Silencing of LINC00467 inhibits cell proliferation in testicular germ cell tumors cells Running Title: Silencing of LINC00467 regulates cell proliferation

doi:10.21203/rs.3.rs-2584712/v1

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Silencing of LINC00467 inhibits cell proliferation in testicular germ cell tumors cells Running Title: Silencing of LINC00467 regulates cell proliferation

https://doi.org/10.21203/rs.3.rs-2584712/v1

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Our previous study showed that LINC00467 expression decreased significantly in testicular germ cell tumors (TGCTs) compared with adjacent tissue. LINC00467 expression in TGCTs was upregulated at stages II/III compared with stage I, and was negatively correlated with the 5-year overall survival and 5-year disease-free survival. However, the role of LINC00467 in the development of TGCTs remains to be elucidated further. LINC00467 expression was silenced in the NCCIT and TCAM-2 cell lines using small interfering RNA (siRNA). The expression level of genes was validated using quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The MTT and CCK8 assays were used to detect cell proliferation. The effects on cell cycle were evaluated using flow cytometry. Western blotting analysis was used to detect the expression level of proteins, while RNA-sequencing and bioinformatics methods were used to explore the mechanism of action of LINC00467 in TGCTs. The silencing of LINC00467 expression decreased cell proliferation, induced S phase arrest, and downregulated the cell cycle-related protein PCNA expression while upregulating the expression of P21. Dihydrotestosterone (DHT) stimulation experiments showed that DHT could upregulate the expression of LINC00467 and that the silencing of LINC00467 could reverse the effect of testosterone on cell proliferation. The Gene Set Enrichment Analysis (GSEA) showed that the P53 signaling pathway was associated with LINC00467. Our study reported that LINC00467 regulated cell proliferation and induced S phase arrest in TGCTs cells through the cell cycle-related proteins, PCNA, and P21. These enriched the mechanism of non-coding RNAs in the development of TGCTs.

Testicular germ cell tumors

LINC00467

Dihydrotestosterone

cell proliferation

cell cycle

Silencing LINC00467 blocks the proliferation of testicular germ tumor cells.
Silencing LINC00467 causes S phase arrest, which may be through regulated PCNA and p21.
LINC00467 is required for the testosterone promotion of cell proliferation.

Testicular cancer is a common malignancy with increasing incidence in young men aged 15 to 44.¹^,² Approximately 98% of testicular cancers are testicular germ cell tumors (TGCTs). TGCTs is a solid tumor that originates from primitive germ cells of the testis. The main histologic subtypes of TGCTs are seminoma and non-seminoma.³ Among them, seminoma originates from the primordial germ cells of the testis and has the morphological characteristics of germ cells. Non-seminomatous tumor cells have two states of differentiation, embryonic and extra-embryonic, including embryonal carcinoma, sarcopenia, yolk sac tumor, and choriocarcinoma. Most patients can be cured using orchiectomy with radiotherapy.^4,5 However, the pathogenesis of TGCTs remain to be elucidated. In addition, there are a series of other problems in TGCT patients who have received treatment.⁶ They may suffer from side effects of radiotherapy and may be twice as likely to develop a second malignancy, such as secondary non-germ cell cancers, cardiovascular disease, or hypogonadism.^7-9 Therefore, a deeper understanding of the pathogenesis of TGCTs is necessary to find more effective treatments and diagnosis for TGCTs.

Long-stranded non-coding RNAs (lncRNAs) are non-coding RNAs that are longer than 200 nt in length, which generally does not encode or rarely encodes a protein. They can be classified as sense lncRNAs, antisense lncRNAs, bidirectional lncRNAs, intragenic lncRNAs, and intergenic lncRNAs. Recently, it has been identified that the vast majority of transcribable sequences in the human genome are lncRNAs.¹⁰ They can regulate target genes through transcriptional regulation, transcription factor trapping, chromatin circularization, and gene methylation.¹¹ Abnormal changes in lncRNAs expression may lead to a variety of diseases, including cancers.¹² For example, several studies have reported that lncRNA MALAT-1 indicated the poor prognosis of oral squamous cell carcinoma and non-small cell lung cancer by promoting tumor growth and metastasis.^13,14 However, there are only a few reports on whether lncRNA is involved in the development of TGCTs.

