CN114081896B

CN114081896B - Application of let-7i-3p in preparation of medicine for treating colorectal cancer and method

Info

Publication number: CN114081896B
Application number: CN202111387397.0A
Authority: CN
Inventors: 涂飞; 王庆志; 李梦凡; 陈音羽; 楚慧茹; 王姝洁; 海伦; 冯志伟; 赵铁锁; 谢婷; 耿芳芳
Original assignee: Xinxiang Medical University
Current assignee: Xinxiang Medical University
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2023-03-14
Anticipated expiration: 2041-11-22
Also published as: CN114081896A

Abstract

The invention relates to the fields of molecular biology and biomedicine, in particular to application of let-7i-3p in preparation of a medicine for treating colorectal cancer and a method thereof. The invention provides an application of any substance of the following substances 1) -2) in preparing a medicine for treating colorectal cancer: 1) let-7i-3p; 2) let-7i-3p expression promoter; the medicine can inhibit apoptosis, cell cycle, proliferation, migration and invasion of colorectal cancer cells by down-regulating CCND1, and the research result provides a new idea for treating colorectal cancer.

Description

Application of let-7i-3p in preparation of medicine for treating colorectal cancer and method

Technical Field

The invention relates to the fields of molecular biology and biomedicine, in particular to application of let-7i-3p in preparation of a medicine for treating colorectal cancer and a method thereof.

Background

Colorectal cancer (CRC) poses a serious threat to human life and health, as it is the third most common cancer and the fourth most cancer cause of death worldwide. Patients with advanced colon cancer cannot receive surgical treatment due to the development of liver and lung metastases. Therefore, there is a need to elucidate the pathogenesis and underlying molecular mechanisms of CRC tumor metastasis, which helps to find potential therapeutic targets for CRC.

MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs with a regulatory effect in eukaryotes, and are about 20 to 25 nucleotides in length. miRNAs can down-regulate the expression of target genes by cleavage or translational inhibition of mRNA. In recent years, numerous studies have shown that miRNAs are involved in a variety of cellular processes, such as the regulation of cell proliferation, invasion, migration and cell cycle progression. For example, miR-BART10-3p directly targets DKKl to regulate the proliferation and migration of EBVaGC cells. LncRNADST 1-AS1 regulates cell proliferation, migration, invasion and apoptosis by regulating miR-605-3 p. Transient activation of miR-294 results in low cardiac muscle cell cycle reactivation. let-7i down-regulates GREBI to inhibit the progression of esophageal cancer. let-7i inhibits invasion and metastasis of gastric cancer by targeting COL1 Al. However, the molecular mechanism and specific biological function of let-7i-3p in colorectal cancer remains largely unknown.

Cyclin D1 is encoded by the CCND1 gene, is a promoter of the cell cycle, and is involved in tumorigenesis of many cancers. Previous studies have shown that high levels of CCND1 are associated with a poor prognosis for patients with colorectal cancer. Therefore, understanding the regulatory mechanisms of CCND1 may help to develop strategies for treating colorectal cancer cell migration and invasion.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of let-7i-3p in preparing a medicament for treating colorectal cancer and a method for inhibiting the development of colorectal cancer cells.

Based on the above purpose, the invention provides an application of let-7i-3p in preparing a medicine for treating colorectal cancer, wherein the nucleotide sequence of let-7i-3p is shown in SEQ ID NO. 1. Further, the treatment includes inhibiting cell cycle, proliferation, invasion and migration of colorectal cancer cells.

Based on the same inventive concept, the invention also provides application of the let-7i-3p expression promoter in preparing a medicine for treating colorectal cancer.

Further, the medicament treats the colorectal cancer by down-regulating CCND1 gene and/or expression product thereof.

Based on the same inventive concept, the invention also provides a medicine for treating colorectal cancer, which comprises the let-7i-3p or the expression promoter thereof.

The invention also provides a method for inhibiting the development of colorectal cancer cells for non-diagnosis and treatment purposes, which is characterized in that the let-7i-3p or the expression promoter thereof is introduced into the developing colorectal cancer cells;

further, the inhibition is in vitro inhibition, and the development comprises cell cycle, proliferation, invasion and migration.

Further, the colorectal cancer cell is a HCT116 cell.

The invention has the beneficial effects that:

the invention discovers for the first time that let-7i-3p can inhibit apoptosis, cell cycle, proliferation, migration and invasion of colorectal cancer cells by down-regulating the CCND1 gene and/or expression products thereof, and the effect is better than that of the CCND1 gene which is directly interfered by siRNA, and the experimental result provides a new idea for treating colorectal cancer.

