Clinical relevance of miR-mediated HLA-G regulation and the associated immune cell infiltration in renal cell carcinoma

Figures & data

Figure 1. Expression of HLA-G and HLA-G-specific miRs in human tumor cell lines. (A) Detection and quantification of HLA-G mRNA. The HLA-G-specific PCR products and different splice variants could be detected in JEG-3, MZ2733RC and MZ2905RC according to Real and co-authors.⁵² (B) Detection of HLA-G protein by Western blot analysis. Western blot analysis of selected cell lines were performed as described in Materials and Methods using the anti-HLA-G mAb MEM-G/4. Staining with the β-actin mAb served as loading control. The cell lines JEG-3 and MZ2733RC revealed HLA-G protein expression. (C) Heterogeneous expression of selected miRs in RCC cell lines. An inverse expression pattern of the HLA-G-specific miR candidates miR-152 and miR-148A and HLA-G protein was detected in the HLA-G⁺ cell lines JEG-3 and MZ2733RC. The HLA-G non-relevant miR-141 did not show an inverse expression pattern. (D) miR-148 family members were enriched and miR-133A only marginal enriched using the miTRAP assay as recently described.³⁴ The results are expressed in absolute copy number and as mean values of three. The HLA-G non-relevant miR-141 (present in the input control) could not be enriched.

Figure 2. Association of miR-152-mediated regulation of HLA-G expression with altered NK and LAK cell cytotoxicity (A) Characterization of the murine NIH/3T3 transfection system expressing human HLA-G (CDS and 3'-UTR) and miR-152 by Western Blot. (B & C) CD107a degranulation assay applying human NK cells (B) and LAK cells (C) as immune effector cells and a murine NIH/3T3-based transfection system expressing human HLA-G and miR-152 as target cells. The NIH/3T3 transfectants are lysed HLA-G dependent by the immune effector cells (E:T ratio is 10:1).

Figure 3. Inverse correlation of HLA-G and HLA-G relevant miR expression in RCC tumor lesions. (A) Representative immunohistochemical staining for a HLA-G⁻ RCC lesion (sample II5, WHO grade: G2). (B) Representative immunohistochemical staining for a HLA-G⁺ RCC lesion (sample II39, WHO grade: G2). (C, D, E) Quantification of the expression of miR-148A (C), miR-152 (D) and miR-133A (E) by qPCR in 36 HLAG⁻ and 36 HLA-G⁺ RCC lesions (listed in Table 1), respectively. The absolute copy number determination is visualized as Box–Whisker plot. Statistical analyses were performed as described in Materials and Methods.

Table 1. Characteristics of the 72 RCC lesions (36 HLA-G⁻ and 36 HLA-G⁺) analyzed for miR-148A, miR-152 and miR-133A