LINC00467 is a 2028 bp full-length lncRNA located on chromosome 1 that is also known as C1orf97. The expression of LINC00467 is highly expressed in the normal testis, but is lower or not expressed in other tissues.¹⁵ The mechanism by which its expression is regulated is still unknown. LINC00467 was first reported to promote neuroblastoma survival and inhibit apoptosis by cis-regulating the expression of its neighboring gene, RD3.¹⁶ LINC00467 targets microRNA‑451a, which promotes carcinogenesis and metastasis in human colorectal cancer.¹⁷ Another study demonstrated the critical role played by the LINC00467/miR-9-5p/PPARA pathway for the occurrence and progression of hepatocellular carcinoma.¹⁸ Overall, LINC00467 is likely to play an important role in many other cancers. Our previous study showed that LINC00467 expression decreased significantly in TGCTs compared with adjacent tissue.¹⁵ LINC00467 expression in TGCTs was upregulated at stages II/III compared with stage I, and was negatively correlated with the 5-year overall survival and 5-year disease-free survival.¹⁵ Therefore, in this study, we conducted an in-depth study on the specific function and molecular mechanism of LINC00467 in TGCTs by knocking down LINC00467 in TGCT cell lines. The results of this study may provide novel ideas for the diagnosis and targeted therapy of TGCTs, and may also have implications for the development of diagnostic markers and therapeutic targets for other tumors.

Cell Culture and siRNA Transfection

The seminoma cell line TCAM-2 (seminoma) was donated by Professor Tang Yuxin.¹⁹ The human embryonic carcinoma cells line NCCIT (non-seminoma) was purchased from the American Type Culture Collection (ATCC, VA, USA). TCAM-2 cells and NCCIT cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, GIBCO, USA) and RPMI-1640 medium (GIBCO, USA), respectively. All cells were cultured in a medium containing 10% fetal bovine serum (FBS, GIBCO, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO) at 37°C and in a 5% CO2 incubator. Transfection was performed when the cells had grown to 60-80% confluence. The instructions provided with the lipofectamine 3000 transfection kit (cat no. L3000015, ThermoFisher) were strictly followed. The LINC00467 siRNA sequences were as follows: LINC00467-siRNA-1: GTCTTCAGGAAGCCAGACA, LINC00467-siRNA-2: GATGCTCTGTAAACCACAT. Both of The nucleotide sequence of the non-target negative control (NC) and LINC00467 siRNA sequences were synthesized by Ribobio (Guangzhou, China).

RNA extraction and qRT-PCR

The TRIzol method was used to extract RNA (cat no. 15596018, ThermoFisher). 1-2 μl of RNA was used to determine its concentration and quality using a spectrophotometer, and another 1-2 μl of RNA was used for gel electrophoresis to determine the quality of the RNA. A Roche reverse transcription kit (Transcriptor First Strand cDNA Synthesis Kit, cat no. 4897030001, Roche) was used for the reverse transcription PCR reaction. Levels of target mRNAs were measured using qRT-PCR (LightCycler 480 SYBR Green I Master, 10 x, cat no. roche04887352001, Roche) after transfection for 48 hours, and β-actin was used for normalization. The primers sequences see in Table 1.

MTT assay

The cells were seeded into 96-well plates at a concentration of 2000 cells per well in 3 replicate wells after transfection 48 hours. At 24, 48, 72, 96 and 120 hours, 20 μl of MTT solution (5 mg/ml) was added to each well. After incubation in a 5% CO₂ atmosphere at 37°C for 4 h, the supernatant in each well was discarded. Then, 150 μl of DMSO was added to each well, and placed on a shaker for 1 min. The absorbance of each well was measured at 492 nm using a microplate reader. The absorbance value was used as the ordinate and the interval time was used as the abscissa, while the MTT cell growth curve was drawn using GraphPad Prism 5.0 software.

CCK8 assay

The cells were seeded into 96-well plates at a concentration of 2000 cells per well in 3 replicate wells after transfection 48 hours. At 24, 48, 72 and 96 hours, 10 μl of CCK-8 Solution (Sangon Biotech) was added to each well. After incubation in a 5% CO₂ atmosphere at 37°C for 2 hours, the mixture was shaken for 1 min on a shaker in the dark. Then, absorbance at 450 nm was measured using a microplate reader. The absorbance value was used as the ordinate and the interval time was used as the abscissa. The CCK8 cell growth curve was drawn using GraphPad Prism 5.0 software.

