Transcriptional Levels of MBOATs in Patients with liver hepatocellular carcinoma
We selected eleven common MBOAT family genes by using Oncomine databases. We contrasted the transcriptional levels of 11 MBOATs in tumors with those in normal samples (Figure 1). The mRNA expression levels of MBOAT1 were significantly raised in patients with hepatocellular carcinoma in two datasets. In Guichard Liver dataset, MBOAT1 was overexpressed in hepatocellular carcinoma versus normal tissue with a fold change of 1.077 (Table 1). In The Cancer Genome Atlas breast statistics (Table 1), MBOAT1 was also overexpressed in hepatocellular carcinoma with a fold change of 1.102, MBOAT2 was found to be higher expressed (fold change = 1.037). Guichard et al.37 showed that MBOAT2 was also increased in hepatocellular carcinoma (fold change = 1.025) in comparation to normal samples. LPCAT3 was found higher expressed in Chen Liver (fold change = 1,214)38 and Roessler Liver 2 (fold change = 1.100).39 In The Cancer Genome Atlas (Table 1), MBOAT7 was overexpressed in hepatocellular carcinoma (fold change = 1.032), Guichard liver dataset (fold change = 1.010)37, Wurmbath Liver dataset (fold change = 2.243)36 and Chen Liver dataset (fold change = 1.471)38 compared to counterparts. What’s more, Roessler et al.39 reported that MBOAT7 was overexpressed both in dataset (fold change = 1.407, 1.267). Compared to normal samples SOAT1 increased in all six of the eight databases. In Guichard’s dataset,37 SOAT1 was found higher expressed (fold change = 1.107) and (fold change =1.113).
Table 1.TheSignificant Changes of MBOATExpressioninTranscriptionLevelbetweenDifferentTypesof Liver hepatocellular carcinomaandNormalLiver Tissues (Oncomine Database)
|
Type of Breast Cancer versus Normal Breast Tissue
|
samples
|
p Value
|
t Test
|
Fold Change
|
Source and/or Reference
|
|
Hepatocellular Carcinoma
|
75
|
0.474
|
0.065
|
1.014
|
Wurmbach Liver Statistics36
|
|
Hepatocellular Carcinoma
|
185
|
2.11E-9
|
6.439
|
1.077
|
Guichard Liver Statistics37
|
MBOAT1
|
Hepatocellular Carcinoma
|
212
|
1.72E-7
|
5.477
|
1.102
|
TCGA
|
|
Hepatocellular Carcinoma
|
197
|
0.292
|
0.549
|
1.049
|
Chen Liver Statistics38
|
|
Hepatocellular Carcinoma
|
445
|
1.000
|
-3.554
|
-1.059
|
Roessler Liver Statistics39
|
|
Hepatocellular Carcinoma
|
115
|
0.999
|
-3.528
|
-1.194
|
Mas Liver Statistics40
|
|
Hepatocellular Carcinoma
|
52
|
0.009
|
2.507
|
1.025
|
Guichard Liver 2 Statistics37
|
|
Hepatocellular Carcinoma
|
212
|
4.66E-4
|
3.416
|
1.037
|
TCGA
|
MBOAT2
|
Hepatocellular Carcinoma
|
197
|
0.957
|
-1.726
|
-1.186
|
Chen Liver Statistics38
|
|
Hepatocellular Carcinoma
|
75
|
0.822
|
-0.963
|
-1.123
|
Wurmbach Liver Statistics36
|
|
Hepatocellular Carcinoma
|
445
|
0.991
|
-2.384
|
-1.062
|
Roessler Liver Statistics39
|
|
Hepatocellular Carcinoma
|
115
|
0.945
|
-1.625
|
-1.077
|
Mas Liver Statistics40
|
|
Hepatocellular Carcinoma
|
185
|
0.344
|
0.404
|
1.002
|
