IncRNA VIM-AS1 expression is positively correlated with the prognosis and immune infiltration in lung adenocarcinoma

doi:10.21203/rs.3.rs-1990949/v1

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IncRNA VIM-AS1 expression is positively correlated with the prognosis and immune infiltration in lung adenocarcinoma

https://doi.org/10.21203/rs.3.rs-1990949/v1

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Background

Studies have reported that Long Non-coding RNA Vimentin antisense RNA1(VIM-AS1) is related to progression and prognosis in several cancers. Although the relationship between VIM-AS1 and the clinical features of lung adenocarcinoma has been described, their studies are incomplete. Therefore, a comprehensive analysis was performed to identify the role and potential clinical value of VIM-AS1 in LUAD progression.

Methods

The expression of VIM-AS1 in LUAD was identified based on Cancer Genome Atlas database (TCGA) and genotypic tissue expression (GTEx). Survival analysis and COX regression analysis were performed to evaluate the clinical value of VIM-AS1 in the prognosis of LUAD patients, and to construct a prognostic nomogram. Correlation and COX regression analysis were performed to filter prognosis-related VIM-AS1 co-expression genes, and to construct the correlation column chart and the prognostic risk model. Correlation analysis was also used to explore the relationship between VIM-ASI expression and LUAD immune microenvironment.

Results

VIM-AS1 expression levels were significantly downregulated in LUAD tissues and significantly associated with short OS, DSS, significant PFI, late T and pathological staging, lymph node metastasis, gender male and complete resection in LUAD patients. Decreased expression of VIM-AS1 was an independent risk factor for poor prognosis in LUAD patients. VIM-AS1 co-expressed genes SLC15A2, ZNF56, FAM76A, GNG7, UCK2, and ADIPOR2 were significantly associated with OS, DSS, and PFI in LUAD patients. The nomogram and risk models constructed based on VIM- AS1 co-expressed genes were associated with the prognosis of LUAD patients. K-M survival analysis showed that high-risk patients were significantly associated with short OS, DSS, and PFI in LUAD patients. VIM- AS1 expression was related to the estimate, immune and stromal scores, and highly associated with immune cells -TFH, Th1 cells, T cells, Tcm, B cells, T helper cells, cytotoxic cells, macrophages, pDC, iDC, aDC, mast cells, DC, Tem, NK CD56dim cells, Tgd and Th2 cells, and significantly correlated with levels of immune cell markers HLA-DPB1, HLA-DRA, CCR7, and other markers.

Conclusion

VIM-AS1 was significantly downregulated in LUAD tissues, which was significantly associated with poor prognosis and immune microenvironment in LUAD patients. The nomogram and risk models of VIM-AS1 were expected to be tools to assess the prognosis of LUAD patients.

VIM-AS1

lung adenocarcinoma (LUAD)

prognosis

co-expressed genes

immune microenvironment

bioinformatics analysis.

It is well known that lung adenocarcinoma (LUAD) remains one of the highly prevalent tumors. Recent studies have shown that the development of LUAD involves long-stranded coding RNA changes ^{[1– 6]}. LINC01006 expression levels are significantly increased in LUAD tissues and cells. Inhibition of LINC01006 expression reduces the ability of LUAD cells to proliferate, migrate, and epithelial-mesenchymal transition (EMT), thereby inhibiting tumor progression. LINC01006 inhibits miR-129-2-3p expression to enhance CTNNB1 expression levels, which in turn promotes LUAD development by activating the Wnt/β-catenin signaling pathway ^[2]. Ribonuclease H2 subunit A (RNASEH2A) is a nucleotide-degrading enzyme gene that has a significant impact on cell proliferation, DNA replication, and genomic stability. The expression level of RNASEH2A is significantly upregulated in LUAD tissues. The increased expression level of RNASEH2A is associated with short survival of LUAD patients, while downregulation of RNASEH2A expression level inhibits proliferation and induces apoptosis of LUAD cells. The long-stranded non-coding RNA (lncRNA) LINC01287 is a competitive endogenous RNA that can promote the proliferation of cancer cells and inhibits apoptosis by competitively binding with miR-3529-5p to upregulate RNASEH2A expression at the post-transcriptional level ^[4].

