C1QTNF6 promotes Oral squamous cell carcinoma tumorigenesis though enhancing proliferation and inhibiting apoptosis of OSCC cells

Immunohistochemistry of OSCC tissue and data from TCGA both implied that C1QTNF6 was closely related to OSCC. We constructed lentivirus to knockdown C1QTNF6 in CAL27 cells and SCC-9 cells. Then the change of C1QTNF6 mRNA expression was detected with qRT-PCR, and the Western blot analysis was performed to detect changes in protein expression. Furthermore, Cell Cycle Analysis and Cell apoptosis analysis was measured. 4-week-old female BALB/c nude mice were purchased to observe the In vivo tumorigenicity. Finally, Pathway Analysis was performed. following


Abstract
Background C1QTNF6 (CTRP6), a member of the CTRP family, has been recently implied to play a role in tumorigenesis. However, the expression status and the role of C1QTNF6 in oral squamous cell carcinoma (OSCC) remains unclear.

Methods
Immunohistochemistry of OSCC tissue and data from TCGA both implied that C1QTNF6 was closely related to OSCC. We constructed lentivirus to knockdown C1QTNF6 in CAL27 cells and SCC-9 cells. Then the change of C1QTNF6 mRNA expression was detected with qRT-PCR, and the Western blot analysis was performed to detect changes in protein expression. Furthermore, Cell Cycle Analysis and Cell apoptosis analysis was measured. 4-week-old female BALB/c nude mice were purchased to observe the In vivo tumorigenicity. Finally, Pathway Analysis was performed.

Results
In this study, we found that C1QTNF6 was overexpressed in OSCC tissues and cell lines, and the cellular proliferation was signi cantly decreased in C1QTNF6 knockdown OSCC cells. Knockdown of C1QTNF6 resulted in cell cycle arrest at the G2/M phase and enhanced apoptosis in OSCC cell lines. Further assays showed that C1QTNF6 silencing inhibits tumor growth of OSCC in vivo. Moreover, microarray analysis revealed that C1QTNF6 silencing results in signi cant alteration of many genes. Ingenuity Pathway Analysis (IPA) revealed that the Acute Phase Response signaling pathway was signi cantly activated following C1QTNF6 silencing.

Conclusions
These results suggested that C1QTNF6 play a promoting role in OSCC tumorigenesis, which may be a promising therapeutic target for OSCC treatment.

