LRRN4 is highly expressed in CRC and correlates with some clinicopathological features
To investigate the potential significance of LRRN4 in CRC patients, the relationship between LRRN4 expression and clinicopathological characteristics was analyzed. As shown in Fig. 1, the expression level of LRRN4 is much higher in CRC compared to normal colorectal tissue (p < 0.001). Moreover, the LRRN4 expression was statistically different in different pathological stage (p = 0.037), lymphatic invasion (p = 0.029), patients' progression-free survival (PFS) status (p = 0.009), and overall survival (OS) status (p = 0.002). No statistical difference was observed in the rest of the clinicopathological parameters, including age, gender, location, microsatellite MSI status, histological types, perineural invasion and venous invasion.
High LRRN4 mRNA expression is an independent prognostic factor for poor PFS and OS in CRC patients
To investigate the potential survival significance of the expression LRRN4, we performed Kaplan-Meier survival analysis. The patients first were classified into low- and high-LRRN4 expression groups using X-tile plots to generate the optimal cut-off score. The survival analysis showed that high LRRN4 expression was associated with poor PFS (Fig. 2A, p = 0.001) and OS (Fig. 2B, p = 0.030).
To further assess whether the LRRN4 expression could independently predict PFS and OS in CRC patients, both univariate and multivariate Cox regression analyses were performed by adjusting for gender, age, stage, MSI status, lymphatic invasion, venous invasion and adjuvant chemoradiotherapy as covariates. Univariate analyses showed that high LRRN4 expression was significantly associated with poor PFS (Table 1, p = 0.015) and OS (Table 2, p = 0.005). In multivariate analyses, high expression of LRRN4 remained a strong prognostic value for PFS (Table 1, HR = 1.797, 95%CI = 1.009-3.200, p = 0.047) and OS (Table 2, HR = 1.733, 95%CI = 1.014-2.961, p = 0.044), even after adjusting for other covariates, indicating its potential prognostic values for PFS and OS in CRC patients.
Table 1
Univariate and multivariate Cox proportional hazards regression analyses for PFS of LRRN4 and clinical features
Variables
|
Uni-variable Cox
|
Multi-variable Cox
|
HR
|
95%CI
|
p value
|
HR
|
95%CI
|
p value
|
LRRN4 (High vs. Low)
|
1.992
|
1.143-3.469
|
0.015
|
1.797
|
1.009-3.200
|
0.047
|
MSI(High vs. Low)
|
0.677
|
0.316-1.448
|
0.314
|
1.209
|
0.524-2.792
|
0.656
|
Age
|
1.346
|
0.753-2.407
|
0.316
|
1.020
|
0.997-1.044
|
0.092
|
Gender (Male vs. Female)
|
1.348
|
0.772-2.354
|
0.294
|
1.118
|
0.619-2.020
|
0.712
|
Histological type (Non-mucinous vs. Mucinous)
|
0.394
|
0.121-1.278
|
0.121
|
0.425
|
0.128-1.410
|
0.162
|
Pathologic stage (III/IV vs. I/II)
|
2.900
|
1.620-5.192
|
0.000
|
1.707
|
0.814-3.578
|
0.157
|
Lymphatic invasion (Yes vs. No)
|
2.348
|
1.346-4.095
|
0.003
|
1.067
|
0.423-2.690
|
0.890
|
Perineural invasion (Yes vs. No)
|
1.932
|
1.093-3.418
|
0.024
|
1.268
|
0.637-2.522
|
0.499
|
Venius invasion (Yes vs. No)
|
2.045
|
1.170-3.575
|
0.012
|
1.215
|
0.523-2.824
|
0.650
|
Adjuvant chemoradiotherapy (No vs.Yes)
|
3.540
|
1.184-6.652
|
0.000
|
2.781
|
1.289-6.000
|
0.009
|
HR, hazard ratio, CI, confdence interval p value below 0.05 was considered signifcant and highlighted (bold)
|
Table 2
Univariate and multivariate Cox proportional hazards regression analyses for OS of LRRN4 and clinical features
Variables
|
Uni-variable Cox
|
Multi-variable Cox
|
HR
|
95%CI
|
p value
|
HR
|
95%CI
|
p value
|
