lncRNA MIR4435‐2HG promoted clear cell renal cell carcinoma malignant progression via miR‐513a‐5p/KLF6 axis

Abstract Long non‐coding RNAs (lncRNAs) take various biological effects in clear cell renal cell carcinoma (ccRCC) mostly through sponging with microRNAs (miRNAs). lncRNA MIR4435‐2HG is found to promote tumour progression in gastric cancer, glioblastoma and hepatocellular carcinoma. However, the role of lncRNA MIR4435‐2HG in ccRCC progression remains unknown. The purpose of this research was to investigate the potential molecular mechanism of lncRNA MIR4435‐2HG regarding the regulation of ccRCC initiation and progression. In this study, we found the up‐regulation of MIR4435‐2HG in ccRCC tissues and cell lines. Functionally, overexpression of MIR4435‐2HG promoted the proliferation as well as the metastasis in ccRCC cell lines, whereas knockdown of MIR4435‐2HG inhibited the above changes. Then, bioinformatic analysis and luciferase reporter assays confirmed the negative regulation effect of MIR4435‐2HG on miR‐513a‐5p. And further investigations showed that KLF6, which collected from the intersection of databases, was the potential conjugated mRNAs of miR‐513a‐5p. Finally, the rescue experiments revealed the relation among MIR4435‐2HG and KLF6, which showed that KLF6 could reverse the promoting effect of MIR4435‐2HG on ccRCC in vitro and in vivo. Therefore, our findings provided insight into the mechanisms of MIR4435‐2HG in ccRCC and revealed an alternative target for the clinical diagnosis and treatment of ccRCC.

identify the molecular mechanisms of ccRCC progression, which is useful in diagnosing and treating.

Recent findings have uncovered the transcription of long non-cod-
ing RNAs (lncRNAs) from human genome with the development of high-throughput sequencing. [7][8][9] lncRNAs, which are more than 200 nucleotides long, have no or limited protein-coding capacity. 10 And lncRNAs can be classified into five types including sense, antisense, intergenic, bidirectional and intronic according to its location. 11 Emerging evidence has documented that lncRNAs were identified to play diverse roles as a molecular modulator in regulating various physiological and pathological processes, such as organ development, immunity, tumorigenesis and tumour progression. [12][13][14] Previous studies revealed that lncRNAs exerted their roles in all aspects of gene regulation and protein function, such as tethers, decoys, guides and scaffolds. [15][16][17][18] Nowadays, increasing studies have reported that a number of lncRNAs might play multiple roles in the initiation and progression of ccRCC. 19,20 MIR4435-2HG, which reported with aberrant expressions in some malignant tumours, could regulate tumorigenesis and progression. [21][22][23] Meanwhile, we found the abnormal expression of MIR4435-2HG in ccRCC by TCGA data analysis. Nevertheless, the mechanisms and functions of MIR4435-2HG in ccRCC were poorly known. Therefore, we aimed to investigate the potential molecular mechanism of lncRNA MIR4435-2HG in cell proliferation and invasion of ccRCC.

| Clinical specimens
A total of 40 matched samples of ccRCC specimens and adjacent non-cancerous tissues were obtained for this investigation. None of the patients had received chemotherapy or radiotherapy before surgery. All the samples were frozen immediately in liquid nitrogen after surgical resection and stored at −80°C until further analysis (Table 1). The clinical and pathological characteristics were obtained from our hospital. The Institutional Review Board approved the use of the tumour samples and animals in this study.
In addition, all cell lines were cultured in a standard humidified atmosphere with 5% CO 2 at 37°C.

| In situ hybridization
Paraffin-embedded sections of ccRCC tissues were deparaffinized with xylene and rehydrated with 100, 90, 70 and 50% ethanol (5 minutes each) at room temperature. The samples were digested with proteinase K and fixed in 4% paraformaldehyde for 10 minutes at room temperature, followed by hybridization with the MIR4435-2HG probe (Servicebio, Wuhan, China) at 55°C overnight and subsequent incubation with HRP-conjugated secondary antibody for 30 minutes at 4°C. Diaminobenzidine was used to develop the stain with a colorimetric reaction for 30 minutes at room temperature, and then, the sections were observed under light microscope. qRT-PCR were listed as followed:

| Colony formation assays
Cells were transfected in a six-well plate at a density of 200 cells/ well. The cells were incubated for nearly 2 weeks at 37°C and 5% CO 2 to fulfil the colony formation assay. Colonies were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet and the number of colonies was counted using ImageJ.

| TCGA analysis
The RNA-seq data of ccRCC and matched normal samples were downloaded from The Cancer Genome Atlas (TCGA) Data Portal (https://tcgadata.nci.nih.gov/tcga/). Data quantification for the expression of miRNAs of MIR4435-2HG was performed by the customized data analysis pipeline, including a series of steps that quality control, alignment and expression quantification. We used fold change ≥2.0 and a P-value ≤ .05 as threshold to screen up-regulated or down-regulated genes.

