miR-933 accelerates the growth of liver cancer cells by enhancing pyruvate kinase isoform M2

increases the interaction between PKM2 and CARM1. Then, miR-933 enhances the complex formation of H3K36me3-Rad51-hMSH6-XRCC6-POLB-PARP1 dependent on PKM2-CARM1 and then increases the DNA damage repair ability, which enhances the expression of ERBB2, N-Ras, CDK2, CyclinD1, and YB1. Ultimately, miR-933 accelerates the growth of liver cancer cells by enhancing PKM2 expression and DNA damage repair.


RAPID COMMUNICATION
miR-933 accelerates the growth of liver cancer cells by enhancing pyruvate kinase isoform M2 MicroRNAs (miRNAs) have been found to play an important role in human tumorigenesis.A study indicates that the plasma level of miR-933 was elevated in patients with dementia. 1Notably, miR-933 (RS79402775) may contribute to the reduction of gastric cancer susceptibility. 2Moreover, the study found that the miR-933 expression level in superficial diffuse melanoma was lower than in nodular melanoma. 3Furthermore, miR-933 has become a reliable prognostic tool for tumor progression. 4Strikingly, single nucleotide polymorphisms of miR-933 were found to be associated with the risk of papillary thyroid cancer. 5In this study, we demonstrate that miR-933 accelerates the growth of liver cancer cells by enhancing the expression of pyruvate kinase isoform M2 (PKM2) and increasing DNA damage repair.These results provide a basis for research on liver cancer prevention.
To further investigate how miR-933 affects the expression of PKM2 and the interaction between PKM2 and CARM1 in human liver cancer, we explored whether several important genes were involved in the regulation of this function.First, the binding ability of H4K16Ac to the promoter region of CREB1 was increased in the rLV-miR-933 group in contrast with the rLV group (Fig. 1H).The binding ability of RNAPolII to the promoter-enhancer loop of CREB1 was increased in the rLV-miR-933 group in contrast with the rLV group (Fig. 1I).Therefore, the transcriptional ability of CREB1 was increased in the rLV-miR-933 group in contrast with the rLV group (Fig. 1J).The expression of CREB1 was increased in the rLV-miR-933 group in contrast with the rLV group (Fig. 1K).Next, the binding ability of CREB1 to the promoter region of PKM2 was increased in the rLV-miR-933 group in contrast with the rLV group (Fig. 1L).Moreover, the binding ability of RNAPolII to the promoter-enhancer loop of PKM2 was increased in the rLV-miR-933 group in contrast with the rLV group (Fig. 1M).The binding ability of RNAPolII to the PKM2 promoter probe was increased in the rLV-miR-933 group in contrast with the rLV group (Fig. S7A).The transcriptional activity of PKM2 was increased in the rLV-miR-933 group in contrast with the rLV group (Fig. S7B).The transcriptional ability of PKM2 was increased in the rLV-miR-933 group in contrast with the rLV group (Fig. 1M).The expression of PKM2 was increased in the rLV-miR-933 group in contrast with the rLV group (Fig. 1O).Ultimately, the interaction between PKM2 and CARM1 was increased in the rLV-miR-933 group in contrast with the rLV group (Fig. 1P).Collectively, these observations suggest that miR-933 could enhance the expression of PKM2 via CREB1 and the interaction between PKM2 and CARM1 in human liver cancer.