Guichard Liver Statistics37
|
HHATL
|
Hepatocellular Carcinoma
|
52
|
0.575
|
-0.191
|
-1.002
|
Guichard Liver 2 Statistics37
|
|
Hepatocellular Carcinoma
|
212
|
0.901
|
-1.296
|
-1.015
|
TCGA
|
|
Hepatocellular Carcinoma
|
75
|
0.869
|
-1.183
|
-1.016
|
Wurmbach Liver Statistics36
|
|
Hepatocellular Carcinoma
|
52
|
0.998
|
-3.135
|
-1.075
|
Guichard Liver 2 Statistics37
|
GOAT
|
Hepatocellular Carcinoma
|
212
|
1.000
|
-8.673
|
-1.274
|
TCGA
|
|
Hepatocellular Carcinoma
|
185
|
1.000
|
-9.590
|
-1.155
|
Guichard Liver Statistics37
|
|
Hepatocellular Carcinoma
|
197
|
0.016
|
2.177
|
1.214
|
Chen Liver Statistics38
|
|
Hepatocellular Carcinoma
|
445
|
0.010
|
2.328
|
1.100
|
Roessler Liver 2 Statistics39
|
|
Hepatocellular Carcinoma
|
43
|
0.754
|
-0.696
|
-1.083
|
Roessler Liver Statistics39
|
LPCAT3
|
Hepatocellular Carcinoma
|
115
|
0.998
|
-3.149
|
-1.295
|
Mas Liver Statistics40
|
|
Hepatocellular Carcinoma
|
52
|
0.845
|
-1.036
|
-1.013
|
Guichard Liver 2 Statistics37
|
|
Hepatocellular Carcinoma
|
75
|
0.990
|
-2.588
|
-1.614
|
Wurmbach Liver Statistics36
|
|
Hepatocellular Carcinoma
|
212
|
0.993
|
-2.515
|
-1.042
|
TCGA
|
|
Hepatocellular Carcinoma
|
185
|
0.997
|
-2.811
|
-1.026
|
Guichard Liver Statistics37
|
|
Hepatocellular Carcinoma
|
197
|
1.88E-8
|
5.753
|
1.471
|
Chen Liver Statistics38
|
|
Hepatocellular Carcinoma
|
43
|
2.30E-5
|
4.600
|
1.402
|
Roessler Liver Statistics39
|
|
Hepatocellular Carcinoma
|
75
|
0.004
|
2.874
|
1.263
|
Wurmbach Liver Statistics36
|
MBOAT7
|
Hepatocellular Carcinoma
|
445
|
9.59E-14
|
7.610
|
1.267
|
Roessler Liver 2 Statistics39
|
|
Hepatocellular Carcinoma
|
52
|
0.046
|
1.735
|
1.010
|
Guichard Liver 2 Statistics37
|
|
Hepatocellular Carcinoma
|
212
|
0.004
|
2.684
|
1.032
|
TCGA
|
|
Hepatocellular Carcinoma
|
115
|
0.825
|
-0.944
|
-1.062
|
Mas Liver Statistics40
|
|
Hepatocellular Carcinoma
|
185
|
0.180
|
0.919
|
1.009
|
Guichard Liver Statistics37
|
|
Hepatocellular Carcinoma
|
52
|
4.03E-7
|
6.467
|
1.107
|
Guichard Liver 2 Statistics37
|
|
Hepatocellular Carcinoma
|
185
|
3.57E-19
|
10.938
|
1.113
|
Guichard Liver Statistics37
|
|
Hepatocellular Carcinoma
|
197
|
0.002
|
2.067
|
1.169
|
Chen Liver Statistics38
|
SOAT1
|
Hepatocellular Carcinoma
|
212
|
4.38E-20
|
11.510
|
1.274
|
TCGA
|
|
Hepatocellular Carcinoma
|
75
|
0.073
|
1.503
|
1.236
|
Wurmbach Liver Statistics36
|
|
Hepatocellular Carcinoma
|
445
|
6.87E-35
|
13.780
|
1.781
|
Roessler Liver 2 Statistics39
|
|
Hepatocellular Carcinoma
|
43
|
0.002
|
3.173
|
1.461
|
Roessler Liver Statistics39
|
|
Hepatocellular Carcinoma
|
115
|
0.680
|
-0.470
|
-1.052
|
Mas Liver Statistics40
|
|
Hepatocellular Carcinoma
|
445
|
1.98E-11
|
6.894
|
1.395
|
Roessler Liver 2 Statistics39
|
|
Hepatocellular Carcinoma
|
43
|
0.014
|
2.325
|
1.436
|
Roessler Liver Statistics39
|
|
Hepatocellular Carcinoma
|
75
|
0.015
|
2.256
|
1.697
|
Wurmbach Liver Statistics36
|
SOAT2
|
Hepatocellular Carcinoma
|
52
