CN115247176B - Long-chain non-coding RNA and application thereof - Google Patents

Long-chain non-coding RNA and application thereof Download PDF

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CN115247176B
CN115247176B CN202210046462.1A CN202210046462A CN115247176B CN 115247176 B CN115247176 B CN 115247176B CN 202210046462 A CN202210046462 A CN 202210046462A CN 115247176 B CN115247176 B CN 115247176B
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liver cancer
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CN115247176A (en
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张金平
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First Affiliated Hospital of Zhengzhou University
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Abstract

The invention discloses application of long-chain non-coding RNA in preparing a reagent for inhibiting liver cancer proliferation and metastasis. Based on the long-chain non-coding RNA molecules with differential expression in liver cancer cells and tissues beside the cancer, the long-chain non-coding RNA is found to have differential expression of LINC00376. The potential target gene of miR-488-3p possibly LINC00376 is predicted by miRBase and confirmed by luciferase experiment. By further converting the long-chain non-coding RNA LINC00376 and miR-488-3p into liver cancer cells, the proliferation of the liver cancer cells can be promoted, and the proliferation of the liver cancer cells can be inhibited by converting the interfering molecules of the long-chain non-coding RNA LINC00376 and the inhibitor of the miR-488-3p into the liver cancer cells. Therefore, the invention discovers a novel composition capable of regulating and controlling the proliferation or metastasis of liver cancer cells, and provides novel evidence for further treating liver cancer and researching pathogenesis of the liver cancer.

Description

Long-chain non-coding RNA and application thereof
Technical Field
The invention relates to the field of tumor cell biology, in particular to a long-chain non-coding RNA LINC00376 and application thereof.
Background
Liver cancer is one of the most common malignant tumors of the digestive system, and the death rate related to the liver cancer is high due to the characteristics of low early diagnosis rate, poor late prognosis and the like. As one of the tumors with high morbidity and mortality, primary liver cancer requires a timely and effective treatment regimen. Primary liver cancer mainly includes 3 different pathological types of hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and HCC-ICC mix, among which hepatocellular carcinoma is the most common pathological type in liver cancer, and is considered to be the main cause of death in liver cancer patients. Hepatocellular carcinoma (hepatocellular carcinoma, HCC) is one of the most common malignant tumors worldwide today and one of the major causes of death in human cancer patients, of which more than half occur in china. Although liver transplantation and hepatectomy are currently the two most prominent treatments for HCC, the survival rate of patients 5 years after surgery is still relatively low, and the recurrence rate is high, with no significant improvement in the long-term efficacy. Because HCC is asymptomatic or atypical in early stages and progresses rapidly, most patients are diagnosed as already in advanced cancer. Because of the incomplete understanding of HCC pathogenesis, patients are difficult to diagnose and treat effectively in early stages, and there is an urgent need to study the molecular mechanisms of HCC development in depth, seeking targets for early diagnosis or accurate treatment of HCC.
Although a large number of tumor-associated genes have been discovered, their underlying molecular mechanisms of action have not been fully elucidated. Non-coding RNAs (ncrnas) have been shown to play a critical role in various diseases, particularly in the development of tumors. Ncrnas are RNAs that do not encode any protein, and can be divided into two classes by length: long non-coding RNAs (LncRNA) and short non-coding RNAs (micrornas, mirnas). LncRNA is ncRNA that is more than 200 nucleotides in length and is widely present during genome transcription. Research shows that miRNA can directly target the 3' -UTR end of a gene and inhibit transcription of the target gene. The lncRNA can be combined with more miRNAs through the action of molecular sponge, so that the inhibition of the miRNAs on the downstream target genes is relieved, and the target genes are promoted to play a role. Recent studies have found that lncRNA plays a variety of different biological functions in HCC and is therefore considered a novel regulator in HCC biological processes, providing a new direction for diagnosis and treatment of HCC.
MiRNA, one of the species in ncRNA, has also been widely studied by scholars and has been shown to play an important role in the development of HCC. Recent studies have shown that there is a regulatory relationship between lncRNA and miRNA. LncRNA can act as a competitive endogenous RNA (competing endogenous RNA, ceRNA) to regulate expression of miRNA and its biological functions, competitively adsorb miRNA and regulate target gene expression downstream thereof.
To date, many oncogenes have been identified as targets of HCC-associated lncRNA. For example, lncRNAs can inhibit proliferation and metastasis of cancer cells by acting on related genes such as HBx, AKT1, GSK3B Cdc25A, E2F1, hnRNP U, PCAF, GF, PCAF, DMR, ICR, IGF2, and p53, etc. By regulating these related genes, lncRNAs can effectively play a role in regulating HCC tumorigenesis and development. However, the number of known target genes increases rapidly, indicating a complex regulatory network of lncRNAs. In general, lncRNAs can affect the behavior of almost all central laws within a cell, from transcription to translation to affecting protein function, through a variety of mechanisms including regulation of epigenetic changes, binding and regulation of RNA, DNA and protein, regulation of genetic variation, and the like.