The latest discovery of IncRNA Vimentin antisense RNA1(VIM-AS1) has important biological functions in cancer progression ^[7–10]. For example, VIM-AS1 is highly expressed in bladder cancer and significantly associated with bladder cancer metastasis. VIM-AS1 promotes ZEB1 expression by competitively binding miR-655 to facilitate the EMT process and enhance prostate cancer cell metastasis ^[7]. lncRNAs play a crucial role in the development and progression of various malignancies. In addition, VIM-AS1 was found to be significantly highly expressed in high-grade lymph node metastasis and vascular invasion colorectal cancer. Inhibition of VIM-AS1 expression can block the cell cycle by inducing apoptosis and senescence in colorectal cancer cells. VIM-AS1 promotes tumor growth and metastasis by inducing the EMT process in colorectal cancer cells ^[8]. Another study shows that the expression level of VIM-AS1 is significantly elevated in glioma tissues. VIM-AS1 overexpression is positively correlated with short overall survival (OS). Inhibition of VIM-AS1 expression inhibits glioma growth, colony formation, and migration in vitro and slows down glioma growth in vivo, and promotes apoptosis by inhibiting cyclin A1, PCNA, vimentin, N-cadherin, and Bcl-2 expression and increasing Bax and e-cadherin expression ^[10]. Currently, no study has confirmed the biological role of VIM-AS1 in LUAD progression. Therefore, this study is proposed to explore the expression of VIM-AS1 in LUAD tissues by bioinformatics, and to identify the relationship between VIM-AS1 expression levels and the clinicopathological characteristics and prognosis of LUAD patients. Prognosis-related VIM- AS1 co-expression genes were identified by correlation analysis and COX regression analysis, and a VIM-AS1-related prognostic nomogram was constructed. An experimental model of VIM-AS1 overexpression in LUAD cells was constructed to confirm the effect of VIM-AS1 overexpression on the growth and migration of LUAD cells. In addition, the potential role of VIM-AS1 expression levels in the immune microenvironment of LUAD was assessed by comprehensive analysis.

Identification of the expression level of VIM-AS1 in LUAD tissues

Gene expression levels of LUAD patients were obtained from Cancer Genome Atlas database (TCGA) and genotypic tissue expression (GTEx). The data were TPM and FPKM types. VIM-AS1 expression data were extracted by Perl language, and whether there was a significant difference in VIM-AS1 expression levels in normal lung tissue and LUAD tissue was identified. Furthermore, patient source matching of normal lung tissue and LUAD tissue samples was performed to identify whether there was a significant difference in the expression levels of VIM-AS1 in tissues from the same LUAD patients.

Identification of the relationship between VIM-AS1 expression levels and prognostic and clinicopathological characteristics of LUAD patients

LUAD patients were grouped according to the median of VIM-AS1 expression values from TPM and FPKM data to group LUAD patients, Kaplan-Meier (K-M) survival analysis was performed to identify the relationship between VIM-AS1 expression levels and OS, Disease-Specific Survival (DSS), and Progress Free Interval (PFI) in LUAD patients, and to investigate the expression levels of VIM-AS1 in the clinical characteristics (T-stage, N-stage, distant metastasis, pathological stage, age, gender, OS, DSS, and PFI) of LUAD patients.

Identification of the relationship between VIM-AS1 expression levels and prognosis of a subgroup of LUAD patients

Based on the data of clinical characteristics such as T-stage, N-stage, distant metastasis, pathological stage, age, gender, and smoking history of LUAD patients, and the median value of VIM-AS1 expression in FPKM data were used to group LUAD patients, the relationship between VIM-AS1 expression level and OS, DSS and PFI of subgroup LUAD patients was investigated by K-M survival analysis.

Construction of VIM-AS1 nomogram

Univariate and multifactorial COX regression analyses were performed to analyze the relationship between T-stage, N-stage, distant metastasis, pathological stage, age, gender, smoking history, and VIM-AS1 expression levels with OS, DSS, and PFI in LUAD patients. P-value < 0.05 was the criterion for significance, and VIM-AS1 nomograms were constructed for the inclusion criteria of the prognostic nomogram.

Biological functions and signaling mechanisms of VIM-AS1 co-expressed genes

Correlation analysis was performed to obtain VIM-AS1 co-expressed genes in 535 LUAD tissues ^[11], and the absolute value of the correlation coefficient of 0.4 and p-value < 0.001 were used as filtering conditions, and this condition was defined as VIM-AS1 significantly associated co-expressed genes. GO annotation was used to understand the biological processes, cellular components, and molecular functions involved in VIM-AS1 co-expressed genes, and the signaling pathways involved in VIM-AS1 co-expressed genes were identified by KEGG pathway analysis.

Construction of VIM-AS1 co-expression gene nomogram

OS, DSS, and PFI-related genes in LUAD patients were obtained using COX regression analysis with the p-value < 0.01, and the intersection genes of LUAD prognosis-related genes and VIM-AS1 co-expressed genes were shown by VENN plots. The relationship between the expression levels of the intersecting genes SLC15A2, ZNF56, FAM76A, GNG7, UCK2, and ADIPOR2 and the OS, DSS, and PFI of LUAD patients were identified by K-M survival analysis, and the VIM-AS1 co-expression gene-related nomogram were constructed.

Construction of a risk model associated with VIM-AS1 co-expression gene

The relationship between SLC15A2, ZNF56, FAM76A, GNG7, UCK2, and ADIPOR2 expression levels and OS, DSS, and PFI in LUAD patients was assessed by LASSO regression analysis, and the model’s coefficients and trajectories were determined. The relationship between high and low risk and OS, DSS, and PFI in LUAD patients was assessed by K-M survival analysis.

