non-alcoholic fatty liver disease, alcoholic fatty liver disease, liver fibrosis, hepatocellular carcinoma, and other liver pathological processes, which are particularly critical to the occurrence and progression of liver diseases^{(7, 8)}. Currently, the mechanism of LSECs capillarization remains elusive, but it is clear that hepatic sinus capillarization is related to several down- or up-regulated genes, such as lyve-1, CD32b, stabilin-2 down-regulated and endothelin-1, CD31, CD34 up-regulated^(9–11). Therefore, it is of great significance to explore genes that show expression changes during LSECs capillarization, which can provide research insights into the occurrence and progression of liver diseases.

Here, we reported that CLEC4G was abundantly expressed in LSECs, whereas analysis of the TCGA database demonstrated that the expression of CLEC4G was significantly down-regulated in liver cancer tissues. Accordingly, we deduced that CLEC4G may be a crucial molecule associated with the occurrence of liver diseases induced by LSECs capillarization. Therefore, our interest was to establish the expression changes of CLEC4G in liver diseases, and to explore whether it is correlated with CD34, clinical-pathological characteristics, and survival in HCC patients.

Bioinformatics analysis

The hepatocellular carcinoma data set (ENSG00000182566) was retrieved from the TCGA database in Genomic Data Commons of the National Center for Biotechnology Information (NCBI). For bioinformatics analysis, we used RNA sequencing data of CLEC4G obtained from 100 non-tumor liver tissues and 100 HCC cancer tissues.

Clinical samples and data

We acquired a set of archived samples including 40 pairs of primary and corresponding adjacent nontumoral liver tissues, 10 normal liver tissues, 10 cirrhosis tissues from the Zhuzhou Central Hospital since 2016. Blood specimens from 30 healthy controls, 30 Chronic HBV patients, 30 Cirrhosis patients, and 40 HCC patients were collected in 2020. Detailed clinical data of HCC patients were recorded. The research protocol conformed to the Clinical Trial Ethics Committee of Zhuzhou Central Hospital with the ethical approval no (2019) ethics approval no (03004). All subjects gave and signed informed consent to the study.

RNA extraction and real-time qPCR

Total tissue RNA was extracted using TRIzol Reagent (Invitrogen) followed by the synthesis of cDNA from 1mg of total RNA using an iScript cDNA Synthesis kit (Bio-Rad). Relative gene expression levels were evaluated using FastStart Universal SYBR Green Master Mix (Roche Diagnostics) with GAPDH mRNA as an endogenous control. Values represented the mean ± the SDs of three independent experiments. The expression values of target genes were calculated via the 2^−△△Ct method. We used the following primers pairs: CLEC4G, forward, 5’- ACAGTCCTTTGGGCTGTGAT-3’, reverse, 5’-TTTGTCCTCAGCAGGTCGT-3’; CD34, forward, 5’-ATGTAAGTAATAAATGTCCAGACAGAACA-3’, reverse, 5’-TAAGTAATGTCAACAGAATGATCAGAACA-3’; GAPDH, forward, 5’- GGAGCGAGATCCCTCCAA AAT-3’, reverse, 5’-GGCTGTTGTCATACTTCTCATGG-3’.

Immunohistochemistry and staining score

Paraffin-embedded sections were applied to immunohistochemical analysis. To retrieve the antigen, tissue sections were deparaffinized, rehydrated, as well as heated in the pressure cooker with sodium citrate buffer (10 mmol/L, pH 6.0). The slides were then incubated with primary antibodies, anti-CLEC4G; 1:200, #181196 (Abcam) and anti-CD34; 1:100 #ZA-0550 (Zsbio). Diaminobenzidine (DAB; Dako) staining was used to detect immunoreactivity. Hematoxylin was used as a counterstain. Immunohistochemical scores were evaluated according to the positive rate (PR) and staining intensity (SI) based on the Yu method⁽¹²⁾.

Enzyme-linked immunosorbent assay (ELISA)

Serum levels of CLEC4G in healthy controls, chronic HBV patients, cirrhosis patients, and HCC patients were assessed using CLEC4G ELISA kits (Catalog no. JL48366, Jianglai Industrial Limited By Share Ltd, Shanghai, China) according to the manufacturer’s instructions.