Therefore, the search of new lncRNA molecules capable of regulating proliferation, migration and metastasis of HCC cells is helpful for further understanding of the pathogenesis of HCC, and has important female significance for deeply researching the molecular mechanisms of HCC occurrence and development and seeking targets for early diagnosis or accurate treatment of HCC.
Disclosure of Invention
The invention aims to provide a LINC00376 of long-chain non-coding RNA and application thereof, which are used for solving the problems of the prior art, and can obviously inhibit the growth and proliferation of tumor cells by reducing the expression of the long-chain non-coding RNA, wherein the LINC00376 is a potential inhibitor of various cancer cells and is expected to be used for treating cancers.
In our earlier experiments, it has been found that there are differential expression of a variety of long non-coding RNAs in hepatoma cells and paracancerous tissues.
The invention discloses application of long-chain non-coding RNA in preparation of a reagent for inhibiting proliferation and metastasis of liver cancer, wherein the long-chain non-coding RNA is LINC00376.
The invention discloses an application of long-chain non-coding RNA in preparing a reagent for diagnosing liver cancer, wherein the long-chain non-coding RNA is LINC00376.
Preferably, the long-chain non-coding RNA realizes proliferation and metastasis of liver cancer through combination with miR-488-3p.
The invention discloses application of a long-chain non-coding RNA and miR-488-3 as a combination in preparation of a reagent for inhibiting liver cancer proliferation and metastasis, wherein the long-chain non-coding RNA is LINC00376.
The invention discloses a composition, which comprises long-chain non-coding RNA LINC00376 and miR-488-3p.
Preferably, the coding sequence of the long-chain non-coding RNA LINC00376 is shown in SEQ ID NO. 1, and specifically comprises the following steps:
acaattagactccaccaatgtcagactgctaaaatgtaaaaaatggccatcaggattttctagggcagcataattattctgcgagtaggtaaaggagttttgattgagatgaagaggtcatagaagtgagaagcactgaaagacacagaagtttgttccttccagtgggttcgtggtctcactgacttcaagaatgaagccacggaccctcgcgaaaggaccagaaccaggggcaaaatctcacagctgcagtatcatttaagactcagagacagcctatccttctgggcaaaggggctgaaaagaaggaccctgtgatccaaagagttggggaattcttcggatccgtattgaagtgattcagctagaaattgataatcttcattttaacactgctctgagttaatggctgctcttgaaaaggtattgaatccacctggaatttccttgtgtttatggttcaagctgttcgtgagtattataccacttgacgactgaggcagaatcaaagaaagtactaatgctcttcagtaaaatatacaaaactacagacaatggcagtgccttattttgtaaagtatatagctatgagcctttgctactccttgctgacatttggaatttaactgaagtgtcttgttaaaaaacatttatggaaataaaaagaatatctttcctttttagggatataaa。
the invention discloses an siRNA molecule, which interferes with long-chain non-coding RNA LINC00376, and the sequence of the siRNA is shown as SEQ ID NO. 7.
Preferably, the siRNA has the sequence 5'gcuucauucu ugaagucagu3'.
The invention discloses an application of the composition and/or the siRNA molecule in preparing a reagent for inhibiting liver cancer proliferation and metastasis.
Preferably, the liver cancer is HepG2 cells.
The invention discloses a kit, which comprises the composition and/or the siRNA molecule.
Preferably, the sequence of the miR-488-3p is shown as SEQ ID NO. 6.
Preferably, the sequence of miR-488-3p is 5 'UUGAAAGGCUAUUUUCUUGGUC 3'.
The invention discloses a primer sequence of an amplified gene LINC00376, which comprises the following steps:
an upstream primer: 5 'acaattagac tccaccaa' as shown in SEQ ID NO. 2;
a downstream primer: 5 'tttatatccctaaaaagg' as shown in SEQ ID NO. 3.
The invention discloses an amplification primer sequence of an internal reference gene GAPDH, which comprises the following steps:
an upstream primer: 5'-GTCAGCCGCATCTTCTTTTG-3', as shown in SEQ ID NO. 4;
a downstream primer: 5'-GCGCCCAATACGACCAAATC-3', as shown in SEQ ID NO. 5.
