LAMC1 expression in human pan-cancers

GTEx datasets showed various levels of LAMC1 gene expression in humans and a low expression level in brain tissues (Fig. 1A). However, a relatively high expression level of LAMC1 was found in CNS tumors (Fig. 1B). In TCGA data, LAMC1 was highly expressed in CHOL, ESCA, GBM, HNSC, KIRC, KIRP, LIHC, LUAD, LUSC, STAD, and THCA. Conversely, LAMC1 expression was downregulated in tumors in BLCA, BRCA, and KICH relative to normal tissue (Fig. 1C). After adding normal tissues from the GTEx dataset as controls, we found enhanced expression of LAMC1 in DLBC, LGG, and THYM (Fig. 1D).

Fig. 1

Differential expression of the LAMC1 gene in pan-cancers. A LAMC1 expression in normal human tissues. B LAMC1 expression in human tumor cell lines. C Comparison of LAMC1 expression between tumor and normal samples from TCGA database. *P < 0.05, **P < 0.01, ***P < 0.001. D Integrated GTEX and TCGA databases to analyze LAMC1 expression between tumor and normal samples of DLBC, LGG, and THYM. *P < 0.05

Multifaceted prognostic features of LAMC1 across pan-cancers

Cox proportional risk model analysis showed that LAMC1 expression levels were significantly correlated to OS of patients with KIRP, LGG, MESO, and UVM (P < 0.001) and many other cancer types (P < 0.05) (Fig. 2A). LAMC1 expression was associated with DSS of various cancers, including BLCA, LGG, MESO, and UVM (P < 0.001) (Fig. 2B). High LAMC1 expression was correlated to DFI in patients with BLCA, CESC, OV, and PAAD (P < 0.05) (Fig. 2C). In terms of the association between high LAMC1 expression levels and PFI, a forest map showed a poor prognosis of patients with ACC, BLCA, LGG, and UVM (P < 0.001) (Fig. 2D). Kaplan–Meier survival analysis also showed that LGG patients with high LAMC1 levels had poor OS, DSS, and PFI (P < 0.001, Fig. 3).

Fig. 2

Association between LAMC1 expression and OS, DSS, DFI, and PFI in pan-cancers. A Forest plot of the association of LAMC1 expression with OS in TCGA tumors. B Forest plot of the association of LAMC1 expression with DSS in TCGA tumors. C Forest plot of the association of LAMC1 expression with DFI in TCGA tumors. D Forest plot of the association of LAMC1 expression with PFI in TCGA tumors

Fig. 3

Kaplan–Meier analysis of the association between LAMC1 expression and OS, DSS, and PFI of LGG patients. A Association between LAMC1 expression and OS of LGG patients. B Association between LAMC1 expression and DSS of LGG patients. C Association between LAMC1 expression and PFI of LGG patients

Clinicopathological analysis of LAMC1 expression in Chinese glioma patients

Bioinformatics analysis of LAMC1 expression in gliomas was performed using the CGGA database [17]. ROC curve analysis showed that LAMC1 expression was a predictor of 1-year (AUC = 0.727), 3-year (AUC = 0.781), and 5-year (AUC = 0.797) survival with a good predictive value (Fig. 4A). Kaplan–Meier survival analysis showed that high LAMC1 expression was associated with a poor prognosis of patients with glioma (Fig. 4B). Univariate Cox analysis showed that LAMC1 expression, primary-recurrent-secondary (PRS) type, histology, grade, age, and chemotherapy were high-risk factors (HR > 1), and IDH mutation and 1p19q codeletion were low-risk factors (HR < 1) (Fig. 4C). Multivariate Cox analysis showed that LAMC1 expression (P < 0.001, HR = 1.163), PRS type (P < 0.001, HR = 1.918), grade (P < 0.001, HR = 2.619), IDH mutation (P = 0.003, HR = 0.691) and 1p19q codeletion (P < 0.001, HR = 0.402) may also be independent prognostic factors (Fig. 4D).

