Microrna-605-3p Inhibited the Growth and Chemoresistance of Osteosarcoma Cells via Negatively Modulating RAF1.

BACKGROUND: Osteosarcoma (OS) is the leading cancer-associated mortality in childhood and adolescence. Increasing evidence has demonstrated the key function of microRNAs (miRNAs) in OS development and chemoresistance. Among them, miRNA-605-3p acted as an important tumor suppressor and was frequently down-regulated in multiple cancers. However, the function of miR-650-3p in OS has not been reported. OBJECTIVE: The aim of this work is to explore the novel role of miR-605-3p in osteosarcoma and its possible involvement in OS chemotherapy resistance. METHOD: The expression levels of miR-605-3p in OS tissues and cells were assessed by reverse transcription quantitative PCR (RT-qPCR). The relevance of miR-605-3p with the prognosis of OS patients was determined by the Kaplan-Meier analysis. Additionally, the influence of miR-605-3p on OS cell growth was analyzed using the cell counting kit-8, colony formation assay, and flow cytometry. The mRNA and protein expression of RAF1 were detected by RT-qPCR and western blot. The binding of miR-605-3p with the 3'-UTR of RAF1 was confirmed by dual-luciferase reporter assay. RESULTS: Our results showed that miR-605-3p was markedly decreased in OS tissues and cells. A lower level of miR-605-3p was strongly correlated with lymph node metastasis and poor 5-year overall survival rate of OS patients. In vitro assay found that miR-605-3p suppressed OS cell proliferation and promoted cell apoptosis. Mechanistically, the proto-oncogene RAF1 was seen as a target of miR-605-3p and strongly suppressed by miR-605-3p in OS cells. Restoration of RAF1 markedly eliminated the inhibitory effect of miR-605-3p on OS progression, suggesting RAF1 as a key mediator of miR-605-3p. Consistent with the decreased level of RAF1, miR-605-3p suppressed the activation of both MEK and ERK in OS cells, which are the targets of RAF1. Moreover, lower levels of miR-605-3p were found in chemoresistant OS patients, and downregulated miR-605-3p increased the resistance of OS cells to therapeutic agents. CONCLUSION: Our data revealed that miR-605-3p serves as a tumor suppressor gene by regulating RAF1 and increasing the chemosensitivity of OS cells, which provided the novel working mechanism of miR-605-3p in OS. Engineering stable nanovesicles that could efficiently deliver miR-605-3p with therapeutic activity into tumors could be a promising therapeutic approach for the treatment of OS.

Long non-coding RNA HANR modulates the glucose metabolism of triple negative breast cancer via stabilizing hexokinase 2.

Increasing evidence has demonstrated the oncogenic roles of long non-coding RNA (lncRNA) hepatocellular carcinoma (HCC)-associated long non-coding RNA (HANR) in the development of HCC and lung cancer; however, the involvement of HANR in triple-negative breast cancer (TNBC) remains largely unknown. Our results demonstrated the significant overexpression of HANR in TNBC tissues and cells. Higher HANR levels significantly correlated with the poorer phenotypes in patients with TNBC. HANR down-regulation inhibited the proliferation and cell cycle progression and increased the apoptosis of TNBC cells. Mechanistically, immunoprecipitation-mass spectrometry revealed hexokinase II (HK2) as a direct binding target of HANR. HANR binds to and stabilizes HK2 through the proteasomal pathway. Consistent with the important role of HK2 in cancer cells, HANR depletion represses the glucose absorbance and lactate secretion, thus reprogramming the metabolism of TNBC cells. An in vivo xenograft model also demonstrated that HANR promoted tumor growth and aerobic glycolysis. This study reveals the role of HANR in modulating the glycolysis in TNBC cells by regulating HK2 stability, suggesting that HANR is a potential drug target for TNBC.

Circ_0059457 Promotes Proliferation, Metastasis, Sphere Formation and Glycolysis in Breast Cancer Cells by Sponging miR-140-3p to Regulate UBE2C.