Background
Oral squamous cell carcinoma (OSCC) is the most common epithelial malignancy in the oral cavity, which accounts for almost 90% of all oral malignancies [1]. Despite the improvements in surgical and other therapeutic strategies, the 5-year survival rate of OSCC patients is still disappointing [2,3]. So far, limited information about the driving forces and molecular pathways in OSCC tumor formation is available, and the molecular events have not been de ned precisely. Thus, a better understanding of the molecular carcinogenesis of OSCC may provide new insights on the development of novel diagnostic and therapeutic strategies, and the improvement of OSCC patients' prognosis.
C1q/tumor necrosis factor-related proteins (CTRPs/C1QTNF), including 16 members, consisting of four different domains: an N-terminal signal peptide, a collagenous-like domain, a short variable region, and a C-terminal globular domain [4]. CTRPs share a common structural domain with adiponectin [5]. C1QTNF6 (CTRP6) is the sixth member of the CTRP family and is widely expressed in many tissues of rodents and primates, including adipose tissue, brain, spleen, lung, liver, muscle, ovary, and so on [6][7][8][9][10], and each CTRP has a unique tissue expression pro le [11]. C1QTNF6 is secreted in serum-forming oligomeric forms [12] and is involved in diverse physiological processes ranging from metabolism, to host defense and organogenesis [13]. It has been reported that C1QTNF6 can stimulate fatty acid oxidation via activation of AMPK [14]. By generating C1QTNF6-/-mice, it has been demonstrated that C1QTNF6 is a novel regulator of the complement alternative pathway, and can be used to treat rheumatoid arthritis in clinical [15]. One study showed that CTRP4 promoted tumor survival through upregulation of IL-6 and TNF-alpha [16]. Recent researches suggested that CTRP6 may play a role in carcinogenesis and cancer progression. Recent studies showed that C1QTNF6 was overexpressed in clear cell renal cell carcinoma and elevated C1QTNF6 expression correlated with clinical progression [17].It has been con rmed that C1QTNF6 is involved in promoting the proliferation, migration, and in reducing the apoptosis of gastric carcinoma cells and NSCLC [18,19]. A study showed that inhibition of C1QTNF6 induced G2-M cell cycle arrest and cell apoptosis [18]. And it has been reported that C1QTNF6-related signaling pathways activated in ccRCC were mainly enriched in DNA replication, Cell Cycle, EMT, and angiogenesis signaling pathway [17]. In particular, the overexpression of C1QTNF6 has been shown in hepatocellular carcinoma tissues and many HCC cell lines, and promotes neovascularization in transplanted HCC cells [20]. These researches suggested a potential regulatory role of C1QTNF6 in tumorigenesis, which makes great sense to further study the function and mechanism of C1QTNF6 in other cancers.
In this study, we con rmed the high expression of C1QTNF6 in OSCC tissues by immunohistochemical analysis and cell lines by RT-PCR. Then we analyzed The Cancer Genome Atlas (TCGA) and found that C1QTNF6 might be a potential tumor-associated regulator involved in carcinogenesis of OSCC. Furthermore, the C1QTNF6 knockdown was performed in OSCC cell lines to explore the pathological relevance between C1QTNF6 and OSCC. We found that C1QTNF6 promoted the proliferation and suppressed the apoptosis of OSCC cells in vitro. Moreover, the xenograft tumor growth was inhibited by C1QTNF6 silence in vivo. In addition, the signal pathway was analyzed to explore the possible molecular mechanism underlying the regulatory function of C1QTNF6 in OSCC by IPA. The data showed that the Acute Phase Response signal pathway might play a role in C1QTNF6-mediated promotion of OSCC

TCGA data processing
For expression analysis, we selected RNAseq and RNAseqV2 paired sample data for analysis. The raw sequencing data and pathological information were downloaded from the TCGA database (https://cancergenome.nih.gov/). For Head and Neck squamous cell carcinoma (HNSCC), there were 528 samples with available data in the TCGA database, including 520 RNAseqV2 samples, and 40 pairs with paired sample data and pathological information. Our expression pro le analysis was based on these 40 paired samples of RNAseqV2 data. The Trimmed Mean of M-values (TMM) method was applied to data standardization. To avoid errors caused by inappropriate sample grouping, BCV biological coe cient of variation was observed for quality control.
Firstly, we estimated the dispersion of multiple pairs of samples and then used a general linear model to estimate whether there is a difference in genes between different groups. Genes with a statistical test P value less than 0.05 are considered differentially expressed genes. The fold changes (FCs) in the expression were calculated and differentially expressed DEGs with log2|FC|≥1.0 (Cancerrmal) were considered to be signi cant, remaining genes are ltered. Besides, several other criteria were adopted to lter genes: the genes which had been reported have functional and clinical relation in HNSCC; genes of multiple transmembrane proteins; gene annotation is not clear (such as open reading frame); genes reported in more than 100 articles in Pubmed. The nal gene list was randomly concentrated, and C1QTNF6 was selected for further experimental research.
Lentivirus Construction and lentiviral transfection of CAL27 cells and SCC-9 cells C1QTNF6-targeting short hairpin RNA (shRNA) oligonucleotides sequences TGTGTGAGATCCCTATGGT were designed and were synthesized and cloned into the pGV115-GFP vector by GeneChem Corporation (Shanghai, China). The sequence of scrambled shRNA (TCTCCGAACGTGTCACGT) used as the negative control (NC) was also inserted into the pGV115-GFP vector. Lentivirus transfection was performed according to the manufacturer's recommended protocol. The CAL27 cells and SCC-9 cells were seeded into a six-well plate (~1×10 5 cells per well) and incubated at 37℃ with 5% CO 2 until they reached ~70% con uence, then lentivirus was added as MOI 1. After 72 h, cells were observed under a uorescence microscope and collected for the following experiments.