Identification of the relationship between VIM-AS1 and the immune microenvironment of LUAD

Immune microenvironment components were calculated from 535 LUAD tissues using ssGSEA and estimate algorithms. Correlation analysis was performed to explore the relationship between VIM-AS1 and estimatescore, immunescore, stromalscore, TFH, Th1 cells, T cells, Tcm, B cells, T helper cells, Cytotoxic cells, Macrophages, pDC, iDC, aDC, Mast cells, DC, Tem, CD8 T cells, Th17 cells, Eosinophils, NK cells, TReg, NK CD56bright cells, Neutrophils, NK CD56dim cells, Tgd and Th2 cell levels. Identified whether there were statistically significant relationships between estimatescore, immunescore, stromalscore, TFH, Th1 cells, T cells, Tcm, B cells, T helper cells, Cytotoxic cells, Macrophages, pDC, iDC, aDC, Mast cells, DC, Tem, CD8 T cells, Th17 cells, Eosinophils, NK cells, TReg, NK CD56bright cells, Neutrophils, NK CD56dim cells, Tgd and Th2 cells in VIM-AS1 overexpression and low expression groups.

Identification of VIM-AS1 in relation to LUAD immune cell markers

The expression data of immune cell markers and VIM-AS1 from 535 LUAD tissues were extracted using Perl language. The relationship between the expression level of VIM-AS1 and the level of immune cell markers in LUAD was explored by correlation analysis.

Statistical analysis

The expression of VIM-AS1 in LUAD tissues was investigated by the Wilcoxon rank sum test, and prognostic factors in LUAD patients were assessed through K-M survival analysis and COX analysis. The t-test was used to investigate statistical differences between immune cells in the high and low VIM-AS1 expression groups. Correlation analysis was performed to identify the relationship between VIM-AS1 co-expressed genes and the immune microenvironment in LUAD. P-value < 0.05 was the criterion for the significance of the study.

VIM-AS1 expression level was significantly downregulated in LUAD tissues

Based on TCGA and GTEx database, TPM and FPKM data showed significantly decreased expression levels of VIM-AS1 in unpaired LUAD tissues (Figs. 2A-C). The expression level of VIM-AS1 was significantly decreased in paired LUAD tissues after the pairing of patient TPM and FPKM data (Figs. 2D and E).

Decreased VIM-AS1 expression levels correlated with poor clinical features in LUAD patients

Survival analysis revealed that decreased levels of VIM-AS1 expression were statistically significantly associated with short OS, DSS, and PFI in LUAD patients (Figs. 3A- C). VIM-AS1 expression was significantly decreased in tissues of LUAD patients within T2 and T3 stages compared to T1 (Figs. 4A and B). VIM-AS1 expression was significantly decreased in the tissues of LUAD patients in the N2 stage compared with the N0 stage (Fig. 4C). VIM- AS1 expression was significantly decreased in tissues of LUAD patients with Stage II-IV compared to Stage I (Figs. 4D-F). VIM-AS1 expression was significantly decreased in the tissues of Male patients compared to Female patients (Fig. 4G). VIM-AS1 expression was significantly decreased in the tissues of R1 LUAD patients compared to ROLUAD patients (Fig. 4H). Under OS, DSS, and PFI conditions, VIM-AS1 expression was significantly increased in tissues of Alive LUAD patients compared with the Dead LUAD patients (Figs. 4I-K).

Decreased VIM-AS1 expression levels were significantly associated with poor prognosis in a subgroup of LUAD patients

Subgroup analysis showed that in the T1-2 stage, T1-3 stage, T2 stage, T2-3 stage, T2-4 stage, T3 stage, T3-4 stage, T4 stage, N0 stage, N1-3 stage, M0 stage, Stage I-II, Stage I-III, Stage II-III, Stage II-IV, the Stage III-IV, Male, Age < = 65, Age > 65, R0, No-smoke and Smoke patients, decreased VIM-AS1 expression levels were significantly associated with short OS in subgroups of LUAD patients (Fig. 4and S1). In T1-2 stage, T1-3 stage, T2 stage, T2-3 stage, T2-4 stage, T3 stage, T3-4 stage, N0 stage, M0 stage, Stage I stage, Stage I-II stage, Stage I- III stage, Stage II-IV stage, Female, Male, Age < = 65, Age > 65, R0, No-smoke, and Smoke LUAD patients, decreased levels of VIM-AS1 expression were significantly associated with short DSS in subgroups of LUAD patients (Fig. 6 and S2). In T1-2 stage, T1-3 stage, T2 stage, T2-3 stage, T2-4 stage, N0 stage, M0 stage, Stage I, Stage I-II, Stage I-III, Stage II-III, Stage II-IV, Stage III-IV, Female, Male, Age < = 65, Age > 65, R0, and Smoke LUAD patients, decreased VIM-AS1 expression levels were associated with short PFI in subgroups of LUAD patients (Fig. 7).

