Thirty pairs of RCC specimens and adjacent normal tissues were obtained from patients who underwent surgical treatment from October 2015 to August 2016 in Tai'an City Central Hospital. Written informed consent was obtained prior to resection from patients and the study was approved by the Ethics Committee of Tai'an City Central Hospital.

RCC cell lines 786-O, Caki-1, Ketr-3, RT112 and T24, as well as the normal renal epithelial cells Ect1/E6E7 were purchased from the American Type Culture Collection (ATCC, Gaithersburg MD, USA). The 786-O, Ketr-3, RT-112 and T24 cells were cultured in RPMI-1640 medium (Gibco, Rockville, MD, USA) containing 10% fetal bovine serum (FBS; Gibco). The Caki-1 cells were cultured in McCoy's 5A medium containing 10% FBS, and Ect1/E6E7 cells were cultured in EMEM medium (Gibco) containing 10% FBS. All cells were cultured in incubators at 37°C in a 5% CO₂ atmosphere.

Total RNA was extracted from cells and tissues using the TRIzol kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions and cDNA was synthesized using Reverse Transcription kit (Thermo Fisher Scientific). Quantitative analysis of cDNA (Thermo Fisher Scientific) was performed using a Fluorescence Quantitative PCR kit (Sangon Biotech Co., Ltd., Shanghai, China). The amplification primers used were as follows: MALAT1 sense, 5′-CTCACTAAAGGCACCGAAGG-3′ and antisense, 5′-GGCAGAGAAGTTGCTTGTGG-3′; miR-22-3p sense, 5′-AAGCTGCCAGTTGAAGAACTGTA-3′ and antisense, 5′-CTCGCTTCGGCAGCACA-3′.

Caki-1 cells were seeded in 24-well plates at 1×10⁵ cells/well and were transfected with specified fragments for 24 h, when the cell aggregation rate was over 80%. Mimics/inhibitors specific for miR-22-3p and short hairpin RNA (shRNA)/scramble fragments targeting MALAT1 were designed and purchased from Hanheng Biotechnology Co., Ltd. (Shanghai, China). Recombinant lentiviral vector for knocking out MALAT1 was constructed and named LV-MALAT1 shRNA and the control group was named LV-MALAT1 scramble group.

Caki-1 cells were transfected with shRNA- MALAT1 or MALAT1 scramble, and cell viability was evaluated according to the instructions of the CCK-8 assay kit (Sigma-Aldrich, St. Louis, MO, USA).

Five parallel lines were evenly drawn on the back of the 6-well plate using marker pens and the Caki-1 cells were grown in 6-well plates at a concentration of 2×10⁵ cells/ml. After incubation for 24 h, the tip was scratched perpendicularly to the horizontal line on the back of the 6-well plate. Destroyed cells were rinsed off with phosphate-buffered saline (PBS) and cultured in serum-free medium for another 24 h. Cell migration was observed and imaged at 0 and 24 h with a digital camera (Olympus Corp., Tokyo, Japan).

Protein samples were extracted from cells and tissues with protein lysate, and the protein concentration was assessed using a BCA kit (Pierce, Rockford, IL, USA). The protein was separated by SDS-PAGE and the isolated protein was transferred to the polyvinylidene fluoride (PVDF) membrane by semi-dry method and then was blocked in 5% skim milk at room temperature, for 2 h. The membrane was incubated with the primary antibodies (anti-Ki-67, cat. no. ab16667; anti-PCNA, cat. no. ab29; anti-MMP3, cat. no. ab53015; anti-MIIP, cat. no. ab167197; p-PI3K, cat. no. ab182651; anti-p-AKT, cat. no. ab131443; anti-AKT, cat. no. ab179463; all at the dilution 1:1,000) and the corresponding HRP-conjugated secondary antibodies (cat. nos. ab6728 and ab6721 at the dilution 1:5,000; obtained from Abcam, Cambridge, UK) and was extensively washed several times with PBST. The proteins were detected using a ChemiDoc XRS imaging system and were analysed through Quantity One analysis software (Bio-Rad Laboratories, San Francisco, CA, USA). GAPDH was used as the internal control.

