Abstract:

Objective To discuss the effect of microRNA （miR）-30c-5p on the proliferation， migration， and invasion of the human prostate cancer cells （LNCap）， and to clarify its possible mechanism. Methods The LNCap cells were divided into LNCap group （without plasmid transfection）， miR-30c-5p mimic group （transfected with miR-30c-5p mimic）， mimic NC group （transfected with miR-30c-5p mimic NC）， sh-DNA damage inducible transcript 4 （DDIT4） group （transfected with sh-DDIT4）， sh-NC group （transfected with sh-DDIT4 NC）， miR-30c-5p mimic+pc-DNA3.1-NC group （co-transfected with miR-30c-5p mimic and pc-DNA3.1 empty vector）， and miR-30c-5p mimic+pc-DNA3.1-DDIT4 group （co-transfected with miR-30c-5p mimic and pc-DNA3.1-DDIT4 over-expression plasmid）. The RWPE-1 cells were cultured normally.Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of miR-30c-5p and DDIT4 mRNA in the cells in various groups； Western blotting method was used to detect the expression levels of DDIT4 protein in the cells in various groups； CCK-8 method was used to detect the proliferation rates of the LNCap cells in various groups； Transwell assay was used to detect the numbers of the invasion LNCap cells in various groups； Scratch assay was used to detect the scratch healing rates of LNCap cells in various groups； dual-luciferase reporter assay was used to detect the targeting relationship between miR-30c-5p and DDIT4.In the in vivo tumor formation experiment， 18 male BALB/c nude mice were divided randomly into blank group， agomiR-NC group （transfected with agomiR-30c-5p NC）， and agomiR-30c-5p group （transfected with agomiR-30c-5p）； there were six mice in each group.The mice in agomiR-NC group and agomiR-30c-5p group were subcutaneously injected with LNCap cells， while the mice in blank group were given an equal volume of physiological saline. The volumes of tumor of the mice in various groups were detected. HE staining was used to observe the morphology of prostate cancer tissue the mice of in various groups； RT-qPCR method and immunofluorescence staining were used to detect the expression levels of miR-30c-5p and DDIT4 mRNA and the fluorescence intensities of DDIT4 protein in prostate cancer tissue of the mice in various groups. Results The In vitro prostate cancer cell experiment results showed that compared with RWPE-1 cells， the expression level of miR-30c-5p in the prostate cancer LNCap cells was decreased （P<0.01）， and the expression levels of DDIT4 mRNA and protein were increased （P<0.05 or P<0.01）. After 48 of transfection， compared with LNCap group and mimic NC group， the expression level of miR-30c-5p in the LNCap cells in miR-30c-5p mimic group was increased （P<0.01）. Compared with LNCap group and sh-NC group， the expression level of DDIT4 mRNA in the LNCap cells in sh-DDIT4 group was decreased （P<0.01）. Compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group， the expression level of miR-30c-5p in The LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was decreased （P<0.01）； compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group， the expression level of DDIT4 mRNA in the LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased （P<0.01）； compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group， the expression level of DDIT4 protein in the LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased （P<0.05）. The CCK-8 method results showed that compared with LNCap group and mimic NC group， the proliferation rate of the LNCap cells in miR-30c-5p mimic group was decreased （P<0.01）； compared with LNCap group and sh-NC group， the proliferation rate of the LNCap cells in sh-DDIT4 group was decreased （P<0.01）； compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group， the proliferation rate of the LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased （P<0.01）. The Transwell assay results showed that compared with LNCap group and mimic NC group， the number of the invasion LNCap cells in miR-30c-5p mimic group was decreased （P<0.01）； compared with LNCap group and sh-NC group， the number of invasion LNCap cells in sh-DDIT4 group was decreased （P<0.01）； compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group， the number of the invasion LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased （P<0.01）.The scratch assay results showed that compared with LNCap group and mimic NC group， the scratch healing rate of the LNCap cells in miR-30c-5p mimic group was decreased （P<0.01）； compared with LNCap group and sh-NC group， the scratch healing rate of the LNCap cells in sh-DDIT4 group was decreased （P<0.01）； compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group， the scratch healing rate of the LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased （P<0.01）. The dual-luciferase reporter assay results showed that compared with the LNCap cells co-transfected with WT-DDIT4 and mimic NC， the luciferase activity of the LNCap cells co-transfected with WT-DDIT4 and miR-30c-5p mimic was decreased （P<0.01）. The in vivo nude mouse tumor formation experiment results showed that on the 3 rd， 6 th， 9 th， 12 th， and 15th days after cell injection， compared with blank group and agomiR-NC group， the tumor volumes of the nude mice in agomiR-30c-5p group were decreased （P<0.05）. The HE staining results showed that in prostate cancer tissue of the mice in blank group and agomiR-NC group， the cell nuclei were enlarged， and nucleoli were prominent and deformed. In the mice in agomiR-30c-5p group， some regions of prostate cancer tissues results showed neatly arranged cells with normally shaped nuclei. The RT-qPCR and immunofluorescence staining showed that compared with agomiR-NC group， the expression level of miR-30c-5p in prostate cancer tissue of the mice in agomiR-30c-5p group was increased （P<0.01）. Compared with blank group and agomiR-NC group， the expression level of DDIT4 mRNA in prostate cancer tissue of the mice in agomiR-30c-5p group was decreased （P<0.01）. DDIT4 protein was mainly expressed in the cytoplasm. Compared with blank group and agomiR-NC group， the fluorescence intensity of DDIT4 protein in prostate cancer tissue of the mice in agomiR-30c-5p group was decreased （P<0.01）. Conclusion The expression level of miR-30c-5p in the prostate cancer LNCap cells is decreased， and it inhibits the proliferation， migration， and invasion of the prostate cancer cells by targeting downregulation of DDIT4， thereby participating in the occurrence and development of prostate cancer.

Key words: Prostate tumor, Micro RNA-30c-5p, DNA damage-inducible transcript 4, Cell proliferation, Cell migration