The invention predicts that miR-488-3p is a potential target gene of LINC00376 through miRBase based on long-chain non-coding RNA molecules with differential expression in liver cancer cells and tissues beside cancer, and is verified by luciferase experiments. By further transforming long-chain non-coding RNA LINC00376 and miR-488-3p into liver cancer cells, the proliferation of liver cancer cells can be promoted, and the proliferation of liver cancer cells can be inhibited by transforming interfering molecules of long-chain non-coding RNA LINC00376 and inhibitors of miR-488-3p into liver cancer cells. Therefore, the invention discovers a novel molecular composition capable of regulating and controlling proliferation or metastasis of liver cancer cells, and provides novel evidence for further treating liver cancer and researching pathogenesis of the liver cancer.
Drawings
FIG. 1 is a graph of trypan blue staining of HepG2 cells.
FIG. 2 is a diagram of RT-PCR expression of LINC00376 in HepG2 cells and paracancerous tissues.
FIG. 3 is a predicted map of LINC00376 and miR-488-3p binding sites.
FIG. 4 is a luciferase assay in which LINC00376 binds to miR-488-3p.
FIG. 5 is a proliferation assay of HepG2 cells transformed by LINC00376 molecule and miR-488-3p.
FIG. 6 is a proliferation assay of HepG2 cells transformed with interfering siRNA of LINC00376 molecule and inhibitor of miR-488-3p.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Example 1 culture and enumeration of HCC cell line HepG2
The human HCC cell line HepG2 was kept for the family room. The culture conditions are as follows: cells were added to RPMI1640 medium containing 10% Fetal Bovine Serum (FBS) and incubated in a 5% co2, 37 ℃ incubator.
The cell counting operation is as follows: and (5) turning on the ultraviolet lamp of the safety cabinet in advance and irradiating for 30min for disinfection. Observing that cells are in a good logarithmic growth phase, collecting the cells and adding 1ml of culture medium, gently blowing and mixing uniformly, adding 10 mu l of trypan blue dye solution into a new EP tube (1.5 ml tube), adding 10 mu l of cell suspension into the EP tube, blowing and mixing uniformly, immediately taking 10 mu l of mixed solution, adding to a counting plate, and finally detecting and counting by using an automatic cell counter. The procedure was repeated 3 times and the average was taken.
The cultured cell line HepG2 was stained with trypan blue as shown in fig. 1.
Example 2 expression of the Long non-coding RNA LINC00376 in cell lines HepG2 and paracancerous tissues
The total RNA of the cell line HepG2 and the paracancerous tissue is respectively extracted and prepared by the Trizol method. TaKaRa PrimeScript kit (TaKaRa, dalian, china) was reversed to prepare cDNA. The analysis of the relative expression level of the target gene LINC00376 was performed by using a relative amount method and an internal reference GAPDH.
The primer sequence of the amplified gene LINC00376 is as follows:
an upstream primer: 5 'acaattagac tccaccaa' as shown in SEQ ID NO. 2;
a downstream primer: 5 'tttatatccctaaaaagg' as shown in SEQ ID NO. 3.
The amplification primer sequence of the reference gene GAPDH is as follows:
an upstream primer: 5'-GTCAGCCGCATCTTCTTTTG-3', as shown in SEQ ID NO. 4;
a downstream primer: 5'-GCGCCCAATACGACCAAATC-3', as shown in SEQ ID NO. 5.
Real time PCR was performed using SYBR Green Realtime PCR Master Mix kit instructions. Firstly, preparing a 20 mu l reaction system: 1. Mu.l of upstream specific PCR primer, 1. Mu.l of downstream specific PCR primer, 2. Mu.l of cDNA template, SYBR Green Real time PCR Master Mix. Mu.l of RNase-FreeH2O 6. Mu.l.
Reaction conditions: denaturation at 94℃for 30 s; 94 ℃ for 15s;58 ℃,60 seconds, 35 cycles of reaction.
The results of RT-PCR of expression of LINC00376 in cell lines HepG2 and paracancestor tissues are shown in FIG. 2. It can be seen that the expression of LINC00376 was higher in the cell line HepG2 than in the paracancerous tissue.
Example 3 prediction and validation of LINC00376 action target
And predicting a potential target gene of miR-488-3p by adopting miRBase, wherein the sequence of miR-488-3p is 5 'UUGAAAGGCUAUUUUCUUGGUC 3', and is shown as SEQ ID NO. 6. The results show that the region of LINC00376 has sites that form complementary binding with miR-488-3p (FIG. 3).
Nucleotide mutations were performed on the binding site portion in LINC00376 (fig. 3), and luciferase reporter experiments were performed. The pmiR-RB-REPORT ™ dual-luciferase REPORT vector uses a Renilla luciferase gene (hRluc) as REPORT fluorescence, a firefly luciferase gene (hluc) as correction fluorescence, a wild-type and mutant LINC00376 gene sequence is cloned to the downstream of the hluc gene, miRNA acts on a target gene through a region, so that the miRNA and the constructed REPORT gene vector are co-transferred, and the interaction between the miRNA and the target gene is verified through the down regulation of the relative fluorescence value of the REPORT gene.
