Exploring the Prognostic Value and Biological Function of CPNE1 Gene in Hepatocellular Carcinoma


 BackgroundLiver hepatocellular carcinoma (LIHC), the major histology subtype of primary liver cancer, accounts for 70-80% proportion of total liver cancer cases. Copine1 (CPNE1), the first discovered CPNE1 family member, participates in the process of carcinogenesis and development of diverse tumors. Our study aimed to investigate the expression and prognostic value of CPNE1 gene in hepatocellular carcinoma (HCC), to explore its functional network in HCC and its effects on biological behaviors such as proliferation, migration and invasion of HCC cells, and to explore its related signaling pathways.METHODSHCCDB, CCLE and HPA online databases were used to explore the expression of CPNE1 gene in HCC tissues; LinkedOmics online database was used to analyze the co-expression network of CPNE1 in hepatocellular carcinoma, and gene set enrichment analysis (GSEA) was used for GO functional annotation, KEGG pathway enrichment analysis, kinase target enrichment, miRNA target enrichment and transcription factor target enrichment analysis. The expression levels of CPNE1 in normal hepatocytes and several hepatocellular carcinoma cell lines were detected by RT-qPCR, and finally HepG2 and MHCC-97H cells were selected to construct CPNE1 knockdown cell lines by transfection with siRNA, and the knockdown efficiency was detected by Western Blot and RT-qPCR. The effect of CPNE1 knockdown on the proliferation of hepatocellular carcinoma cells was examined by CCK8 assay and clone formation assay; the effect of CPNE1 knockdown on the migration ability of hepatocellular carcinoma cells was assessed by cell scratch assay and Transwell cell migration assay; finally, the expression of related signaling pathway proteins was examined by Western Blot. The correlation of CPNE1 expression with immune infiltration and immune checkpoint molecules in HCC tissues was analyzed using TIMER online database.RESULTSAnalysis in several databases showed that CPNE1 was highly expressed in HCC tissues and significantly correlated with sex, age, cancer stage and tumor grade. Overall survival (OS) was significantly lower in patients with high CPNE1 expression than in patients with low CPNE1 expression, and CPNE1 could be used as an independent prognostic indicator for HCC. GSEA analysis showed that co-expressed genes of CPNE1 were mainly involved in biological processes such as establishment of protein localization to membrane, ribonucleoprotein complex biogenesis and lipid localization. Q-PCR showed that CPNE1 expression was upregulated in HCC cells compared with normal hepatocytes, and knockdown of CPNE1 gene inhibited the AKT/P53 pathway, resulting in decreased proliferation, migration and invasion of HCC cells. The level of CPNE1 expression in HCC was significantly and positively correlated with the level of infiltration of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells (p<0.001), and with the expression of immune checkpoint molecules PDCD1, CD274, CTLA4, LAG3, HAVCR2, and TIGIT.CONCLUSION﻿The expression of CPNE1 was significantly higher in HCC tissues than in normal liver tissues, and high CPNE1 expression was associated with poor prognosis. Knockdown of CPNE1 inhibited AKT/P53 pathway activation and suppressed HCC cell proliferation and migration. There was a significant correlation between CPNE1 expression and tumor immune infiltration in HCC.

