NL2037006A - Method for inhibiting slc16a8 from inducting colorectal cancer under hypoxia and application - Google Patents

Method for inhibiting slc16a8 from inducting colorectal cancer under hypoxia and application Download PDF

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NL2037006A
NL2037006A NL2037006A NL2037006A NL2037006A NL 2037006 A NL2037006 A NL 2037006A NL 2037006 A NL2037006 A NL 2037006A NL 2037006 A NL2037006 A NL 2037006A NL 2037006 A NL2037006 A NL 2037006A
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slc16a8
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Peng Hong
Huang Shan
Zhu Yanbin
Meng Changyuan
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The Affiliated Nanchong Center Hospital Of North Sichuan Medical College
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Abstract

U I T T R E K S E L Disclosed is a method for inhibiting SLC16A8 from inducing colorectal cancer (CRC) under hypoxia and an application, falling within the technical field of CRC. It is found from the above studies that hypoxia can accelerate proliferation, epithelial— 5 mesenchymal transition (EMT), metastasis, angiogenesis, and glycolysis of CRC cells, and SLCl6A8 silencing can reverse these processes. In addition, inhibiting SLCl6A8 can inhibit the growth, EMT, glycolysis of a tumor in a CRC nude mouse model and change the pathogeny structure of the tumor. The malignant biological 10 properties of SLCl6A8 participating in CRC cells under hypoxia provides a preliminary theoretical basis for the treatment and diagnosis of CRC.