Circular RNA (circRNA) has been confirmed to regulate breast cancer (BC) progression. However, the role of circ_0059457 in BC progression is still unclear.The expression of circ_0059457, taspase 1 (TASP1), microRNA (miR)-140-3p and ubiquitin-binding enzyme E2C (UBE2C) was detected by quantitative real-time PCR. Cell proliferation, migration, invasion and sphere formation ability were assessed by cell counting kit-8 assay, EdU assay, wound healing assay, transwell assay and sphere formation assay. Cell glycolysis was assessed by detecting glucose uptake, lactate levels and ATP/ADP ratio. Dual-luciferase reporter assay, RIP assay, RNA pull-down assay were used to validate RNA interaction. Xenograft tumor model to assess the effect of circ_0059457 on BC tumor growth in vivo. Circ_0059457 had elevated expression in BC tissues and cells. Circ_0059457 knockdown inhibited BC cell proliferation, metastasis, sphere formation ability, and glycolysis. In terms of mechanism, circ_0059457 sponged miR-140-3p, and miR-140-3p targeted UBE2C. MiR-140-3p inhibition reversed the effect of circ_0059457 knockdown on BC cell malignant behaviors. Besides, miR-140-3p overexpression inhibited BC cell proliferation, metastasis, sphere formation ability and glycolysis, and these effects were abrogated by UBE2C enhancement. Furthermore, circ_0059457 regulated UBE2C expression through sponging miR-140-3p. Additionally, circ_0059457 knockdown obviously inhibited BC tumor growth in vivo. Circ_0059457 promoted BC progression via miR-140-3p/UBE2C axis, which provided potential target for the treatment of BC.

LncRNA BCRT1 depletion suppresses cervical cancer cell growth via sponging miR-432-5p/CCR7 axis.

Breast cancer-related transcript 1 (BCRT1), a lncRNA that is overexpressed in several human cancers, facilitates the progression of breast cancer and osteosarcoma. Nevertheless, the function of BCRT1 in cervical cancer (CC) still remains unknown. In this study, BCRT1 was significantly overexpressed in CC tissues and correlated with the advanced stage of CC patients. BCRT1 depletion dampened CC cell proliferation, and drives cell apoptosis and cell cycle inhibition. Mechanistically, BCRT1 bound miR-432-5p and negatively modulated miR-432-5p's expression in CC cells. Reduced miR-452-3p expression was observed in CC tissues and exerted tumor suppressive function in CC cell growth. Further mechanism study revealed that CCR7 was clarified as a target of miR-432-5p and was inhibited following BCRT1 depletion. CCR7 transfection could recover CC cell growth that was suppressed with BCRT1 down-regulation. These results revealed the novel function of BCRT1/miR-432-5p/CCR7 pathway in CC, suggesting BCRT1 might be a potential biomarker and target for CC treatment. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03863-x.

Cancer cell-derived exosomal miR-20a-5p inhibits CD8+ T-cell function and confers anti-programmed cell death 1 therapy resistance in triple-negative breast cancer.

Circulating miRNAs (cirmiRNAs) can be packaged into the exosomes, participating in intercellular communication, which affects the malignant progression and therapy resistance of triple-negative breast cancer (TNBC). Currently, immune checkpoint inhibitors that regulate T-cell function, especially antibodies against programmed cell death 1 (PD-1) or its ligand PD-L1, are emerging as new promising therapy for TNBC patients. However, only very limited patients showed complete or partial response to anti-PD-1 treatment. Dysfunction of CD8+ T cells is one of the key reasons for the immune escape of TNBC. The regulation of exosome-derived cirmiRNAs on CD8+ T cells in TNBC deserves more investigation. Here, the cirmiR-20a-5p level was significantly upregulated in the plasma of TNBC patients and culture supernatant of TNBC cells. High abundance of cirmiR-20a-5p was correlated with a worse prognosis of TNBC. cirmiR-20a-5p was secreted in the form of exosomes by TNBC cells. Exosomal cirmiR-20a-5p was internalized into CD8+ T cells and resulted into the dysfunction of CD8+ T. A mechanism study uncovered that cirmiR-20a-5p targeted the nuclear protein ataxia-telangiectasia (NPAT) and decreased NPAT expression in CD8+ T cells. An in vivo xenograft mouse model showed that cirmiR-20a-5p conferred TNBC to anti-PD-1 treatment resistance. Collectively, these findings indicated that cirmiR-20a-5p released by TNBC cells via exosome promotes cancer cell growth and leads to the immunosuppression by inducing CD8+ T cell dysfunction. This study suggests that targeting cirmiR-20a-5p might be a novel strategy for overcoming the resistance of TNBC to anti-PD-1 immunotherapy.

(R)-9bMS Inhibited the Protein Synthesis and Autophagy of Triple Negative Breast Cancer Cells via Regulating miR-4660/mTOR Axis.