Drawings

FIG. 1 shows the differential expression of let-7i-3p and CCND1 in CRC and FHC cells; a is the expression level of let-7i-3p, B is the expression level of CCND1 gene, C is a CCND1 protein band detected by Western Blot, and D is the expression level of CCND1 protein.

FIG. 2 is a graph of let-7i-3p expression (A) in HCT116 cells transfected with let-7i-3p mimics and negative control NC, as well as the effects on apoptosis (B), cell cycle (C), proliferation (D), migration (E) and invasion (F) of HCT116 cells.

FIG. 3 shows the effect of let-7i-3p on the CCND1 gene and its encoded protein; a is a pmirGLO-CCND1 Wild Type (WT) and Mutant (MUT) 3-UTR dual-luciferase reporter recombinant plasmid, B is the relative luciferase activity of HCT116 cells co-transfected with the recombinant plasmid and let-7i-3p, C is the relative luciferase activity of 293T cells co-transfected with the recombinant plasmid and let-7i-3p, D is the expression level of CCND1 gene in HCT116 cells co-transfected with the recombinant plasmid and let-7i-3p, and E is the expression level of CCND1 protein in HCT116 cells co-transfected with the recombinant plasmid and let-7i-3p.

Fig. 4 shows the expression level of CCND1 gene (a) in HCT116 cells knocking down CCND1, and the effects on apoptosis (B), cell cycle (C), proliferation (D), migration (E) and invasion (F) of HCT116 cells.

FIG. 5 shows the CCND1 gene expression level (A) and CCND1 protein expression level in HCT116 cells overexpressing CCND1, and the effects on HCT116 cell cycle (B), proliferation (C), migration (D), and invasion (E).

FIG. 6 shows the effect of let-7i-3p and CCND1 on the ERK signal path; a is a Western Blot detection band of Erk1/2, p-Erk1/2 and alpha-tubulin protein after transfection of HCT116 cells with let-7i-3p and NC; b is the expression level of p-Erk1/2 after the let-7i-3p and NC transfect HCT116 cells; c is the expression level of p-Erk1/2 after HCT116 cells are transfected by let-7i-3p and NC; d is a Western Blot detection band of p-Erk1/2 and GAPDH protein after siCCND1 and NC transfect HCT116 cells; e is the expression level of p-Erk1/2 after transfection of siCCND1 and NC on HCT116 cells, F is the Western Blot detection band of p-Erk1/2 and alpha-tubulin protein after transfection of pcDNA3.1 and pcDNA3.1-CCND1 on HCT116 cells; f is the expression level of p-Erk1/2 after transfection of HCT116 cells with pcDNA3.1 and pcDNA3.1-CCND 1.

Detailed Description

The invention is described in detail below with reference to the drawings and specific examples, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.

Materials and methods

Cell lines and cell culture human intestinal cancer cell lines SW480, HCT116, loVo, RKO, HT29, normal colonic epithelial cell lines FHC and 293T were all from the Chinese academy of sciences (Shanghai, china). All cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, USA) containing 10% Fetal Bovine Serum (FBS) and 1% double antibody (100U/ml penicillin and 100mg/ml streptomycin.) they contained 5% CO ₂ And incubated at 37 ℃ in a humid atmosphere.

The oligonucleotide transfection let-7i-3p mimics (named let-7i-3 p), the negative control double strand (named NC) and the siRNA (named siCCNDl) resisting CCND1 are synthesized by the company Jima (Shanghai, china) for transfection. Cells were transfected using liposome 3000 reagent (Invitrogen, carlsbad, CA, USA) according to the product instructions. These sequences are listed in table 1.

TABLE 1 oligonucleotide sequences

^a F, an upstream primer; r, a downstream primer.

Example 1: differential expression of let-7i-3p and CCND1 in CRC and FHC cells

1. Experimental methods

The method comprises the steps of detecting the expression quantity of let-7i-3p and CCND1 genes in human colorectal cancer cell lines (HCT 116, SW480 and LoVo) and normal colon epithelial cells FHC by utilizing a qRT-PCR technology;

the expression level of the CCND1 protein in human colorectal cancer cell lines (HCT 116, SW480, loVo, RKO and HT 29) and normal colon epithelial cell FHC is detected by using a Western-bolt technology.