Cell cycle assays

The cells were mixed with 95% ice-cold ethanol and fixed at 4°C for 16 hours after transfection 48 hours. Then, the cells were centrifuged at 300 g for 3 min and the supernatant was discarded. The cells were washed by resuspending them in ice-cold PBS. Then, 0.4 ml of propidium iodide staining solution (Cell Cycle and Apoptosis Analysis Kit, cat no. FXP021-200, Beijing 4A Biotech Co., Ltd) was added to each tube of cells slowly and the cell pellet was fully resuspended and incubated at 37°C for 30 minutes in the dark. The red fluorescence was detected at an excitation wavelength of 535 nm using a BD flow cytometry analyzer. The light scattering pattern was detected at the same time. Cell cycle analysis was performed using Modfit software.

Cell apoptosis assay

The cells were resuspended in a 1×Annexin-binding buffer and the cell concentration was adjusted to 1×10⁶ cells/mL. Then, 100 µL of the cell suspension from each sample was taken and 5 µL of Alexa Fluor® 488annexin V and 1 µL of PI (100 µg/mL) (FITC Annexin V Apoptosis Detection Kit I, cat no. 556547, BD) was added. The mixture was kept to incubate at room temperature for 15 minutes in the dark. Then, 400 µL of 1×annexin-binding buffer was added to each sample and mixed by vortexing, and then a BD C6 flow cytometer was used for detection. Cell apoptosis analysis was performed using Flowjo 10 software.

DHT-treated cells

The cells were seeded into 6-well plates at an appropriate density. After the cells had reached 70% confluence overnight, the cells were washed twice with 1×PBS, and mediums containing 0 nM/mL, 1 nM/mL, and 5 nM/mL DHT (cat no. S4757, Selleck) were prepared using the complete medium as previously described. Then, 2 mL of complete medium was added at the above concentrations to each well on the six-well plate using three wells for each concentration. The cells were cultured in a 5% CO₂atmosphere at 37°C for 36 h and then the cells were collected.

Western blotting analysis

The protein precipitates were extracted using a RIPA protein lysate (cat no. 89900, ThermoFisher) containing a protease inhibitor phenylmethylsulfonyl fluorid (cat no. 36978, ThermoFisher, USA). Protein concentrations were determined using a BCA kit (ThermoFisher, USA). Then, the protein was subjected to SDS polyacrylamide electrophoresis and transferred onto a PVDF membrane (cat no. IPVH00010, Millipore). The PVDF membrane was blocked with 5% skimmed milk at room temperature. Then, the PVDF membranes were incubated overnight at 4°C with primary antibodies diluted with 5% skimmed milk. The primary antibodies used were mouse anti-PCNA Monoclonal antibody (1:2000, cat no. ab29, Abcam, USA) and rabbit anti-P21 Polyclonal antibody (1:1000, cat no. 10355-1-AP, Proteintech, Chian). Rabbit anti-Vinculin Monoclonal antibody (1:5000, cat no. ab129002, Abcam, USA) was used as the loading control. Then, the appropriate secondary antibody (CoWin Biosciences) was incubated with the PVDF membranes based on the genetic origin of the primary antibody. A gel imaging system was used to scan the protein bands using ECL reagents, and Vinculin was used as an internal reference.

RNA-sequencing

RNA sequencing was performed through de-ribosomal strand-specific library sequencing by first removing rRNA from total RNA to retain the mRNAs and ncRNAs. The enriched mRNAs and ncRNAs were fragmented into short fragments. The cDNA fragments were purified using a QiaQuick PCR extraction kit to repair the ends, add poly(A), and ligate the Illumina sequencing joints. The second-strand cDNA was digested with UNG (uracil-N-glucanase). Agarose gel electrophoresis was conducted to determine the size of the digested products. Then, the products were obtained and amplified using PCR and sequenced using an Illumina HiSeqTM 4000 platform (Gene Denovo, Guangzhou, China). The resulting cDNA library was sequenced using an Illumina Novaseq6000 system (Gene Denovo Biotechnology Co., Guangzhou, China).

Gene Set Enrichment Analyses

GSEA determines whether a pre-defined set of genes shows statistically significant differences in the gene expression profiling of the negative control group vs the LINC00467 silencing group. An annotated gene set (https://www.gsea-msigdb.org/gsea/msigdb/genesets.jsp?collection=CP:KEGG) was selected as the reference gene set. The Hiplot (https://hiplot.com.cn/) was used for the enrichment analysis at default parameters.