VIM-AS1 prognostic nomogram

COX regression analysis showed that T stage, N stage, distant metastasis, pathological stage, and VIM-AS1 were adverse factors affecting OS and DSS in LUAD patients, and decreased VIM- AS1 expression level was an independent factor influencing OS and DSS in LUAD patients (Tables 1 and 2). COX regression analysis showed that T stage, N stage, pathological stage, and VIM-AS1 were adverse factors affecting adverse factors of PFI in LUAD patients (Table 3). TNM stage had an important role in assessing the prognosis of LUAD patients and combined with the results of COX regression analysis, we constructed a VIM-AS1 correlation column line graph (Fig. 8).

Table 1

Factors influencing OS in patients with LUAD
Characteristics	N	HR (95% CI)	P	HR (95% CI)	P
T stage	523
T1	175	Reference
T2	282	1.521 (1.068–2.166)	0.02	1.580 (1.003–2.489)	0.049
T3	47	2.937 (1.746–4.941)	< 0.001	3.236 (1.591–6.585)	0.001
T4	19	3.326 (1.751–6.316)	< 0.001	2.045 (0.931–4.490)	0.075
N stage	510
N0	343	Reference
N1	94	2.381 (1.695–3.346)	< 0.001	2.227 (1.167–4.249)	0.015
N2	71	3.108 (2.136–4.521)	< 0.001	1.980 (0.826–4.746)	0.126
N3	2	0.000 (0.000-Inf)	0.994	0.000 (0.000-Inf)	0.993
M stage	377
M0	352	Reference
M1	25	2.136 (1.248–3.653)	0.006	1.773 (0.808–3.891)	0.153
Pathologic stage	518
Stage I	290	Reference
Stage II	121	2.418 (1.691–3.457)	< 0.001	0.896 (0.449–1.791)	0.757
Stage III	81	3.544 (2.437–5.154)	< 0.001	1.420 (0.537–3.759)	0.48
Stage IV	26	3.790 (2.193–6.548)	< 0.001
Gender	526
Female	280	Reference
Male	246	1.070 (0.803–1.426)	0.642
Age	516
<=65	255	Reference
> 65	261	1.223 (0.916–1.635)	0.172
Smoker	512
No	72	Reference
Yes	440	0.894 (0.592–1.348)	0.591
VIM-AS1	526
Low	260	Reference
High	266	0.577 (0.431–0.773)	< 0.001	0.613 (0.432–0.868)	0.006

Table 2

Factors influencing DSS in LUAD patients
Characteristics	N	HR (95% CI)	P	HR (95% CI)	P
T stage	488
T1	168	Reference
T2	262	1.701 (1.085–2.668)	0.021	1.519 (0.853–2.705)	0.156
T3	43	2.846 (1.453–5.572)	0.002	2.563 (0.977–6.726)	0.056
T4	15	2.770 (1.061–7.230)	0.037	1.805 (0.582–5.597)	0.306
N stage	475
N0	327	Reference
N1	83	2.751 (1.808–4.185)	< 0.001	2.110 (0.925–4.809)	0.076
N2	63	2.762 (1.698–4.493)	< 0.001	1.916 (0.551–6.666)	0.307
N3	2	0.000 (0.000-Inf)	0.995	0.000 (0.000-Inf)	0.996
M stage	344
M0	323	Reference
M1	21	2.455 (1.269–4.749)	0.008	2.279 (0.922–5.633)	0.074
Pathologic stage	483
Stage I	277	Reference
Stage II	112	3.017 (1.931–4.715)	< 0.001	1.090 (0.447–2.658)	0.849
Stage III	72	3.326 (2.028–5.457)	< 0.001	1.292 (0.325–5.143)	0.716
Stage IV	22	4.632 (2.371–9.050)	< 0.001
Gender	491
Female	262	Reference
Male	229	0.989 (0.687–1.424)	0.954
Age	481
<=65	243	Reference
> 65	238	1.013 (0.701–1.464)	0.944
Smoker	477
No	69	Reference
Yes	408	1.040 (0.602–1.796)	0.889
VIM-AS1	491
Low	240	Reference
High	251	0.495 (0.340–0.721)	< 0.001	0.523 (0.329–0.832)	0.006