Description

P1975 /NLpd
METHOD FOR INHIBITING SLC16A8 FROM INDUCTING COLORECTAL CANCER
UNDER HYPOXIA AND APPLICATION
Technical field
The present invention relates to the technical field of colo- rectal cancer (CRC), in particular to a method for inhibiting
SLC16A8 from inducing CRC under hypoxia and an application.
Background
CRC is a common malignant tumor of the gastrointestinal tract worldwide, and CRC in most cases is not caused by a single factor.
Most CRC patients have no obvious symptoms in the early stage, and about 40%-50% of the patients are in advanced stage with metasta- sis when the diagnosis is confirmed. The most common sites of me- tastasis are livers, peritoneums and lungs. Currently, the treat- ment is mostly based on radiotherapy, but the recurrence and me- tastasis rates after surgery are higher. Therefore, studying the development mechanism of CRC is convenient for the accurate diag- nosis, accurate treatment and early prevention of the disease, and is an effective way to reduce the mortality rate caused by CRC.
Cancer cells can realize growth, survival, proliferation and long-term maintenance through metabolic reprogramming. This metab- olism occurs when mitochondrial function is impaired and the cells provide energy by enhancing anaerobic metabolism, and the glucose is metabolized to pyruvate which is transformed to lactic acid by lactate dehydrogenase to be expelled out of the cells, and this phenomenon is known as the Warburg effect. Tumor cells proliferate faster and have a higher rate of metabolic uptake compared to healthy cells. This metabolism of tumor cells leads to significant depletion of metabolites in the local microenvironment, which re- sults in resource constraints. In addition, waste products from tumor cell metabolism may hinder the growth of neighboring cells, and excess lactic acid produces an acidic tumor microenvironment that promotes tumor migration and invasion. Therefore, molecules that attenuate the Warburg effect in CRC cells play a key role in the treatment of CRC.
The SLClé gene family consists of 14 members and is also known as monocarboxylic acid transporter (MCT) family. Members of the SLC16 family are involved in a variety of metabolic pathways including energy metabolism, gluconeogenesis, t-lymphocyte activa- tion, intestinal metabolism, spermatogenesis, pancreatic p-cell dysfunction, thyroid hormone metabolism, and drug transport in brain, skeletal muscle, heart, and tumor cells. SLC16A8, as the gene family, is primarily responsible for the transport of the monocarboxylic acid metabolites of pyruvate, lactic acid-lactic acid, and ketone bodies. SLCL16A8 can also be involved in intercel- lular lactic acid transmembrane transport. However, it is unclear whether SLC16A8 promotes CRC by altering the Warburg effect.
Summary
An objective of the present invention is to provide a method for inhibiting SLC16A8 from inducing CRC under hypoxia and an ap- plication, determining that SLCl6A8 can accelerate the prolifera- tion, epithelial-mesenchymal transition (EMT), metastasis, angio- genesis and glycolysis of CRC cells under hypoxia, and inhibition of SLC16A8 may weaken the Warburg effect and thus achieve the therapeutic effect of CRC.
In order to realize the above objective, the present inven- tion provides an application of inhibiting SLC16A8 in the prepara- tion of a medication for the treatment of hypoxia-induced CRC.
Therefore, the present invention adopts the method for inhib- iting SLC16A8 from inducing CRC under hypoxia and an application described above to determine that SLC16A8 can accelerate the pro- liferation, EMT, metastasis, angiogenesis and glycolysis of CRC cells under hypoxia, and inhibition of SLC16A8 may weaken the War- burg effect and thus achieve the therapeutic effect of CRC.
Technical solutions of the present invention are described further in detail by reference to the example below.
Detailed description
Technical solutions of the present invention are described further by reference to an example below.
Unless otherwise defined, technical or scientific terms used in the present invention have the ordinary meaning understood by those of ordinary skill in the field to which the present inven-
tion belongs.
Example 1
Sl: hypoxia-induced CRC cells were constructed by the follow- ing steps. (I) Acquisition of CRC tissue samples
CRC tissues and paired paracancerous tissues were collected from August 2015 to June 2016.
Inclusion criteria: no preoperative radiotherapy or chemo- therapy; and CRC confirmed by postoperative pathological examina- tion.
Exclusion criteria: incomplete medical records; preoperative neoadjuvant therapy; concomitant other systemic malignant tumors; and severe dysfunction of other organs.
All tissues were rapidly stored at -80°C. All patients signed informed consent forms approved by the Ethics Committee of the hospital. The study complied with medical ethics regulations. (IT) CRC cells were cultured in a Roswell Park Memorial In- stitute (RPMI)-1640 medium with 10% fetal bovine serum (FBS) (Sig- ma) at 37°C and 5% COs. (ITI) Cells treatment
CRC cells were transfected with SLC16A8 siRNA#1, SLCI6A8 siR-
NA#2, SLC16A8 siRNA#3 and negative control (NC) after 1, 6 and 12 h of hypoxia, respectively. The above oligonucleotides were trans- fected via liposome 3000 for 48 h.
Experimental testing (I) gRT-PCR
Total RNAs were isolated from cells and milled tissues using
