Overexpression of MTFR1 promotes cancer progression and drug-resistance on cisplatin and is related to the immune microenvironment in lung adenocarcinoma

Objective: The roles of MTFR1 in the drug resistance of lung adenocarcinoma (LAC) to cisplatin remain unexplored. In this study, the expression, clinical values and mechanisms of MTFR1 were explored, and the relationship between MTFR1 expression and immune microenvironment was investigated in LAC using bioinformatics analysis, cell experiments, and meta-analysis. Methods: MTFR1 expression and clinical values, and the relationship between MTFR1 expression and immunity were explored, through bioinformatics analysis. The effects of MTFR1 on the growth, migration and cisplatin sensitivity of LAC cells were identified using cell counting kit-8, wound healing and Transwell experiments. Additionally, the mechanisms of drug resistance of LAC cells involving MTFR1 were investigated using western blotting. Results: MTFR1 was elevated in LAC tissues. MTFR1 overexpression was associated with sex, age, primary therapy outcome, smoking, T stage, unfavourable prognosis and diagnostic value and considered an independent risk factor for an unfavourable prognosis in patients with LAC. MTFR1 co-expressed genes involved in the cell cycle, oocyte meiosis, DNA replication and others. Moreover, interfering with MTFR1 expression inhibited the proliferation, migration and invasion of A549 and A549/DDP cells and promoted cell sensitivity to cisplatin, which was related to the inhibition of p-AKT, p-P38 and p-ERK protein expression. MTFR1 overexpression was associated with stromal, immune and estimate scores along with natural killer cells, pDC, iDC and others in LAC. Conclusions: MTFR1 overexpression was related to the unfavourable prognosis, diagnostic value and immunity in LAC. MTFR1 also participated in cell growth and migration and promoted the drug resistance of LAC cells to cisplatin via the p-AKT and p-ERK/P38 signalling pathways.


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remains unsatisfactory [4][5][6].Therefore, it is of great clinical and scientific significance to elucidate the unfavourable prognostic biomarkers involved in the progression of LAC.
Mitochondrial fission regulator (MTFR) 1 was a member of the MTFR family.Mitochondrial abnormality has been related to the progression of cancer and other diseases [7][8][9][10].Zeng et al. reported that Faciogenital Dysplasia 1 (FGD1) was upregulated in hepatocellular carcinoma (HCC), and increased FGD1 expression correlated with the progression and unfavourable prognosis of patients with HCC.Moreover, knocking out FGD1 in cancer cells significantly inhibited the malignant behaviour of HCC, causing mitochondrial dysfunction and highlighting its oncogenic properties [10].MTFR1 was a mitochondrial regulator that played a role in regulating mitochondrial fission and cell progression [11][12][13][14][15][16].Wang et al. reported that miR-324-5p reduced mitochondrial fission, myocardial cell apoptosis and myocardial infarction by inhibiting MTFR1 [11].Similarly, Huang et al. found that myocardial ischemia-reperfusion injury increased the expression of the long-chain non-coding RNA (lncRNA) GPR19.The inhibition of lncRNA GPR19 expression in C57BL/6 mice improved the cardiac function and reduced the apoptosis and myocardial fibrosis scar formation.Additionally, lncRNA GPR19 reduced oxidative stress and apoptosis in neonatal rat ventricular cardiomyocytes induced by oxygen-glucose deprivation/recovery by miR-324-5p/MTFR1 [14].Wang et al. observed that MTFR1 could be used to classify tumours based on the extracapsular spread status.In cancer patients with negative lymph nodes, the overall survival (OS) of patients with extracapsular spread-positive tumours was significantly worse [15].Consistently, Li et al. reported that MTFR1 was overexpressed in LAC and associated with unfavourable prognosis of patients with cancer.MTFR1 could enhance the proliferation, invasion, migration and glycolysis of LAC cells [16].However, the roles and pathways of MTFR1 in the resistance of LAC cells to cisplatin remain unreported.Therefore, this study investigates the roles and mechanisms of MTFR1 in LAC progression using comprehensive analysis, and basic research to evaluate whether MTFR1 has the potential as a therapeutic marker for patients with LAC.

MTFR1 expression was increased in LAC tissues
The Cancer Genome Atlas (TCGA) and XENA database analysis revealed that MTFR1 expression was increased in LAC tissues (Figure 1 and Supplementary Figure 1).In detail, the levels of MTFR1 increased significantly in unpaired and 57 paired LAC patents from TCGA and XENA transcripts per million (TPM) type data (Figure 1A-1C), and in unpaired and 57 paired LAC patents from TCGA fragments Per Kilobase Million (FPKM) type data (Figure 1D, 1E).Additionally, MTFR1 expression levels were also significantly increased in LAC tissues based on the meta-analysis of the TCGA and Gene Expression Omnibus (GEO) databases (Supplementary Figure 1).