2. Results

To confirm that let-7i-3p is aberrantly expressed in CRC cells, we analyzed the expression levels of let-7i-3p in 3 CRC cells (HCT 116, SW480, loVo) and normal colonic epithelial cell line (FHC) (FIG. 1A). Our qRT-PCR results show a significant reduction in let-7i-3p expression in CRC cells compared to FHC cells. Considering that let-7i-3p mainly acts through its target gene, we screened potential target genes through targetScan 7.2. Combined with data analysis and literature reviews, it is predicted that CCND1 may be the target gene for let-7i-3p. To evaluate the relationship of let-7i-3p to CCND1 in colorectal cancer. Similarly, we also tested the expression level of CCND1 in three CRC cells and FHC. As shown in fig. 1B, expression of CCND1 was up-regulated in CRC cells compared to FHC cells. The expression of CCND1 protein in CRC cells and FHC was detected by immunoblotting. The results show that the relative expression levels of CRC cells (HCT 116, SW 480) are significantly up-regulated compared to FHC cells (fig. 1C and 1D). These results further support CCND1 as a potential target gene for let-7i-3p regulation.

Example 2: potential role of let-7i-3p in CRC cells

1. Method of producing a composite material

The method comprises the steps of transfecting let-7i-3p mimics and negative control NC in six-hole plate HCT116 cells by using a liposome lipo3000 according to the operation of an instruction, collecting cell RNA after 24 hours, carrying out reverse transcription on the cell RNA to form cDNA, and detecting the transfection efficiency of the let-7i-3p by using qRT-PCR;

transfecting cells according to the method, collecting cell suspension after 48 hours, fixing the cells, dyeing and mechanically detecting apoptosis according to the operation of an apoptosis detection kit instruction;

thirdly, transfecting cells according to the method, collecting cell suspension after 48 hours, fixing the cells according to the operation of the cycle detection kit instruction, dyeing and detecting the cell cycle on a computer;

fourthly, transfecting cells according to the method, collecting cell suspension after 48 hours, planting the cells in a 96-well plate with 4000 cells/well, detecting OD values by using CCK8 reagent after 24 hours, 48 hours, 72 hours, 96 hours and 120 hours, planting the cells in 800 cells/well and 6-well plate, changing liquid after one week, fixing the cells by using 4% formaldehyde after 2 weeks, then staining the cells by using crystal violet, photographing and detecting the formation of cell colonies, thereby judging the cell proliferation condition;

carrying out transfection on cells according to the method, collecting cell suspension after 48 hours, and carrying out transfection on the cells according to the method of 3X 10 ⁵ After 24 hours, scratching is carried out on each cell/hole by using a 100ul gun head, a serum-free culture medium is replaced, the scratch width is recorded by taking a picture under a microscope, and then, the scratch width is recorded at the same interval of 24 hours for 3 times continuously, so that the migration change of the cells is judged;

sixthly, transfecting cells according to the method, collecting cell suspension after 48 hours, and adjusting the density of the cell suspension to be 1 × 10 ⁵ And (2) each cell/ml, adding 200ul of serum-free cell suspension into an upper chamber of a 24-well plate chamber, adding 600ul of complete culture medium into a lower chamber, taking out the chamber after 24 hours, washing the chamber by PBS, fixing by 4% formaldehyde, staining by crystal violet, and observing the number of cells penetrating through a membrane at the bottom of the chamber under a microscope, thereby judging the invasion condition of the cells.

2. Results

HCT116 was transfected with let-7i-3p or NC. After transfection, let-7i-3p transfection significantly increased expression of let-7i-3p compared to non-transfected HCT116 (FIG. 2A). To verify the biological function of let-7i-3p in HCT116 cells, we used CCK8, colony formation assay, flow, scratch migration, and transwell assay. The Annexin-V/7-AAD staining of the kit is not different, which shows that let-7i-3p has no influence on apoptosis (FIG. 2B). However, in cells overexpressing let-7i-3p, G1 phase cells were significantly increased and S phase cells were significantly decreased compared to the control group (fig. 2C). Furthermore, CCK-8 and colony formation experiments showed that the proliferative capacity of HCT116 cells transfected with let-7i-3p was significantly inhibited (FIG. 2D). Furthermore, as shown in FIGS. 2E-2F, CRC cells overexpressing let-7i-3p had significantly wider wound widths compared to control cells. Consistent with the data of the wound healing experiment, the transwell experiment result shows that over-expression of let-7i-3p inhibits the invasion capacity of HCT116 cells. Taken together, these data indicate that let-7i-3p overexpression inhibits cell cycle, proliferation, migration and invasion, but has no effect on apoptosis.