| Western blotting
Total protein was prepared from 769-P and Caki-1 cells using RIPA buffer with a proteinase inhibitor and phosphatase inhibitors. The lysates were centrifuged on ice for 30 minutes and then centrifuged at 13400×g for 15 minutes at 4°C. The protein concentration was measured by a BCA Kit (Beyotime Biotechnology, Beijing, China). We separated the extracted proteins with SDS-PAGE, which were transferred to PVDF membranes. 5% of non-fat milk was used to block the membrane, which was incubated with primary antibodies against KLF6 at 4°C overnight. Subsequently, the membranes were incubated with the corresponding secondary antibody at room temperature for 1 hour.
The expression of GAPDH was used as loading control. According to the manufacturer's instructions, a Chemiluminescence Reagent (ECL) Kit (Beyotime Biotechnology) was utilized to visualize protein bands. All antibodies were purchased from Servicebio (Wuhan, China).

| Tumorigenicity assays in nude mice
The indicated stable cell lines (2 × 10 6 ) were subcutaneously injected into the right flank of BALB/c (nu/nu) 4-to 6-weekold female nude mice. Tumour size was measured once per 4 days, and mice were killed to analyse the tumour burden after 4 weeks, and the tumour volume (V) was calculated using the formula: V = 1/2(length × width 2 ). All procedures of animal experiments were performed in accordance with the Nanjing Medical University's Animal Ethics Committee.

| Statistical analysis
The measurement data were expressed as the mean ± SD and were analysed in GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA). One-way ANOVA analysis or two-tailed Student's t tests were performed for P-value analysis, as appropriate. The data of control group were chosen as y-axis normalization controls in this manuscript. Unless otherwise noted, each experiment was carried out at least in triplicate. P < 0.05 was considered statistically significant.

| Identification of MIR4435-2HG as an lncRNA up-regulated in ccRCC
Initially, several online databases were used to probe the lncRNAmediated initiation and progression of ccRCC. Based on the analysis of gene expression profiling interactive analysis (GEPIA) data, we found that MIR4435-2HG was highly expressed in various cancers ( Figure 1A). What is more, significant up-regulation of MIR4435-

| Long non-coding RNA MIR4435-2HG increased proliferation and invasion abilities of ccRCC cells
To  Figure 2C). Collectively, these results revealed that MIR4435-2HG had tumour-inductive activity in ccRCC progression.

| MIR4435-2HG interacted with miR-513a-5p and repressed its expression in ccRCC
In sequence, we attempted to obtain a better understanding of the mechanism of MIR4435-2HG in promoting ccRCC progression. It has been known that competitive endogenous RNAs (ceRNAs), which sponge corresponding miRNAs to realize their modulating effects on target mRNAs, are the most well-known mechanism of cytoplasmic lncRNAs, 18 so we speculated that MIR4435-2HG could regulate ccRCC through this way as well.

F I G U R E 4 KLF6 was a direct target of miR
And the expression of miR-513a-5p in 769-P and ACHN cells was down-regulating compared with that in HK2 cells as we expected ( Figure 3D). Furthermore, dual-luciferase reporter gene assay was applied to assess whether MIR4435-2HG acts on miR-513a-5p. The results showed that compared with NC group, the luciferase activity of cells transfected with miR-513a-5p mimic in MIR4435-2HG-WT group decreased significantly (P < 0.01), whereas there was no significant change in luciferase activity in cells transfected with miR-513a-5p mimic in MIR4435-2HG-MUT group ( Figure 3E). We further performed co-localization experiment to confirm the above results, which was consistent with the interaction of MIR4435-2HG and miR-513a-5p ( Figure 3F).

| miR-513a-5p inhibited proliferation and invasion abilities of ccRCC cells
To elaborate the function of miR-513a-5p in proliferation and invasion abilities of ccRCC cells, the experiments in vitro were conducted. Different transfection groups were constructed in 769-P and ACHN cells. The up-regulation of KLF6 expression in ccRCC cells could reverse the promotion on cell viability ( Figure 5A,B) and invasive capacity ( Figure 5C) caused by miR-513a-5p up-expression.
Accordingly, the down-regulation of KLF6 could reverse inhibition on cell viability ( Figure 5A,B) and invasive capacity caused by miR-513a-5p knockdown Figure 5C). In addition, KLF6, the target gene of miR-513a-5p, in different transfection groups of 769-P and ACHN cells, was detected by Western blot, and the results are displayed in Figure 5D. Moreover, tumours of miR-513a-5p antagonists groups presented higher positive rate of KLF6 compared with NC antagonist groups ( Figure 5E). These results highlighted that miR-513a-5p could suppress tumorigenesis of ccRCC by down-regulating KLF6.