MTFR1 overexpression correlated with diagnosis, sex, age, therapy outcome, smoking and T stage in patients with LAC
Receiver operating characteristic (ROC) analysis revealed that the area under the curves of MTRF1 in normal tissues and LAC tissues were 0.915, 0.744, and 0.941 based on the TPM and FPKM data from TCGA and XENA database, respectively (Figure 2).This indicated that MTRF1 has significant diagnostic values in LAC.MTFR1 expression correlated with sex, age, primary pathological outcome, smoking and the T stage of patients with LAC (Figure 3).Furthermore, in the high-and low-expression groups of MTFR1, the expression levels of MTFR1 were significantly correlated with T stage, N stage, sex and age (Table 1).

MTFR1 overexpression correlated with unfavourable prognosis in patients with LAC
Kaplan Meier (K-M) survival analysis revealed that increased MTFR1 expression was significantly associated with unfavourable OS, disease-specific survival (DSS), and progression-free interval (PFI) in patients with LAC based on the data of the FPKM type (Figure 4).Consistently, the Lung cancer explorer (LCE) database analysis also revealed that patients with high MTFR1 expression had an unfavourable prognosis (Figure 5).Additionally, univariate COX regression analysis revealed that T stage, N stage, M stage, pathological stage and MTFR1 overexpression were the risk factors for unfavourable OS (Table 2).Moreover, the T stage, N stage, M stage and pathological stage were the risk factors for unfavourable DSS (Table 3).Additionally, T stage, N stage, pathological stage and MTFR1 overexpression were the risk factors for unfavourable PFI (Table 4).The MTFR1-related nomograms in the OS and PFI were established based on the results of Cox analysis to assess the survival time of patients with cancer (Figure 6).

Inhibition of MTFR1 expression reduces LAC cell growth and migration
A total of 391 MTFR1 co-expressed genes were positively related genes and 106 MTFR1 co-expressed AGING genes were reversely related.Kyoto Encyclopedia of Genes and Genomes (KEGG) results showed that MTFR1 co-expressed genes were involved in the cell cycle, oocyte meiosis, DNA replication and other functions (Table 5).Figure 7A, 7B display the successfully constructed cell model.Additionally, the  suppression of MTFR1 expression inhibited A549 cell proliferation, migration and invasion (Figure 7C-7E).Thus, these findings suggest that MTFR1 could reduce LAC cell growth and migration.

Inhibition of MTFR1 expression promotes the sensitivity of LAC cells to cisplatin through p-AKT and p-ERK/p38 mechanisms
In our cell model, cell Counting Kit-8 (CCK-8), migration and invasion assays revealed that the suppression of MTFR1 expression could inhibit A549/DDP cell proliferation, migration and invasion and promote the sensitivity of A549/DDP cells to cisplatin (Figure 8A-8F).Furthermore, the inhibition of MTFR1 expression downregulated the phosphorylation levels of extracellular regulated protein kinases (ERK), AKT serine/threonine kinase 1 (AKT) and P38 in A549 and A549/DDP cells, whereas the effects on the ERK, AKT and P38 were not significant, as shown using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting (Figure 9).This suggests that MTFR1 expression promotes cell progression and the drug resistance of LAC cells to cisplatin through p-AKT and p-ERK/P38 mechanisms.Abbreviations: LAC: lung adenocarcinoma; OS: overall survival; DSS: disease-specific survival; PFI: progression-free interval.

