SNHG12 Regulated by KMT2B Participates in the Pathogenesis of Renal Cell Carcinoma via E2F1/CEP55

Jia-fu Feng (  fengjiafu@uestc.edu.cn ) Department of Clinical Laboratory,Mianyang Central Hospital,School of Medicine,University of Electronic Science and Technology of China https://orcid.org/0000-0002-1714-4110 Jun Wang Department of Medical Technology Institute,Chengdu University of Traditional Chinese Medicine Gang Xie Department of Pathology,Mianyang Central Hospital,School of Medicine,University of Electronic Science and Technology of China Yao-dong Wang Department of Urology Surgery,Mianyang Central Hospital,School of Medicine,University of Electronic Science and Technology of China Xiao-han Li Department of Medical Laboratory,A liated Hospital of Southwest Medical University Wen-yu Yang Department of Medical Technology Institute,Chengdu University of Traditional Chinese Medicine Yu-wei Yang Department of Clinical Labortory,Mianyang Central Hospital,School of Medicine,University of Electronic Science and Technology of China Bin Zhang Department of Clinical Laboratory,Mianyang Central Hospital,School of Medicine,University of Electronic Science and Technology of China


Introduction
Renal cell carcinoma (RCC) is the main pathological type of kidney cancer, accounting for 70-90% [1]. Epidemiological survey shows that its morbidity and mortality have both been on the rise worldwide in recent years [2]. RCC is a highly concealed malignant tumor originating from the renal tubular epithelium. Only 10% of patients present with the "classic triad" ( ank pain, gross hematuria, and a palpable renal mass) [3]. Due to the lack of biomarkers for early diagnosis and prognosis of RCC, most RCC patients are diagnosed at the middle and late stages, accompanied by local spread and distant metastasis [4]. The main treatment of RCC is radical or partial nephrectomy followed by chemotherapy and/or radiotherapy. In addition, 20% -40% of patients have recurrence and/or distant metastasis after surgery. Although progresses have been made in the diagnosis and treatment of RCC in the past decades, RCC is still one of the most drug-resistant malignancies and a common cause of cancer-related deaths [5]. Therefore, it is urgent to explore the molecular mechanism of RCC occurrence and development, to identify new and reliable biomarkers of RCC and to develop new therapeutic targets for early diagnosis and treatment of RCC.
Long non-coding RNA (lncRNA) is a type of non-coding RNA with a length greater than 200 nucleotides. It is involved in the multi-level regulation of gene expression and its abnormal expression and mutation are Page 3/21 usually closely related to tumorigenesis and metastasis [6][7][8]. In addition, lncRNA can be speci cally expressed in cancer and stably exist in body uids [9][10][11], which can be used as a new type of cancer biomarkers and therapeutic targets. Some lncRNAs can encode small nucleolar RNA and are called small nucleolar RNA host genes (SNHGs). Of them, SNHG12 has been reported to be up-regulated in human endometrial cancer [12], bladder cancer [13], nasopharyngeal cancer [14], colorectal cancer [15], lung adenocarcinoma [16], breast cancer [17], liver cancer [18], and clear cell RCC [19], and plays an important role in proliferation and migration of cancer cells. The methylation of lncRNAs promoter can regulate expression of lncRNA, which is related to the occurrence of many diseases. For example, in clear cell RCC, the methylation status of two CpG sites is negatively correlated with the expression of SNHG3 and SNHG15, suggesting that DNA hypomethylation may play an important role in promoting the transcription of SNHG3 and SNHG15 [20]. SNHG11 binds to the HRE site in the gene promoter, and promotes gene transcription and tumor invasion and metastasis of colorectal cancer through the SNHG11/HIF-1α pathway [21]. CpG methylation in the promoter region of SNHG12 promotes the competitive binding of SNHG12 with miR-129-5p, regulates the MAPK/ERK pathway and G1/S cell cycle transition, thereby affecting the resistance of glioblastoma cells to temozolomide [22]. However, the role of SNHG12 regulated by DNA methylation in RCC is still unclear.
In this study, we explored the role of SNHG12 promoter methylation in RCC development through series lossand gain-of-function experiments. Our ndings may provide evidence for identifying new molecular targets for the diagnosis and treatment of RCC.
Study cohort RCC tissues and the corresponding precancerous tissue samples were collected from RCC patients (n=46) who were treated in Mianyang Central Hospital from January 2017 to January 2019. All patients were con rmed to have RCC by surgery and pathological analysis. The inclusion criteria: 1) Patients with pathologically con rmed RCC cases; 2) Patient did not receive any chemotherapy, radiotherapy, or other antitumor treatment before operation; 3) Patients had complete clinical data. The exclusion criteria: 1) Patients without pathological con rmation; 2) Patients with recurrence and distant metastasis after treatment; 3) Patients with a history of mental illness; 4) Patients with dysfunction of the heart, liver, pancreas, and other important organs; 5) Patients with respiratory and circulatory diseases; 6) Patients with non RCC tumors. Prior written and informed consent were obtained from every patient and the study was

CCK-8
After 48 h of transfection, cells were seeded into 96-well plates at 1.0×10 5 cells/ml (100 µL/well). After routine culture overnight, the cells were treated according to CCK-8 kit (Beyotime, Shanghai, China), and the cell viability was detected at 24 h, 48 h, and 72 h. The OD490 was detected with a microplate reader.

