Junhui Hu

LncRNA MALAT1 functions as a competing endogenous RNA to regulate ZEB2 expression by sponging miR-200s in clear cell kidney carcinoma

Long non-coding RNA (lncRNAs) play a critical role in the development of cancers. LncRNA metastasis-associated lung adenocarcinoma transcript 1(MALAT1) has recently been identified to be involved in tumorigenesis of several cancers such as lung cancer, bladder cancer and so on. Here, we found that MALAT1 exist a higher fold change (Tumor/Normal) in clear cell kidney carcinoma (KIRC) from The Cancer Genome Atlas (TCGA) Data Portal and a negative correlation with miR-200s family. We further demonstrated MALAT1 promote KIRC proliferation and metastasis through sponging miR-200s in vitro and in vivo. In addition, miR-200c can partly reverse the MALAT1′s stimulation on proliferation and metastasis in KIRC. In summary we unveil a branch of the MALAT1/miR-200s/ZEB2 pathway that regulates the progression of KIRC. The inhibition of MALAT1 expression may be a promising strategy for KIRC therapy.

MicroRNA-10b promotes migration and invasion through KLF4 and HOXD10 in human bladder cancer

The present study was performed to investigate the effect of microRNA-10b (miR-10b) on cell migration and invasion in human bladder cancer (BC). Real-time PCR was performed to detect the expression of miR-10b in BC cell lines. miR-10b mimics, the negative control for mimics, miR-10b inhibitor and the negative control for inhibitor were transfected into BC cell lines and the effects of miR-10b on the migration and invasion of cells were investigated through Transwell assay. Meanwhile, protein levels of KLF4, HOXD10, E-cadherin and MMP14 were measured. Luciferase assays were also performed to validate KLF4 and HOXD10 as miR-10b targets. In vivo metastasis assay was performed to validate if miR-10b can promote BC cell line metastasis in vivo. miR-10b is significantly upregulated in BC cell lines and metastatic tissues. Increased miR-10b expression significantly enhanced BC cell migration and invasion, while decreased miR-10b expression reduced cell migration and invasion. In vivo metastasis assay demonstrated that overexpression of miR-10b markedly promoted BC metastasis. Moreover, KLF4 and HOXD10 were identified as direct targets of miR-10b in BC cells. Silencing of KLF4 or HOXD10 recapitulated the pro-metastatic function. Furthermore, we found that E-cadherin and MMP14 may be the downstream factors of KLF4 and HOXD10 in the suppression of BC metastasis by miR-10b. These data suggest that miR-10b may function as oncogenes in BC cells. Targeting these novel strategies, inhibition of miR-10b/KLF4/E-cadherin axis and miR-10b/HOXD10/MMP14 axis may be helpful as a therapeutic approach to block BC cell metastasis.

Up‐regulation of p21WAF1/CIP1 by miRNAs and its implications in bladder cancer cells

We have previously reported that synthetic dsRNA can activate p21 expression by targeting the p21 promoter, thereby suppressing the proliferation of human bladder cancer cells. As complementarity between dsRNA and its target sequences is necessary for RNA activation, miRNAs may also trigger p21 expression through the same mechanism. Here, the expression levels of three miRNAs (miR‐370, miR‐1180 and miR‐1236) decreased in bladder cancer tissues compared to healthy controls and the levels of these mRNAs positively correlated with p21 mRNA levels. The three miRNAs induced nuclear p21 expression through p21‐promoter binding. Overexpression of the three miRNAs inhibited the proliferation of bladder cancer cells mainly by regulating p21. Therefore, these miRNAs could be candidates for anti‐cancer drugs.

miR-490-5p suppresses tumour growth in renal cell carcinoma through targeting PIK3CA.

