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. Author manuscript; available in PMC: 2019 Apr 15.
Published in final edited form as: Cancer Res. 2018 Sep 5;78(20):5877–5890. doi: 10.1158/0008-5472.CAN-18-1011

Figure 5. HNF1A-AS1 enhanced the expression of cell cycle regulators and promoted p21 degradation by ubiquitination.

Figure 5.

(A). The expression levels of CDK2, CDK4 and cylcinE1 in HNF1A-AS1 over-expressed MKN-45 cells and BGC-823 cells were detected by Western Blotting assay.

(B-C). Western Blotting assay was used to detect the expression levels of CDC34 and P21 in HNF1A-AS1 over-expressed MKN-45 cells (B) and BGC-823 cells (C).

(D-G). MKN-45 cells (D-E) and BGC-823 cells (F-G) transfected with HNF1A-AS1 overexpression vector and control cells treated with Actinomycin D (ActD 5ug/ml) for the indicated periods of time. P21 mRNA levels were analyzed by RT-qPCR.

(H-K). MKN-45 cells (H-I) and BGC-823 cells (J-K) transfected with HNF1A-AS1-targeting shRNA and control cells treated with Actinomycin D (ActD 5ug/ml) for the indicated periods of time. P21 mRNA levels were analyzed by RT-qPCR.

(L-M). MKN-45 cells (L) and BGC-823 cells (M) transfected with HNF1A-AS1 overexpression vector and control cells were treated with cycloheximide (CHX 5ug/ml) or vehicle for the indicated periods. P21 protein levels were analyzed by western blotting.

(N-O). MKN-45 cells (N) and BGC-823 cells (O) transfected with HNF1A-AS1 overexpression vector and control cells were treated with MG132 (5 μM) or vehicle for 24 h. Cell lysates were analyzed by western blotting.

(P-Q). MKN-45 cells (P) and BGC-823 cells (Q) transfected with HNF1A-AS1-targeting shRNA and control cells were treated with MG132 (5 μM) or vehicle for 24 h. Cell lysates were analyzed by western blotting.

(R). HNF1A-AS1 overexpression group or control group were treated with MG-132 (10 um) for 24h. Cell lysates were immunoprecipitated with antibody against P21. The ubiquitination of p21 was notably increased in MKN-45 cells overexpressing HNF1A-AS1 compared with the control group.

(S). LV-HNF1A-AS1 enhanced the expression of CDK2, CDK4, cyclinE1, and decreased the expression of p21 in mice tumor xenografts, compared to that of the LV-NC group.

ActD and CHX is respectively short for Actinomycin D and Cycloheximide. Three independent experiments were performed, and data are shown as mean ± SD.