Tumor sample	HLA-G protein		RCC Subtype	WHO grade	Metastases
	Membranous “staining intensity (percentage of positive tumor cells)”	Cytoplasmic “Staining intensity (percentage of positive tumor cells)”
I 1	0(0)	0(0)	Chromophobe	G2	n.d.
I 3	0(0)	0(0)	Chromophobe	G2	n.d.
I 5	0(0)	0(0)	Clear cell	G2	no
I 6	0(0)	0(0)	Clear cell	G1	n.d.
I 7	0(0)	0(0)	Clear cell	G3	n.d.
I 9	0(0)	0(0)	Chromophobe	G2	n.d.
I 10	0(0)	0(0)	Clear cell	G2	n.d.
I 12	0(0)	0(0)	Chromophobe	G2	n.d.
I 14	0(0)	0(0)	Clear cell	G2	n.d.
I 26	0(0)	0(0)	Chromophobe	G2	n.d.
I 32	0(0)	0(0)	Clear cell	G2	n.d.
I 41	0(0)	0(0)	Papillary/clear cell	n.d.	n.d.
I 42	0(0)	0(0)	Chromophobe	G2	n.d.
I 51	0(0)	0(0)	Clear cell	G2	n.d.
I 56	0(0)	0(0)	Clear cell	G2	n.d.
I 59	0(0)	0(0)	Papillary	G2	n.d.
II 2	0(0)	0(0)	Clear cell	G2	n.d.
II 5	0(0)	0(0)	Clear cell	G2	no
V 13	0(0)	0(0)	Clear cell	G3	n.d.
V 15	0(0)	0(0)	Clear cell	G2	n.d.
V 19	0(0)	0(0)	Chromophobe	G2	n.d.
V 20	0(0)	0(0)	Clear cell	G3	yes
V 21	0(0)	0(0)	Clear cell	G2	n.d.
V 28	0(0)	0(0)	Clear cell	G2	n.d.
V 35	0(0)	0(0)	Clear cell	G3	n.d.
V 51	0(0)	0(0)	Chromophobe	G2	n.d.
V 54	0(0)	0(0)	Clear cell	G2	no
VI 3	0(0)	0(0)	Clear cell	G2	no
VI 5	0(0)	0(0)	Clear cell	G3	no
VI 7	0(0)	0(0)	Sarcomatoid	G3	no
VI 8	0(0)	0(0)	Clear cell	G2	no
VI 9	0(0)	0(0)	Clear cell	G2	no
VI 10	0(0)	0(0)	Chromophobe	G2	no
VI 12	0(0)	0(0)	Clear cell	G2	no
VI 13	0(0)	0(0)	Clear cell	G2	no
VI 20	0(0)	0(0)	Clear cell	G3	no
I 8	2(100)	2(80)	Clear cell	G3	n.d.
I 25	3(100)	2(80)	Clear cell	G2	n.d.
I 29	3(100)	2(100)	Clear cell	G2	n.d.
I 37	3(100)	2(90)	Clear cell	G2	n.d.
I 44	3(100)	3(100)	Clear cell	G3	n.d.
I 46	3(100)	−	Papillary/clear cell	G2	n.d.
II 4	3(100)	2(80)	n.o.s	n.d.	n.d.
II 10	3(100)	1(100)	Clear cell	G3	no
II 39	3(100)	2(100)	Clear cell	G2	no
II 58	3(80)	2(40)	Clear cell	G2	n.d.
III 2	3(100)	2(100)	Clear cell	G3	n.d.
III 11	3(80)	2(80)	Clear cell	G3	n.d.
III 20	3(100)	2(50)	Clear cell	G3	n.d.
III 31	3(100)	2(40)	Clear cell	G2	yes
III 33	3(90)	3(90)	Clear cell	G2	n.d.
III 36	3(90)	2(90)	Clear cell	G3	n.d.
III 39	3(90)	3(50)	Clear cell	G3	n.d.
III 45	3(100)	2(80)	Clear cell	G2	n.d.
IV 2	3(100)	3(100)	Clear cell	G3	yes
IV 10	3(100)	2(80)	Clear cell	G2	n.d.
V 16	3(100)	−	Clear cell	G1	n.d.
V 24	3(100)	2(50)	Clear cell	G2	n.d.
V 26	3(100)	1(100)	Clear cell	G2	n.d.
V 39	3(100)	3(100)	Clear cell	G2	n.d.
V 43	3(70)	−	Clear cell	G2	n.d.
V 55	3(100)	1(100)	Clear cell	G3	yes
V 56	3(100)	2(100)	Clear cell	G3	yes
VI 16	3(100)	2(100)	Clear cell	G3	yes
VI 23	3(80)	2(60)	Clear cell	G2	no
VI 26	3(100)	2(100)	Clear cell	G2	no
VI 46	3(100)	2(80)	Clear cell	G3	n.d.
VII 1	3(100)	2(100)	Clear cell	G2	yes
VII 21	3(100)	−	Clear cell	G1	n.d.
VIII 1	3(100)	−	Clear cell	G2	n.d.
VIII 24	3(100)	1(100)	Papillary	G2	n.d.
VIII 27	3(100)	1(50)	Clear cell	G2	n.d.

The expression of HLA-G as well as the respective clinical parameters were analyzed in 72 selected RCC lesions (36 HLA-G⁻ and 36 HLA-G⁺) using immunohistochemistry and qPCR, respectively. Scoring was performed as described in Materials and Methods. Staining intensity of HLA-G positive tumors was categorized to negative (0), weak (1), moderate (2) and strong (3) staining. Percentages of HLA G positive tumor cells are listed in brackets.

Table 3. Correlation of HLA-G expression with WHO grade

	HLA-G expression	WHO grade G1 [%]	WHO grade G2 [%]	WHO grade G3 [%]
HLA-G membranous	HLA-G negative	13.4	63.6	23.0
	HLA-G weak	16.2	71.6	12.2
	HLA-G moderate	17.1	63.2	19.7
	HLA-G strong	10.6	56.1	33.3
HLA-G cytoplasmic	HLA-G negative	17.5	62.3	20.1
	HLA-G weak	8.6	69.1	22.2
	HLA-G moderate	6.2	70.8	23.1
	HLA-G strong	15.8	36.8	47.4

Distribution of membranous and cytoplasmic HLA-G expression over different categories of WHO grade in RCC tumors, as found in HLA-G immunohistochemistry. Only staining intensity is shown. Strong cytoplasmic expression significantly accumulates in G3 tumors (p = 0.014).

