MicroRNA-335 suppresses the proliferation,migration, and invasion of breast cancer cells by targeting EphA4

MicroRNAs (miRNAs) are small noncoding RNAs that exert their functions by targeting specific mRNA sequences. Many studies have demonstrated that miRNAs are crucial for cancer progression, during which they can act as either oncogenes or tumor suppressors. Previous research has shown that miR-335 is downregulated in breast cancer, and it has been shown to be a breast cancer suppressor. In addition, emerging evidence indicates that erythropoietin-producing hepatocellular A4 (EphA4) is implicated in cancer cell proliferation, migration, and invasion. However, little is known about the relationship between miR-335 and EphA4 in breast cancer. In the present study, we used bioinformatic and biochemical analyses to demonstrate that EphA4 is a direct downstream target of miR-335 in human breast cancer MCF-7 and MDA-MB-23 cells and revealed that miR-335 negatively regulates the expression of EphA4 in these cells. Further investigation revealed that miR-335 overexpression inhibits MCF-7 and MDA-MB-231 cell proliferation and that this inhibition is attenuated by EphA4 coexpression. Similarly, miR-335 overexpression also inhibited growth and downregulated EphA4 expression in tumors in nude mice. Moreover, our results demonstrated that miR-335 overexpression suppresses migration and invasion in MCF-7 and MDA-MB-231 cells, an effect that was reversed by EphA4 overexpression. These findings confirmed that EphA4 is a direct target gene of miR-335 and that miR-335 suppresses breast cancer cell proliferation and motility in part by directly inhibiting EphA4 expression.     

Long Non-coding RNA FEZF1-AS1 Promotes Growth and Reduces Apoptosis Through Regulation of miR-363-3p/PAX6 Axis in Retinoblastoma

Retinoblastoma is the most common malignancy in children's eyes with high incidence. Long non-coding RNAs (lncRNAs) play important roles in the progression of retinoblastoma. LncRNA FEZF1 antisense RNA 1 (FEZF1-AS1) has been found to stimulate retinoblastoma. However, the mechanism of FEZF1-AS1 underlying progression of retinoblastoma is still unclear. In current study, FEZF1-AS1 was up-regulated in retinoblastoma tissues and cells. FEZF1-AS1 overexpression enhanced retinoblastoma cell viability, promoted cell cycle, and inhibited apoptosis. Conversely, FEZF1-AS1 knockdown reduced cell viability, cycle, and elevated apoptosis. The interaction between FEZF1-AS1 and microRNA-363-3p (miR-363-3p) was confirmed. FEZF1-AS1 down-regulated miR-363-3p and up-regulated PAX6. PAX6 was a target gene of miR-363-3p. EZF1-AS1 promoted retinoblastoma cell viability and suppressed apoptosis via PAX6. Further, we demonstrated that FEZF1-AS1 contribute to tumor formation in vivo. In conclusion, FEZF1-AS1 elevated growth and inhibited apoptosis by regulating miR-363-3p/PAX6 in retinoblastoma, which provide a new target for retinoblastoma treatment.

     

Long noncoding RNA OIP5-AS1 causes cisplatin resistance in osteosarcoma through inducing the LPAATβ/PI3K/AKT/mTOR signaling pathway by sponging the miR-340-5p