Table 3

Factors influencing PFI in patients with LUAD
Characteristics	N	HR (95% CI)	P	HR (95% CI)	P
T stage	523
T1	175	Reference
T2	282	1.758 (1.276–2.422)	< 0.001	1.515 (1.090–2.105)	0.013
T3	47	3.495 (2.199–5.556)	< 0.001	2.450 (1.332–4.508)	0.004
T4	19	1.113 (0.444–2.791)	0.819	0.505 (0.166–1.534)	0.228
N stage	510
N0	343	Reference
N1	94	1.540 (1.118–2.122)	0.008	1.215 (0.691–2.136)	0.499
N2	71	1.498 (1.018–2.205)	0.04	0.510 (0.178–1.465)	0.211
N3	2	0.906 (0.127–6.485)	0.922	0.781 (0.088–6.921)	0.825
M stage	377
M0	352	Reference
M1	25	1.513 (0.855–2.676)	0.155
Pathologic stage	518
Stage I	290	Reference
Stage II	121	2.013 (1.478–2.742)	< 0.001	1.298 (0.734–2.297)	0.37
Stage III	81	1.831 (1.257–2.669)	0.002	2.872 (0.940–8.769)	0.064
Stage IV	26	2.086 (1.189–3.657)	0.01	2.006 (1.010–3.985)	0.047
Gender	526
Female	280	Reference
Male	246	1.172 (0.901–1.526)	0.236
Age	516
<=65	255	Reference
> 65	261	1.023 (0.784–1.335)	0.867
Smoker	512
No	72	Reference
Yes	440	0.968 (0.658–1.426)	0.87
VIM-AS1	526
Low	260	Reference
High	266	0.646 (0.496–0.841)	0.001	0.761 (0.577–1.004)	0.053

Biological functions and signaling mechanisms of VIM-AS1 co-expressed genes

Correlation analysis revealed 214 VIM-AS1 co-expressed genes (Table S1). GO annotation and KEGG analysis showed that VIM-AS1 co-expressed genes were involved in cilium movement, axoneme assembly, positive regulation of NF-kappaB transcription factor activity, peptidase activator activity, cysteine-type endopeptidase activator activity involved in the apoptotic process, peptidase activator activity involved in the apoptotic process, and Herpes simplex virus 1 infection (Table 4).

Table 4

Biological functions and signaling mechanisms of VIM-AS1 co-expressed genes
Type	ID	Description	p
BP	GO:0003341	cilium movement	3.02E-09
BP	GO:0044458	motile cilium assembly	8.48E-07
BP	GO:0044782	cilium organization	2.14E-06
BP	GO:0007018	microtubule-based movement	2.54E-06
BP	GO:0060271	cilium assembly	6.67E-06
BP	GO:0001578	microtubule bundle formation	7.57E-06
BP	GO:0035082	axoneme assembly	9.20E-06
BP	GO:0003351	epithelial cilium movement	3.47E-05
BP	GO:0060287	epithelial cilium movement involved in determination of left/right asymmetry	5.51E-05
BP	GO:0070286	axonemal dynein complex assembly	0.000111749
BP	GO:0051092	positive regulation of NF-kappaB transcription factor activity	0.000179556
BP	GO:0061371	determination of heart left/right asymmetry	0.000184336
BP	GO:0036159	inner dynein arm assembly	0.000203004
BP	GO:0036158	outer dynein arm assembly	0.000299906
BP	GO:0007368	determination of left/right symmetry	0.000359552
BP	GO:0098581	detection of external biotic stimulus	0.000422459
BP	GO:0032495	response to muramyl dipeptide	0.000494152
BP	GO:0009855	determination of bilateral symmetry	0.000530414
BP	GO:0009799	specification of symmetry	0.00055273
BP	GO:0006919	activation of cysteine-type endopeptidase activity involved in apoptotic process	0.000589812
BP	GO:0009595	detection of biotic stimulus	0.000754505
CC	GO:0005930	axoneme	7.67E-07
CC	GO:0097014	ciliary plasm	8.21E-07
CC	GO:0044441	ciliary part	1.28E-06
CC	GO:0031514	motile cilium	1.67E-05
CC	GO:0032838	plasma membrane bounded cell projection cytoplasm	5.45E-05
CC	GO:0097729	9 + 2 motile cilium	0.001257541
MF	GO:0016504	peptidase activator activity	0.000257015
MF	GO:0008656	cysteine-type endopeptidase activator activity involved in apoptotic process	0.000279009
MF	GO:0016505	peptidase activator activity involved in apoptotic process	0.000474164
MF	GO:0045503	dynein light chain binding	0.001085694
KEGG	hsa05168	Herpes simplex virus 1 infection	7.27E-10

Screening for key VIM-AS1 co-expressed genes

The VENN plot shows six intersecting genes of prognosis-related genes and VIM-AS1 co-expressed genes in LUAD patients. The intersecting genes were SLC15A2, ZNF56, FAM76A, GNG7, UCK2, and ADIPOR2. Figure 9 shows that VIM-AS1 expression levels were significantly correlated with SLC15A2, ZNF56, FAM76A, GNG7, UCK2, and ADIPOR2 expression levels.