Introduction Liver cancer, one of the top ve deadliest cancers globally, has the high mortality [1,2]. Liver hepatocellular carcinoma (LIHC), the major histology subtype of primary liver cancer, accounts for 70-80% proportion of total liver cancer cases and is chie y related to hepatitis C virus (HCV), hepatitis B virus (HBV) and alcoholism [3,4]. Surgical resection or liver transplantations is the common treatment choice in patients with early hepatocellular carcinoma. However, many cases are usually refractory to treat surgically due to initial diagnosis at an advanced stage. Although advanced LIHC exists multiple treatments, including surgical excision, transarterial embolization, chemotherapy and radiofrequency ablation, the treatments effects are limited and LIHC still has a rate of recurrence as high as 70% [5]. In brief, patients with LIHC have a poor overall survival. Despite the risk factors (HBV, HCV, alcohol-related cirrhosis, smoking, diabetes, fatty liver disease, obesity, iron overload and multiple diet exposure) of LIHC is well known, the precise mechanism underlying development and progression of LIHC remains unclear [6,7]. Therefore, in-depth studies exploring novel biomarkers and delineating its mechanism are urgently needed.
Copines family, a widely distributed and highly conserved throughout evolution phospholipid-binding protein, shares common structural features: 2 C2 domains in the N terminus, 1 von Willebrand factor A (VWA) domain in the C terminus [8,9]. C2 domains possess properties of Ca2+ dependence and phospholipid-binding and may be associated with signal transduction and cell membranes transport.
VWA domain could mediate interactions among extracellular proteins and may be related to recruitment of target proteins and regulating activity of speci c proteins [10,11]. In mammals, it has been identi ed that Copines family contains 9 members named sequentially as CPNE1 9 in order of discovery [9].
Copine1 (CPNE1), the rst discovered CPNE1 family member [8], is located on human chromosome 20q11.21, encodes 537 amino acids and has multiple splice forms [12]. CPNE1 is observed to be upregulated in multiple tumor tissue compared to normal tissues. Studies have highlighted that CPNE1 involves in various cellular biology process, such as apoptosis, growth control, autophagy, mitotic, in ammation, exocytosis and cytoskeletal organization and gene transcription [13]. Meanwhile, CPNE1 participates in the process of carcinogenesis and development of breast cancer [14], non-small cell lung cancer [15], prostate cancer [16], liver cancer [17], thyroid cancer [18] and osteosarcoma [19]. The expression of CPNE1 is associated with TNM staging, lymph node metastasis and distant metastasis of lung adenocarcinoma [15]. The expression of CPNE1 is higher in prostate cancer tissue and castrationresistant prostate cancer tissue than that in normal prostatic tissues and noncastrated-resistant prostate cancer tissue, respectively. Also, CPNE1 is signi cantly correlated with the tumor stage, Gleason score and recurrence-free survival of prostate cancer and is positively correlated with expression of TRAF2 as a prognostic marker in prostate cancer [16]. CPNE1 is linked to chromosome deletion of 13q in hepatic carcinoma cells and mediates the process of occurrence and progression by regulating the dedifferentiation, cell cycle and proliferation in liver cancer [17]. CPNE1 can act as potential biomarker to identify well-differentiated thyroid cancer tissue and normal thyroid tissues, which simpli es the process of early thyroid cancer diagnosis [18]. And yet, role of CPNE1 in LIHC is unclear.
Using multiple databases, we detected the expression level of CPNE1 and relationship between CPNE1 and clinicopathologic parameters of LIHC patients. Then, we established CPNE1 knockdown cell lines to explore effects of CPNE1 on malignant phenotypes of LIHC cell. Our results may provide theoretical basis for targeted therapy strategy of LIHC.

Expression analysis and survival analysis
We searched for the gene symbol 'CPNE1' using the HCCDB database. HCCDB provides visualization of the results of multiple computational analyses, such as differential expression analysis, tissue-speci c and tumor-speci c expression analysis [20]. Then, the expression of CPNE1 in cancer cell lines was validated using the Cancer Cell Line Encyclopedia (CCLE) dataset (https://portals.broadinstitute.org/ccle) [21]. In addition, we validated the protein expression of CPNE1 in the Human Protein Atlas (HPA) database (www.proteinatlas.org) [22].
The UALCAN database (http://ualcan.path.uab.edu) [23] was used for subgroup analysis of CPNE1 mRNA expression. The hepatocellular carcinoma of the liver (LIHC) dataset from The Cancer Genome Atlas (TCGA) was selected for analysis. CPNE1 expression levels (gender, age, cancer stage, tumor grade and TP53 mutation status) in different subgroups were analyzed. Then, we analyzed the prognostic signi cance of CPNE1 in hepatocellular carcinoma using the Kaplan-Meier survival mapping database (http://kmplot.com) [24].