Statistical analysis

Data were expressed as mean ± SD. We applied the Student’s t-test to compare the CLEC4G expression levels in normal liver tissues, cirrhosis tissues, nontumoral liver tissues, and HCC tissues, and to compare serum CLEC4G expression in healthy controls, chronic HBV patients, cirrhosis patients, and HCC patients. Correlations between CLEC4G and individual clinicopathologic parameters were evaluated via the Nonparametric χ² test, whereas correlations between the expression of CLEC4G and CD34 were evaluated using the Spearman’s rank-order test. The Kaplan–Meier method was adopted to estimate survival rates for CLEC4G expression. Equivalences of the survival curves were tested using log-rank statistics. All statistical analyses were performed using SPSS 25.0 software (IBM Corporation).

Bioinformatics analysis of CLEC4G expression in liver and liver cancer tissues.

First, we analyzed the protein levels of CLEC4G in various human organs (Fig. 1A). The results showed that CLEC4G was most abundant in liver. Further, the mRNA expression of CLEC4G in HCC patients was subjected to bioinformatics analysis in TCGA database. Both heat maps (Fig. 1B) and box plots (Fig. 1C) revealed significantly down-regulated expression of CLEC4G in HCC tissues.

CLEC4G mRNA and protein expression levels in the evolution of liver pathogenesis

The expression of CLEC4G in HCC patients was further confirmed by real-time quantitative PCR and immunohistochemistry in clinical HCC specimens, and the expression of CLEC4G in normal liver tissues and cirrhotic tissues was also detected. The PCR results showed that, compared to normal liver tissues, the expression of CLEC4G was gradually lower in cirrhosis and para cancer tissues, as well as HCC tissues. (Fig. 2A). IHC was applied to evaluate the expression and distribution of CLEC4G in the set same subjects as in Fig. 2A. According to the analysis of IHC results, the expression of CLEC4G was higher in normal liver tissues and lower in cirrhosis and para cancerous tissues; however, it was lowest in liver cancer tissues (Fig. 2B). This trend was also confirmed by immunohistochemical staining scores (Fig. 2C).

Serum CLEC4G levels in healthy controls, liver fibrosis patients, and HCC patients.

We detected serum CLEC4G levels in healthy controls, liver fibrosis patients, and HCC patients through ELISA. The serum levels of CLEC4G in healthy controls (median: 479.4 pg/ml) were significantly lower compared to that in liver fibrosis patients (median: 379.8 pg/ml), and HCC patients (median: 342.5 pg/ml). Thus, we implicated CLEC4G as a potential marker in liver pathogenesis (Fig. 3A). Furthermore, ROC curve and AUC analysis were also performed. The AUC of CLEC4G to distinguish liver fibrosis patients from healthy controls was 0.911 (Fig. 3B), and the AUC to distinguish HCC patients from healthy controls was 0.966 (Fig. 3C). In addition, CLEC4G also had an AUC of 0.693 to distinguish HCC patients from liver fibrosis patients (Fig. 3D). The details of ROC curve and AUC analysis were shown in Fig. 3E.

Relationship between CLEC4G and survival rate and clinicopathologic features in HCC patients.

Furthermore, we explored the relationship between CLEC4G and survival rates of HCC patients. First, bioinformatics analysis revealed that patients with low CLEC4G expression levels had significantly shorter 5-year survival rates (Fig. 4A) and disease-free survival (Fig. 4B) than those with high CLEC4G expression. In clinical HCC samples, the Kaplan–Meier analysis also revealed that patients with low CLEC4G expression levels in tissues had significantly shorter 5-year survival rates (P = 0.023) (Fig. 4C) and disease-free survival (P = 0.038) (Fig. 4D) than those with high CLEC4G expression. Notably, CLEC4G levels were highly associated with tumor size(P = 0.023), tumor stage(P = 0.046), and vascular metastasis(P = 0.008) in HCC patients (Table 1).