The abnormal expression of long noncoding RNAs (lncRNAs) plays an important role in the regulation of human cancer progression and drug resistance. The lncRNA OPI5-AS1 is a crucial regulator in some cancers; however, its role in cisplatin resistance of osteosarcoma remains unclear. We found that OIP5-AS1 was significantly upregulated in cisplatin-resistant (CR) osteosarcoma cells MG63-CR and SaOS2-CR compared with the corresponding parental cells. OIP5-AS1 silencing suppressed cell growth in vitro and in vivo, and promoted apoptosis of MG63-CR and SaOS2-CR cells, indicating that knockdown of OIP5-AS1 significantly decreased cisplatin resistance in MG63-CR and SaOS2-CR cells. This conclusion was supported by the decreased expression of the drug resistance-related factors multidrug resistance-associated protein 1 (MRP1) and P-glycoprotein (P-gp) upon OIP5-AS1 silencing. In addition, OIP5-AS1 downregulation suppressed the PI3K/AKT/mTOR signaling pathway. Importantly, we demonstrated that OIP5-AS1 functions as a competing endogenous RNA of miR-340-5p and regulates the expression of lysophosphatidic acid acyltransferase (LPAATβ), which is a target of miR-340-5p. Moreover, downregulation of miR-340-5p partly reversed the inhibitory effect of OIP5-AS1 knockdown on the PI3K/AKT/mTOR pathway and therefore counteracted cisplatin resistance in MG63-CR and SaOS2-CR cells. In conclusion, OIP5-AS1 causes cisplatin resistance in osteosarcoma through inducing the LPAATβ/PI3K/AKT/mTOR signaling pathway by sponging the miR-340-5p. Our results contribute to a better understanding of the function and mechanism of OIP5-AS1 in osteosarcoma cisplatin resistance.     

Evaluation of exosomal miR-9 and miR-155 targeting PTEN and DUSP14 in highly metastatic breast cancer and their effect on low metastatic cells

Breast cancer is one of the most prevalent cancers in women. Triple-negative breast cancer consists 15% to 20% of breast cancer cases and has a poor prognosis. Cancerous transformation has several causes one of which is dysregulation of microRNAs (miRNAs) expression. Exosomes can transfer miRNAs to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. We used gene expression omnibus (GEO) datasets and miRNA target prediction tools to find overexpressed miRNA in breast cancer cells and their target genes, respectively. Exosomes were extracted from MDA-MB-231 and MCF-7 cells and characterized. Overexpression of the miRNAs of MDA-MB-231 cells and their exosomes were analyzed using quantitative Real-time PCR. The target genes expression was also evaluated in the cell lines. Luciferase assay was performed to confirm the miRNAs: mRNAs interactions. Finally, MCF-7 cells were treated with MDA-MB-231 cells’ exosomes. The target genes expression was evaluated in the recipient cells. GSE60714 results indicated that miR-9 and miR-155 were among the overexpressed miRNAs in highly metastatic triple negative breast cancer cells and their exosomes. Bioinformatic studies showed that these two miRNAs target PTEN and DUSP14 tumor suppressor genes. Quantitative Real-time PCR confirmed the overexpression of the miRNAs and downregulation of their targets. Luciferase assay confirmed that the miRNAs target PTEN and DUSP14. Treatment of MCF-7 cells with MDA-MB-231 cells’ exosomes resulted in target genes downregulation in MCF-7 cells. We found that miR-9 and miR-155 were enriched in metastatic breast cancer exosomes. Therefore, exosomal miRNAs can transfer from cancer cells to other cells and can suppress their target genes in the recipient cells.     

An integrated approach of bioinformatic prediction and in vitro analysis identified that miR-34a targets <Emphasis Type="Italic">MET</Emphasis> and <Emphasis Type="Italic">AXL</Emphasis> in triple-negative breast cancer

Background

Breast cancer is the most prevalent cancer among women, and AXL and MET are the key genes in the PI3K/AKT/mTOR pathway as critical elements in proliferation and invasion of cancer cells. MicroRNAs (miRNAs) are small non-coding RNAs regulating the expression of genes.

Methods

Bioinformatic approaches were used to find a miRNA that simultaneously targets both AXL and MET 3′-UTRs. The expression of target miRNA was evaluated in triple-negative (MDA-MB-231) and HER2-overexpressing (SK-BR-3) breast cancer cell lines as well as normal breast cells, MCF-10A, using quantitative real-time PCR. Then, the miRNA was overexpressed in normal and cancer cell lines using a lentiviral vector system. Afterwards, effects of overexpressed miRNA on the expression of AXL and MET genes were evaluated using quantitative real-time PCR.