MTFR1 expression was correlated with LAC immune infiltration
significant in the high-and low-MTFR1 expression groups (Supplementary Figure 3).MTFR1 overexpression significantly correlated with LAC immune cell markers (such as HLA-DPB1, HLA-DQB1, HLA-DPA1, STAT5A, ITGAM, HLA-DRA, PD-1 and others) (Figure 12 and Table 6).studies have described various roles for MTFR1 in the mitochondrial division [11,13,16,19,20].For instance, Wang et al. reported that, in oral squamous cell carcinoma extracapsular spread, MTFR1 could be regarded as an indicator of the prognosis of patients without lymph node metastasis [11].However, to date, there have been no reports on the roles of MTFR1 in drug sensitivity and immunity in LAC.This study revealed that MTFR1 was significantly overexpressed in LAC tissues, and elevated MTFR1 expression was correlated with the sex, age, and T stage in patients with LAC.ROC results revealed that MTRF1 has significant diagnostic values in LAC.K-M survival analysis   and meta-analysis revealed that MTFR1 overexpression was associated with OS, DSS, and progression-free interval (PFI) in patients with LAC.COX regression analysis revealed that MTFR1 overexpression was the risk factors for unfavourable OS and PFI.This indicated that MTFR1 overexpression was a prognostic risk factor for LAC patients.
Cell proliferation is associated with the mechanisms governing the cell cycle.Dysregulated cell cycling leads to the abnormal regulation of cell proliferation and can induce excessive increases in cell number, which leads to unfavourable prognosis in patients with tumours [12,21,22].For example, mini-chromosome maintenance protein 10 (MCM10) strongly compensated for DNA replication pressure and promoted genome replication in S-phase cancer cells, which was more pronounced in cancer stem cell-like cells (CSC) [21].FLAP endonuclease 1 (FEN1) was a structure-specific nuclease that plays a role in DNA replication and repair.
Interference with FEN1 expression could kill defective human cells and induce DNA damage responses in sensitive and resistant cell lines [22].These results indicate that cell cycle and DNA replication play an important role in cancer progression.Now, Wang et al. reported that MTFR1 could inhibit mitochondrial fission and induce apoptosis [11].Similarly, Li et al. reported that MTFR1 was overexpressed in LAC and could promote LAC cell growth and migration [16].Preliminary evidence suggested that MTFR1 was involved in cancer progression as a carcinogenic gene.
In our study, MTFR1 co-expressed genes were involved  and in vivo [26].In keeping with the importance of tumour-associated immune cells, our data revealed that MTFR1 expression also correlated with LAC stromal cells and immune cells, as well as immune cell markers, indicating that MTFR1 might play a vital role in LAC progression and immune escape.
MTFR1 was identified to have the important biological roles in LAC progression using bioinformatics and cell experiments to provide a new candidate molecule for the treatment of patients with LAC.However, the present study has certain limitations.The proliferation, migration, invasion and drug sensitivity of each group of cancer cells after activating p-AKT or p-ERK expression in the cell models that interfered with MTFR1 expression were not examined and should be investigated, and the MTFR1 expression in A549 and A549/DDP cells would be detected in the future.In conclusion, MTFR1 overexpression correlated to the diagnostic value, sex, age, primary therapy outcome, smoking, T stage, prognosis and immune infiltration of patients with LAC.Moreover, MTFR1 participates in AGING cell growth and migration and promotes the resistance of LAC cells to cisplatin via p-AKT and p-ERK/P38 signalling pathways.Thus, it can be considered a potential therapeutic target for the unfavourable prognosis of patients with LAC.

TCGA and XENA databases
In May 2022, the genomic data of 535 cases of LAC and 59 cases of normal lung tissues of FPKM and TPM types were acquired from TCGA database and, the genomic data of 347 cases of normal lung tissues and 515 cases of LAC tissues of TPM types from XENA database.The gene expression data of 57 normal lung and LAC tissue samples, wherein the data belonged to the same 57 patients, were obtained and MTFR1 expression levels were investigated.The clinical data of patients with LAC from TCGA were screened.Furthermore, MTFR1 expression data and clinical data were combined to investigate the potential of MTFR1 in the clinicopathological characterisation and prognosis of patients with LAC.

The expression levels and clinical values of MTFR1 in LAC
The diagnostic value of MTFR1 in normal lung tissues and cancer tissues derived from the TCGA and XENA databases was identified using ROC analysis [27].The expression of MTFR1 in each group was investigated according to the clinical pathological characteristics of patients.Additionally, the relationship between MTFR1 expression level and survival time, disease-specific survival and PFI of patients with LAC were determined using survival analysis after grouping by MTFR1 expression median value.Moreover, in the LCE database, a metaanalysis was conducted using data from the TCGA and GEO databases to investigate the expression and prognostic value of MTFR1 in LAC [28].The clinical data of patients from TCGA were collated, and the factors affecting the prognosis of patients with LAC were analysed using univariate COX regression analysis.Subsequently, the MTFR1-related nomograms were constructed.

MTFR1 co-expressed genes
Spearman correlation coefficient (r) has been used to predict the relationship between two genes [29].MTFR1 co-expressed genes in LAC tissues were filtrated based on P < 0.001 and the absolute values of coefficient >0.4,resulting in identifying the strongly related genes.

Signalling mechanisms of MTFR1 co-expressed genes
KEGG analysed the biological functions and mechanisms of the genes involved [30].The signalling pathways of MTFR1 co-expressed genes were authenticated using KEGG [31], with the cut-off criterion as P < 0.05, which denoted statistical significance.

Cell culture and construction of cell models
Lung cancer cells A549 and A549/DDP were cultured in 10% foetal bovine serum (Gibao, USA) and 1% penicillin and streptomycin RPMI-1640 medium (Gibao, USA) and incubated in 5% CO2 at 37°C.Additionally, appropriate concentrations of cisplatin were added to A549/DDP cells.The A549 and A549/DDP cells were plated in 6-well plates at a 5 × 10 6 concentration.When cell density reached 70%, the media was changed to the serum-free medium with the siRNAs [32].The siRNA interference sequences of MTFR1 were the GCUGGAUUAAGCGCCUAAUTT (siRNA-1), CACUGCAGGUGACUUAGAUTT (siRNA-2), and CUGCUUGAUGAUUAATT (siRNA-3).The expression of MTFR1 was verified using qRT-PCR and western blotting at 24 h.

