High Expression of the Newly Found Long Noncoding RNA Z38 Promotes Cell Proliferation and Oncogenic Activity in Breast Cancer

Abstract

The aberrant expression of long noncoding RNAs (lncRNAs) has great impacts on cancer origination and progression. In the current study, a newly found lncRNA Z38, which was identified through combining experiments of suppression subtractive hybridization (SSH) and reverse dot-blotting, was found to have high expression in breast cancer. More importantly, inhibiting Z38 expression by gene silencing greatly suppressed breast cancer cell proliferation and tumorigenesis, and treatment with Z38 siRNAs significantly induced cell apoptosis and inhibited tumor growth. In conclusion, the newly found lncRNA Z38, which plays important roles in breast cancer, may act as a candidate biomarker and therapeutic target in carcinomas.

Keywords: CLDND1; cell proliferation; lncRNA Z38; oncogene; siRNA.; tumorigenesis.

Figure 1

The structural analysis of RNA gene Z38 and its relative expression level in cancer. (A) The structures of the predicted CLDND1 mRNAs on NCBI. (B) A simulated diagram of Z38 structure. (C) Z38 expression profile of different breast cancer cell lines and breast epithelial cell line HBL-100, assayed by qRT-PCR (Z38 relative expression level of MCF-7 = 1). (D) A (breast paraneoplastic tissues) and C (breast cancer tissues) are images of H&E staining; B (breast paraneoplastic tissues) and D (breast cancer tissues) are images of in situ hybridization. The violet dots and arrows in D represent the expression of Z38. (E) △C_T (C_{T, Target gene}-C_{T, GAPDH}) showing the relative expression level of Z38, Z38b, and Z38d in different cancer cell lines. ND: Not Detected. (F and G) Z38 expression profile of different cancer tissues (Z38 relative expression level of liver cancer tissue = 1), and cancer cell lines (Z38 relative expression level of MCF-7 = 1) and breast epithelial cell line HBL-100, assayed by qRT-PCR.

Figure 2

Non-protein-coding features of Z38. (A) The expression of EGFP in MCF-7 cells after transfection with the following plasmids: pEGFP-C1, pEGFP-C1-Z38, pEGFP-C1-Z38(S), pEGFP-N3, pEGFP-N3-Z38, pEGFP-N3-Z38(S). (B) The transcription level of Z38 in the in vitro translation experiments, assayed by semi-quantitative PCR. PBSIIKS-Z38-P represents the circular plasmid, and PBSIIKS-Z38-F represents the linearized plasmid. +RT represents reverse transcription, and -RT represents non-reverse transcription. (C) The results of in vitro translation assays. Arrow 1 indicates the positive control provided in reagent kit, arrow 2 indicates the nonspecific bands detailed by manufacturer, and arrow 3 indicates the target protein. PstI represents a kind of endonuclease. (D) The expression of fusion proteins of FLAG and CLDND1, assayed by western blotting. (E) The expression of CLDND1 proteins in MDA-MB-231 cells, assayed by immunofluorescence. The normal rabbit IgG was used as the negative control. (F) The expression of CLDND1 proteins in cancer cells, assayed by western blotting.

Figure 3

The inhibitory effects on breast cancer cell proliferation and tumorigenesis after reducing Z38 expression in MCF-7 and MDA-MB-231 cells. (A) Z38 expression level in cells after stable transfection with the following gene silencing plasmids of Z38: shZ38-146, shZ38-227, and the negative control shCtrl. (B) Results of MTT assays. (C) Results of colony formation in agarose. (D and E) Histograms showing the tumor volume and tumor weight of in vivo xenograft experiments, respectively. (F) Histogram showing the tumor growth rate. ^* (p < 0.05) when compared with shZ38-146; ^# (P < 0.05) when compared with shZ38-227.

Figure 4

The inhibitory effects of the specific siRNAs of lncRNA Z38 on breast cancer cell MDA-MB-231. (A) Images showing the effects of siRNAs on cell apoptosis, assayed by TUNEL assays. (B) Images of the colony formation in agarose of MDA-MB-231 after transfection with Z38 siRNAs. (C) Histogram showing the relative colony formation. (D) Images showing the effects of siRNAs on cell apoptosis. Cells were transiently co-transfected using siRNAs and plasmid pEGFP-C1-Z38(S). (E) Semi-quantitative PCR measuring Z38 expression level of MDA-MB-231 cells after transfection with siRNAs. (F) Image showing the influence of Z38 siRNAs on tumor growth, assayed by intratumoral injection. (G) Histogram showing the difference of tumor weight, assayed by intratumoral injection. ^* (p < 0.05) when compared with siRNA-1; ^# (P < 0.05) when compared with siRNA-2.