Fig. 4

Clinicopathological analysis of LAMC1 expression in the CGGA database. A Receiver operator characteristic (ROC) curve of LAMC1 expression. AUC: area under the curve. B Survival analysis of glioma patients in high and low LAMC1 expression groups. P < 0.001. C Univariate analysis of LAMC1 expression. D Multivariate analysis of LAMC1 expression

Correlation between LAMC1 expression and clinical phenotypes of various cancers

Next, we examined differential expression of LAMC1 in patients with each tumor type in accordance with age and the tumor stage in TCGA database. We found that patients older than 65 years of age with BRCA (P = 0.00032), COAD (P = 0.007), KIRC (P = 0.011), LIHC (P = 0.0078), or PAAD (P = 0.0018) had low LAMC1 expression levels. LAMC1 expression was higher in THYM (P = 0.012) and UCEC (P = 0.041) patients over 65 years of age (Additional file 1: Fig. S2A). We also found statistical significance between LAMC1 expression and the partial tumor stage in eight cancer types, including BLCA, COAD, HNSC, KICH, KIRC, LUSC, PAAD, and UVM. Notably, LAMC1 expression increased with the stage in most tumors, suggesting that high LAMC1 expression is associated with tumor progression (Additional file 1: Fig. S2B). Clinicopathological characteristic correlation analysis of the CGGA data demonstrated that the expression level of LAMC1 was remarkably related to age (Fig. 5A), grade (Fig. 5B), PRS type (Fig. 5C), chemotherapy (Fig. 5D), and histology (Fig. 5G) in glioma samples. Recently, it has been widely recognized that IDH mutations and 1p19q codeletion suggest a favorable prognosis of gliomas [18]. In this study, high expression levels of LAMC1 were found in IDH-wildtype and 1p19q non-coding glioma compared with IDH-mutants or 1p19q codeletions (Fig. 5E, F). These findings suggest that LAMC1 participates in the clinical development of glioma.

Fig. 5

Correlation of LAMC1 expression levels with clinical features in glioma patients in the CGGA database. A Age. B Grade. C PRS type. D Chemotherapy. E IDH mutation. F 1p19q codeletion. G Histology

Correlation of LAMC1 protein expression to clinicopathological parameters and prognosis in the glioma cohort

We explored the correlation between LAMC1 protein expression and clinicopathological parameters based on the information of tissue microarray cases. The positive sites for LAMC1 protein expression in the tissue were both cytoplasmic and nuclei. The calculated total immunoreactive scores of LAMC1 expression ranged from 2 to 24 in all samples. We divided the cases into subgroups of high (IHC score > 10) and low (IHC score ≤ 10) LAMC1 expression based on the cutoff point determined by X-tile software related to survival time and status. LAMC1 expression was correlated to the degree of pathological grade and postoperative recurrence of patients (P < 0.05). Additionally, no association was found between LAMC1 expression and gender (P = 0.798) or age (P = 0.314) (Additional file 1: Table S1). Representative images of the IHC staining intensity of LAMC1 in gliomas with different pathological grades and normal brain tissue are shown in Fig. 6. LAMC1 was nearly negative in normal brain tissue (Fig. 6A, B). As shown in Fig. 6C, glioma patients with higher pathological grades (G2–4) had higher total immunoreactive scores for LAMC1 than those with a lower grade (G1) (P < 0.05). Survival analysis by the Kaplan–Meier method with the log-rank test indicated that patients with a high level of LAMC1 had worse outcomes and shorter OS (P = 0.0482) and DFS (P = 0.0033) than those with low LAMC1 expression (Fig. 6D). Age and pathological grade were independent prognostic indicators in both univariate and multivariate Cox analysis models (Additional file 1: Table S2).

Fig. 6

LAMC1 protein expression correlates to clinicopathological parameters of glioma patients based on tissue array analysis. A Representative images of the IHC staining intensity of LAMC1 in gliomas with different pathological grades and normal brain tissue. Bars = 200 μm. B Immune response score (IRS) of LAMC1 in gliomas and normal controls, ****P < 0.0001. C IRS of LAMC1 protein expression in glioma tissues with different grades. *P < 0.05, **P < 0.01. D Kaplan–Meier analysis of OS and DFS of glioma patients in accordance with LAMC1 protein expression levels

LAMC1 knockdown inhibits while overexpression promotes glioma cell proliferation, migration, and invasion

Hs683 cells with highest LAMC1 expression and U251 cells with lowest LAMC1 expression were chosen for subsequent experiments. Fluorescence microscopy showed that lentiviral particles had a higher infection efficiency in glioma cells, and western blotting verified the efficacy in LAMC1 knockdown or overexpression (Additional file 1: Fig. S1). CCK-8 assays showed that cell proliferation in the LAMC1 knockdown group was significantly slower than that in the control group, and LAMC1 overexpression was significantly enhanced the viability compared with the relevant controls (P < 0.001, Fig. 7A), the results of colony formation were consistent with the CCK-8 findings (Fig. 7B). Furthermore, LAMC1 knockdown decreased while LAMC1 overexpression increased the number of cells that migrated or invaded through transwell chambers (P < 0.001, Fig. 7C). Wound-healing assay results were consistent with the migration assay results (Fig. 7D). These data collectively indicate that LAMC1 promotes glioma cell proliferation, migration, and invasion.

