Bioinformatics analysis of TMED2 in LUAD using TIMER2.0, Kaplan-Meier plotter, and GSEA
The predicted role of TMED2 in LUAD was detected using TIMER2.0, Kaplan-Meier plotter, and GSEA, as shown in Fig. 1. TMED2 expression was quantified in a series of common cancers via TIMER2.0, and it was found that the level of TMED2 in LUAD tumor tissues was higher than that in normal tissues (P < 0.05). In addition, for the analyses of overall survival (OS) and post-progressive survival (PPS) using the Kaplan-Meier plotter system, given their high level of TMED2 expression (Fig. 1B-1C), LUAD patients were predicted to have a worse outcome (P < 0.05). Furthermore, the GSEA showed that TMED2 in LUAD was positively correlated with poor survival and was negatively correlated with apoptosis (Fig. 1D-1E).
TMED2 expression in tumor tissues and different LUAD cell lines
Bioinformatics analysis showed that TMED2 may be associated with LUAD; therefore, we detected the TMED2 expression levels in tumor tissues or LUAD cell lines (Fig. 2). The expression of TMED2 in tumor tissues was significantly higher than that in para-tumor tissues (P < 0.05, Fig. 2A-2B). Similar to the results of immunohistochemistry (IHC) (Fig. 2C), the approximate localization of TMED2 was determined to be in the cytoplasm and cell membrane. In addition, the expression level of TMED2 in HCC827 cells was significantly higher (P < 0.05, compared with the other cells (Fig. 2D-2E). Therefore, we chose HCC827 as the LUAD model cell line for the follow-up experiments in our study.
Knocking down TMED2 inhibited the development of LUAD in vitro
Since TMED2 might be correlated with the progression of LUAD, as suggested in our previous results, we attempted to decrease the expression of TMED2 via lentivirus-mediated knockdown (sh-TMED2), as shown in Fig. 3. We observed the proliferation of Con, sh-TMED2, and control-shTMED2 HCC827 cells (Fig. 3A). The OD value at 3 and 4 days, as detected via MTT in the sh-TMED2 group was decreased (P < 0.05). To investigate the apoptotic function of TMED2, we used PI-Hoechst staining (Fig. 3B), and found that apoptosis was increased in the sh-TMED2 group (Fig. 3C, P < 0.05). In addition, we detected the levels of tumor biomarkers in LUAD (Fig. 3D) and found that the levels of CEA, NSE, and EGFR in the sh-TMED2 group were reduced (P < 0.05, Fig. 3E).
Knocking down TMED2 inhibited the development of LUAD in vivo
Knocking down TMED2 could inhibit the development of LUAD in vitro; thus, we determined if this would be consistent in vivo (Fig. 4). First, we observed the tumor volumes of the mice treated with the different cell constructs. The tumor volumes at days 21 and 28 in the sh-TMED2 mice were significantly decreased (P < 0.05, Fig. 4A). Next, the expression of TMED2 and the proliferation index were detected via IHC (Fig. 4B), and it was found that TMED2 and Ki-67 levels were reduced after TMED2 knockdown. Further, to investigate the apoptotic function of TMED2, we used TUNEL staining (Fig. 4C), and found that the incidence of apoptosis in sh-TMED2 mice was increased (Fig. 4D, P < 0.05). Finally, we also detected the levels of tumor biomarker levels in LUAD (Fig. 4E) and found that the expression of CEA, NSE, and EGFR in sh-TMED2 mice was reduced (P < 0.05, Fig. 4F), consistent with the in vitro results.
GSEA of TMED2 with inflammation, TLR4 and NF-κB
To investigate the potential mechanism of TMED2 in regulating the progression of LUAD, we performed GSEA (Fig. 5). The results showed that TMED2 was negatively correlated with inflammation, TLR4, and NF-κB in LUAD (Fig. 5A-5C).
TMED2 knockdown increased inflammation that was regulated through the TLR4/NF-κB signaling pathway in vitro and in vivo
To further confirm the tumor-inhibition effect of TMED2 and whether it was mediated through the TLR4/NF-κB signaling pathway, we used an activator of TLR4 (sparstolonin B) to boost TLR4 expression, and observed the changes in the downstream inflammatory factors using western blotting (Fig. 6). As shown in Fig. 6A, sparstolonin B increased the levels of TLR4, NF-κB, IL-1β, and IL-18 after sh-TMED2 administration (P < 0.05), compared to the sh-TMED2 group (Fig. 6B) in vitro. Furthermore, sparstolonin B did not significantly alter TMED2 levels in vitro. Similar outcomes were observed in vivo, as shown in Fig. 6C-6D.
Bioinformatics analysis of TMED2 in the TME, immune score, TME-associated immune cells, and their target markers in LUAD assessed using a pan-cancer system and TIMER2.0
Since TMED2 was predicted to be an immunoregulatory gene in LUAD, we predicted TMED2 in the TME, immune score, TME-associated immune cells, and their target markers in LUAD. TMED2 was negatively correlated with the TME and was positively correlated with the immune score (Fig. 7A-7B). Further, TMED2 was negatively correlated with regulatory T cells (Tregs), M2 macrophages, memory B cells, monocytes, CD8 + T cells, activated mast cells, follicular helper T cells, eosinophils, and NK cells, and was positively correlated with activated memory T cells, naïve B cells, resting mast cells, and neutrophils (Fig. 7C). Furthermore, TMED2 was negatively correlated with CD56, CD19, CD14, and CD68, and was positively correlated with CD16 and CD32 (Fig. 7D).
Bioinformatics analysis of TMED2 using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) in pan-cancer
In the GO analysis, we found that TMED2 was positively correlated with DNA packaging, keratinization, olfactory receptor activity, protein DNA complex, and sensory perception of smell (Fig. 8A). In the KEGG analysis, TMED2 was negatively correlated with olfactory transduction, porphyrin, and chlorophyll metabolism, and was positively correlated with allograft rejection, asthma, and the intestinal immune network for IgA production (Fig. 8B).
Target gene analysis of TMED2 in LUAD patients
Results of the target gene analysis of TMED2 in LUAD is shown in Fig. 9. At higher expression levels of TMED2 in LUAD patients, mutations in TGM4, SPON1, PLVAP, SCAPER, RTN3, TWISTNB, DET1, FASTKD1, HTRA4, SNIP1, PLEKHM3, SAAL1, KCNK18, ZER1, CKAP4, AFF4, ISOC1, DNAJC3, STIL, TANGO6, CMPK2, EMILIN3, OR51A2, WFIKKN2, PRAMEF20, MICU3, PAOX, MCTP2, and USP38 were found. These results suggest that TMED2 is related to these gene mutations and is involved in LUAD prognosis.
The potential mechanisms of TMED2-induced inflammation in LUAD are shown in Fig. 10. TMED2 probably regulates inflammation via the TLR4/NF-κB signaling pathway, enhancing the proliferation and development of LUAD cells, and is involved in prognosis. Furthermore, TMED2 may regulate TME-associated immune cells in LUAD.