The binding site of MALAT1 on miR-22-3p was predicted through bioinformatic analysis (TargetScan, http://www.targetscan.org/). PCR amplification of MALAT1 fragments contained a fragment of the miR-22-3p binding site, which was inserted into the pMIR-REPORT vector. The mutant plasmid was used as the control group. Caki-1 cells were co-transfected with MALAT1 WT/MALAT1-MUT and/or miR-22-3p mimic for 24 h. Fluorescence intensity was evaluated according to the Dual-Luciferase Reporter kit (Promega Corporation, Madison, WI, USA).

Caki-1 cells were treated with 4% paraformaldehyde for 30 min and 0.5% Triton X-100 for 30 min and then were blocked with 5% goat serum for 2 h at room temperature. The cells were then incubated with the primary antibody (Akt, 1:200; cat. no. ab179463; Abcam) overnight at 4°C. On the second day, the cells were incubated with the corresponding FITC-conjugated secondary antibodies (1:2,000; cat. no. ab6785; Abcam) in the dark for 50 min, and then were stained with DAPI. Cell fluorescence was observed under a fluorescence microscope (Olympus Corp.).

Sixteen male specific-pathogen-free (SPF) nude mice (~20 g) were purchased from the Beijing Experimental Animal Center (Beijing, China). The animal experiments were performed according to the guidelines of the National Institute of Health (NIH). All animal experiments were approved by the Medical Ethics Committee of The Second Hospital Affiliated to Tianjin Medical University (Tianjin, China). An RCC xenograft mouse model was created by subcutaneous injection of 1×10⁷ Caki-1 cells transfected with LV-MALAT1 shRNA or LV-MALAT1 scramble to SPF nude mice. When sacrificed, the weight of mice was ~25 g. The tumor volume was evaluated every 5 days until 30 days after the development of a palpable tumor according to the formula (a × b²)/2, where a and b are the largest diameter and the perpendicular diameter, respectively.

All data were analyzed using the SPSS 19.0 software (IBM Corp., Armonk, NY, USA) and the results were expressed as the mean ± standard deviation (SD). The Student's t-test was used to assess comparisons between two groups. Multiple-group comparisons were made using one-way analysis of variance (ANOVA). P<0.05 was considered to indicate a statistically significant difference.

The expression of MALAT1 and miR-22-3p in RCC tissues and cell lines was detected through qRT-PCR. As displayed in Fig. 1A, the expression level of MALAT1 in the RCC tumor tissue was significantly higher than that in adjacent normal tissues (P<0.01). Concurrently, the expression of MALAT1 in the RCC cell lines was significantly increased compared with the normal renal epithelial cells Ect1/E6E7 (Fig. 1B; P<0.05 and P<0.01). In addition, the expression level of miR-22-3p in the RCC tumor tissues and cell lines was significantly decreased compared with the control tissues and cell line (Fig. 1C and D; P<0.05 and P<0.01). The aberrant expression of MALAT1 and miR-22-3p compared with normal renal epithelial cells indicated a systematic predictable link between MALAT1/miR-22-3p and the progression of RCC.

Previous research has demonstrated that MALAT1 regulates the proliferation and migration of a variety of cancer cells. In the present study, we selected the Caki-1 cell line for the following experiments as it has the relatively highest expression of MALAT1 and the lowest expression level of miR-22-3p in the related cell lines. The expression of MALAT1 was significantly downregulated through transfection with MALAT1 shRNA in Caki-1 cells compared with the scramble group and the control group (Fig. 2A; P<0.01). Transfection with MALAT1 shRNA suppressed cell proliferation compared with MALTA1 scramble group (Fig. 2B; P<0.05 and P<0.01). Furthermore, the wound healing assay exhibited that the migration ability of cells in the MALAT1 shRNA group was much lower than that of the control group (Fig. 2C; P<0.05). The results of the western blot analysis revealed that MALAT1 shRNA significantly suppressed the expression of Ki-67 and proliferating cell nuclear antigen (PCNA) compared with the scramble and the control group (P<0.05) (Fig. 2D). As expected, MALAT1 shRNA significantly inhibited the expression of matrix metalloproteinase-3 (MMP-3) and promoted the expression of migration and invasion inhibitory protein (MIIP) (Fig. 2E; P<0.05). The above-mentioned results indicated that MALAT1 shRNA significantly inhibited the proliferation and migration of RCC cells.