Statistical analysis

The Student’s t-test was used to determine the significance of differences between the two groups. GraphPad Prism Software 8.0 was used to analyze the statistics. A P value of <0.05 was considered to indicate a significant difference.

Silencing LINC00467 inhibits the proliferation of TGCT cells

We used siRNA to silence the expression of LINC00467 in TCAM-2 and NCCIT cells and detected the silencing efficiency using qRT-PCR (Figure 1A, 1B). The cell proliferation of TGCT cells was analyzed using CCK8 (Figure 1C, 1D) and MTT (Figure 1E, 1F) assays. As shown in Figure 1C-1F, LINC00467 silencing inhibited cell proliferation, compared with the negative control group.

Silencing LINC00467 induces S phase arrest but does not affect apoptosis

Flow cytometry assays were used to examine the effect of LINC00467 silencing on the cell cycle (Figure 2A). It showed that LINC00467 silencing affected cell cycle distribution and induced S phase arrest (Figure 2B, 2C), compared with the negative control group. Next, to examine the effect of LINC00467 silencing on cell apoptosis, Annexin V/PI double staining was performed, followed by flow cytometry analysis (Supplementary Figure 1A). LINC00467 silencing had no effect on cell apoptosis, compared with the negative control group (Supplementary Figure 1B, 1C). Together, these results indicate that LINC00467 may exert a cancer-promoting effect.

Silencing LINC00467 regulates cell cycle-related proteins

After identifying the function of LINC00467, we attempted to explore its molecular mechanism. Based on the aforementioned cell proliferation detection and cell cycle detection results, after silencing LINC00467 in TCAM-2 and NCCIT cells, we detected key node molecules, such as P21 and PCNA, in the cell cycle signaling pathway (Figure 3A). We found that the silencing LINC00467 resulted in a decrease in PNCA protein expression in the two cell lines, as well as the upregulation of the P21 protein expression (Figure 3B-3C).

Silencing LINC00467 reverses the effect of testosterone on cell proliferation

Androgens have a robust impact on cell proliferation. Next, we aimed to verify whether testosterone exerts a regulatory effect on LINC00467, which affects cell proliferation. First, we tested the effect of different concentrations of DHT (0-5 nM/ml) on the expression of LINC00467 in cultured NCCIT cell lines using qRT-PCR. Interestingly, both 1 nM/ml DHT and 5 nM/ml DHT upregulated the expression of LINC00467 (Figure 3D). Further, MTT experiments showed that silencing LINC00467 could significantly inhibit the promotional effect of DHT on cell proliferation (Figure 3E). Our results showed that LINC00467 is required for the testosterone promotion of cell proliferation.

GSEA showed that LINC00467 silencing was related to the P53 signaling pathway

To further explore the specific mechanism of LINC00467 on the proliferation of TGCT cells, we used RNA-sequencing to detect the expression profile of TCAM-2 cells after silencing LINC00467 and the expression levels of many genes were found to be regulated (Figure 4A). Based on RNA-sequencing data, we explored the LINC00467 related signal pathway using GSEA. The p53 signaling pathway was enriched in the gene expression dataset following the silencing of LINC00467 (Figure 4B). The network of the p53 signaling pathway genes and their transcription factors based on our RNA-seq data revealed that LINC00467 may be able to regulate the P53 signaling pathway through multiple transcription factors, including the transcription factors, NONAG and HOXA5 (Figure 4C). One of the P53 signaling pathway genes, CCNG1, is associated with the distribution of TGCT pathological grades (Figure 4E). The expression of CCNG1 was also significantly downregulated in the LINC00467 silencing group, which was validated by qRT-PCR (Figure 4D). These results indicate that the silencing of LINC00467 is associated with the P53 signaling pathway and that this pathway may be involved in the progression of TGCT.

