SHMT1 expression is decreased in HCC tissues and cells
To evaluate the expression of SHMT1 in HCC, we first explored the publicly available database compiled at the FireBrowse website (www.firebrowse.org). Among 28 human cancers, 11 cases showed elevated TUFT1 levels, while 17 cases showed decreased SHMT1 levels, compared with the corresponding normal tissues (Fig. 1A). Among these human cancers, HCC (LIHC in Figure1A and Additional file 1: Figure S1A) tissues showed a substantial decrease in SHMT1 levels compared with normal liver. To validate the data obtained from FireBrowse website, we employed the “R2: Genomics Analysis and Visualization Platform” (http://r2.amc.nl) to explore SHMT1 expression in Gene Expression Omnibus (GEO) datasets of HCC. As shown in Fig. 1B and C, 2 GEO datasets (GSE54236 and GSE45436) consistently showed decreased SHMT1 mRNA level in HCC specimens compared with adjacent non-tumor liver specimens (P < 0.05). Moreover, IHC staining data from the Human Protein Atlas website (https://www.proteinatlas.org/) showed that SHMT1 protein in HCC tissues had significantly decreased staining intensity compared with that in normal liver tissues (Fig. 1D).
To further validate the data from online datasets, we measured SHMT1 mRNA levels in 120 pairs of HCC tissues and adjacent non-tumor liver specimens. qRT-PCR showed that SHMT1 mRNA level was significantly decreased in HCC tissues (Fig. 2A, P < 0.05). IHC staining confirmed that HCC tissues had significant decreased staining intensity of SHMT1 protein compared to noncancerous tissues (Fig. 2B, P < 0.05). Furthermore, qRT-PCR and western blot were performed to measure the mRNA and protein level of SHMT1 in HCC cell lines and LO2 cell. Compared with the immortalized hepatocyte LO2, four HCC cell lines showed significantly decreased level of SHMT1 (Fig. 2C and D). Altogether, these data confirm that SHMT1 expression level is significantly decreased in HCC.
Clinical significance of reduced SHMT1 expression in HCC specimens
To investigated the clinical significance of SHMT1 in HCC, we divided HCC patients into low SHMT1 group (n = 71) and high SHMT1 group (n = 49) with the cutoff value defined as the mean level of SHMT1 mRNA. As shown in Table 1, decreased SHMT1 level was associated with venous infiltration (P = 0.009), high tumor grade (P = 0.021) and advanced TNM stage (P = 0.019). We further divided the HCC cohort based on IHC staining of SHMT1 protein on HCC specimens and found that negative staining of SHMT1 in HCC tissues was correlated with higher AFP level (P = 0.016, Additional file 2: Table S1), vascular invasion (P = 0.002, Additional file 2: Table S1) and advanced TNM stage (P = 0.006, Additional file 2: Table S1). TCGA data from “R2: Genomics Analysis and Visualization Platform” (http://r2.amc.nl) indicated that the expression of SHMT1 was reduced in HCCs with vascular invasion compared to HCCs without vascular invasion (P = 0.0441, Additional file 1: Figure S1B). Furthermore, statistical analysis of TCGA data from UALCAN [25] revealed that advanced HCCs had a significant lower expression of SHMT1 compared to early HCCs (P = 0.0003, Additional file 1: Figure S1C). Consistently, high-grade HCCs showed an obvious lower expression of SHMT1 as compared with low-grade HCCs (P < 0.0001, Additional file 1: Figure S1D). Survival analysis showed that patients with low SHMT1 level had significantly decreased overall survival (Fig. 2E, P = 0.0012). Data from TCGA database also validated that decreased SHMT1 level was correlated with poor prognosis of HCC patients (Fig. 2F, P = 0.0093). Taken together, these data indicate that SHMT1 plays tumor suppressive role and may function as prognostic marker in HCC.
SHMT1 inhibits the metastasis, EMT and MMP2 production of HCC cells
Since occurrence of metastasis is an important reason for the poor prognosis of HCC patients, we further investigate whether SHMT1 influences the metastasis of HCC cells. Among the four HCC cells, SHMT1 showed highest level in Hep3B cells and lowest level in HCCLM3 cells. Therefore, we performed SHMT1 overexpression in HCCLM3 cells and SHMT1 knockdown in Hep3B cells. Transfection of SHMT1 expressing vector effectively increased SHMT1 mRNA and protein level in HCCLM3 cells (Fig. 3A and B, P < 0.05). Boyden chamber assay showed that forced expression of SHMT1 suppressed the migration of HCCLM3 cells (Fig. 3C, P < 0.05). Transwell assay demonstrated that SHMT1 overexpression led to significantly decreased invasion of HCCLM3 cells (Fig. 3C, P < 0.05). In contrary, transduction of lentivirus with SHMT1 shRNA into Hep3B cells led to significantly decreased level of SHMT1 (Fig. 3D and E, P < 0.05), subsequently resulted in enhanced migratory and invasive ability of Hep3B cells (Fig. 3F, P < 0.05). To avoid the variability between cell lines, we transfected Hep3B cells with SHMT1 vector and found that forced expression of SHMT1 in Hep3B cells also resulted in significant decrease of cell migration and invasion (P < 0.05, Additional file 3: Figure S2). Additionally, we performed MTT assay to investigate whether SHMT1 alteration influenced cell viability. MTT data showed that overexpression or knockdown of SHMT1 did not have any significant influence on cell viability (Additional file 4: Figure S3).