Fig. 7

Effects of LAMC1 intervention on proliferation, migration, and invasion of glioma cells. A Viability of glioma cells assessed by CCK-8 assays, ** P<0.01, ***P < 0.001. B Proliferation ability glioma cells assessed by colony formation assays. C Migration and invasion abilities of glioma cells were compared by transwell assays. D Migration and repair abilities of glioma cells were compared by wound-healing assay. *P < 0.05, ***P < 0.001

High HIF-1α expression promotes clinical progression and correlates positively to LAMC1 expression in gliomas

HIF-1α expression was significantly upregulated in various TGCA solid tumors, including GBM, GBMLGG, LGG, CESC, ESCA, STES, COAD, STAD, HNSC, LUSC, THCA, PAAD, TGCT, ALL, LAML, and CHOL. Significant downregulation of HIF-1α expression was also observed in KIRP, KIPAN, KIRC, SKCM, ACC, and KICH (Fig. 8A). Moreover, high HIF-1α expression associates with poor prognosis of gliomas (Fig. 8B), as well as the advanced pathological grade, 1p19q codeletion and IDH mutation status (Fig. 8C). Based on clinical glioma tissue, we detected that the expression level of HIF-1α protein increases with the increase of pathological grade (Additional file 1: Fig. S3). Correlation analysis showed that HIF-1α expression in normal brain tissue and glioma was positively correlated to LAMC1 (r = 0.499, P < 0.001; Fig. 8D).

Fig. 8

Expression of HIF-1α in TCGA pan-cancers and its prognosis, clinical pathology and correlation with LAMC1 in gliomas. A Expression of HIF-1α in pan-cancer and control tissues. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. B The relationship between HIF-1α and the OS, DFS and PFI of glioma. C Relationship between HIF-1α expression and the pathological grade, 1p19q codeletion and IDH mutation of glioma patients. D Correlation between HIF-1α and LAMC1 expression in glioma

Effects of hypoxia on LAMC1 expression in glioma

LAMC1 and HIF-1α expression varied with the change in hypoxic exposure time (Fig. 9A, B, Additional file 1: Fig. S4A). After hypoxic treatment for 12 h, LAMC1 and HIF-1α protein expression was the highest. Notably, while confirming that the HIF-1α inhibitor YC-1 has no significant cytotoxicity on glioma cells (Additional file 1: Fig. S5), we have noticed that YC-1 downregulates the expression of LAMC1 while inhibiting HIF-1α expression at the same time (Fig. 9C, D, Additional file 1: Fig. S4B). Bioinformatics predicted four binding sites for HIF-1α protein on the human LAMC1 gene promoter (Fig. 9E). The results of promoter luciferase assay showed that HIF-1α directly regulated activity of the LAMC1 promoter (Fig. 9F). It was noteworthy that we observed strong luciferase activity in cells co-transfected with LAMC1 Pro-luc and HIF-1α-NC plasmids, suggesting other regulators of LAMC1 activation in addition to HIF-1α (Fig. 9F).

Fig. 9

HIF-1α regulates LAMC1 expression in glioma cells. A, B Western blot and semi-quantitative analysis of LAMC1 and HIF-1α protein expression in glioma cells under normoxia (N) and hypoxia (H). C, D Western blot and semi-quantitative analyses of HIF-1α inhibitor YC-1-induced suppression of LAMC1 and HIF-1α protein expression in glioma cells at 12 h of hypoxia. ***P < 0.001. E Prediction of binding sites between the LAMC1 promoter and HIF-1α protein. F Results of luciferase reporter assays. ***P < 0.001; ns, no statistical difference. LAMC1Pro-luc-HIF-1α-OE, LAMC1 promoter plus HIF-1α overexpression; LAMC1Pro-luc-HIF-1α-NC, LAMC1 promoter plus negative control of HIF-1α; LAMC1Pro-luc-NC-HIF-1α-OE, negative control of LAMC1 promoter plus HIF-1α overexpression; LAMC1Pro-luc-NC-HIF-1α-NC, negative control of LAMC1 promoter plus negative control of HIF-1α