Silencing LINC00467 upregulates the expression of immune checkpoints

Immune checkpoints are very important for the development of tumor immunotherapy and immunotherapy drugs. Two immune checkpoints, HAVRC2 and TIGIT, were found to be significantly upregulated in TGCT compared with adjacent tissue by analyzing The Cancer Genome Atlas (TCGA) databases (Figure 5A). After analyzing our RNA-sequencing data, it was found that HAVRC2 and TIGIT were differentially upregulated after the silencing of LINC00467 (Figure 5B). In addition, the expression of HAVRC2 and TIGIT were upregulated by the silencing of LINC00467, as shown through qRT-RNA validation (Figure 5C, 5D). Furthermore, the negative correlation between GDSC drug sensitivity (CH542802 and XMD14-99) with HAVCR2 expression indicated that the higher the expression of LINC00467, the less sensitive the CH542802 and XMD14-99 drugs (Figure 5E). The negative correlation between CTRP drug sensitivity and the expression of HAVCR2 and TIGIT also showed that the higher the expression of LINC00467, the less sensitive CTRP drugs, such as I-BET151, SR-II-138A, and triazolothiadiazine (Figure 5F). The above results are significant for the selection of targeted drugs for the clinical treatment of TGCTs.

TGCTs are malignant tumors with a high genetic component that tends to be clustered into families.²¹ Its genetic risk is polygenic, with approximately 50 susceptibility genes already identified.²² The functional role of these gene products in the development of TGCTs is unclear and the underlying molecular mechanisms are complex. Das and colleagues reported that SPRY4-IT1 acts as an oncogene in TGCT development.²³ The miR-302/367 and miR-371-373 clusters have been found to be highly expressed in tumor tissues as well as in serum samples obtained from TGCT patients.^24–27 AKAP4 and TNP1 are associated with TGCT development by regulating immune cells infiltration in the TGCT immune microenvironment.²⁸ AXIN1 has been shown to act as a tumor suppressor in TGCT cells by reducing PI3K/AKT/mTOR signaling.²⁹ The above results indicate that both genetic factors and epigenetic factors are associated with the development of TGCTs and LncRNAs may act as oncogenes or tumor suppressors in TGCTs.

LINC00467 is a testis-specific lncRNA with an expression pattern that is very similar to testicular tumor antigens, and is therefore very likely to exert important functions. Our previous study showed that LINC00467 could activate the AKT signaling pathway by binding to AZGP1 to promote its degradation, which in turn promotes non-small cell lung cancer progression.³⁰ Furthermore, another study conducted by us showed that LINC00467 is driven by copy number amplification and DNA demethylation, and may be a potential biomarker for breast cancer diagnosis and prognosis.³¹ In this study, we found that the silencing of LINC00467 inhibits the proliferation of TGCTs cells. In addition, the silencing of LINC00467 also induced cell S phase arrest but did not affect apoptosis. To further elucidate the mechanism by which LINC00467 performs its function, we detected two proteins involved in the cell cycle, PCNA and P21. The PCNA protein exists in the nucleus and is a cofactor for DNA polymerase delta, a marker protein for cell proliferation and DNA replication. This protein can participate in the control of eukaryotic DNA replication by increasing the processability of DNA polymerase during leading strand elongation.³² It also induces the strong stimulation of 3'-5' exonuclease and 3'-phosphodiesterase, which increase the processivity of the leading strand during DNA replication. Furthermore, it also plays a key role in DNA damage response (DDR) through its positioning at replication forks to coordinate DNA replication.³² Our Western blotting results also showed that PCNA expression was significantly downregulated following LINC00467 silencing, suggesting that LINC00467 may play a role in DNA synthesis and other stages of cell proliferation. P21 protein binds and inhibits the phosphorylation activity of CDK2 or CDK4 complexes, arresting cell cycle progression at the G1 phase. The expression of this protein is tightly controlled by the tumor suppressor protein, P53, which responds to various stress stimuli, including DNA damage, by mediating p53-dependent G1 arrest of the cell cycle. P21 also inhibits DNA synthesis through the DNA polymerase delta by competing with POLD3 for PCNA binding. Overall, LINC00467 plays a regulatory role in DNA replication and DNA damage repair. Our experimental results showed that the expression of P21 was significantly upregulated after the silencing of LINC00467. Interestingly, we found that the cell cycle was not arrested at the G1 phase, but at the S phase. This suggests that LINC00467 may act through the unconventional P21-dependent cell cycle regulation mode, and its specific mode of action remains to be further elucidated.³³

