Chimeric RNA TNNI2-ACTA1-V1 Regulates Cell Proliferation by Regulating the Expression of NCOA3

Abstract

Chimeric RNA is a crucial target for tumor diagnosis and drug therapy, also having its unique biological role in normal tissues. TNNI2-ACTA1-V1 (TA-V1), a chimeric RNA discovered by our laboratory in porcine muscle tissue, can inhibit the proliferation of Porcine Skeletal Muscle Satellite Cells (PSCs). The regulatory mechanism of TA-V1 in PSCs remains unclear, but we speculate that NCOA3, DDR2 and RDX may be the target genes of TA-V1. In this study, we explored the effects of NCOA3, DDR2 and RDX on cell viability and cell proliferation by CCK-8 assay, EdU staining and flow cytometry. Furthermore, the regulatory pathway of proliferation in PSCs mediated by TA-V1 through NCOA3 or CyclinD1 was elucidated by co-transfection and co-immunoprecipitation (Co-IP). The results revealed that overexpression of NCOA3 significantly increased cell viability and the expression level of CyclinD1, and also promotes cell proliferation by changing cells from the G1 phase to the S phase. In addition, inhibiting the expression of NCOA3 substantially reduced cell viability and inhibited cell proliferation. Overexpression of DDR2 and RDX had no significant effect on cell viability and proliferation. Co-transfection experiments showed that NCOA3 could rescue the proliferation inhibition of PSCs caused by TA-V1. Co-IP assay indicated that TA-V1 directly interacts with NCOA3. Our study explores the hypothesis that TA-V1 directly regulates NCOA3, indirectly regulating CyclinD1, thereby regulating PSCs proliferation. We provide new putative mechanisms of porcine skeletal muscle growth and lay the foundation for the study of chimeric RNA in normal tissues.

Keywords: NCOA3; cell proliferation; chimeric RNA TA-V1; growth; porcine skeletal muscle.

Figure 1

The results of the CCK-8 assay to detect cell viability in seven days.

Figure 2

The effects of NCOA3, DDR2 and RDX on cell proliferation of PSCs at 48 h post-transfection. EdU staining results of PSCs in pCMV-HA-NCOA3, pCMV-HA-DDR2 and pCMV-HA-RDX with magnification 100×.

Figure 3

The effects of overexpression of target gene on EdU staining, cell cycle and expression level of CyclinD1. (A) The percentage of EdU-positive cells in NCOA3, DDR2 and RDX groups. (B) Effect of target gene on cell cycle. Cell samples from each group were collected at 48 h post-transfection. (C) Detection of mRNA expression level of CyclinD1 by qPCR. A value of p < 0.05 was considered statistically significant. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4

The results of siRNA screening, the lower the expression level of NCOA3, the better the inhibitory effect. **p < 0.01.

Figure 5

The effect of interfering expression of NCOA3 on cell viability of PSCs. The assay lasts for seven days.

Figure 6

The effect of interfering expression of NCOA3 on the proliferation of PSCs. The effect of inhibiting the expression of NCOA3 on EdU staining. The results of the percentage of EdU positive cells after interfering expression of NCOA3.

Figure 7

The effects of inhibiting NCOA3 expression on cell cycle and expression level of CyclinD1. (A) The effects on cell cycle after interfering expression of NCOA3. (B) After 48 h of interference expression of NCOA3, cells were collected. The expression levels of CyclinD1 were detected. **p < 0.01.

Figure 8

At post-transfection of pCMV-TA-V1, cells were collected at 24 and 48 h, respectively. The expression levels of NCOA3 and CyclinD1 were detected, respectively, by qPCR. **p < 0.01.

Figure 9

The effects of co-transfection of TA-V1 and NCOA3 on cell viability.

Figure 10

The effects of co-transfection of TA-V1 and NCOA3 on cell proliferation. The effect of co-transfection of TA-V1 and NCOA3 on EdU staining is shown.

Figure 11

The effects of co-transfection of TA-V1 and NCOA3 on EdU staining, cell cycle and expression level of CyclinD1. (A) The percentage of EdU positive cells after co-transfection of TA-V1 and NCOA3. (B) The effects on cell cycle after co-transfection of TA-V1 and NCOA3. (C) The effects of co-transfection of TA-V1 and NCOA3 on the expression level of CyclinD1. The NS means no significant. **p < 0.01.

Figure 12

Identification of the protein-protein interaction between TA-V1 and NCOA3 using Co-immunoprecipitation (Co-IP). HEK293T cells were transfected with pCMV-HA-TA-V1, pCMV-HA-NCOA3 or empty vector pCMV-HA. Cells were treated as described in Materials and Methods. (A) Anti-NCOA3 antibody was used as the primary antibody for Co-IP. (B) Anti-HA antibody was used as the primary antibody for Co-IP.