Figure 4. Immunohistochemical staining of tumor-infiltrating lymphocytes in RCC lesions (TMAs). (A) : Representative immunohistochemical staining of CD3⁺ and CD8⁺ TILs on a HLA-G⁻ and HLA-G⁺ RCC lesion revealing statistical significant higher numbers of CD3⁺ and CD8⁺ T lymphocytes in HLA-G⁺ tumors. (B) Mean numbers of CD3⁺, CD8⁺, CD4⁺, CD56⁺ and CD69⁺ cells per field of view in 36 HLA-G⁻ and 36 HLA-G⁺ RCC lesions (listed in Table 1) visualized as Box–Whiskers-Blots. Only CD3⁺ (p < 0,001) and CD8⁺ (p < 0,001) TILs showed significant differences in the presence in the HLA-G⁺ and HLA-G⁻ cohorts.

Figure 5. Correlation between the presence or absence of TILs and the HLA-G expression with disease-specific survival of the patients visualized as Kaplan–Meier-Plots. Kaplan–Meier-Plots of disease-specific survival in relation to HLA-G expression and tumor-infiltrating lymphocytes. The presence of CD4⁺ cells was associated (but not significant; p = 0.097) with better disease-specific survival. The markers HLA-G, CD3, CD8, CD56, CD4, FOXP3, CD 69 and CD 25 showed no significant correlation to disease-specific survival.

Table 4. Summary of the primer sequences and annealing temeratures

Primer	Application	Sequence (5′→3′)	Condition	Reference
141-RT-Rkt	Stem-loop primer	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCATCT	42°C
141 PCR fw	qRT-PCR/PCR	GCCCTAACACTGTCTGGTAA	60°C
148aRT-Rkt	Stem-loop primer	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAAAG	42°C
148a PCR fw	qRT-PCR/PCR	GCCCTCAGTGCACTACAGA	60°C
152RT-Rkt	Stem-loop primer	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCAAGT	42°C
152 PCR fw	qRT-PCR/PCR	GCCCTCAGTGCATGACAGA	60°C
148bRT-Rkt	Stem-loop primer	GTCGTATCCAGTGCAGGTCCGAGGTATTCGCACTGGATACGACACAAAG	42°C
148b PCR fw	qRT-PCR/PCR	GCCCTCAGTGCATCACAGGA	60°C
133ART-Rkt	Stem-loop primer	GTCGTATCCAGTGCAGGTCCGAGGTATTCGCACTGGATACGACCAGCTG	42°C
133A PCR fw	qRT-PCR/PCR	GCCCTTTGGTCCCCTTCAAA	60 °C
541RT-Rkt	Stem-loop primer	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGTCCA	42°C
541 PCR fw	qRT-PCR/PCR	GCCCTGGTGGGCACAGAATC	60°C
stem loop reverse primer	qRT-PCR/PCR	GTGCAGGGTCCGAGGT	60°C
HLAGEX2se	PCR	GGGTCGGGCGGGTCTCAA	62°C	[43]
HLAGEx2as	PCR	TCCGTGGGGCATGGAGGT	62°C	[43]
HLAGEx3se	PCR	CCCAGACCCTCTACCTGGGAGA	65°C	[43]
HLAGEX3as	PCR	CTCTCCTTGTGCTAGGCCAGGCTGGGAGG	65°C	[43]
HLAGEX4se	PCR	CCATGAGAGATGCAAAGTGCT	55°C	[43]
HLAGEx4as	PCR	TGCTTTCCCTAACAGACATGAT	55°C	[43]
SeqHLAGEx2	Sequencing	ATGGAGGTGGGGGTCGTGATCT		[43]
SeqHLAGEX3	Sequencing	GGTGGGTCCGGGCGAGGGCGAGGCT		[43]
SeqHLAGEX4	Sequencing	CCATGAGAGATGCAAAGTGCT		[43]
G.257	PCR	GGAAGAGGAGACACGGAACA	61°C	[49]
G.1225	PCR	TGAGACAGAGACGGAGACAT	61°C	[49]
HLA-G E6/E8 fw	qRT-PCR/PCR	TTGCTGGCCTGGTTGTCCTT	60°C	[43]
HLA-G E6/E8 rev	qRT-PCR/PCR	TTGCCACTCAGTCCCACACAG	60°C	[43]
HPRT fw	qRT-PCR/PCR	CCCTGGCGTCGTGATTAGTGATGAT	60°C
HPRT rev	qRT-PCR/PCR	TGCTTTGATGTAATCCAGCAGGTCAGC	60°C
3'-UTR HLA-G fw	Cloning	AAAACTAGTAAACAGCTGCCCTGTGT	60°C
3'-UTR HLA-G rev	Cloning	AAAACGCGTAAAGTTCTCATGTCTTCCATTT	60°C
3'-UTR β-Actin fw	Cloning	AAAACTAGTAGGCGGACTATGACTTAGTT	60°C
3'-UTR β-Actin rev	Cloning	AAAACGCGTACTGGTCTCAAGTCAGTGTA	60°C
KlonmiR-152fw	Cloning	AAACTCGAGTTCTGGGTCCGTTTGGAGT	60°C
KlonmiR-152rev	Cloning	AAAGAATTCGTTCTGCCCAGCCCT	60 °C
KlonmiR-541fw	Cloning	AAACTCGAGAGAATTTCCAGAAGCAACAG	60°C
KlonmiR-541rev	Cloning	AAAGAATTCCCAGGATCCCTCAAAGAGTA	60°C
HLA-GBamHIHLA-Gfw	Cloning	AAAGGATCCCCAAGGATGGTGGTCATGG	60°C
HLAGmiTrap fw	Cloning	AAAGAATTCAAACAGCTGCCCTGTGT	60°C
newmiTRAPrev	Cloning	AAACTCGAGCTCTCAAATTTCAGGAATC	60°C
mutPrimerfw	Site-directed mutagenesis	CTCAAATTTGTGGTCCACTCGAGCTATAACTTACTTCTGTATT
mutPrimerrev	Site-directed mutagenesis	AATACAGAAGTAAGTTATAGCTCGAGTGGACCACAAATTTGAG
HLAGrtpcrDBfw	qRT-PCR/PCR	TTGCTGGCCTGGTTGTCCTT	60°C	[50]
HLAGrtpcrDBrev	qRT-PCR/PCR	TTGCCACTCAGTCCCACACAG	60°C	[50]
Forward GAPDH	qRT-PCR/PCR	CAAGGTCATCCATGACAACTTTG	60°C	Fermentas
Reverse GAPDH	qRT-PCR/PCR	GTCCACCACCCTGTTGCTGTAG	60°C	Fermentas
152decoyse1	Hybridization/cloning	GATCCGGACGGCGCTAGGATCATCAACCCAAGTTCTGTCATGACTGACAAGTATT
152decoyse2	Hybridization /cloning	CTGGTCACAGAATACAACCCAAGTTCTGTCATGACTGACAAGATGATCCTAGCGCCGTCTTTTTTG
152decoyas2	Hybridization /cloning	GAATTCAAAAAAGACGGCGCTAGGATCATCTTGTCAGTCATGACAGAACTTGGGTTGTATTCTGTG
152decoyas1	Hybridization /cloning	ACCAGAATACTTGTCAGTCATGACAGAACTTGGGTTGATGATCCTAGCGCCGTCCG

