Table1_Prognosis-related genes participate in immunotherapy of renal clear cell carcinoma possibly by targeting dendritic cells.XLSX

Tumor immunotherapy has become one of the most promising approaches to tumor treatment. This study aimed to screen genes involved in the response of clear cell renal cell carcinoma (ccRCC) to immunotherapy and analyze their function. Based on the Gene Expression Omnibus and The Cancer Genome Atlas datasets, we screened out nine differentially expressed genes (TYROBP, APOC1, CSTA, LY96, LAPTM5, CD300A, ALOX5, C1QA, and C1QB) associated with clinical traits and prognosis. A risk signature constructed by these nine genes could predict the survival probability for patients at 1 year, 3 years, and 5 years. The immune checkpoint blockade response rate in the high-risk group was significantly higher than in the low-risk group (49.25% vs. 24.72%, p ≤ 0.001). The nine prognosis-related genes were negatively correlated with activated dendritic cells in the low-risk group but not in the high-risk group. qRT-PCR, immunohistochemistry, and immunofluorescence showed that the nine prognosis-related genes were associated with dendritic cell activity and the PD-1 positive staining rate. In conclusion, the nine prognosis-related genes have a high prognostic value. The patients in the high-risk group were more likely to benefit from immunotherapy, and the mechanism might be related to the release of dendritic cell-mediated immunosuppression.</p

Table5_Prognosis-related genes participate in immunotherapy of renal clear cell carcinoma possibly by targeting dendritic cells.XLSX

Tumor immunotherapy has become one of the most promising approaches to tumor treatment. This study aimed to screen genes involved in the response of clear cell renal cell carcinoma (ccRCC) to immunotherapy and analyze their function. Based on the Gene Expression Omnibus and The Cancer Genome Atlas datasets, we screened out nine differentially expressed genes (TYROBP, APOC1, CSTA, LY96, LAPTM5, CD300A, ALOX5, C1QA, and C1QB) associated with clinical traits and prognosis. A risk signature constructed by these nine genes could predict the survival probability for patients at 1 year, 3 years, and 5 years. The immune checkpoint blockade response rate in the high-risk group was significantly higher than in the low-risk group (49.25% vs. 24.72%, p ≤ 0.001). The nine prognosis-related genes were negatively correlated with activated dendritic cells in the low-risk group but not in the high-risk group. qRT-PCR, immunohistochemistry, and immunofluorescence showed that the nine prognosis-related genes were associated with dendritic cell activity and the PD-1 positive staining rate. In conclusion, the nine prognosis-related genes have a high prognostic value. The patients in the high-risk group were more likely to benefit from immunotherapy, and the mechanism might be related to the release of dendritic cell-mediated immunosuppression.</p

The number of up- and down-regulated miRNAs which target at Ca2+-CaM, Rho/ROCK, Ras/Raf/MEK/ERK and PKC pathways.

<p>The number of up- and down-regulated miRNAs which target at Ca2+-CaM, Rho/ROCK, Ras/Raf/MEK/ERK and PKC pathways.</p

qRT-PCR measured the relative abundance of some miRNAs at 12 and 24 h after CA07 or PR8 infection.

<p>The relative abundance of each miRNA was determined in triplicate at different time points. *<i>P</i>< 0.05 versus non-infected cells, **<i>P</i>< 0.01 versus non-infected cells, ***<i>P</i>< 0.001 versus non-infected cells.</p

Effect of influenza A virus on transmonolayer electrical resistance (TER) and cytoskeleton in HUVECs.

<p>(A) HUVEC monolayers were infected with PR8 or CA07 at a MOI of 30, and the non-infected cells were used as control. TER values were monitored at 0 h, 2 h, 4 h, 6 h, 12 h, 24 h and 36 h after infection. *<i>P</i>< 0.05 versus non-infected cells, **<i>P</i>< 0.01 versus non-infected cells, ***<i>P</i>< 0.001 versus non-infected cells. (B) At 6, 12, 24 h after 30 MOI of PR8 or (C) CA07 infection, F-actin in HUVECs were examined using confocal microscopy with non-infected cells as control. F-actin cytoskeleton (red) and nuclei (blue) were examined using confocal microscopy. Scale bars represent 30um. PR8 and CA07 infection caused F-actin rearrangement in HUVECs. (D) Apoptosis of HUVECs were determined by TUNEL method via flow cytometry. The average fluorescence intensity was calculated by the Image software from three independent experiments. Three replicates were included. Data are expressed as means ± SEM, n = 3. The flow cytometry showed no significant apoptosis in HUVECs infected by PR8 or CA07.</p

List of the differential miRNAs and their target genes in HUVECs at 12 h and 24 h after CA07 infection.

<p>List of the differential miRNAs and their target genes in HUVECs at 12 h and 24 h after CA07 infection.</p

The infection rate of HUVECs was determined by immunofluorescence staining of nucleoprotein (NP) protein.

<p>The immunofluorescence staining of normal cell, The infection rate of PR8 at MOI of 20, 30 and 40 were 32%, 46%, 51%, respectively at 12 h after infection and 50%, 62%, 68%, respectively at 24 h after infection. NP (green), nuclei (blue) were examined using fluorescent microscope.</p

List of the differential miRNAs and their target genes in HUVECs at 12 h and 24 h after PR8 infection.

<p>List of the differential miRNAs and their target genes in HUVECs at 12 h and 24 h after PR8 infection.</p

Analysis of miRNA profiles in HUVECs infected with influenza A virus.

<p>(A) At 12 h and 24 h after PR8 infection or CA07 infection, miRNA microarray was used to analyze the differential miRNAs between the miRNA profiles of the flu-infected and the non-infected cells. The up-regulated genes were shown in red and the down-regulated were shown in green. Three numbers of biological replicates were conducted. A1 and A2 indicated the miRNAs in non-infected cells at 12 h and 24 h, respectively; B1 and B2 indicated the miRNAs in PR8-infected HUVECs at 12 h and 24 h after infection, respectively; C1 and C2 indicated the miRNAs in CA07-infected HUVECs at 12 h and 24 h after infection, respectively. The degree of similarity between the replicates were demonstrated by Pearson's Correlation Coefficient, with the r values ranged from 0.77 to 0.99. (B) The numbers of coinciding miRNA that differentially expressed at the same time point in PR8- or CA07- infected- HUVECs is shown at the junction of two circles.</p