Example 3: CCND1 is a direct target of let-7i-3p

1. Method of producing a composite material

The method comprises the steps of utilizing a biological informatics software TargetScan Human7.2 and RNA-hybrid to predict binding sites of let-7i-3p and CCND1, mutating a seed sequence to form a mutant target gene;

constructing two recombinant plasmids of a pmirGLO-CCND1 wild type and a mutant type by utilizing a molecular cloning technology, and respectively co-transforming HCT116 cells and 293T cells with let-7i-3p; after 24 hours, collecting cells, and detecting enzyme activity by using a dual-luciferase report system kit;

thirdly, transfecting HCT116 cells according to the method, collecting RNA extracted from the cells after 24 hours, collecting protein extracted from the cells after 48 hours, and detecting the change of the expression level of the CCND1 gene and the protein after transfection by qRT-PCR and Western-blot respectively.

2. As a result, the

The bioinformatics websites TargetScan Human7.2 and RNA-hybrid were used to analyze the let-7i-3p potential target genes. We constructed Wild Type (WT) or Mutant (MUT) 3-UTR dual-luciferase reporter recombinant plasmids of pmirGLO-CCND1 (FIG. 3A). The results show that after co-transfection of lel-7i-3p, the fluorescence ratios in both HCT116 and 293T cells were significantly reduced for the Wild Type (WT) group, while the fluorescence ratios in Mutant (MUT) group cells were not significantly changed (FIGS. 3B and 3C). To verify that CCND1 is the true downstream target of lel-7i-3p, we detected the expression of let-7i-3p on CCND1 by qRT-PCR and Western-blot techniques. As shown in fig. 3D and 3E. let-7i-3p significantly inhibited the mRNA and protein expression levels of CCND 1. These data indicate that CCND1 is a direct target for let-7i-3p.

Example 4: knockdown of CCND1 inhibits cell cycle, proliferation, migration and invasion in HCT116

1. Method for producing a composite material

The method comprises the steps of transfecting siCCND1 and negative control NC in six-hole plate HCT116 cells by using a liposome lipo3000 according to the operation of an instruction, extracting cell RNA after 24 hours, extracting protein after 48 hours, and detecting the knocking efficiency of the siCCND1 by using qRT-PCR and Western-blot;

transfecting siCCND1 and NC with cells according to the method, collecting cell suspension after 48 hours, operating according to the cycle detection kit specification, fixing the cells, dyeing and detecting the cell cycle on a computer;

thirdly, transfecting cells according to the method, collecting cell suspension after 48 hours, planting the cells in a 96-well plate according to 4000 cells/well, detecting OD values by using a CCK8 reagent after 24 hours, 48 hours, 72 hours, 96 hours and 120 hours, planting the cells in 800 cells/well and a 6-well plate, changing liquid after one week, fixing the cells by using 4% formaldehyde after 2 weeks, then dyeing by using crystal violet, and photographing to detect the formation of cell colonies, thereby judging the cell proliferation condition;

fourth, cells were transfected according to the above method, and 48 hours later, a cell suspension was collected and pressed3×10 ⁵ After 24 hours, scratching is carried out on each cell/hole by using a 100ul gun head, a serum-free culture medium is replaced, the scratch width is recorded by taking a picture under a microscope, and then, the scratch width is recorded at the same interval of 24 hours for 3 times continuously, so that the migration change of the cells is judged;

carrying out transfection on the cells according to the method, collecting cell suspension after 48 hours, and adjusting the density of the cell suspension to be 1 × 10 ⁵ And (2) each cell/ml, adding 200ul of serum-free cell suspension into an upper chamber of a 24-well plate chamber, adding 600ul of complete culture medium into a lower chamber, taking out the chamber after 24 hours, washing the chamber by PBS, fixing by 4% formaldehyde, staining by crystal violet, and observing the number of cells penetrating through a membrane at the bottom of the chamber under a microscope, thereby judging the invasion condition of the cells.

2. Results

siCCNDl was transfected in HCT116 cells. Silencing of CCND1 was confirmed by qRT-PCR and WB. The results showed that HCT116 cells were able to efficiently knock down the expression of CCND1 mRNA and protein (fig. 4A and 4B). To further investigate the role of CCND1 in HCT116 cells, the role of siCCNDl in controlling cell proliferation (fig. 4C), cell cycle (fig. 4D), migration (fig. 4E) and invasion (fig. 4F) was analyzed. The results show that silencing of CCND1 results in significant inhibition of cell proliferation, migration and invasion, in a pattern similar to that of let-7i-3p overexpression.