Table 2. Patients' and tumor characteristics

Patients' and tumor characteristics
Gender	Female	166/445 (37.3%)
	Male	279/445 (62.7%)
Age at diagnosis	Mean	63,6
	Median	65
	Minimum	23
	Maximum	92
Age group	≤ 65	196 (43.3%)
	66+	189 (41.7%)
Status	Tumor- specific death	35/380 (9.2%)
	Death of different cause	66/380 (17.4%)
	Alive at last contact (censored)	279/380 (73.45%)
Time to last contact (months)	Minimum	0
	Maximum	160
	Mean	44.7
	Median	36
RCC subtype	Clear cell	345/440 (78.4%)
	Papillary	49/440 (11.1%)
	Chromophobe	29/440 (6.6%)
	Others	17/440 (3.9%)

Summary of the main patients' and tumor characteristics of the tissue samples of the tissue microarray analyzed.

Real LM, Cabrera T, Collado A, Jimenez P, Garcia A, Ruiz-Cabello F, Garrido F. Expression of HLA G in human tumors is not a frequent event. Int J Cancer 1999; 81:512-8; PMID:10225437; http://dx.doi.org/10.1002/(SICI)1097-0215(19990517)81:4%3c512::AID-IJC2%3e3.0.CO;2-O

 PubMed Web of Science ®Google Scholar

Braun J, Misiak D, Busch B, Krohn K, Huttelmaier S. Rapid identification of regulatory microRNAs by miTRAP (miRNA trapping by RNA in vitro affinity purification). Nucleic Acids Res 2014; 42(8):e66; PMID:24510096; http://dx.doi.org/10.1093/nar/gku127

 PubMedGoogle Scholar