Circular RNA Calm4 Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cells Pyroptosis via the Circ-Calm4/miR-124-3p/PDCD6 Axis

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mice and medium from PASMCs. (n=6). H. Verification of the efficacy of circ-Calm4 in knocking down endogenous circ-Calm4 in PASMCs relative to that of the scramble negative control construct circ-Calm4 (n=6-7). I. Transfecting si-circ-Calm4-2 did not alter the expression of lincalm4 in PASMCs exposed to hypoxia. (n=6). J. Verification of circ-Calm4 overexpression at the mRNA level in PASMCs. (n=6). K. Circ-Calm4 overexpression did not alter the expression of lincalm4 in PASMCs exposed to hypoxia as detected by quantitative PCR (n=5). Circ-Calm4 siRNA reversed the increased protein levels of cleaved Caspase-1 induced by hypoxia in PASMCs (n=5). L. Circ-Calm4 siRNA reversed the increased protein levels of cleaved Caspase-1 induced by hypoxia in PASMCs (n=5). M&N. Circ-Calm4 overexpression increased the protein levels of NLRP3, Caspase-1, IL-1β and mRNA levels of Nlrp3,  and Asc in PASMCs, which were detected by qPCR and western blot analysis, respectively. (n=4-6). Each datapoint in the figure represents a unique biological replicate. The data are presented as the means ± SD. Statistical analysis was performed with one-way ANOVA followed by Bonferroni correction and Student's t test for two means. The graph A, B and E were analyzed by the Mann Whitney U test. The graph H, I and M-IL-1β were analyzed by the Kruskal-Wallis test followed by Dunn post-test. *p <0.05, **p<0.01, ***p<0.001. Lincalm4 siRNA did not alter the protein levels of NLRP3, Caspase-1, IL-1β and mRNA levels of Nlrp3, and Asc in PASMCs exposed to hypoxia, which were detected by qPCR and western blot analysis, respectively. (n=4-6). Each datapoint in the figure represents a unique biological replicate. The data are presented as the means ± SD. Statistical analysis was performed with one-way ANOVA followed by Bonferroni correction and Student's t test for two means. The graph A and B were analyzed by the Mann Whitney U test, the graph C was analyzed by the Kruskal-Wallis test followed by Dunn post-test. *p <0.05, **p<0.01. Figure Ⅲ. Effects of circ-Calm4 on echocardiography. A. Mice inhaled AAV9 viral particles carrying shRNA targeting circ-calm4 (sh-circ-calm4) or a scrambled control RNA fragment (sh-Scr) via dropwise intranasal instillation for ten days before establishment of the hypoxia-induced PH model. Specific knockdown of circ-Calm4 by sh-circ-Calm4 in mouse lung tissues was verified (n=8). B & C. The RVSP and RVH index values were lower in hypoxic mice with circ-Calm4 silencing via circ-Calm4 shRNA than in their counterparts (n=5). D. Echocardiographic image showing the increases in PAVTI and PAAT in hypoxic mice infected with AAV-sh-calm4 compared with hypoxic mice infected with NC-AAV-sh-calm4 (n=5). E. Knockdown of circ-Calm4 by sh-circ-Calm4 reversed the hypoxiainduced upregulation of Ki67 in mouse lung tissues. Scale bars=100 µm. Lung sections stained with Ki67 (green), pulmonary smooth muscle stained with α-SMA (red), DAPI (blue) for nuclear staining and HIF-1α (green) was used as positive control. F. Knockdown of circ-Calm4 by sh-circ-Calm4 increased the expression of Caspase3 in mouse lung tissues upon hypoxia. Scale bars=100 µm. Lung sections stained with Caspase3(green), pulmonary smooth muscle stained with α-SMA (red), DAPI (blue) for nuclear staining and HIF-1α (green) was used as positive control. G. Knockdown of circ-Calm4 by sh-circ-Calm4 increased the expression of cleaved-Caspase3 in mouse lung tissues upon hypoxia. Scale bars=100 µm. Lung sections stained with cleaved-Caspase3 (green), pulmonary smooth muscle stained with α-SMA (red), DAPI for nuclear staining. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means ± SD. Statistical analysis was performed with oneway ANOVA followed by Bonferroni correction and Student's t test for two means. *p <0.05, **p<0.01, ***p<0.001.