Western blot analysis
Cell samples were harvested, washed twice with PBS, and Lysed with pre-cooled Lysis Buffer. The protein concentration was determined by BCA Protein Assay Kit (Pierce). Each sample protein concentration is adjusted to 2μg/μL, and saved at -80℃ for later use. The total protein was separated on 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene uoride (PVDF) membranes at 4℃ (300mA for 150min). The PVDF membranes were blocked in 5% skim milk in TBST for 1h and incubated overnight with primary antibodies at 4℃. After washing three times in TBST, the membranes were incubated in secondary antibody coupled to HRP before visualization. Immunohistochemistry 13 paired cancer and paracancerous tissue samples were collected from OSCC patients in Qilu hospital of Shandong University, both preoperative and postoperative pathology of which suggest oral squamous cell carcinoma. All patients gave informed consent before the tissue was collected. The research had obtained the approval of Ethics Committee on Scienti c Research of Shandong University Qilu Hospital and the approval number is KYLL-2017-544. OSCC tissues and normal tissues were immunostained for C1QTNF6 expression using a C1QTNF6 antibody (Invitrogen, USA) at a 1:100 dilution for a 1-h incubation according to the supplier's instructions. The secondary antibody used was a goat anti-rabbit IgG incubated for 1 h at 37℃. Images were obtained at the optical facility.

Cell Cycle Analysis by Flow Cytometry
Lentivirus-transfected cells were seeded in 6-cm dishes for culture. When the cells achieved approximately 80 % con uence, they were trypsinized, washed twice in PBS, and xed with pre-chilled 70% ethanol for at least 1h at 4℃. Then the cells were washed with PBS and stained with PI mixture (40×PI stock (2 mg/ml), 100×RNase stock (10 mg/ml) and 1×PBS buffer at a dilution of 25:10:1,000) for 45 min at 37℃. The cell cycle results were measured by Guava easyCyte HT (Millipore, USA), and all experiments were performed in triplicate.

Annexin V Apoptosis Assay
Cell apoptosis analysis was measured using eBioscience™ Annexin V Apoptosis Detection Kit APC according to the manufacturer's instructions. Brie y, Lentivirus-transfected cells were trypsinized, washed twice in pre-chilled D-hank's, and resuspended in binding buffer. Then 5 μl of annexin V-APC was added into 100μl of the cell suspension and incubated at room temperature in the dark for 15 min. Samples were analyzed by Guava easyCyte HT (Millipore, USA).
In vivo tumorigenicity Twenty 4-week-old female BALB/c nude mice were purchased from the Shanghai Institute for Biological Sciences (Shanghai, China). Twenty BALB/c nude mice were randomly divided into two groups. The lentivirus-transfected cells (1 × 10 7 ) were inoculated into the right side of the axillary of nude mice subcutaneously as previously described. The mice were observed every 5 days after anesthesia by intraperitoneal injection of 0.7% pentobarbital sodium according to the dosage of 10μL per gram of body weight. The length and width of tumors were measured using calipers, and the volume of tumors was calculated using the equation (L × W 2 )/2. On day 53, tumor growth was monitored by luciferase activity detection using the IVIS imaging system. Meanwhile, the mice were euthanized by injection of an overdose of 2% pentobarbital, after which cervical dislocation was performed to con rm death, and the tumors were removed and weighed.
Pathway Analysis (IPA) Firstly, a signal histogram was made to show the signal intensity distribution of all chip probes, and the average Z-score value of all samples in the same signal intensity interval is less than 2. Relative Signal Box Plot showed the medians of all samples, and the Z-score value of the medians was less than 2.
Correlation Analysis was demonstrated by the Pearson correlation coe cient distribution chart, which indicated that the correlation coe cients within both groups were all greater than 0.99. The principal component analysis was then conducted. All the works above con rmed that the data owned ne reliability, repeatability, also signi cant differences between groups, so it met the criteria for continued analysis. The lowest 20% of the signal intensity of the probe set was ltered out as background noise. Secondly, we used the coe cient of variation method to calculate the coe cient of variation of the same probe in the same sample, and ltered out probes with a coe cient of variation greater than 25% in both groups. Finally, we got data of 39,287 probes.
We used a linear model based on empirical Bayes distribution to calculate the signi cant difference level P-value and used the Benjamini-Hochberg method to correct the signi cant difference level. The screening criteria for signi cantly different genes are |Fold Change|>1.5 and FDR<0.05. Hierarchical Clustering was proceeded. The clustering algorithm classi es samples and variables in two dimensions.
We used the Ingenuity Pathways Analysis (IPA, Ingenuity systems, Inc., Redwood City, CA, www.ingenuity.com) tool to examine biological functions and disease as well as functional relationships between genes and gene networks.