Wound healing test
The cells were seeded in a 6-well plate at 2.5×10 4 cells/ml and cultured for 24 h. Next, a 10 µL sterile disposable pipette was used to make a scratch on the cells. The cell images at 0 h and 48 h after the scratch were taken under an inverted microscope. The relative distance of cell migration to scratch area was measured, and the actual migration distance was calculated according to the scratch area distance of cells.

Transwell assay
The Transwell upper chamber (Yanhui Biotechnology, Shanghai, China) was pre-coated with ECM gel (Sigma-Aldrich, USA). After starving culture for 24 h, the cells were added to the upper chamber at 2.5×10 5 cells/ml (0.2 ml in total). In the lower chamber, 700 µL of pre-cooled DMEM medium containing 10% FBS was added.
The chamber was then incubated in a 37°C, 5% CO 2 saturated humidity incubator. After 24 h, the cells in the lower chamber were xed with methanol, stained with 0.1% crystal violet. Invaded cells were photographed and counted using randomly selected 5 visual elds in each chamber under an inverted microscope with a magni cation of 200 ×. The experiment was repeated three times independently.
Matrigel-based capillary-like tube formation in vitro RCC cells were transfected as above described. After 48 h, the cell supernatant was collected. The tumorconditioned medium was prepared according to the ratio of 4:5:1 (tumor supernatant: DMEM medium: FBS). HUVECs were seeded into a 96-well plate pre-coated with Matrigel and incubated with tumor-conditioned medium for 8 h. Finally, 4 elds of view were randomly selected from each well and the tube length was quanti ed under the phase-contrast microscope.
Chromatin immunoprecipitation (ChIP) 786-O cells transfected with sh-NC or sh-KMT2B were xed with 1.0% formaldehyde and subjected to ultrasonic treatment to obtain the DNA fragments. For ChIP, the supernatant was incubated with the negative control antibody rabbit anti-IgG (ab109489, 1:100), H3K4me3 antibody (1: 1000, ab8580), and E2F1 antibody (1: 500, ab179445) (Abcam, Cambridge, UK) at 4°C overnight. The endogenous DNA-protein complexes were precipitated with Protein Agarose/Sepharose (Sangon biotech, Shanghai, China). The DNA fragments were extracted with phenol/chloroform. RT-qPCR was used to detect the enrichment of H3K4me3 in the SNHG12 promoter and E2F1 in the CEP55 promoter.

RNA immunoprecipitation (RIP)
RIP kit (Millipore, USA) was used to detect the binding of SNHG12 and E2F1 protein. The 786-O cells were subjected to lysis with RIPA (P0013B, Beyotime) for 5 min. The antibody-bound magnetic beads, which were prepared by incubating magnetic beads with anti-E2F1 (1:50, ab179445, Abcam) and IgG (ab172730, 1:100, Abcam) for 30 min, were incubated with the supernatant at 4°C overnight. After that, the samples were digested with proteinase K and RNA was extracted for subsequent RT-qPCR detection.

Dual luciferase reporter assay
The potential binding site of E2F1 in the promoter region of CEP55 was analyzed through the bioinformatics website (http://jaspar.genereg.net). After that, the pGL3-CEP55-WT and pGL3-CEP55-MUT plasmids were constructed with wild type (WT) and mutant type (MUT) binding sequences of E2F1, respectively. Then, these plasmids were co-transfected with oe-NC and oe-E2F1 into the HEK-293T cells (ATCC). After 48 h, the Dual-Luciferase Reporter Assay System kit (Promega, USA) and the TD-20/20 Luminometer was used to detect the luciferase activity.

RT-qPCR
The nucleus and cytoplasm of RCC cells were separated using PARIS kit (Life Technologies, Carlsbad, CA, USA). The total RNA of cells and tissues as well as nucleus and cytoplasm of RCC cells was extracted by Trizol (Invitrogen). RT-qPCR was conducted as reported previously [24]. The primers are shown in Table 1. Table 1 RT-qPCR primers.