|
0.156
|
1.022
|
1.009
|
Guichard Liver 2 Statistics37
|
|
Hepatocellular Carcinoma
|
115
|
0.769
|
-0.744
|
-1.039
|
Mas Liver Statistics40
|
|
Hepatocellular Carcinoma
|
212
|
0.343
|
0.407
|
1.004
|
TCGA
|
|
Hepatocellular Carcinoma
|
185
|
0.619
|
-0.304
|
-1.002
|
Guichard Liver Statistics37
|
|
Hepatocellular Carcinoma
|
75
|
2.44E-6
|
10.324
|
3.256
|
Wurmbach Liver Statistics36
|
|
Hepatocellular Carcinoma
|
52
|
1.09E-6
|
6..083
|
1.100
|
Guichard Liver 2 Statistics37
|
|
Hepatocellular Carcinoma
|
212
|
3.46E-18
|
10.619
|
1.254
|
TCGA
|
HHAT
|
Hepatocellular Carcinoma
|
197
|
5.04E-9
|
6.027
|
1.524
|
Chen Liver Statistics38
|
|
Hepatocellular Carcinoma
|
185
|
3.38E-15
|
9.157
|
1.102
|
Guichard Liver Statistics37
|
|
Hepatocellular Carcinoma
|
445
|
2.45E-20
|
9.820
|
1.328
|
Roessler Liver 2 Statistics39
|
|
Hepatocellular Carcinoma
|
115
|
0.046
|
1.738
|
1.091
|
Mas Liver Statistics40
|
|
Hepatocellular Carcinoma
|
43
|
0.008
|
2.551
|
1.203
|
Roessler Liver Statistics39
|
|
Hepatocellular Carcinoma
|
52
|
1.47E-5
|
4.935
|
1.068
|
Guichard Liver 2 Statistics37
|
|
Hepatocellular Carcinoma
|
212
|
2.16E-13
|
8.367
|
1.249
|
TCGA
|
DGAT1 Hepatocellular Carcinoma
|
185
|
1.53E-9
|
6.356
|
1.103
|
Guichard Liver Statistics37
|
|
Hepatocellular Carcinoma
|
43
|
0.025
|
2.030
|
1.298
|
Roessler Liver Statistics39
|
Hepatocellular Carcinoma
|
197
|
0.113
|
1.217
|
1.112
|
Chen Liver Statistics38
|
Hepatocellular Carcinoma
|
445
|
0.028
|
1.913
|
1.098
|
Roessler Liver 2 Statistics39
|
Hepatocellular Carcinoma
|
75
|
0.049
|
1.727
|
1.377
|
Wurmbach Liver Statistics36
|
Hepatocellular Carcinoma
|
115
|
0.703
|
-0.536
|
-1.059
|
Mas Liver Statistics40
|
PORCN
|
Hepatocellular Carcinoma
|
75
|
0.235
|
0.731
|
1.028
|
Wurmbach Liver Statistics36
|
|
Hepatocellular Carcinoma
|
445
|
0.304
|
0.515
|
1.008
|
Roessler Liver 2 Statistics39
|
|
Hepatocellular Carcinoma
|
115
|
0.716
|
-0.578
|
-1.029
|
Mas Liver Statistics40
|
|
Hepatocellular Carcinoma
|
43
|
0.746
|
-0.667
|
-1.041
|
Roessler Liver Statistics39
|
TCGA, The Cancer Genome Atlas.
In The Cancer Genome Atlas data (Table 1), higher expressed SOAT1 was found (fold change = 1.274). Additionally, in Roessler’s dataset,39 SOAT1 was found higher expressed in hepatocellular carcinoma (fold change = 1.461, 1.781), and, in Chen Liver dataset (fold change = 1.169). SOAT2 was overexpressed in both Roessler39 and Wurmbach 36dataset (fold change =1.395, 1.697). Noticeably HHAT were significantly upregulated in patients with hepatocellular carcinoma in all eight datasets, (fold change =3.256, 1.1023, 1.100, 1.524, 1.328, 1.091, 1.254, respectively)36-42. The Cancer Genome Atlas data (Table 1), higher expressed HHAT was found (fold change= 1.254). In The Cancer Genome Atlas and dataset of Wurmbath36, Guichard37, Roessler39, DGAT1was found higher expressed(fold change = 1.294, 1.377, 1.103, 1.068,1.298, 1.098) compared to normal samples (Table 1).