LRRN4 (High vs. Low)
|
2.063
|
1.247-3.413
|
0.005
|
1.733
|
1.014-2.961
|
0.044
|
MSI (High vs. Low)
|
0.913
|
0.463-1.801
|
0.792
|
0.957
|
0.449-2.039
|
0.909
|
Age
|
1.032
|
1.011-1.053
|
0.002
|
1.035
|
1.012-1.059
|
0.003
|
Gender (Male vs. Female)
|
1.667
|
0.993-2.798
|
0.053
|
1.541
|
0.898-2.644
|
0.116
|
Histological type (Mucinous vs. Non-mucinous)
|
1.239
|
0.588-2.611
|
0.573
|
1.254
|
0.570-2.760
|
0.574
|
Pathologic stage (III/IV vs. I/II)
|
2.914
|
1.723-4.927
|
0.000
|
4.691
|
2.333-9.431
|
0.000
|
Lymphatic invasion (Yes vs. No)
|
2.025
|
1.214-3.377
|
0.007
|
0.633
|
0.268-1.496
|
0.298
|
Venius invasion (No vs. Yes)
|
0.990
|
0.597-1.640
|
0.968
|
2.180
|
0.949-5.005
|
0.066
|
Adjuvant chemoradiotherapy (No vs. Yes)
|
2.481
|
1.487-4.138
|
0.001
|
0.540
|
0.291-1.001
|
0.051
|
HR, hazard ratio, CI, confdence interval p value below 0.05 was considered signifcant and highlighted (bold)
|
The prognostic significance of LRRN4 for CRC patients is validated in protein level
Having found the significance of the expression of LRRN4 mRNA in CRC prognosis, we further investigated the expression of LRRN4 protein in samples from 81 CRC patients by immunohistochemistry (Fig. 3A, 3B). We first analyzed the expression levels of LRRN4 on colorectal cancer and normal mucosal tissues. Consistent with the results of the TCGA data analysis, there were more patients (83%) with high-LRRN4 expression in CRC compared to normal colorectal tissue (39%) (Fig. 3C, p༜0.0001). Then the correlation of LRRN4 expression and clinical characteristics in colorectal cancer patients was analyzed. The results showed that high LRRN4 expression was statistically related to pathological stage (Table 3, p = 0.001), lymph nodes metastasis (Table 3, p = 0.006) and OS status (Table 3, p = 0.001). Next, we performed survival analysis and found high LRRN4 expression was correlated with poor PFS (Fig. S1, p = 0.129) and OS (Fig. 3D, p = 0.0003), although no statistical significance was found in PFS. Univariate and multivariate Cox regression analyses were further performed by adjusting for gender, age, location, histological type, pathologic stage, grade and adjuvant chemoradiotherapy as covariates. In univariate analysis, high LRRN4 expression was significantly associated with poor OS (Table 4, p = 0.001). In multivariate analysis, LRRN4 remained a strong prognostic value for OS (Table 4, HR = 3.999, 95%CI = 1.564-10.224, p = 0.004), indicating the significance of LRRN4 expression for outcomes in CRC patients.
Table 3
The Correlation of LRRN4 expression and Clinical Characteristics in CRC Patients
Characteristics
|
LRRN4-Low (%)
|
LRRN4-High (%)
|
p value
|
Gender
|
|
|
|
Male
|
17(53.1)
|
27(55.1)
|
1.000
|
Female
|
15(46.9)
|
22(44.9)
|
|
Age
|
|
|
|
≤65
|
15(46.9)
|
32(65.3)
|
0.113
|
>65
|
17(53.1)
|
17(34.7)
|
|
Overall survival status
|
|
|
|
Alive
|
24(75.0)
|
17(34.7)
|
0.001
|
Death
|
8(25.0)
|
32(65.3)
|
|
Progression-free survival status
|
|
|
|
Progression-free
|
20(62.5)
|
15(45.5)
|
0.216
|
Progression
|
12(37.5)
|
18(54.5)
|
|
Location
|
|
|
|
Colon
|
20(62.5)
|
37(75.5)
|
0.225
|
Rectum
|
12(37.5)
|
12(24.5)
|
|
Histological type
|
|
|
|
Mucinous
|
9(29.0)
|
16(32.7)
|
0.808
|
Non-mucinous
|
22(71.0)
|
33(67.3)
|
|
Pathologic stage-T
|
|
|
|
T 1/2
|
6(18.8)
|
5(10.2)
|
0.328
|
T 3/4
|
26(81.3)
|
44(89.8)
|
|
Pathologic stage-N
|
|
|
|
N0
|
21(65.6)
|
16(32.7)
|
0.006
|
N1/2
|
11(34.4)
|
33(67.3)
|
|
Pathologic stage-M
|
|
|
|
M0
|
31(96.9)
|
42(85.7)
|
0.100
|
M1
|
1(3.1)
|
7(14.3)
|
|
Pathologic stage
|
|
|