BACKGROUND: Unlike other subtypes of breast cancer, triple negative breast cancer (TNBC) exhibits aggressive and metastatic behaviors and a lack of effective targeted therapeutics. (R)-9bMS, a small-molecule inhibitor of the non-receptor tyrosine kinase 2 (TNK2), significantly inhibited TNBC cell growth; however, the functional mechanism of (R)-9bMS in TNBC remains largely unknown. OBJECTIVE: To explore the functional mechanism of (R)-9bMS in TNBC. METHODS: Cell proliferation, apoptosis and xenograft tumor growth assays were performed to evaluate the effects of (R)-9bMS on TNBC. The expression levels of miRNA and protein were detected by RTqPCR or western blot, respectively. Protein synthesis was determined by analyzing the polysome profile and 35S-met incorporation. RESULTS: (R)-9bMS attenuated TNBC cell proliferation, induced cell apoptosis, and inhibited xenograft tumor growth. Mechanism study indicated that (R)-9bMS upregulated the expression of miR-4660 in TNBC cells. The expression of miR-4660 is lower in TNBC samples than that of the non-cancerous tissues. miR-4660 overexpression inhibited TNBC cell proliferation by targeting the mammalian target of rapamycin (mTOR), which reduced mTOR abundance in TNBC cells. Consistent with the downregulation of mTOR, exposure of (R)-9bMS inhibited the phosphorylation of p70S6K and 4E-BP1, which consequently interrupted the total protein synthesis and autophagy of TNBC cells. CONCLUSION: These findings uncovered the novel working mechanism of (R)-9bMS in TNBC by attenuating mTOR signaling via up-regulating miR-4660. The potential clinical significance of (R)- 9bMS in TNBC treatment is interesting to explore.

PsnWRKY70 Negatively Regulates NaHCO3 Tolerance in Populus.

Poplar is an important afforestation and ornamental tree species in Northeast China. The distribution area of saline-alkali land is approximately 765 hm2 in Northeast China. The breeding of saline-alkali-resistant transgenic trees could be an effective method of afforestation in saline-alkali land. WRKY transcription factors play a crucial role in abiotic stress. In this study, we analyzed the genetic stability of the two-year-old PsnWRKY70 transgenic poplars. The results showed that PsnWRKY70 of transgenic poplars had been expressed stably and normally at the mRNA level. The gene interference expression (RE) lines had no significant effect on the growth of PsnWRKY70 under NaHCO3 stress, and the alkali damage index of RE lines was significantly lower than that of WT and overexpression (OE) lines at day 15 under NaHCO3 stress. POD activity was significantly higher in RE lines than in WT. The MDA content of the RE line was lower than that of the WT line. Transcriptome analysis showed that RE lines up-regulated genes enriched in cell wall organization or biogenesis pathway-related genes such as EXPA8, EXPA4, EXPA3, EXPA1, EXPB3, EXP10, PME53, PME34, PME36, XTH9, XTH6, XTH23, CESA1, CESA3, CES9; FLA11, FLA16 and FLA7 genes. These genes play an important role in NaHCO3 stress. Our study showed that the interference expression of the PsnWRKY70 gene can enhance the tolerance of NaHCO3 in poplar.

MicroRNA-137 Inhibits Esophageal Squamous Cell Carcinoma by Downregulating DAAM1.

BACKGROUND: A growing body of evidence demonstrates that miR-137 acts against cancers; however, the biological function of miR-137 in esophageal squamous cell carcinoma (ESCC) remains to be fully understood. OBJECTIVE: The aim of this study is to explore the role of miR-137 in ESCC. METHODS: miR-137 expression was detected by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and target protein expression was detected by western blot. Cell counting, colony formation and flow cytometry were employed to determine the effects of miR-137 on the growth of ESCC cells. Dual-luciferase reporter assay was performed to validate the binding of miR- 137 with a dishevelled-associated activator of morphogenesis 1 (DAAM1) 3'-UTR. RESULTS: miR-137 was shown to be down-regulated in ESCC. miR-137 expression was inversely correlated with the 5-year survival rate of ESCC patients. Up-regulated miR-137 attenuated ESCC proliferation and promoted ESCC cell apoptosis. Meanwhile, to further reveal how miR-137 regulated the malignant behaviors of ESCC, the downstream mRNA binding targets of miR-137 were explored. miR-137 was demonstrated to bind DAAM1 3'-UTR and repressed the expression of DAAM1. The expression of DAAM1 and miR-137 in ESCC was inversely correlated. Additionally, the reintroduction of DAAM1 had the capacity to reverse the negative role of miR- 137 in ESCC cell growth. CONCLUSION: These findings have uncovered the new function of miR-137 in ESCC via negatively regulating DAAM1, suggesting miR-137 as a potent therapeutic candidate for ESCC treatment.