Studies have reported that the upregulation of testosterone promotes the proliferation of epithelial cells and adipocytes.^34,35 In our study, cell experiments confirmed that adding DHT to cells can stimulate the expression of LINC00467. Furthermore, the silencing of LINC00467 could significantly inhibit the promotional effect of DHT on cell proliferation. These results suggest that LINC00467 may be an important link during DHT-regulated cell proliferation, which provides a novel perspective for us to understand the progression of TGCTs. To further explore the mechanism by which LINC00467 affects TGCT cell proliferation, we performed RNA-sequencing of LINC00467-silenced TCAM-2 cells. The enrichment analysis showed that the crucial biological signaling pathway enriched in the LINC00467-silenced group is the P53 signaling pathway. One of the P53 signaling pathway genes, CCNG1, is also associated with the distribution of TGCT pathological grades. It has been suggested that LINC00467 may regulate the cell cycle and inhibit cell proliferation through the P53 signaling pathway. Additionally, our previous study found that LINC00467 played a key role in the invasion and metastasis of TGCTs,¹⁵ indicating that LINC00467 regulates the invasion and metastasis of TGCTs, while the resulting cell proliferation participates in TGCT development.

Immune checkpoints play monumental roles in regulating the immune system and retain key potentials in hindering the development and autoimmunity of malignancies.³⁶ At present, immune checkpoint therapy is used as a cancer therapy in addition to radiation, chemotherapy and surgery, and includes the blocking of CTLA-4 and PD-1.³⁷ To explore whether LINC00467 regulates the expression of immune checkpoints, we analyzed our RNA-sequencing data and performed qRT-PCR validation. The results showed that LINC00467 silencing leads to increased expression of HAVRC2 and TIGIT. Both immune checkpoints were found to be significantly upregulated in TGCT as shown by the TCGA data analysis. According to our previous study, the higher the expression of LINC00467, the lower the rate of TGCT patient overall survival and progression-free survival.¹⁵ These results indicate that LINC00467 directly regulates the expression of HAVRC2 and TIGIT, which can provide novel insights for developing new treatment strategies for TGCT.

A number of recent studies have shown that extracellular nucleic acids (Circulating nucleic acid, CNA), which is found in the blood (serum or plasma), urine, seminal plasma and other body fluids, are closely related to the physiological and pathological states of the human body.^38–40 Therefore, the detection of circulating nucleic acid, as a minimally invasive and convenient method for detecting biomarkers, is of great significance for disease diagnosis and prognosis prediction. In the next step, we will further study and clarify the expression level of LINC00467 in the serum and other body fluids of TGCT patients and its clinical significance, to establish a more minimally invasive and convenient novel method for disease diagnosis, classification, and prognosis prediction based on LINC00467 expression.

In summary, we found that the silencing of LINC00467 performs an important role by inhibiting cell proliferation through the regulation of proteins, such as P21 and PCNA. The testosterone-stimulated expression of LINC00467 may promote the development of TGCTs. This study also provides a theoretical and experimental basis for drug research that targets LINC00467 for the treatment of TGCTs.

AUTHOR CONTRIBUTIONS

H. B. and L. F. conceived and planned the experiments. D. Z., F. Z., and J. F. acquired the data. H. B., F. Z., and Z. L. analyzed and interpreted the data. F. Z. drafted the manuscript. J. F. and Q. Z. proofed the manuscript. H. B. and L. F. revised and confirmed the final manuscript. All authors approved this version of the article.

ACKNOWLEDGMENTS

This research was funded by the National Key Research and Development Program of China (Nos. 2016YFC1000600); Changsha Municipal Natural Science Foundation (Nos. kq2014033); Hunan Provincial Grant for Innovative Province Construction (Nos. 2019SK4012); and the Fundamental Research Funds for the Central Universities of Central South University (Nos. 2021zzts0322).

DISCLOSURE STATEMEN

The authors report there are no competing interests to declare.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the

corresponding authors upon reasonable request.

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Table 1 is available in the Supplementary Files section.

No competing interests reported.

Table1.pdf
SupplementaryFigure1.jpg
Supplementary Figure 1. Silencing of LINC00467 does not affect the apoptosis of TGCT cells (A) Apoptosis was determined using flow cytometry analysis of Annexin V/PI double-stained TCAM-2 and NCCIT cell lines. (B&C) Apoptosis rate in the two cell lines (n.s: no significance, siRNA group vs. NC group). n.s: not statistically significant.

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Silencing of LINC00467 inhibits cell proliferation in testicular germ cell tumors cells Running Title: Silencing of LINC00467 regulates cell proliferation

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