LinkedOmics
LinkedOmics (http://www.linkedomics. org/login.php) is a public portal containing multi-omics data from 32 cancers in TCGA [28]. In the "LinkFinder" module, we performed co-expression statistical analysis of CPNE1 using Spearman's test, and the results are displayed as volcano and heat maps. In the "LinkInterpreter" module, we performed gene ontology (GO), Kyoto Gene and Genome Encyclopedia (KEGG) pathway, kinase-target enrichment, miRNA-target enrichment and transcription factor-target enrichment analysis by gene set enrichment analysis (GSEA). The screening criteria were set as false discovery rate (FDR) < 0.05, and the number of simulations was 1000.

RNA extraction and real-time PCR assay
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, USA) and the manufacturer's manual was followed. Complementary DNA for reverse transcription was synthesized by the Prime Script RT kit (Takara, Tokyo, Japan). real-time PCR analysis was then performed. The 2-ΔΔCt method was applied to determine differences between multiple samples. CPNE1 primer sequence: sense strand, 5'-ACCCACTCTGCGTCCTT-3'; antisense strand, 5'-TGGCGTCTTGTTGTCTATG-3'.

CCK8 and clone formation
Cell proliferation capacity was measured by Cell Counting Kit 8 (CCK-8, Promotor, Wuhan, China) according to the instructions. After adding CCK-8 reagent to 96-well plates, the cells were incubated for 2 hours. The absorbance at 450 nm (OD450) was recorded. Clone formation assay was used to assess the clonogenic ability of HCC cells. Monolayers (2 × 10 3 /well) were inoculated into 6-well plates. Afterwards, cells were continuously cultured in DMEM (promoter, Wuhan, China), which was spiked with fetal bovine serum (10%, Gibco, Grand Island, NY, USA). 2 weeks later, colonies were xed in an incubator at 37°C for 15 min using methanol and then stained with crystal violet (0.5%, Promoter, Wuhan, China) for 15 min. The number of colonies was counted under an optical microscope.
Transwell migration assay Hepatocellular carcinoma cells (5× 10 4 /ml), digested with 0.25% trypsin and conditioned with serum-free DMEM to a density of 1× 10 5 cells/ml, were transferred to the upper chamber. DMEM medium (600 μl) containing 10% FBS was added to the lower chamber. After incubation in a 5% CO2, 37°C incubator for 24 h, the cells remaining on top of the Transwell membrane were removed with a cotton swab, and the cells migrating to the lower surface of the cells were xed with methanol for 10 min and then stained with 0.1% crystal violet staining solution for 20 min. Images of migrating cells were taken by inverted microscopy. Five elds of view were randomly selected and the stained calls were counted. Repeat the experiment three times.

Statistical analysis
All statistical analyses were performed by R 3.6.3 software and GraphPad Prism 8.0 software. the Wilcoxon and Kruskal-Wallis tests were used to compare differences in the expression of CPNE1; logistic regression was used to analyze the relationship between clinicopathological characteristics and CPNE1; Kaplan-Meier survival analysis and log-rank test were used to compare the survival differences between the two groups; correlation analysis was performed according to Spearman's correlation coe cient. Other experimental data were compared between the two groups by t-test, and differences were considered statistically different at p < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001.