Results

By applying bioinformatic software and programs, miRNAs that target the 3′-UTR of both AXL and MET mRNAs were determined, and according to the scores, miR-34a was selected for further analyses. The expression level of miR-34a in MDA-MB-231 and SK-BR-3 was lower than that of MCF-10A. Furthermore, AXL and MET expression in SK-BR-3 and MDA-MB-231 was lower and higher, respectively, than that of MCF-10A. After miR-34a overexpression, MET and AXL were downregulated in MDA-MB-231. In addition, MET was downregulated in SK-BR-3, while AXL was upregulated in this cell line.

Conclusions

These findings may indicate that miR-34a is an oncogenic miRNA, downregulated in the distinct breast cancer subtypes. It also targets MET and AXL 3′-UTRs in triple-negative breast cancer. Therefore, it can be considered as a therapeutic target in this type of breast cancer.

     

Breast cancer stem cell-derived extracellular vesicles transfer ARRDC1-AS1 to promote breast carcinogenesis via a miR-4731-5p/AKT1 axis-dependent mechanism

ObjectivesDeregulation of long non-coding RNAs (lncRNAs) has been frequently reported in breast cancer (BC). This goes to show the importance of understanding its significant contribution towards breast carcinogenesis. In the present study, we clarified a carcinogenic mechanism based on the ARRDC1-AS1 delivered by breast cancer stem cells-derived extracellular vesicles (BCSCs-EVs) in BC.MethodsThe isolated and well characterized BCSCs-EVs were co-cultured with BC cells. The expression of ARRDC1-AS1, miR-4731-5p, and AKT1 was determined in BC cell lines. BC cells were assayed for their viability, invasion, migration and apoptosis in vitro by CCK-8, Transwell and flow cytometry, as well as tumor growth in vivo after loss- and gain-of function assays. Dual-luciferase reporter gene, RIP and RNA pull-down assays were performed to determine the interactions among ARRDC1-AS1, miR-4731-5p, and AKT1.ResultsElevation of ARRDC1-AS1 and AKT1 as well as miR-4731-5p downregulation were observed in BC cells. ARRDC1-AS1 was enriched in BCSCs-EVs. Furthermore, EVs containing ARRDC1-AS1 enhanced the BC cell viability, invasion and migration and glutamate concentration. Mechanistically, ARRDC1-AS1 elevated the expression of AKT1 by competitively binding to miR-4731-5p. ARRDC1-AS1-containing EVs were also found to enhance tumor growth in vivo.ConclusionCollectively, BCSCs-EVs-mediated delivery of ARRDC1-AS1 may promote the malignant phenotypes of BC cells via the miR-4731-5p/AKT1 axis.     

MicroRNA-221/222对乳腺癌MDA-MB-231/DOX细胞阿霉素耐药性的影响

目的:探讨微小RNA-221/222(miR-221/222)对乳腺癌MDA-MB-231/阿霉素(DOX)细胞DOX耐药性的影响。方法:采用脂质体法转染miR-221/222抑制物(miR-221/222 inhibitor)至MDA-MB-231/DOX细胞内(Inhibitor组),同时设立空白对照组和转染无关序列的阴性对照组,采用实时荧光定量PCR (qRT-PCR)检测MDA-MB-231细胞株及MDA-MB-231/DOX细胞株的miR-221/222表达水平及转染效率;CCK-8法检测转染48 h后MDA-MB-231/DOX细胞对DOX药物敏感性的变化;流式细胞术(FCM)检测转染MDA-MB-231/DOX细胞的细胞凋亡率;蛋白免疫印迹实验(WB)检测转染后MDA-MB-231/DOX细胞内促凋亡蛋白p53上调凋亡调控因子(PUMA),Bcl2蛋白修饰因子(BMF)以及细胞周期蛋白激酶抑制因子p27(p27Kip1)的表达情况。结果:MDA-MB-231/DOX细胞中的miR-221/222表达水平高于亲本MDA-MB-231细胞(P0.05);MDA-MB-231/DOX细胞转染miR-221/222 inhibitor 96 h后,miR-221/222的表达水平低于空白对照组和阴性对照组(P0.05);与空白对照组相比,MDA-MB-231/DOX细胞转染miR-221/222 inhibitor 48h后,DOX继续处理48 h后,细胞的凋亡率明显升高,且细胞内的促凋亡蛋白PUMA,BMF以及p27Kip1的表达均增加(P0.05);DOX对inhibitor组耐药细胞的半数抑制浓度(IC50)显著低于空白对照组细胞及阴性对照组(P0.05)。结论:miR-221/222能够增加MDA-MB-231/DOX细胞对DOX的耐药性,这可能与下调促凋亡蛋白的表达有关;降低miR-221/222水平可诱导MDA-MB-231/DOX凋亡,并且上调促凋亡蛋白的表达,从而部分逆转MDA-MB-231/DOX对DOX的耐药性。     