Trizol (Invitrogen), and the extracted part of RNAs were treated with RNaseR (2 U/ug) for 10 min at 37°C, and the control was treated with an equal amount of double-distilled water. After re- verse transcription, the gene was amplified, and the amplified gene was calculated and analyzed by 27%%°%, (IT) Cell proliferation
After CRC cells (4x10° per well to perform purpose ul CCK-8 experiment (Tokyo, Japan)) were transfected for 48 h, CCK-8 was added for 2 h to detect an optical density (OD) value at 450 nm.
After EdU staining was performed, the CRC cells were fixed with 4%
paraformaldehyde, and the fixed CRC cells were destained with 2 mg/mL glycine and 0.5% TritonX-100 for 10 min. Samples were photo- graphed under a fluorescence microscope after the cells are added to the EdU cell proliferation detection kit (GV-CA1170) to be made into sections. (III) Transwell
Transfected CRC cells were resuspended in a serum-free medium for migration. 5x10° cells were added to an upper chamber of each
Transwell (8 um, Corning), and 500 pL of medium containing 10% FBS was placed in a lower chamber. After routine culture was performed for 24 h, the medium was removed and the cells were washed 3 times, and the washed cells were fixed with 4% paraformaldehyde for 20 min. The liquid in the upper chamber was removed with a cotton swab, and the cells were stained with 1% crystal violet for 10 min. After the cells were washed, 5 chambers were randomly se- lected to be photographed under the microscope to obtain the re- sults. The infected, pre-cooled matrix gel was diluted with a se- rum-free medium at a ratio of 1:8. 80 pL of diluted matrix gel was applied to the upper chambers across the wells, and evenly covered the bottom of the chambers for 60 min at 37°C to form a gel. The remaining steps were the same as in the migration experiment. (IV) Tube formation
Treated CRC cells were co-cultured with human umbilical vein endothelial cells (HUVECs). The matrix gel, after being thawed in advance at 4°C for staying overnight, was diluted with FBS-free medium. 2x10° cells were inoculated on an surface of the matrix gel of 37°C for placing for 24 h. The results were recorded using an inverted microscope. (V) Glucose and lactic acid content determination
According to the specification of Sigma-Aldrich, the glucose consumption and lactic acid synthesis levels in CRC cells were monitored using a glucose kit and a lactic acid kit. (VI) Extracellular acidification rate (ECAR) detection
On this basis, ECAR of CRC cells was determined using the seahorse XF glycolysis on the seahorse XFe96 extracellular flux analyzer or cell water schizosome stress test kit (Seahorse Bio- science).
(VII) Western blot
Total proteins were collected by radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) and monitored with bi- cinchoninic acid (BCA) kit (Invitrogen). Proteins (40 ug) were 5 transferred onto a polyvinylidene fluoride (PVDF) membrane (mi- croporous membrane) after being isolated in a sodium dodecyl sul- fate polyacrylamide gel electrophoresis (SDS-PAGE) gel. After be- ing sealed, the membrane was exposed to primary antibody (Abcam) at 4°C overnight, followed by treating with secondary antibody (Abcam) for 1 h, and developing by dropwise adding with an en- hanced chemiluminescence (ECL) color development solution. After
ECL chemiluminescence was performed, proteins were developed on a gel-imager (Bio-Rad). (VIII) In vivo xenograft model
CRC cells transfected with shSLC16A8 or control constructs were subcutaneously implanted into the right sides of individual mice (5x10%/mouse), and after the tumors were approximately 100 mm, the mice were injected intra-tumorally with shCTRL or shSLC16A8°, and the tumors were monitored with a vernier caliper every three days. Tumor volumes were measured weekly (volume = length x width)? x 1/2). (IX) H&E staining
CRC tissues of mice were fixed with 4% paraformaldehyde, the fixed CRC tissues were dehydrated with gradient ethanol, the dehy- drated CRC tissues were embedded in paraffin, and the embedded CRC tissues were sliced into 4 um sections. After baking, the sections were dewaxed and hydrated with xylene and gradient alcohol, cell nucleus were stained with hematoxylin, cytoplasm were stained with eosin, ethanol dehydration and xylene transparent were performed, and finally the sections were sealed with neutral resin. Morpho- logical changes of brain tissues were observed under a microscope. {X) Immunohistochemical (IHC) test
Paraffin sections were subjected to microwave antigenic heat repair with 0.01 mol/L sodium citrate, and endogenous enzymes were blocked by incubation with 3% hydrogen peroxide. After washing, the sections were closed with 5% bovine serum albumin (BSA) for 30 min. The closed sections were incubated with goat secondary anti-
body (Abcam) for 1 h after being incubated with diluted primary antibody (Ki-67, Abcam) overnight at 4°C. The incubated sections were sealed with neutral resin after being restained with diamino- benzidine (DAB) and hematoxylin. The staining results were ob- served under a microscope. Five high magnification chambers (x200) per mouse were selected for IHC analysis.
The measured data were analyzed using SPSS22.0 software (SPSS, Inc.), and student t-test or ANOVAP < 0.05 were used to in- dicate statistically significant results.
The results were as follows. PCR in cancerous and paracan- cerous tissues of clinical CRC patients showed that SLC16A8 was significantly up-regulated in cancerous tissues. Online databases showed that patients with high expression of SLC16A8 had signifi- cantly poorer prognosis. IHC results showed that SLC16A8 expres- sion was up-regulated in CRC tissues. The expression level of