Construction of pMIR-LINC 00376-wt (expression of wild-type LINC 00376) and pMIR-LINC 00376-Mut (expression of mutant LINC 00376) plasmids T24 cells were transformed and miR-488-3p mimetic and empty vector miR-Nc were further transformed as controls, medium was aspirated after 48h of transfection, 35. Mu.L/well of luciferagent was added, shaking was performed for 10min, transferred to LUMITRAC ™ and 200 well white cell culture plates, and fluorescence values were determined. As a result, miR-488-3p was found to significantly inhibit the wild-type pMIR-LINC 00376-wt cells, while pMIR-LINC 00376-Mut had no significant effect on inhibition of luciferase activity (FIG. 4). The downstream target molecule indicating the action of LINC00376 molecule might be miR-488-3p.
EXAMPLE 4 proliferation assay of HepG2 cells transformed by LINC00376 molecule and miR-488-3p
The plasmid molecules pcDNA-LINC00376, empty vector and miR-488-3p for constructing and expressing LINC00376 are respectively transformed into HepG2 cells, and simultaneously the plasmid molecules pcDNA-LINC00376 and miR-488-3p are transformed into the plasmid molecules, so that the cells are subjected to cell proliferation experiments, are cultured in a 6-hole plate for 9 days, the culture solution is replaced every three days, and the cells are counted after being dyed by trypan blue. Wherein, the cell count of the transformed corresponding vector was compared with HepG2 blank cells with the cell count of the originally cultured HepG2 as 1. The counting result is shown in fig. 5. When we over-expressed LINC00376 molecule and miR-488-3p in the cell, the proliferation capacity of HepG2 cell line was significantly improved.
EXAMPLE 5 proliferation assay of HepG2 cells transformed by interfering siRNA of LINC00376 molecule and inhibitor of miR-488-3p
The corresponding siRNA is designed according to the sequence of LINC00376 molecule, the sequence is 5'gcuucauucu ugaagucagu' and is shown as SEQ ID NO. 7. An inhibitor molecule of miR-488-3p is designed, the sequence of the inhibitor molecule is 5'gaccaagaaaauagccuuucaa 3', the sequence is shown as SEQ ID NO. 8, and the base of the sequence is subjected to 2' -O-methyl modification. siRNA of LINC00376 molecule and inhibitor molecule of miR-488-3p are respectively transformed into HepG2 cells, cell proliferation experiments are carried out, the cells are cultured in a 6-hole plate for 9 days, culture solution is replaced every three days, and the cells are counted after trypan blue staining. Wherein, the cell count of the transformed corresponding vector was compared with HepG2 blank cells with the cell count of the originally cultured HepG2 as 1. The counting result is shown in fig. 6. Proliferation potency of HepG2 cell lines was significantly down-regulated when we silenced LINC00376 molecules and miR-488-3p expression in cells by interference and inhibitors.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.
Sequence listing
<110> Zhengzhou university first affiliated hospital
<120> a long non-coding RNA and use thereof
<130> 2022
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acaattagac tccaccaatg tcagactgct aaaatgtaaa aaatggccat caggattttc 60
tagggcagca taattattct gcgagtaggt aaaggagttt tgattgagat gaagaggtca 120
tagaagtgag aagcactgaa agacacagaa gtttgttcct tccagtgggt tcgtggtctc 180
actgacttca agaatgaagc cacggaccct cgcgaaagga ccagaaccag gggcaaaatc 240
tcacagctgc agtatcattt aagactcaga gacagcctat ccttctgggc aaaggggctg 300
aaaagaagga ccctgtgatc caaagagttg gggaattctt cggatccgta ttgaagtgat 360
tcagctagaa attgataatc ttcattttaa cactgctctg agttaatggc tgctcttgaa 420
aaggtattga atccacctgg aatttccttg tgtttatggt tcaagctgtt cgtgagtatt 480
ataccacttg acgactgagg cagaatcaaa gaaagtacta atgctcttca gtaaaatata 540
caaaactaca gacaatggca gtgccttatt ttgtaaagta tatagctatg agcctttgct 600
actccttgct gacatttgga atttaactga agtgtcttgt taaaaaacat ttatggaaat 660
aaaaagaata tctttccttt ttagggatat aaa 693
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acaattagac tccaccaa 18
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tttatatccc taaaaagg 18
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gtcagccgca tcttcttttg 20
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uugaaaggcu auuuucuugg uc 22
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gcuucauucu ugaagucagu 20

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  1. The application of siRNA molecules in preparing a reagent for inhibiting HepG2 cell proliferation is characterized in that the sequence of the siRNA is shown as SEQ ID NO. 7.
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