MicroRNA-3646 Contributes to Docetaxel Resistance in Human Breast Cancer Cells by GSK-3β/β-Catenin Signaling Pathway

Acquisition of resistance to docetaxel (Doc) is one of the most important problems in treatment of breast cancer patients, but the underlying mechanisms are still not fully understood. In present study, Doc-resistant MDA-MB-231 and MCF-7 breast cancer cell lines (MDA-MB-231/Doc and MCF-7/Doc) were successfully established in vitro by gradually increasing Doc concentration on the basis of parental MDA-MB-231 and MCF-7 cell lines (MDA-MB-231/S and MCF-7/S). The potential miRNAs relevant to the Doc resistance were screened by miRNA microarray. We selected 5 upregulated miRNAs (has-miR-3646, has-miR-3658, has-miR-4438, has-miR-1246, and has-miR-574-3p) from the results of microarray for RT-qPCR validation. The results showed that expression level of miR-3646 in MDA-MB-231/Doc cells was significantly higher than in MDA-MB-231/S cells. Compared to MCF-7/S cells, miR-3646 expression was up-regulated in MCF-7/Doc cells. Further studies revealed that transfection of miR-3646 mimics into MDA-MB-231/S or MCF-7/S cells remarkably increased their drug resistance, in contrast, transfection of miR-3646 inhibitors into MDA-MB-231/Doc or MCF-7/Doc cells resulted in significant reduction of the drug resistance. By the pathway enrichment analyses for miR-3646, we found that GSK-3β/β-catenin signaling pathway was a significant pathway, in which GSK-3β was an essential member. RT-qPCR and Western blot results demonstrated that miR-3646 could regulate GSK-3β mRNA and protein expressions. Furthermore, a marked increase of both nuclear and cytoplasmic β-catenin expressions (with phosphorylated-β-catenin decrease) was observed in MDA-MB-231/Doc cells compared with MDA-MB-231/S cells, and their expression were positively related to miR-3646 and negatively correlated with GSK-3β. Taken together, our results suggest that miR-3646-mediated Doc resistance of breast cancer cells maybe, at least in part, through suppressing expression of GSK-3β and resultantly activating GSK-3β/β-catenin signaling pathway.     

Long non-coding RNA OIP5-AS1 promotes pancreatic cancer cell growth through sponging miR-342-3p via AKT/ERK signaling pathway

Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1), a long non-coding RNA (lncRNA), has been reported to link with the progression of some cancers. However, its biological functions and underlying molecular mechanisms in pancreatic cancer are largely unknown. The aim of this study was to investigate the role of lncRNA OIP5-AS1 in pancreatic cancer. Quantitative real-time PCR analysis revealed that OIP5-AS1 is highly expressed in pancreatic cancer tissues versus adjacent non-tumor tissues. In vitro functional assays showed that downregulation of OIP5-AS1 or overexpression of miR-342-3p inhibited the proliferation, decreased Ki67 expression, and induced cell cycle arrest in pancreatic cancer cells. The expression of cyclinD1, CDK4, and CDK6 was decreased by knockdown of OIP5-AS1. Moreover, we found that OIP5-AS1 acted as a miR-342-3p sponge to suppress its expression and function. Dual-luciferase assay confirmed the interaction of OIP5-AS1 and miR-342-3p and verified anterior gradient 2 (AGR2) as a direct target of miR-342-3p. Results showed that depletion of miR-342-3p abolished the inhibitory effects of OIP5-AS1 knockdown on pancreatic cancer cell growth. The expression of Ki67, AGR2, cyclinD1, CDK4, CDK6, p-AKT, and p-ERK1/2 was reversed by silencing of miR-342-3p in pancreatic cancer cells with OIP5-AS1 knockdown. Further, knockdown of OIP5-AS1 suppressed tumor growth in a xenograft mouse model of pancreatic cancer. OIP5-AS1 induced pancreatic cancer progression via activation of AKT and ERK signaling pathways. Therefore, we demonstrate that OIP5-AS1 functions as oncogene in pancreatic cancer and its downregulation inhibits pancreatic cancer growth by sponging miR-342-3p via targeting AGR2 through inhibiting AKT/ERK signaling pathway.