Construction of VIM-AS1 co-expression gene nomogram and risk model

K-M survival analysis showed that ADIPOR2 and UCK2 overexpression were significantly associated with short OS, DSS, and PFI in LUAD patients, and decreased expression levels of SLC15A2, ZNF56, FAM76A, and GNG7 were significantly associated with short OS, DSS, and PFI in LUAD patients (Fig. 10 and S3), and a prognostic nomogram was constructed for SLC15A2, ZNF56, FAM76A, GNG7, UCK2 and ADIPOR2 expression associated with LUAD patients in a prognostic nomogram (Fig. 11). LASSO regression analysis to assess the relationship between SLC15A2, ZNF56, FAM76A, GNG7, UCK2, and ADIPOR2 expression levels and OS, DSS, and PFI in LUAD patients showed that SLC15A2, ZNF56, FAM76A, GNG7, UCK2, and ADIPOR2 were all risk factors associated with OS, DSS, and PFI in LUAD patients (Fig. 12). K-M survival analysis showed that patients at high risk were significantly associated with short OS, DSS, and PFI in LUAD patients (Fig. 12).

VIM-AS1 expression is significantly associated with the LUAD immune microenvironment

Correlation analysis showed that VIM-AS1 expression levels correlated with the estimate, immune, and stromal scores (Fig. 13A-C), while estimate, immune, and stromal scores were statistically significantly different in the high and low VIM-AS1 expression groups (Fig. 13D-F). VIM-AS1expression levels were significantly correlated with TFH, Th1 cells, T cells, Tcm, B cells, T helper cells, cytotoxic cells, macrophages, pDC, iDC, aDC, mast cells, DC, Tem, NK CD56dim cells, Tgd and Th2 cells (Table 5 and Figure S4). Immune cells aDC, B cells, CD8 T cells, cytotoxic cells, DC, pDC, mast cells, iDC, macrophages, NK cells, T cells, T helper cells, Tcm, TFH, Tgd, Th1 cells, and Th2 cells had statistically significant differences between the high and low VIM-AS1 expression groups (Figure S5). Decreased levels of VIM-AS1 expression were associated with decreased levels of HLA-DPB1, HLA-DRA, CCR7, CD1C, HLA-DPA1, HLA-DQB2, STAT4, HLA-DQB1, CD2, CTLA4, ITGAX, CD19, CD3E, NRP1, CD3D, STAT5A, IRF5, TBX21, CD8A, BCL6, CD86, ITGAM, HAVCR2, TNF, CSF1R, IL13, IFNG, CCL2, PDCD1, IL10, CD79A, CCR8, FOXP3, IL21, MS4A4A, CEACAM8, CD8B, LAG3, CD163, GATA3, HLA-DQB3, IL17A, and VSIG4 (Table 6 and Fig. 14).

Table 5

VIM-AS1 expression is significantly associated with LUAD immune cells
Immune cells	Cor	P
TFH	0.267272247	3.33802E-10
Th1 cells	0.266029341	4.05515E-10
T cells	0.242464462	1.34636E-08
Tcm	0.207492169	1.29276E-06
B cells	0.203072331	2.18494E-06
T helper cells	0.192651297	7.1989E-06
Cytotoxic cells	0.185816115	1.52108E-05
Macrophages	0.170959356	7.05723E-05
pDC	0.15843427	0.000233886
iDC	0.131256264	0.002349839
aDC	0.12929564	0.002733602
Mast cells	0.120902643	0.005106716
DC	0.116470932	0.0069996
Tem	0.110935101	0.010232703
CD8 T cells	0.061074858	0.158339421
Th17 cells	0.05245251	0.225808701
Eosinophils	0.0501771	0.246613144
NK cells	0.032017347	0.459894417
TReg	0.025375581	0.558103771
NK CD56bright cells	-0.022328852	0.606326567
Neutrophils	-0.071372248	0.099129777
NK CD56dim cells	-0.103737265	0.01638089
Tgd	-0.259285997	1.14551E-09
Th2 cells	-0.321998221	2.26364E-14

Table 6

VIM-AS1 expression levels significantly correlate with immune cell marker levels
Gene markers	Cor	P
HLA-DPB1	0.348	< 0.001
HLA-DRA	0.347	< 0.001
CCR7	0.337	< 0.001
CD1C	0.337	< 0.001
HLA-DPA1	0.335	< 0.001
HLA-DQB2	0.319	< 0.001
STAT4	0.318	< 0.001
HLA-DQB1	0.309	< 0.001
CD2	0.302	< 0.001
CTLA4	0.302	< 0.001
ITGAX	0.282	< 0.001
CD19	0.273	< 0.001
CD3E	0.261	< 0.001
NRP1	0.255	< 0.001
CD3D	0.243	< 0.001
STAT5A	0.242	< 0.001
IRF5	0.236	< 0.001
TBX21	0.225	< 0.001
CD8A	0.206	< 0.001
BCL6	0.206	< 0.001
CD86	0.205	< 0.001
ITGAM	0.201	< 0.001
HAVCR2	0.197	< 0.001
TNF	0.196	< 0.001
CSF1R	0.19	< 0.001
IL13	0.184	< 0.001
IFNG	0.174	< 0.001
CCL2	0.173	< 0.001
PDCD1	0.172	< 0.001
IL10	0.171	< 0.001
CD79A	0.17	< 0.001
CCR8	0.17	< 0.001
FOXP3	0.153	< 0.001
IL21	0.145	< 0.001
MS4A4A	0.131	0.002
CEACAM8	0.127	0.003
CD8B	0.121	0.005
LAG3	0.107	0.013
CD163	0.106	0.015
GATA3	0.092	0.034
HLA-DQB3	0.091	0.035
IL17A	0.088	0.043
VSIG4	0.086	0.046
STAT5B	0.085	0.05
STAT1	0.075	0.084
STAT6	0.071	0.102
GZMB	0.033	0.441
CD68	0.024	0.582
PTGS2	0.021	0.622
KIR2DS4	-0.027	0.526
KIR3DL1	-0.031	0.467
NOS2	-0.041	0.346
KIR3DL3	-0.05	0.252
KIR2DL4	-0.053	0.224
KIR2DL3	-0.056	0.199
KIR3DL2	-0.09	0.037
KIR2DL1	-0.119	0.006
STAT3	-0.13	0.003