C1QTNF6 is high expressed in OSCC tissue and cell lines
We downloaded and analyzed 40 paired data from The Cancer Genome Atlas (TCGA) and found that C1QTNF6 might be a potential tumor-associated regulator involved in the carcinogenesis of OSCC (Fig.  1a). Furthermore, we collected 13 paired OSCC tissue and normal tissue and discovered the high expression of C1QTNF6 in OSCC tissues by RT-PCR.
Then we examined the expression of C1QTNF6 in human OSCC tissue by immunohistochemical analysis and cell lines Cal-27, SCC-9 by RT-PCR. Compared with normal tissues, the expression of C1QTNF6 was higher in the OSCC tissues (Fig. 1b&c). The high levels of C1QTNF6 mRNA in Cal-27 and SCC-9 were observed (Fig. 1d).
The e ciency of C1QTNF6 knockdown by Lentiviral-Based shRNA in human oral squamous cell carcinoma cell lines To explore the biological role of C1QTNF6 in OSCC tumorigenesis, the lentiviral-based shRNA was constructed to knockdown C1QTNF6 in human OSCC cell lines cal-27 and SCC-9. More than 90% of siRNA-transfected cells expressed GFP under a uorescent microscope at 48h after lentivirus infection, which suggested a high infection e ciency (data not shown). The E ciency of the C1QTNF6 knockdown was con rmed by qRT-PCR and western blot. C1QTNF6 mRNA levels were signi cantly reduced in both cell lines after C1QTNF6-shRNA transfection, showing approximately 60-80% knockdown e ciency (Fig.  2a). Western blot analysis showed the dramatically decreased C1QTNF6 protein expression in C1QTNF6-shRNA transfected cells compared to that in Ctrl-shRNA transfected cells (Fig. 2b).

Cell proliferation was inhibited by C1QTNF6 knockdown in human oral squamous cell carcinoma cell lines
Sustained proliferation is a hallmark of cancer cells. To detect the effect of the C1QTNF6 gene on cell proliferation of OSCC cell lines, we performed two different assays to evaluate cell proliferation. For Celigo assay, the viable cells infected with LV-C1QTNF6-shRNA or LV-Ctrl-shRNA were counted every day for ve consecutive days by Celigo Cytometer. Silencing of C1QTNF6 lowered the cell growth curve from day 3 to day 5 in Cal-27 (Fig. 3a) and SCC-9 cells (Fig. 3b). And the inhibition of cell growth by C1QTNF6 silence was more pronounced over time. Additionally, an MTT assay was carried out to detect cell viability. We found that optical density at 490nm increased by 3.3-fold in Cal-27 cells treated with Ctrl-shRNA and 1.1-fold in those treated with C1QTNF6-shRNA at the 4 days after transfection in Cal-27 (Fig.  3c). Similar results were observed in SCC-9 cells (Fig. 3d). Together, the data indicated that the knockdown of C1QTNF6 signi cantly inhibited cell proliferation in human oral squamous cell carcinoma cell lines.