| MIR4435-2HG promoted tumours in ccRCC cells via modulating KLF6
The KLF6 expression of 769-P and ACHN cells in different transfection groups was detected by qRT-PCR, and the results are displayed in Figure 6A. Compared with sh-NC+pcDNA3.1 group, the expression level of KLF6 was notably reduced in sh-MIR4435-

| D ISCUSS I ON
RCC is well-recognized malignancies of the urinary system, which accounts for 4% of adult malignancies, and annual estimates of newly diagnosed cases are in rising trend year by year steadily. 24 About 70% of ccRCC patients have been diagnosed clinically localized disease, and the mainstay of treatment is surgical resection; however, post-operative metastatic diseases occur in 20%-30% of ccRCC patients. 25 So, because of the unsatisfaction of relatively poor detection and prognosis, numerous researches on tumour treatmentrelated factors have been receiving increasing attention. 24,25 It has been proved that lncRNAs have been clarified by growing evidence as active biological molecules, which played an extensive modulatory role in ccRCC carcinogenesis and progression. [26][27][28][29] According to the location, lncRNAs could realize their function through impli- The tumour sections from different transfected groups of xenograft mouse models were subjected to H&E staining and immunohistochemistry staining using antibodies against KLF6 (400×). All of the data were analysed from three independent experiments. * P < 0.05; ** P < 0.01 lncRNAs HOXA11-AS could exert their function through regulating MMP16 expression. 31 In tumour biology, MIR4435-2HG was typically up-regulated in malignant tissues in gastric cancer, glioblastoma and hepatocellular carcinoma. [21][22][23] In the present study, we identified the lncRNA MIR4435-2HG as a highly overexpression lncRNA in ccRCC. Nevertheless, the molecular mechanisms and potential function of MIR4435-2HG in ccRCC were still unclear.
Initially, the key findings in our study were that the expression of MIR4435-2HG was significantly up-regulated in ccRCC tissues relative to corresponding paracancerous normal tissues from TCGA analysis, which was in compliance with the result of clinical ccRCC specimens. Moreover, we characterized the tumorigenic role of MIR4435-2HG in ccRCC cells by gain-and loss-of-function assays.
In vitro, the results shed light that knocking down MIR4435-2HG in 769-P cells would significantly inhibit cell proliferation and invasion.
In contrast, overexpression of MIR4435-2HG in ACHN cells robustly increased ccRCC cell proliferation and invasion. Therefore, our investigations shed a lot of light on MIR4435-2HG severed as an oncogene in ccRCC and could be explored as a potential diagnostic and treatment indicator for ccRCC.
In the subsequent study, we aimed to uncover the potential mechanism of how MIR4435-2HG affected tumorigenesis and development of ccRCC. lncRNAs exert their functions may depend on where they are. 32 In the cytoplasm, lncRNAs may function as decoys for miRNAs and their target genes, 33 whereas in the nucleus, ln-cRNAs may be involved in guiding RNA-binding protein of transcription factors or epigenetic regulation. 34 In our study, we revealed that MIR4435-2HG was mainly localized in the cytoplasm but not in the nucleus, indicating its potential to regulate the expression of downstream gene through the way of 'decoy'.
In our investigation, bioinformatic analysis showed that miR-513a-5p bound to MIR4435-2HG, which was confirmed by qRT-PCR and dual-luciferase reporter assay. To further explore the potential conjugated mRNAs of miR-513a-5p, we collected the data from MiRDB, StarBase, TargetScan and PITA and found that KLF6 was included in the intersection of the databases. KLF6, also called Kruppel-like factor 6, was demonstrated to enhance the activity of mRNAs that maintain carcinogenic transcriptional networks in ccRCC. For instance, Syafruddin SE et al revealed that KLF6 supported the expression of lipid metabolism genes and promoted the expression of PDGFB, which enhanced transcriptional networks by activating mTOR signalling. 35 Bioinformatic analysis showed that miR-513a-5p bound to KLF6, which was also confirmed by qRT-PCR and dual-luciferase reporter assay. The results revealed that miR-513a-5p could interact with KLF6, therefore modulated the expression of KLF6.
To demonstrate that the axis that MIR4435-2HG promoted KLF6 expression by sponging to miR-513a-5p in ccRCC, we designed a rescue experiment to reveal the role of MIR4435-2HG/miR-513a-5p/ KLF6 axis in ccRCC. The results showed that the change in KLF6 expression could reverse the function of MIR4435-2HG on ccRCC in vitro and in vivo. These findings provided the evidence that MIR4435-2HG was a potential diagnostic biomarker and a critical molecular target for tumour progression for ccRCC.

| CON CLUS ION
In conclusion, our study illustrated that lncRNA MIR4435-2HG could serve as an oncogene in ccRCC by facilitating ccRCC cell proliferation and invasion. Mechanistically, we found that MIR4435-2HG directly sponged miR-513a-5p, which was down-regulated in ccRCC, and promoted KLF6 expression by disrupting the interaction between miR-513a-5p and KLF6 in ccRCC. Together, characterization of the MIR4435-2HG/miR-513a-5p/KLF6 axis might provide innovative insights into the prevention and treatment progress for ccRCC.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest that pertain to this work.

AUTH O R CO NTR I B UTI O N S
All authors have read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.