SLC16A8 was positively correlated with the expression of hypoxia- inducible factor (HIF)-la, suggesting that SLC16A8 may be involved in CRC through HIF-lo-mediated hypoxia.
The expression levels of SLC16A8 in four CRC cell lines were detected by gPCR, and the mechanism of action of SLC16A8 and HIF- la in CRC was clarified. The highest expression of SLC16A8 was found in LoVo and RKO cell lines. Subsequently, hypoxia was ap- plied to LoVo and RKO cell lines, and HIF-lx was maximum at 12 h, while SLC16A8 was maximum at 24 h. Subsequent experiments were performed under selected hypoxic conditions of 12 h. The results of ECAR showed that the ECAR of CRC cell lines was significantly increased after hypoxia. Lactic acid detection experiments also showed that the extracellular lactic acid level gradually in- creased with the increase of hypoxia time. Western blot results showed that the expression of the key enzymes in metabolic repro- gramming, PKM2 and PDHA, gradually increased with the increase of hypoxia time. Meanwhile, glucose consumption gradually increased.
The expression of RKO under different hypoxia time was de- tected to clarify the malignant biological behavior of CRC cell lines RKO and LoVo under hypoxic conditions. The cell prolifera- tion activity was significantly enhanced with the prolongation of hypoxia time and was positively correlated with time. The further
Transwell chamber experiments showed that hypoxia also enhanced the migration and invasion ability of RKO and LoVo cells and re- sulted in up-regulation of the expression levels of E-calcium dherin, N-cadherin, and vimentin. CRC cells and endothelial cells from each group were co-cultured. The results showed that the an- giogenic capacity of the corresponding endothelial cells gradually increased with the prolongation of hypoxia time.
The siRNA targeting SLC 16 A8 was synthesized to further ex- plore the mechanism of action of SLC16A8 in CRC cells. After
SLC16A8 siRNA was transfected into RKO and LoVe cells, qPCR and
Western blot results showed that all three siRNAs inhibited the expression of SLC16A8. siRNA #2 had the highest knockdown effi- ciency, so siRNA #2 will be selected for subsequent experiments.
Hypoxia-treated CRC cells were further interfered by SLC16AS8 to clarify the regulation of SLC16A8-mediated metabolic reprogram- ming of tumors by hypoxia. SLC16A8 siRNA significantly down- regulated the up-regulation of hypoxia treatment-induced SLC16A8 expression. The results of ECAR showed that the modification of
SLC16A8 siRNA could alleviate hypoxia-induced extracellular acidi- fication, along with a significant decrease in extracellular lac- tic acid content. Meanwhile, SLC16A8 siRNA reversed the signifi- cant up-regulation of hypoxia-induced PKM2 and LDHA expression, and glucose consumption was suppressed.
The disruption of SLC16A8 in hypoxia-treated CRC cells iden- tified a role for hypoxia in SLCléA8-mediated regulation of EMT.
SLC16A8 siRNA significantly inhibited the increase in prolifera- tive activity induced by hypoxia treatment. SLC16A8 siRNA signifi- cantly reversed the enhanced migration and invasion of RKO and
LoVo cells induced by hypoxia, along with changes in expression of
E-Cadherin, n-cadherin and vimentin. Finally, each group of CRC cells were co-cultured with vascular endothelial cells. Hypoxia- induced CRC cells could significantly induce vascular endothelial cells to form tubes; and in contrast, SLC16A8 siRNA treatment of
CRC cells significantly reversed the ability of endothelial cells to form tubes.
The effect of SLC16A8 on tumor growth was investigated by es- tablishing a nude mouse model with tumors. Down-regulation of
SLC16A8 gene significantly inhibited tumor growth and significant- ly suppressed tumor weight. Western blot and IHC results showed that knockdown of SLC16A8 effectively suppressed the expression level of SLC16A8 in tumor tissues. Meanwhile, the down-regulation of SLC16A8 gene resulted in suppression of tumor proliferation signals and enhancement of apoptosis signals, along with a dra- matic decrease in serum lactic acid content. Western blot results showed that the expression of EMT-related proteins was changed.
The expression level of e-cadherin was up-regulated in the SLC16A8 knockout group, while the expression levels of n-cadherin and vi- mentin were significantly down-regulated. The expression of the key metabolic reprogramming enzymes PKM2 and LDHA was also signif- icantly down-regulated.
Therefore, the present invention adopts the method for inhib- iting SLC16A8 from inducing CRC under hypoxia and an application described above, determining that SLC16A8, as an oncogene, can ac- celerate the proliferation, EMT, metastasis, angiogenesis and gly- colysis of CRC cells under hypoxia. The inhibition of SLC16A8 may weaken the Warburg effect and thus achieve the therapeutic effect of CRC.
Finally, it is to be noted that: the example described above is merely illustrative of the technical solutions of the present invention, and not to limit the present invention. Although the present invention is described in detail by reference to the pre- ferred example, it is to be understood by those ordinary skilled in the art that: the technical solutions of the present invention can still be modified or replaced equivalently, and these modifi- cations or equivalent substitutions can not make the modified technical solution out of the spirit and scope of the technical solutions of the present invention.