High expression of CPNE1 in LIHC
To evaluate the expression level of CPNE1 in HCC tissue and adjacent normal tissue, we analysed 10 HCC cohorts in HCCDB database and found the mRNA level of CPNE1 in HCC tissue was obviously higher than in adjacent normal tissues ( Figure 1a). CPNE1 is overexpressed in HCC cell lines compared with most tumor types, which was obtained by the Cancer Cell Line Encyclopedia (CCLE) (Figure 1b). Moreover, we used HPA database to explore the protein expression of CPNE1. Compared to normal liver tissue, HCC tissue exhibited CPNE1 strong positive staining ( Figure 1c). Here, we presented the images of HCC liver (Patient IDs: 2556) and normal liver (Patient IDs: 2429). All results suggested the expression level of CPNE1 was signi cantly upregulated in HCC.
To enhance the credibility of the above results, we evaluated the high expression of CPNE1 in LIHC sample from TCGA through the UALCAN database. Compared with the normal samples (n = 50), the mRNA level of CPNE1 was higher in the LIHC samples (n = 371) (Figure 2a). By Subgroup analysis, we found that CPNE1 was also highly expressed in the subgroups of sex and age (Figure 2b, e). In terms of tumour stage and cancer grade, we found CPNE1 was highly expressed in grades 1-4 and stages 1-4 ( Figure 2c, d). Furthermore, CPNE1 was evidently linked to TP53 mutation and was markedly upregulated in LIHC patients with TP53 mutations (Figure 2f). Collectively, these data implicated that the overexpression of CPNE1 was strongly linked to LIHC progression.  (Table 2). Furthermore, compared to hepatitis patients, the HR values for PFS and OS in patients without hepatitis indicated signi cant statistic differences (Table  1). These results showed that overexpression of CPNE1 may decline survival in patients without hepatitis.

Mutations of CPNE1 in LIHC
In the cBioPortal database, we assessed the mutation frequency of CPNE1 in LIHC. In the 366 LIHC patients, CPNE1 gene alteration was identi ed in only 2 LIHC patients and the somatic mutation rate was 0.5% (Figure 4a). These alterations largely contained ampli cation and missense mutation (Figure 4a). The somatic mutation rate of CPNE1 was so low that we failed to nd a correlation between CPNE1 mutation and the survival of LIHC patients. In addition, we further evaluated the mutation types of CPNE1 in COSMIC database. As shown, approximately 23.33% of the samples were found missense substitutions (Figure 4b). The substitution mutations occurred primarily at C > G (28.

Co-expression genes of CPNE1 and enrichment analysis in patients with LIHC
To further elucidate the importance of CPNE1 in LIHC, we explored coexpression patterns of CPNE1 using LinkFinder module in LinkedOmics. The result showed 5,896 genes (dark red dots) were related positively to CPNE1, while 3,780 genes (dark green dots) were related negatively to CPNE1 in LIHC (FDR<0.05) (Figure 5a) . Additionally, the top 50 genes clearly related (positively and negatively) to CPNE1 were displayed in Fig 5b and Fig 5c. CPNE1 (Figure 5f). By contrast, among the top 50 negatively correlated genes, there were 12/50 genes with low HR (p < 0.05) (Figure 5g).
Subsequently, we conducted Functional Enrichment Analysis. GO term revealed that CPNE1 and its coexpressed genes were primarily involved in the establishment of protein localization to membrane, ribonucleoprotein complex biogenesis, lipid localization and response to xenobiotic stimulus (Figure 5d).
KEGG results showed these genes were mainly enriched in ribosome, spliceosome, metabolic pathways and bile secretion (Figure 5e).