|
Stage I
|
5(15.6)
|
3(6.1)
|
0.006
|
Stage II
|
16(50.0)
|
10(20.4)
|
|
Stage III
|
10(31.3)
|
29(59.2)
|
|
Stage IV
|
1(3.1)
|
7(14.3)
|
|
Adjuvant chemoradiotherapy
|
|
|
|
No
|
9(28.1)
|
19(38.8)
|
0.351
|
Yes
|
23(71.9)
|
30(61.2)
|
|
Differentiation
|
|
|
|
Low/Middle
|
29(93.5)
|
42(89.4)
|
0.527
|
High
|
2(6.5)
|
5(10.6)
|
|
HR, hazard ratio, CI, confdence interval p value below 0.05 was considered signifcant and highlighted (bold)
|
Table 4
Univariate and multivariate Cox proportional hazards regression analyses for OS of LRRN4 and clinical features
Variables
|
Uni-variable Cox
|
Multi-variable Cox
|
HR
|
95%CI
|
p value
|
HR
|
95%CI
|
p value
|
LRRN4 (High vs. Low)
|
3.773
|
1.735-8.206
|
0.001
|
3.999
|
1.564-10.224
|
0.004
|
Age
|
1.012
|
0.987-1.037
|
0.351
|
1.018
|
0.990-1.047
|
0.219
|
Gender (Female vs. Male)
|
0.892
|
0.478-1.664
|
0.720
|
0.867
|
0.433-1.738
|
0.687
|
Location(Rectum vs. Colon)
|
0.749
|
0.366-1.532
|
0.428
|
1.189
|
0.527-2.679
|
0.677
|
Histological type (Non-mucinous vs. Mucinous)
|
0.528
|
0.280-0.995
|
0.048
|
0.496
|
0.229-1.075
|
0.076
|
Pathologic stage (III/IV vs. I/II)
|
2.099
|
1.066-4.132
|
0.032
|
1.357
|
0.596-3.093
|
0.467
|
Adjuvant chemoradiotherapy(Yes vs. No)
|
0.705
|
0.374-1.328
|
0.279
|
0.865
|
0.422-1.777
|
0.694
|
Differentiation(Low/Middle vs.High)
|
0.883
|
0.271-2.876
|
0.837
|
0.559
|
0.156-2.000
|
0.371
|
HR, hazard ratio, CI, confdence interval p value below 0.05 was considered signifcant and highlighted (bold)
|
LRRN4 promotes cell proliferation in colorectal cancer cell lines
In light of our above results in CRC patients suggesting that LRRN4 was closely correlated with stage and prognosis of CRC, thus we further conduct cell experimental to explore the potential biological function of LRRN4 in CRC cells. We initially investigated the LRRN4 expression in CRC cell lines (Caco2, SW480, HCT-116, and LoVo, Fig. 4A). LRRN4 was highly expressed in Caco2 and SW480 compared to in HCT-116 and LoVo cell lines. Therefore, we generated sublines of Caco2 and SW480 silencing LRRN4, which were infected with lentivirus expressing two different LRRN4-shRNA (Caco2-kd1 and Caco2-kd2; SW480-kd1 and SW480-kd2) and corresponding control (Caco2-scr, SW480-scr), respectively. Meanwhile, HCT-116 and LoVo were infected with lentivirus overexpressing LRRN4 (HCT-116-OE and LoVo-OE) and corresponding control (HCT-116-EV and LoVo-EV) respectively. The knockdown of LRRN4 in Caco2, SW480 and overexpression of LRRN4 in HCT-116 and LoVo cells were validated by real-time quantitative PCR (Fig. 4B-C).
To determine the role of LRRN4 in cell proliferation, we carried out EdU assay. As shown in Fig. 4D-E, knockdown of LRRN4 significantly suppressed the cell proliferation of Caco2 and SW480 cells (Caco2-kd1, Caco2-kd2, SW480-kd1 and SW480-kd2 vs. Caco2-scr, SW480-scr), while the LRRN4 overexpression promoted the proliferation of HCT-116 and LoVo cells (HCT-116-OE, LoVo-OE vs. HCT-116-EV, LoVo-EV). Moreover, we investigated the capacity of colony formation in the cell lines with different LRRN4 expression. The results showed that knockdown of LRNN4 significantly decreased colony formation rate in CRC cells (Caco2-kd1, Caco2-kd2, SW480-kd1 and SW480-kd2 cells vs. Caco2-scr, SW480-scr) (Fig. 4F). However, an increase in the size and number of colonies was observed in HCT-116 and LoVo cells with LRRN4 overexpression compared to corresponding control cells (HCT-116-EV and LoVo-EV) (Fig. 4G), which further verified the promotive effect of LRRN4 on cell proliferation.