Western Blot
The total protein of tissues or cells was extracted with RIPA (P0013B, Beyotime
KMT2B up-regulates SNHG12 by regulating the H3K4me3 modi cation of the SNHG12 promoter As shown in Figure 3A, there was a large amount of H3K4me3 enrichment in the SNHG12 promoter. It is reported that KMT2B mediates the transcriptional activation of H3K4me3 modi cation [26]. Moreover, through ENCORI database analysis, KMT2B was highly expressed in RCC ( Figure 3B) and positively correlated with SNHG12 expression ( Figure 3C). Therefore, KMT2B may regulate SNHG12 by mediating the H3K4me3 modi cation of its promoter region.
Furthermore, overexpression of SNHG12 promoted binding of E2F1 with CEP55 promoter region, whereas SNHG12 inhibited this binding (oe-NC vs. oe-SNHG12: q=8.724, p=0.001; sh-NC vs. sh-SNHG12: q=11.861, p<0.001) ( Figure 4G). Then, we predicted the binding site between E2F1 and CEP55 promoter region through affect the luciferase activity of the oe-E2F1 group, while sites 2 and 3 were mutated, the luciferase activity increased ( Figure 4H and 4I). The above results revealed that site 1 was the main site for E2F1 to act on the CEP55 promoter region. q=8.970, p=0.001) ( Figure 5F).

Knockdown of SNHG12 inhibits RCC growth and angiogenesis in vivo
As shown in Figure 6A q=38.692, all p<0.001) and weight (q=20.071, p<0.001) .

Discussion
RCC is a heterogeneous tumor that originates from the renal parenchyma and is one of the deadliest malignant tumors in the urinary system [27]. So far, the mechanism underlying the occurrence and development of RCC is still not fully understood. RCC-related biomarkers are less studied, and RCC early diagnosis is di cult. In addition, RCC responds poorly to conventional chemotherapy and radiotherapy, and there is a lack of targeted therapy drugs for RCC, resulting in a poor prognosis and a low 5-year survival rate for RCC patients with advanced stage IV and later [28]. However, if diagnosed early, patients with local RCC can be treated by nephrectomy (partial or total nephrectomy). The treatment effect of TI and T2 stage surgery is better, which can not only improve the quality of life, but also the 5-to 10-year survival rate of RCC patients [29]. Therefore, further understanding of the pathogenesis of RCC may help the diagnosis and treatment of RCC patients. Here, in this study, we explored the regulation of SNHG12 by KMT2B and the downstream factors of SNHG12 (including E2F1 and CEP55) involved in its effect on RCC.
Here, in this study, the biological function and mechanism of SNHG12 in the occurrence and development of RCC were investigated. We rst found that SNHG12 was highly expressed in RCC tissues and cells. In

Conclusion
In summary, KMT2B up-regulates SNHG12 through the modi cation of H3K4me3 in the SNHG12 promoter region, which in turn recruits the transcription factor E2F1, and ultimately promotes expression of CEP55, and the proliferation, migration, and invasion of RCC cells, as well as the angiogenesis ability of HUVECs ( Figure  7). These all ultimately promote RCC growth and angiogenesis.    SNHG12 recruits transcription factor E2F1 to affect CEP55 transcription.
A: The correlation between SNHG12 and CEP55 in RCC was analyzed through ENCORI database. B: The expression of E2F1 in RCC was analyzed through the ENCORI database (RCC=535, Normal=72). C: The correlation between E2F1 and CEP55 in RCC was analyzed through the ENCORI database. D: The level of SNHG12 in nuclear and cytoplasm was analyzed by RT-qPCR. E: The binding of SNHG12 and E2F1 was detected by RIP. F: Detection of E2F1 binding to the CEP55 promoter region by CHIP. G: After overexpression of SNHG12, the binding of E2F1 to the CEP55 promoter region was detected by CHIP. H: The binding of transcription factor E2F1 to the promoter region of the target gene CEP55 was detected by dual luciferase reporter assay. I: Three sites in the promoter region of CEP55. J: The expression of CEP55 in each group was detected by RT-qPCR. *P <0.05. All experiments were repeated three times.

Figure 5
The effect of SNHG12/E2F1/CEP55 on RCC cell proliferation, migration, invasion and HUVEC angiogenesis. All experiments were repeated three times.

Figure 6
The effect of SNHG12/E2F1/CEP55 on RCC growth and angiogenesis in vivo.  Schematic diagram of the mechanism of SNHG12 in RCC.
KMT2B up-regulates SNHG12 through the modi cation of H3K4me3, which in turn recruits the transcription factor E2F1, and ultimately promotes the expression of CEP55, and related factors such as VEGF, MMP-2 and MMP-9.