Table 1

Correlative analysis of CLEC4G levels with clinicopathologic features.
Clinicopathologic parameters	Number of specimens	CLEC4G levels		P-value
		Low	High
Age (mean ± SD)		52.6 ± 14.6	54.1 ± 12.3	.681
Sex
Male	28	18	10	.722
Female	12	7	5
a-fetoprotein (AFP)
≤ 40 ng/mL	18	9	9	.140
> 40 ng/mL	22	16	6
Hepatitis B virus
Negative	12	6	6	.285
Positive	28	19	9
Tumor size
≤ 3	15	6	9	.023
> 3	25	19	6
Tumor number
1	26	16	10	.864
≥ 2	14	9	5
Grade
IཞII	18	12	6	.622
IIIཞIV	22	13	9
Stage
I	16	7	9	.046
IIཞIII	24	18	6
Vascular invasion
No	16	6	10	.008
Yes	24	19	5

CLEC4G is negatively correlated with CD34 in HCC tumor tissues

CD34 is a highly specific and sensitive marker of vascular endothelial cells, which successively increased in the evolution of liver pathogenesis⁽¹³⁾. Interestingly, we found that mRNA (Fig. 5A) and protein (Fig. 5B) levels between CLEC4G and CD34 exhibited a negative correlation (R=-0.830, P < 0.001; R=-0.817, P < 0.001, respectively). Co-expression of CLEC4G and CD34 in the same tissues showed an inverse correlation; that is, lower CLEC4G expression implied higher CD34 expression and vice versa (Fig. 5C).

Although numerous pieces of research are diverting their focus on tumor cell processes, such as proliferation and invasion, the tumor microenvironment also plays a crucial role before or during tumor progression and metastasis^(14–16). In the liver, LSECs are the primary non-parenchymal cells, which exert crucial effects in the pathological process of the liver and the regulation of the occurrence and progression of liver diseases^{(17, 18)}. Several research groups have explored the mechanisms underlying LSEC differentiation and loss of fenestrae ^{(9, 19, 20)}. However, reports on underlying mechanisms of LSEC capillarization should be further elucidated. In the present study, we focused on the expression of CLEC4G in the LSECs membrane, which critically mediates immune-conditioning and viral infection ^{(21, 22)} The results strongly demonstrated that CLEC4G is largely expressed in normal liver tissues, but is significantly reduced with the occurrence and progression of liver diseases. Thus, there is speculation that CLEC4G may be an important molecule for the maintenance of normal physiological function of LSECs. Also, it potentially contributes to the capillarization process of LSECs. Moreover, we found that, as the disease progressed to liver cancer, the expression of CLEC4G protein was significantly minimal, particularly in the central portion of the tumor. Thus, with the aggravation of the liver disease, the capillarization of LSECs is more aggravated and CLEC4G levels are subsequently reduced.

Angiogenesis, an integral part of the tumor progression⁽²³⁾ also significantly plays a role in liver chronic inflammation⁽²⁴⁾. CD34 and vascular endothelial growth factor (VEGF) are well-known mediators for angiogenesis in chronic liver disease^{(25, 26)}. Herein, we revealed that CD34 had a high positive rate for 38/40 (95.0%) in hepatocellular carcinoma patients, which concurred with a previous report⁽²⁷⁾. Interestingly, the expressions of CLEC4G and CD34 exhibited significant negative correlation both at the mRNA and protein levels in the same specimen. Also, based on these findings, it is evident that low expression of CLEC4G is associated with elevated expression of CD34, which induces angiogenesis in tumor progression.

To our knowledge, we were the first to report the expression changes of CLEC4G in the progression of liver disease and the potential role of CLEC4G in angiogenesis. However, owing to the limitations of experimental conditions, we could not comprehensively explore the specific mechanism of CLEC4G associated with the progression of liver diseases and angiogenesis. Further elucidation of the clinical significance of CLEC4G in patients with liver cancer revealed that low CLE4CG expression was associated with increased tumor size, tumor stage, and vascular invasion. HCC patients expressing low CLE4CG levels showed a significant correlation with poor overall survival rate. These clinical findings implied that the lower the expression of CLEC4G was linked to higher malignancy. Collectively, CLE4CG potentially exerts an endogenous protective effect against the progression of liver disease. Thus, the present findings are valuable for the elucidating the mechanism of LSEC capillarization and may provide a research basis for the occurrence and progression of liver diseases.

The present study demonstrated that CLEC4G is involved in the process of LSEC capillarization. Notably, the expression of CLEC4G exhibited a gradual reducing trend in the malignant process of liver disease and was rarely expressed in hepatocellular carcinoma tissues, which was contrary to the expression of CD34. Thus, CLEC4G is a promising marker for pathological diagnosis of hepatocellular carcinoma.