LRRN4 promotes cell DNA synthesis and inhibits apoptosis in CRC cells
The role of LRRN4 in CRC cell proliferation could be regulating the cell cycle or cell apoptosis. Therefore, the cell cycle and apoptosis were analyzed using flow cytometry. The cell cycle distribution analysis showed significantly decreased cell populations in the S phase in both Caco2 and SW480 with knockdown of LRRN4 (Caco2-kd1, Caco2-kd2, SW480-kd1 and SW480-kd2) compared to their corresponding control cells (Caco2-scr, SW480-scr) (Fig. 5A). On the contrary, overexpression of LRRN4 induced a higher percentage of S phase distribution in HCT-116 and LoVo cells compared to their control (Fig. 5B). We next analyzed whether LRRN4 exerted any impacts on cell apoptosis. When compared to their corresponding control, knockdown of LRNN4 exhibited a higher percentage of apoptotic cells in Caco2 and SW480 cells (Fig. 5C). On the contrary, the rates of apoptotic cells were significantly decreased in CRC cells with overexpression of LRRN4 (HCT-116-OE and LoVo-OE) compared to the control cells (HCT-116-EV and LoVo-EV) (Fig. 5D). Taken together, these data indicated that LRRN4 plays a functional role in promoting DNA synthesis and inhibiting cell apoptosis.
LRRN4 accelerates cell migration
To further explain the correlation between LRRN4 and pathological stage in CRC patients from cell level, wound healing assay was employed to evaluate the effect of LRRN4 on cell migration. Knockdown of LRRN4 significantly impaired the migratory capacity of Caco2 and SW480, resulting in impaired wound closure (Fig. 6A) at two different time points (24h and 48h). To confirm the above findings, cell migration was determined in CRC cells with overexpression of LRNN4. The results showed that overexpression of LRRN4 significantly accelerates wound closure compared to control, indicating a promotive effect of LRRN4 on cell migration in HCT-116 and LoVo cells (Fig. 6B).
LRRN4 accelerates xenograft tumor growth in vivo
After having demonstrated the positive impact of LRRN4 on CRC cells in vitro, we sought to confirm its role in a more physiologically relevant in vivo model. A xenograft model was utilized to investigate whether LRRN4 impacts tumor growth in vivo. Cells were injected subcutaneously into SCID mice. As shown in Fig. 7A, the sizes of xenograft tumors of cells with knockdown of LRRN4 (Caco-kd1 and Caco2-kd2) were much smaller than that of control (Caco2-scr). In the growth curve analysis (Fig. 7B, C), the tumor growth was significantly inhibited by knockdown of LRRN4, indicating the promotion role of LRRN4 in CRC xenograft tumors.
LRRN4 affects several cancer-related biological functions and pathways
To investigate the potential molecular mechanisms of LRRN4 involved in CRC, we first used WGCNA to identify gene co-expression modules and link them to LRRN4 expression. We found significant correlations between module eigengenes and the following traits, including LRRN4 expression, histological type, lymphatic invasion and perineural invasion. The magenta module was positively associated with LRRN4 expression and several clinicopathological phenotypes, which would be further analyzed (Fig. 8A). Next, we calculated the relationship between module membership of magenta module and gene significance. For most traits, strong correlations were observed (LRRN4: cor = 0.27, p = 9.3e−128; histological type: cor = 0.3, p = 1.1e−158; lymphatic invasion: cor = 0.45, p < 1e−200; perineural invasion: cor = 0.43, p < 1e−200), indicating that the genes most representative of the magenta module’s overall expression profile were those most strongly related to LRRN4 expression and clinicopathological traits (Fig. 8B).
To attribute biological meaning to the magenta module, we enriched a module significance for the gene ontology and pathway annotations returned from DAVID database. In total, 8 cell components, 13 biological processes, and 5 molecular functions were enriched (Fig. 8C-8E). The enriched biological processes included cell adhesion, response to hypoxia, cell migration and negative regulation of cysteine-type endopeptidase activity involved in apoptotic process, which was linked to malignant features of CRC cells. Microtubule binding, ion channel binding, laminin binding, fibroblast growth factor binding and potassium ion transmembrane transporter activity were highly enriched molecular functions. Meanwhile, the LRRN4-related genes in the magenta module were enriched in several cancer-related pathways, including Ras signaling pathway, Proteoglycans in cancer and Glutamatergic synapse, which are reported to be implicated in tumorigenesis and tumor progression including CRC (Fig. 8F).