Correlation between MBOATs mRNA level and clinicopathological parameters in patients with hepatocellular carcinoma
we compared the mRNA expression of MBOAT family genes between liver hepatocellular carcinoma and normal liver tissues with the GEPIA. The results indicated that the expression levels of MBOAT1, MBOAT2, MBOAT7, SOAT1, SOAT2, HHAT, DGAT1 and PORCN were higher in liver hepatocellular carcinoma tissues than in normal tissues, while the expression level of HHATL and GOAT have no statistically significant differences in the former than in the latter (Figure 2). By using GEPIA(Gene Expression Profiling Interactive Analysis) dataset (http://gepia.cancer-pku.cn/), we found MBOAT2, GOAT,MBOAT7 have significantly relationship between MBOAT expression and Tumor stage in hepatocelluer patients(Figure 3). We performed RNA-seq to test MBOAT RNA expression in hepatocellular carcinoma tissues. RNA-seq data is reported as median FPKM (number Fragments Per Kilobase of exon per Million reads), generated by the The Cancer Genome Atlas (TCGA). We found the average FPKM of LPCAT3, SOAT1, SOAT2, MBOAT7 and DGAT up to 4.7, 4.9, 5.2, 10.1, 20, respectively(Figure 4).
Diagnostic and prognostic value of MBOATs with Hepatocellular Carcinoma
We further explored the key efficiency of MBOATs in the survival of patients with Hepatocellular Carcinoma. Kaplan- Meier Plotter tools were used to analyze the relationship between the mRNA levels of MBOATs and the survival of patients with Hepatocellular Carcinoma by using publicly available datasets (2015 version; http://kmplot.com/analysis/index.php?p=service&cancer=liver).
The Kaplan-Meier curve and log rank test analyses revealed that the increased MBOAT1, 2, 7, SOAT1, PORCN mRNA levels and the decreased HHATL, HHAT and LPCAT3 mRNA levels were highly associated with the overall survival (OS), relapse free survival (RFS), distant metastasis free survival (DSS) and post-progression survival (PFS) (p < 0.05) (Figure 5) of the whole patients with hepatocellular carcinoma. The patients with hepatocellular carcinoma with high mRNA levels of the MBOAT1, 2, 7, SOAT1, PORCN factors or inferior mRNA levels of HHATL, HHAT and LPCAT3 were predicted to have high OS, RFS, PFS and DSS.(Figure 5) of the whole patients with hepatocellular carcinoma. The patients with hepatocellular carcinoma with high mRNA levels of the MBOAT1, 2, 7, SOAT1, PORCN factors or inferior mRNA levels of HHATL, HHAT and LPCAT3 were forcasted to have high OS, RFS, PFS and DSS.
We also analyzed the MBOAT alterations, correlations, and networks by using the cBioPortal online tool for liver Hepatocellular Carcinoma (The Cancer Genome Atlas, Provisional;(http://www.cbioportal.org/index.do?session_id=5b4c1773498eb 8b3d566f7b8) to analysis the alters in MBOAT family genes and their Frequently Altered Neighbor Genes in Patients with Hepatocellular Carcinoma. MBOATs were changed in 289 (20%) of 1461 sequences patients (1461 total) (Figure 6AB).
Proteomics analysis of the MBOATs in patients with hepatocellular carcinoma
We analyzed the MBOAT family using proteomic data from the published CELL article. The data of proteome can be viewed in NODE (https://www.biosino.org/node) by pasting the accession (OEP000321) into the text search box or through the URL: (https://www.biosino.org/node/project/detail/OEP000321). Only MBOAT7 and SOAT1 has difference between patients with hepatocellular carcinoma then normal tissue, significantly(Figure 7A).
SOAT1 Knockdown Suppressed the Proliferation and Migration of HCC Cell Lines
we designed three siRNA based on the SOAT1 gene sequence. Three siRNAs were transfected into Huh7 and Hep3B cell lines, and sicontrol was transfected into control group. The inhibition efficiency of the siSOAT1 was detected by Western Blot. The results showed that siSOAT1-3 had the best inhibition efficiency(Figure 7B), then we only use this siRNA to finish the following proliferation and migration tests . We use CCK8 assay was conducted to examine HCC cell proliferation. In this study, the siRNA inhibitors of SOAT1 expression could significantly inhibit the proliferation of Huh7 and Hep3B (Figure 7C). Cell migration assay was used to evaluate the migration ability. In the cell migration experiment of Huh7 and Hep3B cells, SOAT1 knockdown could significantly reduce the migration ability of tHCC cells (Figure 7D).