Multiple projects have shown that lncRNAs are associated with the prognosis and the growth of cancer cells ^[12–15]. Glioma tissues and cells showed significant downregulation of p53 expression and a significant increase in lncRNA PVT1 expression. p53 inhibits glioma cell proliferation, migration and invasion,which stops tumor growth, and induces apoptosis by downregulating PVT1 expression. PVT1 binds to TGF-β to activate the TGF-β/Smad signaling pathway to promote glioma progression ^[12]. ITGB1-DT is overexpressed in gastric adenocarcinoma tissues. Elevated levels of ITGB1-DT expression are associated with T-stage, treatment outcome, OS, and PFI in gastric adenocarcinoma patients and are detrimental factors affecting the prognosis of STAD patients. Inhibition of ITGB1-DT expression inhibited proliferation, invasion, and migration of gastric adenocarcinoma cells. ITGB1-DT overexpression correlated with the levels of gastric adenocarcinoma B cells, T cells, T helper cells, and other immune cells ^[14]. LINC01426 was overexpressed in LUAD tissues and cells. High levels of LINC01426 were associated with patient TNM stage, lymphatic metastasis, degree of tumor differentiation, and short OS. Interference with LINC01426 expression reduces the proliferation and metastatic capacity of LUAD H1299 and PC9 cells ^[15]. VIM-AS1 has been shown to be associated with cancer metastasis and poor patient prognosis ^{[7–10, 16–18]}. For example, VIM-AS1 expression levels are significantly downregulated in breast cancer tissues. VIM-AS1 expression levels are associated with obesity ^[16]. VIM-AS1 and vimentin (VIM) expression levels are significantly elevated in oral squamous cell carcinoma, while e-cadherin expression levels are downregulated. VIM-AS1 expression levels are associated with oral squamous cell carcinoma patients with clinicopathological features and have diagnostic value ^[17]. It was found that VIM-AS1 has significant clinical value in LUAD. The expression level of VIM-AS1 was significantly decreased in LUAD tissues. The low VIM-AS1 expression level was significantly associated with short OS, DSS, and PFI in LUAD patients, and late T-stage was significantly associated with pathological stage, lymph node metastasis, Male and R1 resection. The COX regression analysis showed that VIM -AS1 expression level decreased as an independent influencing factor of poor prognosis in LUAD patients. VIM-AS1 nomogram and risk models are expected to assess the prognosis of LUAD patients. Preliminary indicates that VIM-AS1 may be a prognostic biomarker for LUAD patients.

Studies have confirmed VIM in non-small cell lung cancer (NSCLC) metastasis ^{[19, 20]}. Tadokoro et al. reported that VIM positivity was associated with poor prognosis in NSCLC patients. VIM positivity was higher in LUAD than in squamous lung cancer. Cellular assays showed that inhibition of VIM expression decreased the invasiveness of cancer cells ^[20]. VIM-AS1 is similar to VIM and is associated with cancer metastasis ^[7–10]. For example, VIM-AS1 is upregulated in high grade, lymph node metastatic, and vascular invasive tumors. Downregulation of VIM-AS1 expression levels inhibits cell cycle and proliferation by promoting apoptosis and cellular senescence. VIM-AS1 induces VIM protein expression in colorectal cancer cells, which in turn promotes tumor growth and metastasis through the epithelial-mesenchymal transition process ^[8]. VIM-AS1 was significantly overexpressed in prostate cancer tissues compared with normal prostate tissues. High VIM-AS1 expression correlated with tumor size, metastasis, and TNM stage. Downregulation of VIM-AS1 expression inhibited PC3 cell proliferation, migration, and invasion. overexpression of VIM-AS1 promoted PC3 cell proliferation, migration, and invasion. VIM-AS1 promotes VIM protein expression ^[9]. This predicts an important role of VIM-AS1 in the LUAD metastasis process. However, further validation by scratch assay, Transwell assay, and immunoblotting is necessary to verify the role of VIM-AS1 overexpression in LUAD cell metastasis.