CLEC4G

C-type lectin domain family 4 member G

LSECs

liver sinusoidal endothelial cells

qRT-PCR

real-time quantitative reverse transcription PCR

IHC

immunohistochemical

HCC

hepatocellular carcinoma

VEGF

vascular endothelial growth factor

positive rate

staining intensity

Ethics approval and consent to participate.

The research protocol conformed to the Clinical Trial Ethics Committee of Zhuzhou Central Hospital with the ethical approval no (2019) ethics approval no (03004). All subjects gave and signed informed consent to the study.

Consent for publication

Not applicable.

Competing interests

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.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xiang Chen and Zui-ming Jiang contributed equally to this article.

Acknowledgements

Not applicable.

Authors’ contributions

XC, ZJ, and MG conceived and designed the study and drafted the manuscript. ZX, MH, ZC, and MT collected, analyzed, and interpreted the experimental data. XC and MT revised the manuscript for important intellectual content. All authors read and approved the final manuscript.

Funding

This work was supported by Zhuzhou City Health Talents 135 Project Special Fund, the Hunan Science and Technology Department, Provincial Science and Technology Innovation Platform and Talent Plan(2016SK4006).

Availability of data and materials

The datasets used during the current study are available from the corresponding author on reasonable request.

Kanda T, Goto T, Hirotsu Y, Moriyama M, Omata M. Molecular Mechanisms Driving Progression of Liver Cirrhosis towards Hepatocellular Carcinoma in Chronic Hepatitis B and C Infections: A Review. Int J Mol Sci. 2019;20(6).
Nault JC, Ningarhari M, Rebouissou S, Zucman-Rossi J. The role of telomeres and telomerase in cirrhosis and liver cancer. Nat Rev Gastroenterol Hepatol. 2019;16(9):544–58.
Ojima H, Masugi Y, Tsujikawa H, Emoto K, Fujii-Nishimura Y, Hatano M, et al. Early hepatocellular carcinoma with high-grade atypia in small vaguely nodular lesions. Cancer Sci. 2016;107(4):543–50.
Frachon S, Gouysse G, Dumortier J, Couvelard A, Nejjari M, Mion F, et al. Endothelial cell marker expression in dysplastic lesions of the liver: an immunohistochemical study. J Hepatol. 2001;34(6):850–7.
Feng LH, Wang H, Dong H, Zhu YY, Cong WM. The stromal morphological changes for differential diagnosis of uninodular high-grade dysplastic nodule and well-differentiated small hepatocellular carcinoma. Oncotarget. 2017;8(50):87329–39.
Natarajan V, Harris EN, Kidambi S. SECs (Sinusoidal Endothelial Cells), Liver Microenvironment, and Fibrosis. Biomed Res Int. 2017;2017:4097205.
Poisson J, Lemoinne S, Boulanger C, Durand F, Moreau R, Valla D, et al. Liver sinusoidal endothelial cells: Physiology and role in liver diseases. J Hepatol. 2017;66(1):212–27.
Feng YX, Li W, Wen XD, Zhang N, Liu WH, Yang ZY. Sinusoidal Endothelial Cell Progenitor Cells Promote Tumour Progression in Patients with Hepatocellular Carcinoma. Stem Cells Int. 2020;2020:8819523.
Ruart M, Chavarria L, Campreciós G, Suárez-Herrera N, Montironi C, Guixé-Muntet S, et al. Impaired endothelial autophagy promotes liver fibrosis by aggravating the oxidative stress response during acute liver injury. J Hepatol. 2019;70(3):458–69.
Géraud C, Mogler C, Runge A, Evdokimov K, Lu S, Schledzewski K, et al. Endothelial transdifferentiation in hepatocellular carcinoma: loss of Stabilin-2 expression in peri-tumourous liver correlates with increased survival. Liver Int. 2013;33(9):1428–40.
Yokomori H, Ando W, Yoshimura K, Yamazaki H, Takahashi Y, Oda M. Increases in endothelial caveolin-1 and cavins correlate with cirrhosis progression. Micron. 2015;76:52–61.