     

mRNA and microRNA selection for breast cancer molecular subtype stratification using meta-heuristic based algorithms

Cancer subtype stratification, which may help to make a better decision in treating cancerous patients, is one of the most crucial and challenging problems in cancer studies. To this end, various computational methods such as Feature selection, which enhances the accuracy of the classification and is an NP-Hard problem, have been proposed. However, the performance of the applied methods is still low and can be increased by the state-of-the-art and efficient methods. We used 11 efficient and popular meta-heuristic algorithms including WCC, LCA, GA, PSO, ACO, ICA, LA, HTS, FOA, DSOS and CUK along with SVM classifier to stratify human breast cancer molecular subtypes using mRNA and micro-RNA expression data. The applied algorithms select 186 mRNAs and 116 miRNAs out of 9692 mRNAs and 489 miRNAs, respectively. Although some of the selected mRNAs and miRNAs are common in different algorithms results, six miRNAs including miR-190b, miR-18a, miR-301a, miR-34c-5p, miR-18b, and miR-129-5p were selected by equal or more than three different algorithms. Further, six mRNAs, including HAUS6, LAMA2, TSPAN33, PLEKHM3, GFRA3, and DCBLD2, were chosen through two different algorithms. We have reported these miRNAs and mRNAs as important diagnostic biomarkers to the stratification of breast cancer subtypes. By investigating the literature, it is also observed that most of our reported mRNAs and miRNAs have been proposed and introduced as biomarkers in cancer subtypes stratification.     

基于生物信息学分析研究miR-182-5p通过靶向调控EP300促进乳腺癌细胞的侵袭和转移

近期研究表明,miR-182-5p对多种癌症的侵袭和转移具有重要作用,但其在乳腺癌侵袭转移中的研究相对较少。本研究通过网上在线microRNA分析工具下载乳腺癌组织及正常乳腺组织表达比较的数据集,分析发现在GSE4589、GSE38167、GSE61438等3个数据库中,在乳腺癌组织中存在26个相同的microRNA,其中8个上调,而我们实验验证发现hsa-miR-182在8例病理组织中的表达上调差异最显著(P=0.001),选定目的基因hsa-miR-182;qRT-PCR检测细胞中miR-182-5p的表达,结果显示,与MCF-10A相比,miR-182-5p在MDA-MB-231、T47D、MDA-MB-453、MCF-7中表达上调(P<0.05);转染miR-182-5p干扰质粒,qRT-PCR检测细胞中miR-182-5p的表达情况。结果显示,miR-182-5p表达显著降低(P=0.003),提示转染成功;Transwell侵袭结果显示,MDAMB-231细胞敲低miR-182-5p,与对照组相比,体外侵袭能力明显降低(P=0.002);Western印迹检测转染miR-182-5p干扰质粒时,MDA-MB-231中上皮-间质转化(epithelial-mesenchymal transition,EMT)相关标志物的表达情况,结果显示,与对照组相比,敲低miR-182-5p使细胞中上皮-钙黏着蛋白(E-cadherin)表达上调,神经-钙黏着蛋白(N-cadherin)、波形蛋白(vimentin)表达下调。为研究探讨miR-182-5p的靶蛋白,采用在线预测软件预测可能与miR-182-5p结合的靶蛋白,cytoscape构建蛋白质互作网络图并筛选出hub基因;双荧光素酶结果证实,miR-182-5p可与EP300靶向结合(P=0.001);采用qRT-PCR、Western印迹检测转染miR-182-5p干扰质粒后EP300在mRNA及蛋白质水平的表达,结果显示,与对照组相比,在敲低miR-182-5p组中EP300在mRNA及蛋白质的表达上调(P=0.001)。综上所述,miR-182-5p可靶向调节EP300,促进乳腺癌细胞的侵袭与转移。     