Epithelial-mesenchymal transition (EMT) and MMP production have been widely recognized as critical mechanisms of cancer metastasis [26,27,28]. Thus, we further explored whether SHMT1 affected EMT and MMP production of HCC cells. As shown in Fig. 3G and H, SHMT1 overexpression in HCCLM3 cells led to significantly increased mRNA and protein level of E-cadherin (P < 0.05) and decreased expression of N-cadherin, vimentin and MMP2 (P < 0.05). On the other hand, SHMT1 knockdown in Hep3B cells resulted in decreased E-cadherin level and increased expression of N-cadherin, Vimentin and MMP2 (Fig. 3I and J, P < 0.05). Previous studies have confirmed that Twist1, Snail1 and Zeb1 were the critical transcriptional factors regulating EMT of cancer cells. We further explored whether SHMT1 affected the expression of these transcriptional factors. qRT-PCR and western blot showed that SHMT1 overexpression inhibited the expression of Twist1 and Snail1 (Additional file 5: Figure S4A) while its knockdown resulted in increased level of these two factors (Additional file 5: Figure S4B). Neither SHMT1 overexpression nor its knockdown had any influence on Zeb1 expression (Additional file 5: Figure S4). In all, these data indicate SHMT1 suppresses the metastatic ability of HCC cells by inhibiting EMT and MMP2 production.
SHMT1 suppresses the metastasis of HCC cells in vivo
After verifying the functional influence of SHMT1 on cell metastasis in vitro, we further investigated whether SHMT1 affected the metastasis of HCC cells in nude mice. As shown in Fig. 4A, compared with cell in control group, HCCLM3 cells overexpressing SHMT1 showed significantly increased number and rate of lung metastasis in nude mice (P < 0.05). Moreover, SHMT1 knockdown significantly enhanced the metastatic ability of Hep3B cells in nude mice, suggested by significantly increased number of metastatic nodules and lung metastasis rate (Fig. 4B, P < 0.05). The IHC staining data (Fig. 4A and B), which showed positive AFP staining in these metastatic nodules, further validated these nodules were indeed derived from HCC cells. These data confirm that SHMT1 inhibit the metastatic ability of HCC cells in vivo.
ROS production is responsible for the promoting effect of SHMT1 knockdown on cell motility, EMT and MMP2 production
Previous studies showed that SHMT1 is a critical enzyme mediating the one-carbon metabolism [7, 29], which is implicated in nucleotide synthesis, DNA methylation and NADH/NADPH production in cancer cells [8, 30]. Since ROS production is closely associated with cellular NADH/NADPH level and is found to regulate the metastasis of cancer cells [31, 32], we explored whether SHMT1 affected cellular ROS production. DCFH-DA fluorescence assay was performed to evaluate the ROS level in HCC cells. As shown in Fig. 5A, SHMT1 knockdown resulted in increased DCFH-DA fluorescence, indicating increased ROS production in Hep3B cells (P < 0.05). To further investigate whether increased ROS production is responsible for the enhanced metastatic ability of Hep3B cells induced by SHMT1 knockdown, we treated Hep3B cell with ROS inhibitor N-acetylcysteine (NAC). NAC treatment effectively abrogated the increase of ROS production induced by SHMT1 knockdown (Fig. 5B, P < 0.05). Consequently, NAC treatment reversed the increase of migration and invasion induced by SHMT1 knockdown in Hep3B cells (Fig. 5C, P < 0.05). qRT-PCR and western blot showed that the decrease of E-cadherin and the increase of N-cadherin, vimentin and MMP2 induced by SHMT1 knockdown was abrogated by NAC treatment (Fig. 5D and E, P < 0.05). Therefore, these data indicate that increased ROS production was responsible for the enhanced cell motility, EMT and MMP2 expression induced by SHMT1 knockdown.