Regulators of CPNE1 networks in LIHC
To further explore the possible regulators of CPNE1 in LIHC, we analysed networks of transcription factor (TF), miRNA or kinase targets of CPNE1 co-expression genes. Kinases ATR, CHEK1, PLK3, CHEK2 and DAPK1 were the top 5 most important targets (Table 3). Interestingly, CPNE1 co-expression genes were not enriched in any signi cant miRNA targets (Table 3). TF enrichment results revealed CPNE1 coexpression genes were predominantly enriched in E2F transcription factor family, including V$E2F1DP2_01, V$E2F_02, V$E2F1_Q6_01, V$E2F1DP1_01 and V$E2F1DP2_01 (Table 3). Results above suggested that CPNE1 had wide-ranging impact on overall transcriptome in LIHC.
The expression level of CPNE1 in LIHC cell lines and the construction of knockdown cell lines To validate whether CPNE1 was overexpressed in LIHC cell lines, we tested the mRNA expression level of CPNE1 in L02 (a human normal liver cell line) and 5 human hepatoma cell lines (MHCC-97H, HepG2, Hep3B, Huh7 and HLF) by using RT-qPCR. Compared with L02 cell, the expression level of CPNE1 in HepG2, MHCC-97H and huh7 was much higher, which indicated the expression level of CPNE1 in human hepatoma cell lines was higher than that in human normal liver cell line (Figure 6a). The result was consistent with our bioinformatic analysis.
From the results above, we selected HepG2 and MHCC-97H for the subsequent experiments and constructed CPNE1 knockdown cell lines. We chose 3 RNA interference targets (CPNE1-si1, CPNE1-si2, CPNE1-si3) transiently transfected into HepG2 and MHCC-97H cells. The knockdown e ciency of CPNE1 was detected by Western Blot and RT-qPCR. Compared with the negative control group, the expression level of CPNE1 in CPNE1-siRNA transfected cells was signi cantly decreased (Figure 6b-e). Of these, CPNE1-si1 and CPNE1-si2 revealed a higher knockdown e ciency in HepG2 and MHCC-97H cells so we selected CPNE1-si1 and CPNE1-si2 for the subsequent experiments.
Effects of CPNE1 on LIHC cell proliferation CCK8 assay was performed to test the difference of cell viability between negative control group and CPNE1-siRNA transfected group. The result showed the OD value of CPNE1-siRNA transfected cells was much smaller than control group after 48h, which indicated the cell viability of CPNE1 knockdown cell lines was considerably reduced in HepG2 and MHCC-97H cells (Figure 7a, b). In addition, plate clone formation assay revealed the clone numbers of CPNE1-siRNA transfected cells were less than control group in HepG2 and MHCC-97H cells (Figure 7c-e). Above results showed that knockdown of CPNE1 inhibited LIHC cells proliferation.

Effects of CPNE1 on LIHC cell migration
To further explore the correlations between CPNE1 and LIHC cell migration, the scratch assay was performed to test the impact of CPNE1 on LIHC cell migration. The result showed the healing rate of low CPNE1 expression group signi cantly reduced in comparison to control group in HepG2 and MHCC-97H cells (Figure 8a-d). This suggested that CPNE1 was related with the migration ability of HepG2 and MHCC-97H cells. Meanwhile, Transwell assay was conducted to validate the effect of CPNE1 on LIHC cell lines migration and invasion capabilities. Consistent with the scratch assay results, in HepG2 and MHCC-97H cells, the number of CPNE1 knockdown cells traversing to the lower chamber was less than that in control group (Figure 8e, f). These results indicated that CPNE1 participated in the regulation of migration and invasion capabilities in LIHC cell lines.

Effects of CPNE1 on AKT/P53 pathway in LIHC
Western blot experiment was conducted to detect protein expression levels of AKT and P53 for further exploration in LIHC. Compared to the control group, the protein expression level of p-AKT in CPNE1-siRNA group was markedly decreased and the expression level of TP53 was upregulated, while the expression level of total AKT indicated no signi cant change in HepG2 and MHCC-97H cells ( Figure  9a, b). These results showed that the knockdown of CPNE1 may inhibit AKT/P53 pathway, thus suppressing cell proliferation of LIHC cell lines.