LncRNA OIP5-AS1 regulates radioresistance by targeting DYRK1A through miR-369-3p in colorectal cancer cells

Object

This study aimed to investigate the role of lncRNA OIP5-AS1 in regulating radioresistance of colorectal cancer (CRC) cells.

Design and synthesis of chromone-nitrogen mustard derivatives and evaluation of anti-breast cancer activity

Chromone has emerged as one of the most important synthetic scaffolds for antitumor activity, which promotes the development of candidate drugs with better activity. In this study, a series of nitrogen mustard derivatives of chromone were designed and synthesised, in order to discover promising anti-breast tumour candidates. Almost all target derivatives showed antiproliferative activity against MCF-7 and MDA-MB-231 cell lines. In particular, methyl (S)-3-(4-(bis(2-chloroethyl)amino)phenyl)-2-(5-(((6-methoxy-4-oxo-4H-chromen-3-yl)methyl)amino)-5-oxopentanamido)propanoate showed the most potent antiproliferative activity with IC₅₀ values of 1.83 and 1.90 μM, respectively, and it also exhibited certain selectivity between tumour cells and normal cells. Further mechanism exploration against MDA-MB-231 cells showed that it possibly induced G2/M phase arrest and apoptosis by generating intracellular ROS and activating DNA damage. In addition, it also inhibited MDA-MB-231 cells metastasis, invasion and adhesion. Overall, methyl (S)-3-(4-(bis(2-chloroethyl)amino)phenyl)-2-(5-(((6-methoxy-4-oxo-4H-chromen-3-yl)methyl)amino)-5-oxopentanamido)propanoate showed potent antitumor activities and relatively low side effects, and deserved further investigation.     

LncRNA OIP5-AS1/cyrano sponges RNA-binding protein HuR

The function of the vast majority of mammalian long noncoding (lnc) RNAs remains unknown. Here, analysis of a highly abundant mammalian lncRNA, OIP5-AS1, known as cyrano in zebrafish, revealed that OIP5-AS1 reduces cell proliferation. In human cervical carcinoma HeLa cells, the RNA-binding protein HuR, which enhances cell proliferation, associated with OIP5-AS1 and stabilized it. Tagging OIP5-AS1 with MS2 hairpins to identify associated microRNAs revealed that miR-424 interacted with OIP5-AS1 and competed with HuR for binding to OIP5-AS1. We further identified a ‘sponge’ function for OIP5-AS1, as high levels of OIP5-AS1 increased HuR-OIP5-AS1 complexes and prevented HuR interaction with target mRNAs, including those that encoded proliferative proteins, while conversely, lowering OIP5-AS1 increased the abundance of HuR complexes with target mRNAs. We propose that OIP5-AS1 serves as a sponge or a competing endogenous (ce)RNA for HuR, restricting its availability to HuR target mRNAs and thereby repressing HuR-elicited proliferative phenotypes.     

HOXC13-AS promotes breast cancer cell growth through regulating miR-497-5p/PTEN axis