C1QTNF6 knockdown induced cell cycle arrest and apoptosis of human oral squamous cell carcinoma cell lines
To nd out whether the promoting effect of C1QTNF6 on the proliferation of OSCC cell lines was achieved by promoting cell cycle stability or inhibiting apoptosis, we used PI staining to measure cell cycle distribution and Annexin-V staining to assess apoptosis in Cal-27 and SCC-9 cells modi ed by Ctrl-shRNA or C1QTNF6-shRNA. As shown in Fig. 4a&b, Ctrl-shRNA transfected Cal-27 cells had the following cell cycle distribution: G0/G1 55.47%, S 26.28%, G2/M 18.25%; knockdown of C1QTNF6 signi cantly reduced the fraction of cells in the S phase, and signi cantly increased the fraction in the G2/M phase, with the following cell cycle distribution: G0/G1 62.77%, S 14.94%, G2/M 22.92% (all P < 0.05). These data demonstrate cell cycle progression through the G2/M phase was hindered in Cal-27 cells after C1QTNF6 silencing. Similar results were shown in SCC-9 cells. Furthermore, the apoptosis of Cal-27 cells and SCC-9 cells were signi cantly increased by C1QTNF6 knockdown (Fig. 4c&d). These results suggested that C1QTNF6 silencing impeded cell cycle progression and promoted apoptosis in OSCC cell lines.

C1QTNF6 promote human oral squamous cell carcinoma cell growth in vivo
To extend the in vitro observations, we investigated the effects of C1QTNF6 knockdown on tumor growth in vivo. Cal-27 cells were used for subsequent experiments. Cal-27 cells that were stably transfected with either C1QTNF6-shRNA or Ctrl-shRNA lentivirus were injected into nude mice, and the tumor growth was monitored. Tumors formed by C1QTNF6-silenced Cal-27 cells were much smaller during the experiment period than the control tumor (Fig. 5a&b). During necropsy, tumors were excised and weighed. Ctrl-shRNA tumors weighed on an average of 1300mg whereas C1QTNF6-shRNA tumors remained at 400mg (Fig.  5c). Collectively, these results emphasize the promoting role of C1QTNF6 in cancer progression in vivo.
C1QTNF6 knockdown may inhibit the tumorigenesis of OSCC through Acute Phase Response signaling pathway by targeting ID1, BBC3 and DDIT3 The above results suggest that C1QTNF6 played a critical role in the development of OSCC. To elucidate the molecular mechanisms by which C1QTNF6 promotes malignancy of the OSCC cells, microarray analysis was used to compare gene expression between cells infected with Ctrl-shRNA and C1QTNF6-shRNA lentivirus. The data revealed that the expression of 1628 genes was identi ed which showing signi cant differentiation (P<0.05 and |Fold Change| >1.5), including 943 upregulated genes and 685 downregulated genes. The heat map showed hierarchical clustering analysis of differentially expressed genes which revealed that OSCC cells from three samples shared a quite similar gene expression patterns following C1QTNF6 knockdown (Fig. 6a). Next, canonical pathway analysis revealed that the Acute Phase Response signaling pathway was signi cantly activated following C1QTNF6 knockdown according to the expression trend of molecules detected in the experimental results (Fig. 6b). Disease and function prediction analysis results showed that Cancer is signi cantly activated (Fig. 6c). Connections among C1QTNF6 and several closely related molecules were built based on the existing ndings (Fig.  6d). Western blot veri ed the expression variation of several molecules in the network above especially ID1, BBC3, and DDIT3, which are target genes of TNF (Fig. 6e).