Example 5: CCND1 overexpression reverses the effect of let-7i-3p on HCT116 cells

1. Method of producing a composite material

The method comprises the steps of constructing pcDNA3.1-CCND1 recombinant plasmids by using a molecular cloning technology, transfecting HCT116 cells, extracting cell RNA after 24 hours, extracting protein after 48 hours, and detecting the over-expression efficiency of CCND1 by using qRT-PCR and Western-blot; transfecting let-7i-3p + pcDNA3.1-CCND1, let-7i-3p and NC with cells according to the method, collecting cell suspension after 48 hours, fixing the cells according to the operation of the cycle detection kit specification, dyeing and detecting the cell cycle on a computer;

thirdly, transfecting cells according to the method, collecting cell suspension after 48 hours, planting 96-well plates according to 4000 cells/well, detecting OD values by using a CCK8 reagent after 24 hours, 48 hours, 72 hours, 96 hours and 120 hours, planting the cells/well and 6-well plates, changing liquid after one week, fixing by using 4% formaldehyde after 2 weeks, then dyeing by using crystal violet, photographing and detecting the formation of cell colonies, and judging the cell proliferation condition;

fourth, cells were transfected according to the method described above, and after 48 hours, a cell suspension was collected and 3 × 10 cells were selected ⁵ After 24 hours, scratching is carried out on each cell/hole by using a 100ul gun head, a serum-free culture medium is replaced, the scratch width is recorded by taking a picture under a microscope, and then, the scratch width is recorded at the same interval of 24 hours for 3 times continuously, so that the migration change of the cells is judged;

2. Results

To confirm the function of CCND1 in CRC cells, we examined the effects of CCNDl overexpression on cell cycle, proliferation, migration, and invasion. We ectopically expressed CCND1 and let-7i-3p in HCT116 cells. QRT-PCR and Western-blot analysis showed significant increases in CCND1 mRNA and protein levels in pcDNA-CCNDl transfected HCT116 cells (FIGS. 5A and 5B). Furthermore, we performed CCK8 experiments, colony formation experiments, flow cytometry, transwell experiments, and scratch healing migration experiments in HCT116 cells. As expected, the results showed no significant difference between the pcDNA-CCND1+ let-7i-3p group and the control group (FIGS. 5C-5F). Taken together, these results indicate that let-7i-3p inhibits HCT116 cell cycle, proliferation, migration, and invasion by targeting CCND 1.

Example 6: let-7i-3p reduces the ERK signal path by turning CCND1 down

1. Method for producing a composite material

And transfecting let-7i-3p, NC, siCCND1, pcDNA3.1 and pcDNA3.1-CCND1 into cells according to the method, extracting protein after 48 hours, and detecting the expression levels of Erk1/2, p-Erk1/2, GAPDH and alpha-tubulin by using a Western-blot technique.

2. Results

To investigate the mechanisms by which let-7i-3p and CCND1 inhibit CRC cell cycle, proliferation, migration and invasion, we investigated whether these effects are mediated by activation of the ERK signaling pathway. And detecting the expression level and p-ERK of the CCND1 by using Western-blot. As shown in fig. 6A-6C. Over-expression of let-7i-3p resulted in a significant reduction of p-ERK in HCT116 cells compared to the control group. Without significant difference in total ERK expression. Similarly, the same results were obtained by down-regulating CCND1 expression in HCT116 cells (fig. 6D and 6E). We ectopically expressed CCND1 in HCT116 cells, significantly increased p-ERK compared to the control group (fig. 6F and 6G). These results indicate that CCND1 plays a catalytic role in the ERK signaling pathway. These results indicate that let-7i-3p targets CCND1 to regulate colorectal cancer cellular biological functions through the ERK signaling pathway.

Taken together, let-7i-3p acts as a tumor suppressor in the progression of colorectal cancer, which inhibits cell cycle, pro-proliferation, migration, and invasion of HCT116 cells. ERK signaling activity is inhibited by direct inhibition of CCND 1. In summary, we have determined the role and molecular mechanism of let-7i-3p in HCT116 cells, and let-7i-3p may be a very active target for future CRC therapy

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

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Claims

Application of let-7i-3p in preparing a medicine for treating colorectal cancer is characterized in that the nucleotide sequence of let-7i-3p is shown in SEQ ID NO. 1; the medicament treats the colorectal cancer by down-regulating CCND1 gene and/or expression products thereof.
2. Use according to claim 1, wherein the treatment is the inhibition of cell cycle, proliferation, invasion and migration of colorectal cancer cells.