Studies have confirmed that immunotherapy improves the prognosis of cancer patients ^{[21– 23]}. Therefore, the relationship between VIM-AS1 expression levels and LUAD immune cells and cellular marker levels was explored. It was found that VIM-AS1 expression levels correlated with LUAD immune microenvironment components estimate, immune and stromal scores, and with immune cells TFH, Th1 cells, T cells, Tcm, B cells, T helper cells, cytotoxic cells, the macrophages, pDC, iDC, aDC, mast cells, DC, Tem, NK CD56dim cells, Tgd and Th2 cells, and immune cell markers HLA-DPB1, HLA-DRA, CCR7, CD1C, HLA-DPA1, HLA-DQB2, STAT4, HLA-DQB1, CD2, CTLA4, ITGAX, CD19, CD3E, NRP1, CD3D, STAT5A, IRF5, TBX21, CD8A, BCL6, CD86, ITGAM, HAVCR2, TNF, CSF1R, IL13, IFNG, CCL2, PDCD1, IL10, CD79A, CCR8, FOXP3, IL21, MS4A4A, CEACAM8, CD8B, LAG3, CD163, GATA3, HLA-DQB3, IL17A, and VSIG4.

A large sample of TCGA database was utilized to find that VIM-AS1 has an important role in LUAD progression. This requires more investigators to verify the expression of VIM-AS1 and the effect on cell growth and migration by collecting LUAD tissues and cellular experiments. Combined with the reports in the literature, VIM-AS1 deserves to be investigated in depth regarding the mechanism of LUAD cell metastasis. Overall, VIM-AS1 expression levels were significantly downregulated in LUAD tissues and significantly associated with short OS, DSS, significant PFI, late T, and pathological staging, lymph node metastasis, Male and complete resection in LUAD patients. Decreased VIM-AS1 expression levels were an independent risk factor for poor prognosis in LUAD patients.VIM-AS1 co-expressed genes constructed by VIM-AS1 expression were significantly associated with the immune microenvironment in LUAD. The VIM-AS1 nomogram and risk models are expected to be a tool for assessing the prognosis of LUAD patients.

Funding

Project number: WA2020RW10

Project name: Research on the application of standardization of minimally invasive surgery for lung cancer -- Application of artificial intelligence (AI) in VATS lung cancer surgery

Entrusted by: Medical and Health Science and Technology Development Research Center, National Health Commission, the First Affiliated Hospital of Sun Yat-sen University.

Consent for publication

The material in this article has not been previously published elsewhere in either print and/or electronic form and is not currently being considered for publication by any other journal.

Ethics and Consent

All methods were carried out in accordance with relevant guidelines and regulations.

Data availability

All data generated or analysed during this study are from open data source “The Cancer Genome Atlas” (TCGA). All raw data are included in this published article [and its supplementary information files].

Author contributions

JH Kang: Conception and design, Administrative support, Provision of study materials, Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

Maimaiti Abudurufu: Provision of study materials, Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

Shuwei Zhang: Conception and design, Administrative support, Provision of study materials, Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

Honghe Luo: Conception and design, Administrative support, Provision of study materials, Manuscript writing, Final approval of manuscript.

Wei Jiang: Conception and design, Administrative support, Provision of study materials, Manuscript writing, Final approval of manuscript.

Competing interests

All authors do not have any conflict of interest related to this manuscript with any hospital, institution, company, or other institution.

All authors have not received any form of payment, financial or other in relation to this manuscript that may result in a conflict of interest.

Acknowledgements

We gratefully thank the project “Research on the application of standardization of minimally invasive surgery for lung cancer -- Application of artificial intelligence (AI) in VATS lung cancer surgery” for financial support.

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No competing interests reported.

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IncRNA VIM-AS1 expression is positively correlated with the prognosis and immune infiltration in lung adenocarcinoma

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Version 1

Abstract

Figures

Introduction

Materials And Methods

Identification of the expression level of VIM-AS1 in LUAD tissues

Construction of VIM-AS1 nomogram

Biological functions and signaling mechanisms of VIM-AS1 co-expressed genes

Construction of VIM-AS1 co-expression gene nomogram

Construction of a risk model associated with VIM-AS1 co-expression gene

Identification of the relationship between VIM-AS1 and the immune microenvironment of LUAD

Identification of VIM-AS1 in relation to LUAD immune cell markers

Statistical analysis

Results

VIM-AS1 expression level was significantly downregulated in LUAD tissues

Decreased VIM-AS1 expression levels correlated with poor clinical features in LUAD patients

VIM-AS1 prognostic nomogram

Biological functions and signaling mechanisms of VIM-AS1 co-expressed genes

Screening for key VIM-AS1 co-expressed genes

Construction of VIM-AS1 co-expression gene nomogram and risk model

VIM-AS1 expression is significantly associated with the LUAD immune microenvironment

Discussion

Declarations

References

Additional Declarations

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