SHMT1 regulates NOX1 expression in HCC
Mitochondria and NADPH oxidases of the Nox family including NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2 [33], are two important sources of ROS production in cancer cells. Therefore, we first speculated whether SHMT1 affected cellular ROS by influencing mitochondrial ROS production. MitoSox immunofluorescence staining showed that SHMT1 overexpression or knockdown did not have obvious effect on mitochondrial ROS production (Additional file 6: Figure S5A and 5B). Measurement of mitochondrial membrane potential (MMP) demonstrated that SHMT1 alteration did not have significant influence on MMP (Additional file 6: Figure S5C and 5D). This indicate mitochondria is not the key player in SHMT1 regulated ROS production in HCC cells. Thus, we further explore whether SHMT1 regulated ROS production by regulating NADPH oxidase expression. qRT-PCR screening assay was performed to examine the effect of SHMT1 on the expression of NADPH oxidase. qRT-PCR for the 7 NADPH oxidases showed that SHMT1 overexpression led to decreased mRNA level of NOX1 while its knockdown resulted in increased level of NOX1 (Fig. 6A and B, P < 0.05). Alteration of SHMT1 expression did not have any obvious effect on the expression of other NADPH oxidases (Fig. 6A and B). These indicate that NOX1, instead of other NADPH oxidases, is potentially a downstream target of SHMT1. Western blot analysis further confirmed that overexpression of SHMT1 led to decreased protein level of NOX1 in HCCLM3 cells (Fig. 6C, P < 0.05). Knockdown of SHMT1 resulted in significantly increased level of NOX1 in Hep3B cells (Fig. 6D, P < 0.05). To further validate the regulatory effect of SHMT1 on NOX1, we performed IHC staining of NOX1 and SHMT1 in HCC tissues. As shown in Fig. 6E, HCC tissues with high SHMT1 level showed significantly decreased staining intensity of NOX1 protein compared with those with low SHMT1 level. The positive rate of NOX1 staining in HCC tissues with negative SHMT1 staining was significantly higher than that in those with positive SHMT1 staining (Fig. 6F, P < 0.05). Correlation analysis showed that IHC score of SHMT1 protein was negatively correlatively with that of NOX1 protein (Fig. 6G, P < 0.05). In all, these data demonstrate that NOX1 expression is under the regulation of SHMT1 in HCC.
NOX1 promotes the cell motility, EMT, MMP2 and ROS production of HCC cells
To further explore the functional influence of NOX1 on HCC cells, we transfected Hep3B cells with NOX1 vector. qRT-PCR and western blot showed that transfection of NOX1 vector significantly increased NOX1 expression in Hep3B cells (Fig. 7A and B, P < 0.05), and resulted in decreased E-cadherin level and increased level of N-cadherin, vimentin and MMP2 (Fig. 7A and B, P < 0.05). DCFH-DA fluorescence showed that forced expression of NOX1 resulted in increased ROS level (Fig. 7C, P < 0.05). Functionally, NOX1 overexpression led to enhanced migration and invasion of Hep3B cells (Fig. 7D, P < 0.05). On the other hand, NOX1 siRNA effectively reduced NOX1 level in HCCLM3 cells (Fig. 7E and F, P < 0.05), and inhibited EMT and MMP2 production of HCCLM3 cells (Fig. 7E and 7 F, P < 0.05). Subsequently NOX1 knockdown led to decreased ROS level (Fig. 7G, P < 0.05) and inhibited cell migration and invasion (Fig. 7H, P < 0.05). Taken together, these data demonstrate NOX1 can enhance cell motility, EMT, MMP2 and ROS production of HCC cells.
NOX1 mediates the functional influence of SHMT1 on HCC cells
To further explore whether NOX1 mediates the functional influence of SHMT1 on HCC cells, Hep3B cells with SHMT1 knockdown were transfected with NOX1 siRNA. Knockdown of NOX1 in Hep3B cells with SHMT1 knockdown suppressed the increase of ROS production resulted from SHMT1 knockdown (Fig. 8A, P < 0.05). NOX1 knockdown also blocked the enhancement of cell migration and invasion induced by SHMT1 knockdown (Fig. 8B, P < 0.05). Moreover, NOX1 knockdown partly reversed the decrease of E-cadherin and the increase of N-cadherin, vimentin and MMP2 induced by SHMT1 knockdown, as suggested by qRT-PCR and western blot (Fig. 8C and D, P < 0.05). On the other hand, SHMT1 overexpressing HCCLM3 cells were transfected with NOX1 vector. Forced expression of NOX1 reversed the decrease of ROS production resulted from SHMT1 overexpression (Fig. 9A, P < 0.05). Furthermore, restoring NOX1 expression abrogated the inhibitory effect of SHMT1 overexpression on cell migration, invasion (Fig. 9 B, P < 0.05) and EMT as well as MMP2 expression (Fig. 9C and D, P < 0.05). These indicate that SHMT1 regulates ROS production, cell motility, EMT and MMP2 expression of HCC cells through NOX1.