Correlation of CPNE1 expression with immune in ltration and immune markers in LIHC
We explored the relationship of CPNE1 expression and immune in ltration using TIMER. The correlation coe cients between CPNE1 expression and the abundances of multiple immune cells (dendritic cells, neutrophils, CD8 + T cells, macrophages, CD4 + T cells and B cells) were explored using Spearman tests. We found that CPNE1 expression had no correlation with tumour purity (cor =0.051, P = 3.47E-01). Furthermore, CPNE1 expression had signi cant association with all six immune cells in ltration, especially B cells (cor =0.398, P = 1.65E-14), macrophages (cor =0.396, P =3.02E-14) and dendritic cells (cor =0.395, P =3.80E-14) (Figure 10a). CPNE1 mutation did not impact immune in ltration (Figure 10b). Additionally, by using Spearman correlation analysis in TIMER database, we assessed the correlation between CPNE1 expression and 7 immune checkpoint molecules (PDCD1 CD274 CTLA4 LAG3 HAVCR2 TIGIT) and found CPNE1 expression was strikingly positively correlated with 7 immune checkpoint molecules (Figure 10c). Then, we analysed the relationship between CPNE1 expression and immune subtypes, which indicated that CPNE1 expression was signi cantly correlated to immune subtypes in LIHC (P < 0.001) (Figure 10d).
All together, these results suggest that CPNE1 is critically engaged in immune in ltration during the advancement of LIHC.