Given miR-933's role in enhancing the expression of PKM2 and the interaction between PKM2 and CARM1, we considered whether miR-933 resulted in carcinogenic functions dependent on PKM2 in liver cancer.First, PKM2 expression was increased in the rLV-miR-933 group and decreased in the rLV-miR-933þrLV-ShRNA-PKM2 group in contrast with the rLV group, respectively (Fig. 1Q a).miR-933 was increased in the rLV-miR-933 group and rLV-miR-933þrLV-ShRNA-PKM2 group in contrast with the rLV group (Fig. 1Q b).Although the proliferation ability was increased in rLV-miR-933 group in contrast with the rLV group (24 h: P Z 0.0024; 48 h: P Z 0.0088), it was not changed in the rLV-miR-933þrLV-ShRNA-PKM2 group in contrast with the rLV group, respectively (24 h: P Z 0.00996; 48 h: P Z 0.148) (Fig. 1R).Although the colony formation ability was increased in the rLV-miR-933 group in contrast with the rLV group (28.82 % AE 2.69 % vs. 69.06% AE 11.14 %, P Z 0.00017), it was not changed in the rLV-miR-933þrLV-ShRNA-PKM2 group in contrast with the rLV group (28.82 % AE 2.69 % vs. 27.17% AE 4.11 %, P Z 0.094) (Fig. 1S  a, b).Although the weight of transplanted tumors was increased in the rLV-miR-933 group in contrast with the rLV group (0.218 AE 0.029 g vs. 0.675 AE 0.091 g, P Z 0.0000057), it was not changed in the rLV-miR-933þrLV-ShRNA-PKM2 group in contrast with rLV group (0.218 AE 0.029g vs. 0.237 AE 0.054 g, P Z 0.258) (Fig. 1T a, b).Although the appearance time of xenograft tumors was decreased in the rLV-miR-933 group in contrast with the rLV group (9.5 AE 1.05 days vs. 7.17 AE 0.75 days, P Z 0.0043), it was not changed in the rLV-miR-933þrLV-ShRNA-PKM2 group in contrast with the rLV group (9.5 AE 1.05 days vs. 9.83 AE 1.72days, P Z 0.319) (Fig. S8A).As shown in Figure S8B, although the well-differentiated cells were decreased and the poorly differentiated cells were increased in the rLV-miR-933 group in contrast to the rLV group, respectively, it was not significantly changed in the rLV-miR-933þrLV-ShRNA-PKM2 group in contrast with the rLV group.Although the expression of PCNA was increased in the rLV-miR-933 group in contrast with the rLV group (35.39 % AE 4.22 % vs. 74.01% AE 11.76 %, P Z 0.000247), it was not changed in the rLV-miR-933þrLV-ShRNA-PKM2 group in contrast with the rLV group (35.39 % AE 4.22 % vs. 33.54% AE 7.11 %, P Z 0.34 (Fig. S8C).Although the expression of ERBB2, POLD1, N-Ras, CDK2, YB1, and CyclinD1 was increased in the rLV-miR-933 group and the expression of RB1, GADD45, and ZIC1 was decreased in the rLV-miR-933 group in contrast to rLV group, it was not altered in rLV-miR-933þrLV-shRNA-PKM2 group in contrast with rLV group (Fig. 1U, V).Although the interactions between H3K36me3 and Rad51, hMSH6, XRCC6, POLB, and PARP1 were increased in the rLV-miR-933 group in contrast with the rLV group, respectively, it was not changed in the rLV-miR-933þrLV-ShRNA-PKM2 group in contrast with rLV group (Fig. 1W).Although the DNA damage marker gH2AX(S139) was significantly decreased in the rLV-miR-933 group in contrast with the rLV group, it was not changed in the rLV-miR-933þrLV-ShRNA-PKM2 group in contrast with rLV group, respectively (Fig. 1X, Y).Although the expression of ERBB2, N-Ras, CDK2, CyclinD1, and YB1 was increased in the rLV-miR-933 group in contrast with the rLV group, it was not changed in the rLV-miR-933þRucaparib (a DNA damage repair inhibitor) group in contrast with the rLV group (Fig. 1Z).Collectively, these observations suggest that PKM2 regulates the carcinogenic functions of miR-933 in liver cancer.