Claims (4)

CONCLUSIESCONCLUSIONS 1. Werkwijze voor het remmen van SLC16A8 voor het induceren van colorectale kanker (CRC) onder hypoxie, omvattende de volgende stappen: Sl: construeren van door hypoxie geïnduceerde CRC-cellen; S2: uitvoeren van kwantitatieve omgekeerde transcriptie polymera- sekettingreactie (gRT-PCR), celproliferatie en celtranswell-test op de CRC-cellen; S3: uitvoeren van buisvormingstest, glucose- en melkzuurgehaltebe- paling, detectie van extracellulaire verzuring (ECAR) en Western blot bepaling op de CRC-cellen; S4: transplanteren van de CRC-cellen in een muismodel en vooraf uitvoeren van hematoxyline- en eosinekleuring (H&E); S5: uitvoeren van een immunohistochemische (IHC) test; en S6: analyseren van de resultaten van een CRC-celtest.A method of inhibiting SLC16A8 to induce colorectal cancer (CRC) under hypoxia, comprising the following steps: S1: constructing hypoxia-induced CRC cells; S2: performing quantitative reverse transcription polymerase chain reaction (gRT-PCR), cell proliferation and cell transwell assay on the CRC cells; S3: performing tube formation test, glucose and lactic acid content determination, detection of extracellular acidification (ECAR) and Western blot determination on the CRC cells; S4: transplanting the CRC cells into a mouse model and performing hematoxylin and eosin (H&E) staining beforehand; S5: performing an immunohistochemical (IHC) test; and S6: analyzing the results of a CRC cell test. 2. Werkwijze voor het remmen van SLC1l6A8 voor het induceren van CRC onder hypoxie volgens conclusie 1, waarbij in S1 de specifieke stappen voor het construeren van de CRC-cellen als volgt zijn: S11: verkrijgen van CRC weefselmonsters; S12: kweken van de CRC-cellen; en 513: behandelen van de CRC-cellen.The method of inhibiting SLC116A8 to induce CRC under hypoxia according to claim 1, wherein in S1, the specific steps for constructing the CRC cells are as follows: S11: obtaining CRC tissue samples; S12: CRC cell culture; and 513: treating the CRC cells. 3. Werkwijze voor het remmen van SLC16A8 voor het induceren van CRC onder hypoxie volgens conclusie 1, waarbij in S2 de specifieke inhoud voor de celtest als volgt is: S21: gRT-PCR: isoleren van totale ribonucleïnezuren (RNA's) uit cellen en gemalen weefsels met Trizol, en uitvoeren van berekening en analyse met 2 AACT; S22: celproliferatie: snijden van CRC-cellen in coupes na trans- fectie en fotograferen van monsters onder een fluorescentiemicro- scoop; en S23: celtranswell: opnieuw in suspensie brengen van getransfec- teerde CRC-cellen in een serumvrij medium.The method of inhibiting SLC16A8 to induce CRC under hypoxia according to claim 1, wherein in S2 the specific content for the cell assay is as follows: S21: gRT-PCR: isolation of total ribonucleic acids (RNAs) from cells and ground tissues with Trizol, and performing calculation and analysis with 2 AACT; S22: cell proliferation: sectioning CRC cells after transfection and photographing samples under a fluorescent microscope; and S23: cell transwell: resuspension of transfected CRC cells in serum-free medium. 4. Toepassing van SLC16A8 om CRC onder hypoxie te induceren, ten gebruike, onder hypoxie, bij de bepaling van SLC16A8, in de ver- snelling van proliferatie, epitheliale-mesenchymale transitie (EMT), metastase, angiogenese en glycolyse van CRC-cellen, en bij de remming van SLC16A8 om Warburg-effect te verzwakken.4. Use of SLC16A8 to induce CRC under hypoxia, for use, under hypoxia, in the determination of SLC16A8 in the acceleration of proliferation, epithelial-mesenchymal transition (EMT), metastasis, angiogenesis and glycolysis of CRC cells, and in the inhibition of SLC16A8 to attenuate Warburg effect.
NL2037006A 2024-02-12 2024-02-12 Method for inhibiting slc16a8 from inducting colorectal cancer under hypoxia and application NL2037006B1 (en)

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Non-Patent Citations (2)

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JAVAN BITA ET AL.: "Constructing a novel hypoxia-inducible bidirectional shRNA expression vector for simultaneous gene silencing in colorectal cancer gene therapy", CANCER BIOTHERAPY & RADIOPHARMACEUTICALS, vol. 33, no. 3, 1 April 2018 (2018-04-01), US, pages 118 - 123, XP093006354, ISSN: 1084-9785, Retrieved from the Internet <URL:https://www.liebertpub.com/doi/pdf/10.1089/cbr.2017.2401> DOI: 10.1089/cbr.2017.2401 *
MAINA ESTHER N. ET AL.: "Identification of novel VHL target genes and relationship to hypoxic response pathways", ONCOGENE, NATURE PUBLISHING GROUP UK, LONDON, vol. 24, no. 28, 11 April 2005 (2005-04-11), pages 4549 - 4558, XP037738334, ISSN: 0950-9232, [retrieved on 20050411], DOI: 10.1038/SJ.ONC.1208649 *

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