Role of PsnWRKY70 in Regulatory Network Response to Infection with Alternaria alternata (Fr.) Keissl in Populus.

WRKY is an important complex family of transcription factors involved in plant immune responses. Among them, WRKY70 plays an important role in the process of the plant defense response to the invasion of pathogens. However, the defense mechanism of PsnWRKY70 is not clear in Populus nigra. In this study, we showed that PsnWRKY70-overexpression lines (OE) had fewer leaf blight symptoms than PsnWRKY70-repressing lines (RE). PsnWRKY70 activated MAP kinase cascade genes (PsnM2K4, PsnMPK3, PsnM3K18), calcium channel proteins-related genes (PsnCNG3, PsnCNGC1, PsnCNG4), and calcium-dependent protein kinases genes (PsnCDPKL, PsnCDPKW, PsnCDPKS, PsnCDPKQ). Furthermore, 129 genes of PsnWRKY70 putative genome-wide direct targets (DTGs) were identified by using transcriptome (RNA-seq) and DNA affinity purification sequencing (DAP-seq). PsnWRKY70 directly binds to the promoters of homologous genes and LRR domain proteins to promote the expression of WRKY6, WRKY18, WRKY22, and WRKY22-1, LRR domain proteins LRR8, LRR-RLK, ADR1-like 2, NB-ARC, etc. Our study suggests that PsnWRKY70 enhances the resistance of A. alternata in poplar by activating genes in both pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI).

Long non­coding RNA SChLAP1 regulates the proliferation of triple negative breast cancer cells via the miR­524­5p/HMGA2 axis.

Long non­coding RNA (lncRNA) second chromosome locus associated with prostate­1 (SChLAP1), also named LINC00913, has been reported to accelerate the carcinogenesis of prostate cancer. The aim of this study was to explore the role and mechanism of SChLAP1 in triple negative breast cancer (TNBC). The expression of SChLAP1 in TNBC tissues and cells was determined by reverse transcription quantitative PCR. The effects of SChLAP1 on the growth of TNBC cells was evaluated by detecting cell viability, colony formation and apoptosis. The present study determined that SChLAP1 was upregulated in TNBC tissues and was associated with the long­distant lymph node metastasis of patients with TNBC. Knockdown of SChLAP1 significantly inhibited cell viability and colony formation, and triggered apoptosis of TNBC cells. Bioinformatics analysis suggested that SChLAP1 acted as a sponge of microRNA (miR)­524­5p and negatively modulated the expression of miR­524­5p. An inverse correlation was also identified between the expression levels of SChLAP1 and miR­524­5p in TNBC tissues. Furthermore, the results demonstrated that SChLAP1 interacted with miR­524­5p, and subsequently regulated the expression level of High Mobility Group AT­Hook 2 (HMGA2) in TNBC cells. It was also found that the overexpression of HMGA2 rescued the suppressed viability of TNBC cells induced by SChLAP1 knockdown. In conclusion, the present findings demonstrated that SChLAP1 modulated the malignant tumor behaviors of TNBC cells by regulating HMGA2 and subsequently restraining miR­524­5p.

MicroRNA-598 inhibits the growth of triple negative breast cancer cells by targeting JAG1.

Triple-negative breast cancer (TNBC) has an aggressive phenotype and a poor outcome. The discovery that dysregulated microRNAs (miRNAs) play an important role in tumor progression has led to the suggestion that miRNAs (miRs) could be a potential target for the treatment of TNBC. In the present study, it was demonstrated that miR-598 expression was significantly decreased in TNBC tissues and was related to the degree of lymph node metastasis of patients with TNBC. Ectopic expression of miR-598 suppressed viability and colony formation, as well as increased the apoptosis of TNBC cells. To further understand the functional mechanism of action underlying miR-598 in TNBC, targets of miR-598 were predicted with the miRDB bioinformatics tool. Jagged 1 (JAG1) was identified as a direct target of miR-598, possessing a binding site for miR-598 in its 3'-untranslated region. Overexpression of miR-598 inhibited the expression of JAG1 in TNBC cells. In addition, JAG1 was highly expressed in TNBC tissues and its expression was negatively correlated with the expression of miR-598. Overexpression of JAG1 significantly attenuated the inhibitory effects of miR-598 on the proliferation and colony formation of TNBC cells. Collectively, these results provided novel insights into the functional mechanism of action for the miR-598/JAG1 pathway in the development of TNBC.