Dysregulated micro‐RNAs have been reported in many human cancers, including renal cell carcinoma. Recent studies indicated that miR‐490 is involved in tumour development and progression. However, the expression profile and function in renal cell carcinoma remains unknown.

lncRNA PVT1 and its splicing variant function as competing endogenous RNA to regulate clear cell renal cell carcinoma progression

Long non-coding RNAs (lncRNAs) exert critical regulatory roles in the development and progression of several cancers. Plasmacytoma variant translocation 1 (PVT1), an lncRNA, was shown to be upregulated in clear cell renal cell carcinoma (ccRCC) in our study, while Kaplan-Meier curve and Cox regression analysis showed that high expression of PVT1 was associated with poor overall survival (OS) and disease free survival (DFS) in ccRCC patients. In vitro experiments revealed that PVT1 promoted renal cancer cell proliferation, migration, and invasion, while in vivo studies confirmed its oncogenic roles in ccRCC. Further bioinformatic analysis and RNA immunoprecipitation revealed that PVT1 could function as an oncogenic transcript partly through sponging miR-200s to regulate BMI1, ZEB1 and ZEB2 expression. Besides, a novel splicing variant of PVT1 lacking exon 4 (PVT1ΔE4) was found to have a higher expression in ccRCC and could also promote cell proliferation and invasion as the full-length transcript did. Besides, SRSF1 decreased the inclusion of exon 4 of full-length transcript and increased the relative expression of PVT1ΔE4 in ccRCC. Mechanistic investigations indicated that PVT1ΔE4 could also upregulate the expression of BMI1, ZEB1 and ZEB2 through interacting with miR-200s. Our study helps reveal new molecular events in ccRCC and provides promising diagnostic and therapeutic targets for this disease.

CRISPR-Mediated VHL Knockout Generates an Improved Model for Metastatic Renal Cell Carcinoma

Metastatic renal cell carcinoma (mRCC) is nearly incurable and accounts for most of the mortality associated with RCC. Von Hippel Lindau (VHL) is a tumour suppressor that is lost in the majority of clear cell RCC (ccRCC) cases. Its role in regulating hypoxia-inducible factors-1α (HIF-1α) and -2α (HIF-2α) is well-studied. Recent work has demonstrated that VHL knock down induces an epithelial-mesenchymal transition (EMT) phenotype. In this study we showed that a CRISPR/Cas9-mediated knock out of VHL in the RENCA model leads to morphologic and molecular changes indicative of EMT, which in turn drives increased metastasis to the lungs. RENCA cells deficient in HIF-1α failed to undergo EMT changes upon VHL knockout. RNA-seq revealed several HIF-1α-regulated genes that are upregulated in our VHL knockout cells and whose overexpression signifies an aggressive form of ccRCC in the cancer genome atlas (TCGA) database. Independent validation in a new clinical dataset confirms the upregulation of these genes in ccRCC samples compared to adjacent normal tissue. Our findings indicate that loss of VHL could be driving tumour cell dissemination through stabilization of HIF-1α in RCC. A better understanding of the mechanisms involved in this phenomenon can guide the search for more effective treatments to combat mRCC.

Alternative Splicing of EZH2 pre-mRNA by SF3B3 Contributes to the Tumorigenic Potential of Renal Cancer

Purpose: Deregulation or mutation of the EZH2 gene causes various tumors, including clear cell renal cell carcinoma (ccRCC). Although several splice variants of EZH2 have been identified, little is known about how EZH2 splicing is regulated or the contribution of alternative splicing to its protumorigenic functions. Experimental Design: We conducted RT-PCR, Western blot analysis, and IHC techniques to examine EZH2 and its alternative splicing transcript expression in renal cancer tissue and renal cancer cell lines. Proliferation, migration, clonogenicity, and tumorigenicity of renal cancer cells either exhibiting knockdown of EZH2 or its splicing factor SF3B3 were assessed by CCK8, Transwell assay, and murine xenograft experiments. Results: We found that the inclusion of alternative EZH2 exon 14 was significantly increased in ccRCC samples and renal cancer cell lines. In ccRCC lines, enforced expression of EZH2Δ14 inhibited, and EZH2 promoted, cell growth, migration, proliferation, and tumorigenicity in a xenograft model. Mechanistic studies demonstrated that EZH2Δ14 isoform functions as a dominant-negative inhibitor of full-length EZH2. Coexpression of EZH2Δ14 variant with full-length EZH2 not only abrogated DAB2IP and HOXA9 suppression but also inhibited EZH2-driven tumorigenesis. Strikingly, the splicing factor SF3B3 stimulates inclusion of exon14 and has pro-proliferative activity. Importantly, the upregulation of SF3B3 expression observed in clinical ccRCC samples parallels the increased inclusion of EZH2 exon14, and the SF3B3 level is associated with higher tumor stage and poor overall survival. Conclusions: These results suggest SF3B3 as a key regulator of EZH2 pre-mRNA splicing and SF3B3 may represent a novel prognostic factor and potential therapeutic target in ccRCC. Clin Cancer Res; 23(13); 3428–41. ©2016 AACR.