Obviously, our findings are noteworthy that miR-933 affects epigenetic regulation, transcriptome, proteome, and several signaling pathways.In particular, our results suggest that miR-933 enhances the expression of PKM2 and the interaction between PKM2 and CARM1 in liver cancer.To date, we demonstrate that miR-933 accelerates the growth of liver cancer cells by enhancing PKM2 expression and DNA damage repair.That is, miR-933 increases the binding ability of H4K16Ac and RNAPolII to the promoter region of

Figure 1
Figure 1 miR-933 accelerates the growth of liver cancer cells by enhancing PKM2.(A) Quantitative reverse-transcription (RT)-PCR was used to detect the mature miR-933.(B) The xenograft tumor was dissected (a) and the tumor size (g) was put into comparison between groups (b).(C) Hep3B cells infected with rLV-Cas9-miR-933 and miR-933 were detected by real-time RT-PCR.CCK8 method was used to determine the cell proliferation ability.(D) The xenograft tumor was dissected (a) and the tumor size (g) was put into comparison between groups (b).(E) Chromatin immunoprecipitation sequencing (CHIP-Seq) with anti-H4K16Ac (a) and the hierarchical clustering analysis (b); CHIP-Seq with anti-CTCF (c) and the hierarchical clustering analysis (d ).(F) miR-933 affects the transcriptome as shown in the heat map analysis (cluster).(G) miR-933 alters proteomics as shown in the differential protein cluster heatmap.(H) CHIP analysis was performed with anti-H4K16Ac.The PCR amplification was carried out using primers designed according to the DNA of the CREB1 promoter region.(I) 3C-CHIP analysis was performed with anti-RNAPolII.(J) The transcriptional ability of CREB1 was detected by RT-PCR.(K) The translational ability of CREB1 was detected by western blotting.(L) CHIP analysis was performed with anti-CREB1.The PCR amplification was carried out using primers designed according to the DNA of the PMK2 promoter region.(M) 3C-CHIP analysis was performed with anti-RNAPolII.(N) PKM2 was detected by RT-PCR.(O) PKM2 was detected by western blotting.(P) Co-immunoprecipitation analysis with anti-CARM1 and western blot analysis with anti-PKM2.(Q) PKM2 was detected with western blotting (a) and the quantitative RT-PCR was performed to detect the mature miR-933 (b).(R) CCK8 method was used to determine the cell proliferation ability.(S) The colony formation ability.(a) Photos of plate colonies.(b) Colony forming rate (%).(T) The xenograft tumor was dissected (a) and the tumor size (g) was put into comparison between groups (b).(U) RT-PCR analysis for ERBB2, POLD1, N-Ras, CDK2, YB1, CyclinD1, RB1, GADD45, and ZIC1.(V) Western blot analysis for ERBB2, POLD1, N-Ras, CDK2, YB1, CyclinD1, RB1, GADD45, and ZIC1.(W) Immunoprecipitation analysis with anti-H3K36me3, anti-Rad51, anti-hMSH6, anti-XRCC6, anti-POLB, and anti-PARP1.(X) gH2AX(S139) detection.(Y) Western blot analysis with anti-gH2AX(S139).(Z) Western blot analysis with anti-ERBB2, anti-N-Ras, anti-CDK2, anti-CyclinD1, and anti-YB1.(Z1) The schematic diagram.miR-933 accelerates the growth of liver cancer cells and increases the binding ability of H4K16Ac and RNAPolII to the promoter region of CREB1.Thereby, the expression of CREB1 was increased.Next, miR-933 increases the binding ability of CREB1 and RNApolII to the promoter region of PKM2, and the expression of PKM2 was increased.Therefore, miR-933 increases the interaction between PKM2 and CARM1.Then, miR-933 enhances the complex formation of H3K36me3-Rad51-hMSH6-XRCC6-POLB-PARP1 dependent on PKM2-CARM1 and then increases the DNA damage repair ability, which enhances the expression of ERBB2, N-Ras, CDK2, CyclinD1, and YB1.Ultimately, miR-933 accelerates the growth of liver cancer cells by enhancing PKM2 expression and DNA damage repair.