Dysregulated long noncoding RNAs (lncRNAs) remains to be explored in tumorigenesis. LncRNA HOXC13 antisense RNA (HOXC13-AS) has been found as an oncogene in many cancers; however, the role of HOXC13-AS in breast cancer still elusive. In this study, the HOXC13-AS levels and its role in cell proliferation was first measured by real-time quantitative polymerase chain reaction, Cell Counting Kit-8 assay, and colony formation assay. It showed that HOXC13-AS was increased in breast cancer tissues compared with the adjacent normal tissues and upregulated HOXC13-AS promoted the growth of breast cancer cells. Then, we found that the miR-497-5p levels were downregulated in cancer tissues compared with the adjacent tissues and miR-497-5p suppressed breast cancer cell proliferation. Further study showed that HOXC13-AS could function as a “sponge” for miR-497-5p then suppress miR-497-5p expression. Moreover, we next identified that Phosphatase and Tensin homolog (PTEN) is the target of miR-497-5p. Overexpression of miR-497-5p by chemical mimics decreased the expression of PTEN, while downregulation of miR-497-5p by HOXC13-AS rescued the expression of PTEN. Finally, we showed that HOXC13-AS promoted the proliferation of breast cancer cells and tumor growth through miR-497-5p/PTEN axis in vitro and in vivo. Hence, we conclude that HOXC13-AS, which is significantly upregulated in breast cancers, promoted cell proliferation through the suppressed miR-497-5p and further upregulated PTEN.     

Long noncoding RNA complementarity and target transcripts abundance

Eukaryotic mRNA metabolism regulates its stability, localization, and translation using complementarity with counter-part RNAs. To modulate their stability, small and long noncoding RNAs can establish complementarity with their target mRNAs. Although complementarity of small interfering RNAs and microRNAs with target mRNAs has been studied thoroughly, partial complementarity of long noncoding RNAs (lncRNAs) with their target mRNAs has not been investigated clearly. To address that research gap, our lab investigated whether the sequence complementarity of two lncRNAs, lincRNA-p21 and OIP5-AS1, influenced the quantity of target RNA expression. We predicted a positive correlation between lncRNA complementarity and target mRNA quantity. We confirmed this prediction using RNA affinity pull down, microarray, and RNA-sequencing analysis. In addition, we utilized the information from this analysis to compare the quantity of target mRNAs when two lncRNAs, lincRNA-p21 and OIP5-AS1, are depleted by siRNAs. We observed that human and mouse lincRNA-p21 regulated target mRNA abundance in complementarity-dependent and independent manners. In contrast, affinity pull down of OIP5-AS1 revealed that changes in OIP5-AS1 expression influenced the amount of some OIP5-AS1 target mRNAs and miRNAs, as we predicted from our sequence complementarity assay. Altogether, the current study demonstrates that partial complementarity of lncRNAs and mRNAs (even miRNAs) assist in determining target RNA expression and quantity.     

Elevation of Soluble Guanylate Cyclase Suppresses Proliferation and Survival of Human Breast Cancer Cells

Nitric oxide (NO) is an essential signaling molecule in biological systems. Soluble guanylate cyclase (sGC), composing of α1 and β1 subunit, is the receptor for NO. Using radioimmunoassay, we discovered that activation of sGC by treatment with bradykinin or sodium nitroprusside (SNP) is impaired in MCF-7 and MDA-MB-231 breast cancer cells as compared to normal breast epithelial 184A1 cells. The 184A1 cells expressed both sGC α1 and sGCβ1 mRNAs. However, levels of sGCβ1 mRNAs were relatively lower in MCF-7 cells while both mRNA of sGC subunits were absent in MDA-MB-231 cells. Treatment with DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) increased mRNA levels of both sGCα1 and sGCβ1 in MDA-MB-231 cells but only sGCβ1 mRNAs in MCF-7 cells. The 5-aza-dC treatment increased the SNP-induced cGMP production in MCF-7 and MDA-MB-231, but not in 184A1 cells. Bisulfite sequencing revealed that the promoter of sGCα1 in MDA-MB-231 cells and promoter of sGCβ1 in MCF-7 cells were methylated. Promoter hypermethylation of sGCα1 and sGCβ1 was found in 1 out of 10 breast cancer patients. Over-expression of both sGC subunits in MDA-MB-231 cells induced apoptosis and growth inhibition in vitro as well as reduced tumor incidence and tumor growth rate of MDA-MB-231 xenografts in nude mice. Elevation of sGC reduced protein abundance of Bcl-2, Bcl-xL, Cdc2, Cdc25A, Cyclin B1, Cyclin D1, Cdk6, c-Myc, and Skp2 while increased protein expression of p53. Our study demonstrated that down-regulation of sGC, partially due to promoter methylation, provides growth and survival advantage in human breast cancer cells.