Yu J, Ren X, Chen Y, Liu P, Wei X, Li H, et al. Dysfunctional activation of neurotensin/IL-8 pathway in hepatocellular carcinoma is associated with increased inflammatory response in microenvironment, more epithelial mesenchymal transition in cancer and worse prognosis in patients. PLoS One. 2013;8(2):e56069.
Choi WT, Kakar S. Immunohistochemistry in the Diagnosis of Hepatocellular Carcinoma. Gastroenterol Clin North Am. 2017;46(2):311–25.
Abadjian MZ, Edwards WB, Anderson CJ. Imaging the Tumor Microenvironment. Adv Exp Med Biol. 2017;1036:229–57.
Novikova MV, Khromova NV, Kopnin PB. Components of the Hepatocellular Carcinoma Microenvironment and Their Role in Tumor Progression. Biochemistry (Mosc). 2017;82(8):861–73.
Spinelli FM, Vitale DL, Sevic I, Alaniz L. Hyaluronan in the Tumor Microenvironment. Adv Exp Med Biol. 2020;1245:67–83.
Baiocchini A, Del Nonno F, Taibi C, Visco-Comandini U, D'Offizi G, Piacentini M, et al. Liver sinusoidal endothelial cells (LSECs) modifications in patients with chronic hepatitis C. Sci Rep. 2019;9(1):8760.
McMahan RH, Porsche CE, Edwards MG, Rosen HR. Free Fatty Acids Differentially Downregulate Chemokines in Liver Sinusoidal Endothelial Cells: Insights into Non-Alcoholic Fatty Liver Disease. PLoS One. 2016;11(7):e0159217.
Mak KM, Mei R. Basement Membrane Type IV Collagen and Laminin: An Overview of Their Biology and Value as Fibrosis Biomarkers of Liver Disease. Anat Rec (Hoboken). 2017;300(8):1371–90.
Xing Y, Zhao T, Gao X, Wu Y. Liver X receptor α is essential for the capillarization of liver sinusoidal endothelial cells in liver injury. Sci Rep. 2016;6:21309.
Zhang F, Ren S, Zuo Y. DC-SIGN, DC-SIGNR and LSECtin: C-type lectins for infection. Int Rev Immunol. 2014;33(1):54–66.
Yang Z, Li Q, Wang X, Jiang X, Zhao D, Lin X, et al. C-type lectin receptor LSECtin-mediated apoptotic cell clearance by macrophages directs intestinal repair in experimental colitis. Proc Natl Acad Sci U S A. 2018;115(43):11054–9.
Bocca C, Novo E, Miglietta A, Parola M. Angiogenesis and Fibrogenesis in Chronic Liver Diseases. Cell Mol Gastroenterol Hepatol. 2015;1(5):477–88.
Hilmi M, Neuzillet C, Calderaro J, Lafdil F, Pawlotsky JM, Rousseau B. Angiogenesis and immune checkpoint inhibitors as therapies for hepatocellular carcinoma: current knowledge and future research directions. J Immunother Cancer. 2019;7(1):333.
Paschoal JP, Bernardo V, Canedo NH, Ribeiro OD, Caroli-Bottino A, Pannain VL. Microvascular density of regenerative nodule to small hepatocellular carcinoma by automated analysis using CD105 and CD34 immunoexpression. BMC Cancer. 2014;14:72.
Buijs N, Oosterink JE, Jessup M, Schierbeek H, Stolz DB, Houdijk AP, et al. A new key player in VEGF-dependent angiogenesis in human hepatocellular carcinoma: dimethylarginine dimethylaminohydrolase 1. Angiogenesis. 2017;20(4):557–65.
Vasuri F, Renzulli M, Fittipaldi S, Brocchi S, Clemente A, Cappabianca S, et al. Pathobiological and Radiological Approach For Hepatocellular Carcinoma Subclassification. Sci Rep. 2019;9(1):14749.

No competing interests reported.

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C-type lectin domain family 4 member G(CLEC4G) is a negative marker for CD34 in the evolution of liver pathogenesis

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Version 1

Abstract

Figures

Introduction

Materials And Methods

Bioinformatics analysis

Clinical samples and data

RNA extraction and real-time qPCR

Immunohistochemistry and staining score

Enzyme-linked immunosorbent assay (ELISA)

Statistical analysis

Results

CLEC4G mRNA and protein expression levels in the evolution of liver pathogenesis

CLEC4G is negatively correlated with CD34 in HCC tumor tissues

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

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