Discussion
Liver cancer is one of the most frequent and fatal digestive malignancies and leads to over one million deaths every year around the world [29,30]. LIHC is a highly aggressive disease and its 5-year postsurgical survival rate is 30%-40% [4,31]. China has the highest incidence of liver cancer across the world [32]. Intrahepatic dissemination, extrahepatic in ltration and metastasis are the leading reason of poor prognosis in LIHC patients [33,34]. The incidence of LIHC is continually increasing, Nevertheless, there is no successful therapy [35].
CPNE1, a tumor-related gene, plays the role of proto-oncogene to promote tumor development. Similar to other CPNE1 family members, the specialized structures determine the key role of CPNE1 in membranes transport and signal transduction [11]. Via vWA domain, CPNE1 could recruit, modulate transcription factors NF-kB and then activate TNF-α receptor, which in turn regulated TNF-α signaling. Meanwhile, the upregulation of TNF-α in uenced the expression of CPNE1 and a positive feedback mechanism existed between CPNE1 and TNF-α. Also, CPNE1 mediated NF-kB signaling by facilitating TNF-α-dependent Inhibitory-κB (IkB) degradation [36]. Via interacting directly with p65, CPNE1 lead to shear of p65 Nterminus and terminated the transcription of NF-kB, which in turn inhibited transcriptional activation of NF-kB [37]. It had been recognized that NF-kB was linked to multiple behaviors of cancer cells, including cell proliferation, apoptosis, migration and invasion [38] and played pivotal functions in initiation and progression of many malignancies (breast cancer, lung cancer, gastric cancer) [39][40][41][42]. Study had reported that CPNE1 could promote proliferation and multi-differentiation potency of neuronal stem cells by activating AKT/mTOR signaling [43]. CPNE1 may regulate growth, migration and invasion of lung adenocarcinoma cells through AKT and ERK pathways, which could promote nonsmall-cell lung cancer progression [15]. CPNE1 was a target of miR-335-5 and CPNE1 silencing could effectively improve clinical responses of EGFR-tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer [44]. In osteosarcoma, downregulation of CPNE1 not only signi cantly impaired the proliferation and metastasis of Saos-2 cell and enhanced sensitivity to cisplatin and doxorubicin, but also changed the expression of genes related to ECM receptors-associated pathway, MAPK pathway, TGF-β pathway, apoptotic pathway and NOD-like receptor pathway [19]. CPNE1 may promote tumorigenesis and radioresistance of triple-negative breast cancer (TNBC) through AKT pathway activation and so target expression of CPNE1 could be a good strategy to sensitize TNBC to radiotherapy [14]. But the role of CPNE1 in liver cancer is not clear.
The role of a great deal genes is complex in the human body. The development of bioinformatics can markedly improve the accuracy and e ciency of studies target genes and cancer [25,45]. In our study, we con rmed the expression of CPNE1 was higher in LIHC tissue than that in normal tissues. High expression of CPNE1 showed potential clinical signi cance and was linked to poor survival of LIHC patients. These results indicated that CPNE1 was a potential target for LIHC treatment. Except for altered gene expression, we also found the mutational signatures of CPNE1 were predominantly missense mutations. However, the mutation frequency of CPNE1 in LIHC was relatively low (only 0.5%) and we failed to nd the association between these mutations and prognosis. More analysis are needed to con rm the clinical signi cance of CPNE1 mutations. To explore the intrinsic mechanisms of CPNE1 in LIHC, the coexpression network of CPNE1 was constructed and gene set enrichment analysis demonstrated CPNE1 and its coexpressed genes were primarily involved in the establishment of protein localization to membrane, ribonucleoprotein complex biogenesis, lipid localization and response to xenobiotic stimulus.
For exploring regulators potentially responsible for CPNE1 dysregulation, we found that CPNE1 is linked with a network of kinases including ATR, CHEK1, PLK3, CHEK2 and DAPK1 in LIHC. These kinases participate in the regulation of mitosis, DNA damage response, cell cycle and genomic stability, and exhibited survival prognosis and differential expression in LIHC. In fact, ATR, a member of phosphatidylinositol-3-kinase-related kinase family, is the major players of DNA damage response, and represents an attractive target for developing antimitotic agents [46]. In addition, activated ATR is critical in the late G2 and S phases to assure appropriate and replication of the whole genome [47,48]. PLK3 may regulate cell cycle progression, centrosomal functions, mitosis, DNA replication, and Golgi fragmentation [49]. In many human malignancies, PLK3 expression was downregulated, including those in the stomach, kidney, head and neck, lung, colon, liver and rectum. Several studies demonstrated downregulated PLK3 expression may be linked with cancer development [48,49].
Then, the E2F family account for the main transcription factors for CPNE1 dysregulation. E2F1 is one of the major bonds in the cell cycle regulatory network. In the progression of LIHC, activated E2F signaling was common, and studies have indicated that dosage-dependent copy number gains in E2F3 and E2F1 drive LIHC [59]. Our ndings indicate that E2F1 is a critical regulator of CPNE1 and that CPNE1 might function by this factor to modulate the proliferation ability and cell cycle of LIHC.
Here, we revealed that the overexpression of CPNE1 was positively linked to immune in ltration. This nding demonstrates that CPNE1 plays a crucial role in immune in ltration during hepatocarcinogenesis.
As far as we know, we are the rst to explore the association of CPNE1 and immune in ltration in LIHC.
To validate the effect of CPNE1 on cell proliferation, migration and invasion in LIHC cell lines, we constructed CPNE1 knockdown cell lines and results revealed the capabilities of CPNE1 knockdown cell proliferation, migration and invasion decreased compared to control cells, which suggested that CPNE1 participated in the genesis and progression of LIHC. Furthermore, CPNE1 affected AKT/P53 pathway and might function by this pathway to modulate the malignant transformation of LIHC.
However, this study had some limitations. First, our ndings were just con rmed in public databases and not in clinical samples. Second, the precise mechanism of CPNE1 involved in the development of LIHC was not elucidated in our study. Last, some of the ndings may need further validatation. Nonetheless, CPNE1 is a potential molecular target in the therapy of LIHC.

Conclusions
The expression of CPNE1 was signi cantly higher in HCC tissues than in normal liver tissues, and high CPNE1 expression was associated with poor prognosis. Knockdown of CPNE1 inhibited AKT/P53 pathway activation and suppressed HCC cell proliferation and migration. There was a signi cant correlation between CPNE1 expression and tumor immune in ltration in HCC.  Table 3 The Kinases, miRNA and transcription factors-target networks of CPNE1 in LIHC (LinkedOmics).