Discussion
C1QTNF6 is a crucial molecular mediator connecting in ammation, brosis, and metabolism [14], and it has been implied in the involvement of tumor development. The role of C1QTNF6 in tumor cell survival and cell cycle has been investigated in other cancer [18,20]. However, the study focusing on the relationship between C1QTNF6 and OSCC tumor development remains limited. In the present study, we reported high levels of C1QTNF6 expression in OSCC tissue and cell lines. In addition, the C1QTNF6 knockdown delayed the proliferation of OSCC cells and induced cell apoptosis in vitro. Inconsistent with our observation, one study showed that the expression of C1QTNF6 in human gastric carcinoma tissues was higher than that in normal gastric tissue, and C1QTNF6 silence decreased the growth and resulted in G2-M cell cycle arrest in gastric carcinoma cells [18]. And it has been reported that C1QTNF6-related signaling pathways activated in clear cell renal cell carcinoma were mainly enriched in DNA replication, Cell Cycle, EMT, and angiogenesis signaling pathway [17]. In line with this observation, we found that C1QTNF6 interference increased the percentage of cells at the G2-M phase in human OSCC cell lines. To extend the in vitro observations, we investigated the effect of C1QTNF6 on tumor growth in vivo. We found a retarded tumor growth in mice inoculated with C1QTNF6-silenced cells. This result further emphasized the promoting role of C1QTNF6 in OSCC progression, which may stimulate tumor proliferation.
To date, the molecular mechanism underlying the function of C1QTNF6 in OSCC remains unclear. As shown above, we have demonstrated the important role of C1QTNF6 in OSCC development and progression. To elucidate the molecular mechanisms by which C1QTNF6 promotes malignancy of OSCC, microarray analysis was performed. Hundreds of genes showed signi cantly differential expression between C1QTNF6-shRNA and Ctrl-shRNA transfected cells. According to the data of Ingenuity Pathway Analysis (IPA), the Acute Phase Response was the top-activated signaling pathway following C1QTNF6 silencing. This signaling pathway is an important component of anticancer responses among multicellular organisms [21]. The acute phase response is a nonspeci c physiological and biochemical reaction to tissue damage, infection, in ammation, and neoplasia during which the synthesis of several plasma proteins is increased (positive acute-phase proteins) or decreased (negative acute-phase proteins) [22]. The acute phase response is not diagnostic for any particular kind of disease but occurs as a response to several pathological conditions and diseases, including bacterial infections, sepsis, surgery, trauma, myocardial infarction, in ammatory diseases, and cancer [23]. Besides, other pathways showed altered activation status after C1QTNF6 knockdown, such as TREM1 signaling, IL-6 signaling, and Tolllike Receptor signaling, can induce tumor cell apoptosis or activate an anti-tumor immune response, hence be implicated in the development of variant cancers [24][25][26]. What's more, western blot assay showed that ID1 was signi cantly down-regulated by C1QTNF6 knockdown. In our previous study, we have demonstrated that the over-expression of ID1 effectively promoted the carcinogenesis of OSCC [12,13]. ID1 is one of the inhibitors of DNA binding (Id) proteins [27]. During development, the Id proteins play a key role in the regulation of cell-cycle progression and cell differentiation by modulating different cellcycle regulators both by direct and indirect mechanisms [28]. ID1 enhanced cell proliferation, colony formation, and tumor growth by regulating the cell cycle in lung cancer [29]. Based on the studies above, we speculated that knockdown of C1QTNF6 caused the low expression of ID1, which further led to cell cycle arrest in OSCC.
BH3-only protein BBC3(BCL-2-binding component 3) belongs to the Bcl-2 family and is a strongly proapoptotic gene that is subject to transcriptional regulation by multiple cell death-signaling pathways [30]. It has become obvious that BBC3 plays a role in p53 induced apoptotic pathways and can also function independently of p53. DDIT3, also known as C/EBP homologous protein (CHOP) or growth arrest and DNA damage-inducible gene 153 (GADD153), is a member of the CCAAT/enhancer-binding protein (C/EBP) family [31]. DDIT3(DNA-damage inducible transcript 3) promoted mice ovarian cells apoptosis via ER stress activation, and knockdown of DDIT3 suppressed cell apoptosis [32]. DDIT3 is an important transcription factor of apoptosis under ER stress and the DDIT3-mediated apoptosis pathway is one of the most dominant pathways in the apoptosis process [33]. Over-expression of CHOP has been reported to lead to cell cycle arrest and/or apoptosis, and CHOP has been shown to regulate numerous pro-and apoptotic genes as a transcriptional factor [34]. High levels of BBC3, DDIT3, and obvious cell apoptosis after C1QTNF6 knockdown were observed in this study. Thus, we tend to believe that knockdown of C1QTNF6 induced the occurrence of endoplasmic reticulum stress and the overexpression of BBC3 and DDIT3 which leads to apoptosis of OSCC cell lines in vitro. Our results of microarray analysis t well with the known effects of the above-mentioned signaling pathways and genes on tumorigenesis, suggesting that the promotion of tumor development by C1QTNF6 might be mediated through regulating the Acute Phase Response signaling and ID1, BBC3, DDIT3 gene expression.