MiR-133 Targets YES1 and Inhibits the Growth of Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer shows worse outcome compared with other subtypes of breast cancer. The discovery of dysregulated microRNAs and their roles in the progression of triple-negative breast cancer provide novel strategies for the treatment of patients with triple-negative breast cancer. In this study, we identified the significant reduction of miR-133 in triple-negative breast cancer tissues and cell lines. Ectopic overexpression of miR-133 suppressed the proliferation, colony formation, and upregulated the apoptosis of triple-negative breast cancer cells. Mechanism study revealed that the YES Proto-Oncogene 1 was a target of miR-133. miR-133 bound the 3'-untranslated region of YES Proto-Oncogene 1 and decreased the level of YES Proto-Oncogene 1 in triple-negative breast cancer cells. Consistent with miR-133 downregulation, YES1 was significantly increased in triple-negative breast cancer, which was inversely correlated with the level of miR-133. Restoration of YES Proto-Oncogene 1 attenuated the inhibitory effects of miR-133 on the proliferation and colony formation of triple-negative breast cancer cells. Consistent with the decreased expression of YES Proto-Oncogene 1, overexpression of miR-133 suppressed the phosphorylation of YAP1 in triple-negative breast cancer cells. Our results provided novel evidence for the role of miR-133/YES1 axis in the development of triple-negative breast cancer, which indicated miR-133 might be a potential therapeutic strategy for triple-negative breast cancer.

Chidamide suppresses the glycolysis of triple negative breast cancer cells partially by targeting the miR­33a­5p­LDHA axis.

Triple negative breast cancer (TNBC) is one of the most aggressive types of breast cancer and has a poor prognosis. Therefore, the development of novel drugs and understanding the molecular mechanisms that may contribute to the initiation and development of TNBC are urgently required. Chidamide, a histone deacetylase inhibitor, has been reported as possessing anti­cancer properties in several cancers, however, the function of chidamide in TNBC remains to be elucidated. The present study revealed that chidamide inhibited the proliferation, colony formation and migration of TNBC cells. Experiments investigating the underlying mechanism revealed that chidamide upregulated the expression of microRNA (miR)­33a­5p in TNBC cells via RT­qPCR. Luciferase reporter assay demonstrated that miR­33a­5p was bound to the 3'­untranslated region of lactate dehydrogenase A (LDHA) and decreased the expression of LDHA in TNBC cells. In addition, chidamide suppressed the expression of LDHA and significantly decreased the glycolysis of TNBC cells. Collectively, the results of the present study demonstrated that chidamide reprogramed glucose metabolism, partially by targeting the miR­33a­5p/LDHA pathway, in TNBC. These findings indicate that chidamide may be a promising novel drug in the treatment of patients with TNBC.

MiRNA-20a-5p promotes the growth of triple-negative breast cancer cells through targeting RUNX3.

Increasing evidence showed that microRNAs (miRNAs) were abnormally expressed in cancers and made effects on the tumorigenesis. Aberrant expression of miR-20a-5p has been reported in human breast carcinoma. However, the functional mechanism of miR-20a-5p in human breast carcinoma, particularly in triple-negative breast cancer (TNBC), required further investigations. Here, firstly, we determined that miR-20a-5p was highly expressed in both TNBC tissues and cell lines. Then, we explored that the overexpression of miR-20a-5p promoted the migration and invasion of TNBC cells in vitro. The tendency was significantly reversed after the depletion of miR-20a-5p. Consistent result could be obtained with the in vivo nude mice tumorigenesis. Thirdly, the underlying molecular mechanism was investigated. The Runt-related transcription factor 3 (RUNX3) was identified as a target of miR-20a-5p in TNBC cells. High expression of miR-20a-5p significantly decreased both the mRNA and protein levels of RUNX3, as well as its direct downstream targets Bim and p21. These results verified the significance of miR-20a-5p and explored its functional mechanisms in TNBC, suggesting the potential clinical applications of miR-20a-5p in TNBC.