Figure Ⅳ. MiR-124-3p is a downstream target gene of circ-Calm4.
A. Knockdown of lincalm4 by siRNA did not alter the expression of mir-124-3p in PASMCs exposed to hypoxia as detected by quantitative PCR (n=6). B. Verification of lincalm4 overexpression at the mRNA level in PASMCs. (n=8) C. Overexpression of lincalm4 did not alter the expression of mir-124-3p in PASMCs exposed to hypoxia. (n=5); D. Overexpression of circ-Calm4 resulted in down-regulation of mir-124-3p in hypoxic PASMCs as detected by quantitative PCR (n=6). E. Circ-Calm4-siRNA abrogated the hypoxia-induced expression downregulation of mir-124-3p at mRNA level, whereas cotransfection with circ-Calm4 siRNA and lincalm4 siRNA did not alter the expression of miR-124-3p upon hypoxia exposure, as indicated by quantitative PCR (n=5). F. Transfecting miR-124-3p mimic did not altered the expression of circ-Calm4 in hypoxic PASMCs as detected by quantitative PCR (n=6). G. Transfecting AMO-124-3p did not altered the expression of circ-Calm4 in hypoxic PASMCs as detected by quantitative PCR (n=6). H. AMO-124-3p did not alter the expression of lincalm4 in hypoxic PASMCs as detected by quantitative PCR (n=6). I. The mRNA levels of miR-124-3p in the nuclear and cytoplasmic fractions upon hypoxia. (n=4). J. Complementary sequences of circ-Calm4 with miR-124-3p is shown. K. We used the position p-value to describe the potential of base pairing, the computational motif analysis of circ-Calm4 were p-value =1.69e-9 and p-value =3.19e-17 of the two miR-124-3p targets sites. The position p-value is defined as the probability that a random sequence would have a match to the motif under test with a score greater or equal to the largest found in the sequence under test.The combined match p-value is defined as the probability that a random sequence would have position p-values such that the product is smaller or equal to the value calculated for the sequence under test. The combined match p-value of base pairing is 6.42e-29. The E-value of a motif is based on its log likelihood ratio, width, sites, the background letter frequencies and the size of the training set. The MEME's e-value of the targets sites is 1.9e-003. L. MiR-124-3p was pulled down from hypoxia-induced PH mice model and hypoxia-exposed PASMCs lysate by biotin-labeled circ-Calm4 probe, the level of miR-124-3p was detected by quantitative PCR (n=4). M. AGO2 protein-based RNA immunoprecipitation assay for the association between circ-Calm4 and miR-124-3p, the level of circ-Calm4 and miR-124-3p were detected by quantitative PCR (n=4). N. Absolute copies per cell of circ-Calm4, miR-124-3p and Pdcd6 were detected by quantitative PCR (n=4). O. Downregulation of miR-124-3p in mice. Scale bars=100 µm. Lung sections were stained for miR-124-3p probes (green), pulmonary smooth muscle was stained for α-SMA (red), DAPI for nuclear staining and Hif-1α was used as positive control. Each datapoint in the figure represents a unique biological replicate. The data are presented as the means ± SD. Statistical analysis was performed with one-way ANOVA followed by Bonferroni correction and Student's t test for two means. The graph A was analyzed by the Kruskal-Wallis test followed by Dunn post-test. The graph L, M and O were analyzed by the Mann Whitney U test. *p <0.05, **p<0.01, ***p<0.001.

Figure Ⅴ. Mir-124-3p inhibits pyroptosis in PASMCs.
A & B. In PASMCs exposed to hypoxia, transfection of the miR-124-3p mimic decreased the protein levels of NLRP3, Caspase-1, IL-1β and mRNA levels of Nlrp3, and Asc, which were detected by qPCR and western blot analysis, respectively. (n=3-6). Each datapoint in the figure represents a unique biological replicate. The data are presented as the means ± SD. Statistical analysis was performed with one-way ANOVA followed by Bonferroni correction. *p <0.05, **p<0.01. ***p<0.001.

Figure Ⅵ. Transfection of PDCD6.
A. The efficacy of si-PDCD6 in knocking down endogenous Pdcd6 in PASMCs relative to that of the negative control construct NC-si-PDCD6 (n=6). B. Knockdown of endogenous Pdcd6 abrogated the increasing proliferation of PASMCs induced by hypoxia. The survival rate was studied by CCK8 assay. (n=8). Each datapoint in the figure represents a unique biological replicate. The data are presented as the means ± SD. Statistical analysis was performed with one-way ANOVA followed by Bonferroni correction. *p <0.05, **p<0.01. ***p<0.001.

Figure Ⅶ. Correlation analysis of circ-Calm4 and lincalm4
Circ-calm4 expression was positively correlated with lincalm4 expression in hypoxic mice. Statistical analysis was performed with Pearson's correlation test. *p <0.05.

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