Conclusions
There is a balance between the proliferation and apoptosis of tumor cells, and how to regulate this balance determines the development and progression of the tumor. In summary, we speculated that the C1QTNF6 knockdown led to complex changes in OSCC, which might produce an anti-cancer effect through acute phase response both in vivo and in vitro. The microarray analysis suggested that target genes, including but not limited to ID1, BBC3, and DDIT3, the changes in the expression level of which could cause cell cycle arrest and promote apoptosis of OSCC. Our study provided the possible molecular mechanisms underlying C1QTNF6-mediated promotion of OSCC tumorigenesis, which need to be further studied, especially cell cycle factor, apoptosis protein, and ER stress. Our ndings not only provide a novel target gene for OSCC therapy but also add new evidence supporting the relationship between C1QTNF6 and tumorigenesis.

Declarations
Ethics approval and consent to participate The present study was approved by The Ethics Committee of Qilu Hospital of Shandong University (Jinan, China) and informed consent was provided by all the patients prior to the start of the study.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used or analysed during the current study are available from the corresponding author on reasonable request. C1QTNF6 is high expressed in OSCC tissue and cell lines. a Analysis of data from The Cancer Genome Atlas (TCGA) showed that C1QTNF6 was signi cantly highly expressed in OSCC tissue compared to normal tissue. b&c Immunohistochemical staining showed that the expression of C1QTNF6 in human OSCC tissues was higher than that in normal tissues. d Quantitative real-time PCR demonstrated the high levels of C1QTNF6 mRNA in Cal-27 and SCC-9.  Knockdown of C1QTNF6 inhibits cell growth and proliferation. a&b. Cell growth was measured by Celigo assay for ve days. The images of uorescence intensity were shown in Cal-27 cells (a) and SCC-9 cells (b) (magni cation, x200). Cell growth curve of the siC1QTNF6 and shCtrl cells were displayed by cell number fold from days 1 to 5. c The cell proliferation of Cal-27 cells (right panel) and SCC-9 cells (left panel) after Ctrl-shRNA or C1QTNF6-shRNA transfection were measured by the MTT assay. staining were shown. d The percentage of cells in apoptosis were summarized. The rate of apoptosis was represented as mean ± SD. *P < 0.05 Figure 5 Knockdown of C1QTNF6 suppresses tumor growth in vivo. a&b. Xenograft models nu/nu mice were generated with Ctrl-shRNA (n=10) and C1QTNF6-shRNA (n = 10) transfected cells. Tumor volume was measured 7 times for 35 days. c Tumor weight in SCID mice inoculated with Ctrl-shRNA or C1QTNF6-shRNA transfected cells was summarized. Data represented the mean ± SD (n = 10). (*P < 0.05).

Figure 6
The knockdown of C1QTNF6 activated Acute Phase Response signaling pathway then targeted ID1, BBC3, DDIT3. a Hierarchical Clustering analysis heat map of DEGs, red indicates upregulation, green indicates downregulation, black means no obvious difference, gray means the gene not detected. b Signaling pathways enriched among differentially expressed genes. The Y-axis represents the -log (10) P value for enrichment, with the threshold drawn at P=0.05. c Showed the signi cant enrichment of differential genes in diseases and functions. The abscissa is the disease or function name, and the ordinate is the signi cance level of enrichment (negative logarithmic transformation with base 10). d Interaction network of several molecules closely connected to C1QTNF6. e Western blot veri ed the express changes.