Western blotting
Total proteins were extracted using RIPA lysate and quantified using BCA protein quantification.Proteins were separated using 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PALL, USA) membranes [33,34].After blocking with 5% non-fat dry milk, the membranes were incubated with 1:1000 MTFR1, p-AKT, AKT, p-P38, P38, p-ERK1/2 and ERK1/2 antibodies overnight at 4°C and followed by 1:1000 secondary antibodies for 1 h at room temperature.After TBST washing, the membranes were visualised.The experiment was repeated three times.AGING

Cell counting kit-8 (CCK-8)
When the A549 and A549/DDP cells were at the logarithmic phase, 2500 cells/well were seeded in 96well plates, placed in 5% CO2 and incubated at 37°C.Following this, 10 ul/well CCK-8 reagent was added into the 96-well plates at 0 h, 24 h, 48 h, 72 h and 96 h and incubated at 5% CO2, 37°C for 1 h to detect cell viability and drug sensitivity.The absorbance values of the cancer cells were detected at 450 nm using a microplate reader.The experiment was repeated thrice.

Cell migration and invasion
A549 and A549/DDP cells at approximately 90% confluence after transfection were scratched with 0.2 ml sterile pipette heads, and the medium in the 6-well plates was changed to serum-free medium after phosphate-buffered saline (PBS) washing.Subsequently, the cancer cells were photographed.The migration distances of the cells in the two groups were observed in real time and photographed under a microscope, and the degree of migration was subsequently calculated.The model cell concentration was adjusted as follows: 200 μl cell suspension was added into the upper of the Transwell membrane and 600 μl complete medium was added into the lower chamber.The no migrated cancer cells on the Transwell membrane were washed thrice using PBS after 5% CO2 and 37°C incubation for 24 h.The cells were subsequently stained using a cell chamber Staining Kit (Biosharp, China) and photographed under a microscope.The experiment was repeated thrice.

Immune microenvironment analysis
ESTIMATE and single sample gene set enrichment analysis (ssGSEA) analysis were the bioinformatics algorithm that calculates the composition of immune cells, immune, ESTIMATE and stromal scores [35,36].The ratio of immune cells in LAC tissues was explored using ssGSEA analysis [35].The immune, ESTIMATE and stromal scores were statistically analysed using the ESTIMATE algorithm [35,36].The relationship between MTFR1 expression and LAC immune infiltration was investigated using Spearman correlation analysis.

The relationship between MTFR1 expression and immune cell markers
The data of immune cell markers and MTFR1 expression were extracted from the data of the LAC tissues in TCGA using Perl.The data were then sorted out, and correlation analysis was used to investigate the relationship between MTFR1 expression level and CD8A, CD8B, PDCD1 and other immune cell markers.

Statistical analysis
The Wilcoxon signed-rank test and chi-square test were applied to investigate the relationship between MTFR1 expression and clinicopathological characteristics in LAC.K-M survival analysis was applied to authenticate the correlation between MTFR1 expression and poor prognosis in patients with LAC.The t-test (mean  standard deviation) was used to identify whether the cell proliferation and migration experiments were statistically significant.P < 0.05 was considered statistically significant.

Figure 1 .
Figure 1.MTFR1 expression levels in LAC tissues from the TCGA and XENA databases.(A-C) The data of TPM type; (D, E) The data of FPKM type.Abbreviations: LAC: lung adenocarcinoma; TCGA: The Cancer Genome Atlas; TPM: transcripts per million; FPKM: Fragments Per Kilobase Million; *** P < 0.001.

Figure 2 .
Figure 2. Diagnostic values of MTFR1 in LAC from the TCGA and XENA databases.(A, B) The data of TPM type; (C) The data of FPKM type.Abbreviations: LAC: lung adenocarcinoma; TCGA: The Cancer Genome Atlas; TPM: transcripts per million; FPKM: Fragments Per Kilobase Million.

Figure 5 .
Figure 5. MTFR1 over-expression was related to the overall survival time in patients with LAC using meta-analysis.Abbreviations: LAC: lung adenocarcinoma.

Figure 9 .
Figure 9. Inhibition of MTFR1 expression could promote the sensitivity to cisplatin of LAC cells via the p-AKT and p-ERK/P38 signalling pathways.(A) mRNA expression in A549 cells; (B) mRNA expression in A549/DDP cells; (C) Protein expression in A549 and A549/DDP cells; (D) Protein expression of statistics analysis.Abbreviation: LAC: lung adenocarcinoma.