To investigate the related mechanism by which MALAT1 regulates the progression of RCC cells, we found a binding site for MALAT1 on miR-22-3p through bioinformatic analysis (TargetScan, http://www.targetscan.org/) (Fig. 3A). Concurrently, there were marked negative relevant relations between the expression of MALAT1 and miR-22-3p in RCC tissues (Fig. 3B). As displayed in Fig. 3C, MALAT1 shRNA significantly elevated the expression of miR-22-3p in RCC cells (P<0.05), but then this effect was reversed through the co-transfection with miR-22-3p inhibitor (P<0.05). Furthermore, the inhibiting effect of MALAT1 shRNA on the expression of MALAT1 was partially reversed by the co-transfection of miR-22-3p inhibitor, identifying that miR-22-3p inhibitor elevated the level of MALAT1 in turn (Fig. 3D; P<0.05 and P<0.05, respectively). Subsequently, we used the luciferase reporter assay to further examine the targeting relationship between MALAT1 and miR-22-3p. The results demonstrated that the luciferase signal in the lncR-MALAT1 MUT and the lncR-MALAT1 WT group remained at the same level, but the addition of miR-22-3p mimic significantly inhibited the luciferase activity by binding to MALAT1 WT fragments. However, the regulatory relationship disappeared when binding to the MALAT1 MUT fragments, indicating that there existed a targeting relationship between MALAT1 and miR-22-3p (Fig. 3E; P<0.01). Furthermore, miR-22-3p inhibitor significantly reversed the inhibitory effect of MALAT1 shRNA on the proliferation and migration of Caki-1 cells (Fig. 3F and G; P<0.05, P<0.01 and P<0.05). These results indicated that MALAT1 may promote the proliferation and migration of RCC cells by targeting miR-22-3p.

Recent research has indicated that PI3K/Akt signaling pathway is closely related to the proliferation and migration of cancer cells (24,25). We have also demonstrated that MALAT1 shRNA inhibited the proliferation and migration of Caki-1 cells in the above-mentioned experiments. Therefore, we further investigated the effect of MALAT1 shRNA on the PI3K/Akt pathway. As displayed in Fig. 4A, MALAT1 shRNA significantly inhibited the expression of p-PI3K and p-Akt, but the addition of miR-22-3p inhibitor into the MALAT1 shRNA-treated Caki-1 cells significantly reversed the inhibitory effect of MALAT1 shRNA on the expression of p-PI3K and p-Akt (P<0.01, P<0.05). In addition, the expression of Akt in the MALAT1 shRNA group was much lower than that in the control group, indicating that MALAT1 shRNA inhibited the activation of Akt (Fig. 4B). Concurrently, miR-22-3p inhibitor reversed the inhibitory effect of MALAT1 shRNA on Akt accumulation on the cell membrane (Fig. 4B). All these results revealed that MALAT1 shRNA inhibited the activation of PI3K/Akt signaling pathway through the upregulation of the expression of miR-22-3p.

To further validate the above results in vivo, we replicated the xenograft animal model of RCC and explored the effect of MALAT1 shRNA on tumor growth and metastasis. The results demonstrated that MALAT1 shRNA inhibited the growth of RCC tumor and elevated the expression of miR-22-3p in vivo (Fig. 5A and B; P<0.05, P<0.01). Concurrently, the expression of PCNA and MMP-3 in the shRNA-MALAT1 group was significantly lower compared with the control group. The ratio of p-Akt/Akt was also significantly lower than that of the control group (Fig. 5C and D, P<0.01). These results further indicated that MALAT1 shRNA can also inhibit the growth and metastasis of RCC tumors by inhibiting the PI3K/Akt signaling pathway activation in vivo.