Regulation of glucose metabolism by p62/SQSTM1 through HIF1α

ABSTRACT The signaling adaptor sequestosome 1 (SQSTM1)/p62 is frequently overexpressed in tumors and plays an important role in the regulation of tumorigenesis. Although great progress has been made, biological roles of p62 and relevant molecular mechanisms responsible for its pro-tumor activity remain largely unknown. Here, we show that p62 knockdown reduces cell growth and the expression of glycolytic genes in a manner that depends on HIF1α activity in renal cancer cells. Knockdown of p62 decreases HIF1α levels and transcriptional activity by regulating mTORC1 activity and NF-κB nuclear translocation. Furthermore, p62 interacts directly with the von Hippel-Lindau (VHL) E3 ligase complex to modulate the stability of HIF1α. Mechanistically, p62 binds to the VHL complex and competes with HIF1α. Expression of p62 inhibits the interaction of DCNL1 (also known as DCUN1D1) with CUL2 and attenuates the neddylation of CUL2, and thus downregulates the VHL E3 ligase complex activity. Functionally, HIF1α expression is required for p62-induced glucose uptake, lactate production and soft agar colony growth. Taken together, our findings demonstrate that p62 is a crucial positive regulator of HIF1α, which is a facilitating factor in p62-enhanced tumorigenesis. Highlighted Article: p62 is a crucial positive regulator of HIF1α, which is a facilitating factor in p62-enhanced tumorigenesis.

Endoglin Is Essential for the Maintenance of Self-Renewal and Chemoresistance in Renal Cancer Stem Cells.

Summary Renal cell carcinoma (RCC) is a deadly malignancy due to its tendency to metastasize and resistance to chemotherapy. Stem-like tumor cells often confer these aggressive behaviors. We discovered an endoglin (CD105)-expressing subpopulation in human RCC xenografts and patient samples with a greater capability to form spheres in vitro and tumors in mice at low dilutions than parental cells. Knockdown of CD105 by short hairpin RNA and CRISPR/cas9 reduced stemness markers and sphere-formation ability while accelerating senescence in vitro. Importantly, downregulation of CD105 significantly decreased the tumorigenicity and gemcitabine resistance. This loss of stem-like properties can be rescued by CDA, MYC, or NANOG, and CDA might act as a demethylase maintaining MYC and NANOG. In this study, we showed that Endoglin (CD105) expression not only demarcates a cancer stem cell subpopulation but also confers self-renewal ability and contributes to chemoresistance in RCC.

A Non-integrating Lentiviral Approach Overcomes Cas9-Induced Immune Rejection to Establish an Immunocompetent Metastatic Renal Cancer Model

The CRISPR-based technology has revolutionized genome editing in recent years. This technique allows for gene knockout and evaluation of function in cell lines in a manner that is far easier and more accessible than anything previously available. Unfortunately, the ability to extend these studies to in vivo syngeneic murine cell line implantation is limited by an immune response against cells transduced to stably express Cas9. In this study, we demonstrate that a non-integrating lentiviral vector approach can overcome this immune rejection and allow for the growth of transduced cells in an immunocompetent host. This technique enables the establishment of a von Hippel-Lindau (VHL) gene knockout RENCA cell line in BALB/c mice, generating an improved model of immunocompetent, metastatic renal cell carcinoma (RCC).